WorldWideScience

Sample records for conferring kanamycin resistance

  1. Novel plasmid conferring kanamycin and tetracycline resistance in the turkey-derived Campylobacter jejuni strain 11601MD.

    Science.gov (United States)

    Crespo, M D; Altermann, E; Olson, J; Miller, W G; Chandrashekhar, K; Kathariou, S

    2016-07-01

    In Campylobacter spp., resistance to the antimicrobials kanamycin and tetracycline is frequently associated with plasmid-borne genes. However, relatively few plasmids of Campylobacter jejuni have been fully characterized to date. A novel plasmid (p11601MD; 44,095nt) harboring tet(O) was identified in C. jejuni strain 11601MD, which was isolated from the jejunum of a turkey produced conventionally in North Carolina. Analysis of the p11601MD sequence revealed the presence of a high-GC content cassette with four genes that included tet(O) and a putative aminoglycoside transferase gene (aphA-3) highly similar to kanamycin resistance determinants. Several genes putatively involved in conjugative transfer were also identified on the plasmid. These findings will contribute to a better understanding of the distribution of potentially self-mobilizing plasmids harboring antibiotic resistance determinants in Campylobacter spp. from turkeys and other sources. Copyright © 2016 Elsevier Inc. All rights reserved.

  2. Kanamycin resistance during in vitro development of pollen from transgenic tomato plants

    NARCIS (Netherlands)

    Bino, R.J.; Hille, J.; Franken, J.

    1987-01-01

    Effects of kanamycin on pollen germination and tube growth of pollen from non-transformed plants and from transgenic tomato plants containing a chimaeric kanamycin resistance gene were determined. Germination of pollen was not affected by the addition of kanamycin to the medium in both genotypes.

  3. Controversy Associated With the Common Component of Most Transgenic Plants – Kanamycin Resistance Marker Gene

    Directory of Open Access Journals (Sweden)

    Srećko Jelenić

    2003-01-01

    Full Text Available Plant genetic engineering is a powerful tool for producing crops resistant to pests, diseases and abiotic stress or crops with improved nutritional value or better quality products. Currently over 70 genetically modified (GM crops have been approved for use in different countries. These cover a wide range of plant species with significant number of different modified traits. However, beside the technology used for their improvement, the common component of most GM crops is the neomycin phosphotransferase II gene (nptII, which confers resistance to the antibiotics kanamycin and neomycin. The nptII gene is present in GM crops as a marker gene to select transformed plant cells during the first steps of the transformation process. The use of antibiotic-resistance genes is subject to controversy and intense debate, because of the likelihood that clinical therapy could be compromised due to inactivation of the oral dose of the antibiotic from consumption of food derived from the transgenic plant, and because of the risk of gene transfer from plants to gut and soil microorganisms or to consumer’s cells. The present article discusses these possibilities in the light of current scientific knowledge.

  4. Cross-resistance of Mycobacterium tuberculosis isolates among streptomycin, kanamycin and amikacin.

    Science.gov (United States)

    Sugawara, I; Zhang, J; Li, C

    2009-06-01

    Seventy-four streptomycin (SM)-resistant M. tuberculosis clinical isolates were subjected to cross-resistance drug testing against two major aminoglycosides, kanamycin (KM) and amikacin (AMK). Among them, 15 clinical isolates (20.3%) were resistant to both KM and AMK. Fifteen (80%) of 19 KM-resistant isolates were AMK-resistant. Fifteen SM, KM, and AMK resistant isolates harbored rrs mutation, but only two had rrs and rpsL double mutations. Low-level SM resistance was associated with rpsL mutation, whereas high-level SM resistance was linked to rrs mutation.

  5. Covalently linked kanamycin - Ciprofloxacin hybrid antibiotics as a tool to fight bacterial resistance.

    Science.gov (United States)

    Shavit, Michal; Pokrovskaya, Varvara; Belakhov, Valery; Baasov, Timor

    2017-06-01

    To address the growing problem of antibiotic resistance, a set of 12 hybrid compounds that covalently link fluoroquinolone (ciprofloxacin) and aminoglycoside (kanamycin A) antibiotics were synthesized, and their activity was determined against both Gram-negative and Gram-positive bacteria, including resistant strains. The hybrids were antagonistic relative to the ciprofloxacin, but were substantially more potent than the parent kanamycin against Gram-negative bacteria, and overcame most dominant resistance mechanisms to aminoglycosides. Selected hybrids were 42-640 fold poorer inhibitors of bacterial protein synthesis than the parent kanamycin, while they displayed similar inhibitory activity to that of ciprofloxacin against DNA gyrase and topoisomerase IV enzymes. The hybrids showed significant delay of resistance development in both E. coli and B. subtilis in comparison to that of component drugs alone or their 1:1 mixture. More generally, the data suggest that an antagonistic combination of aminoglycoside-fluoroquinolone hybrids can lead to new compounds that slowdown/prevent the emergence of resistance. Copyright © 2017 Elsevier Ltd. All rights reserved.

  6. Exogenous alanine and/or glucose plus kanamycin kills antibiotic-resistant bacteria.

    Science.gov (United States)

    Peng, Bo; Su, Yu-Bin; Li, Hui; Han, Yi; Guo, Chang; Tian, Yao-Mei; Peng, Xuan-Xian

    2015-02-03

    Multidrug-resistant bacteria are an increasingly serious threat to human and animal health. However, novel drugs that can manage infections by multidrug-resistant bacteria have proved elusive. Here we show that glucose and alanine abundances are greatly suppressed in kanamycin-resistant Edwardsiella tarda by GC-MS-based metabolomics. Exogenous alanine or glucose restores susceptibility of multidrug-resistant E. tarda to killing by kanamycin, demonstrating an approach to killing multidrug-resistant bacteria. The mechanism underlying this approach is that exogenous glucose or alanine promotes the TCA cycle by substrate activation, which in turn increases production of NADH and proton motive force and stimulates uptake of antibiotic. Similar results are obtained with other Gram-negative bacteria (Vibrio parahaemolyticus, Klebsiella pneumoniae, Pseudomonas aeruginosa) and Gram-positive bacterium (Staphylococcus aureus), and the results are also reproduced in a mouse model for urinary tract infection. This study establishes a functional metabolomics-based strategy to manage infection by antibiotic-resistant bacteria. Copyright © 2015 Elsevier Inc. All rights reserved.

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

    Science.gov (United States)

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

    2015-06-01

    Antibiotic-resistant bacteria are a great threat to human health and food safety and there is an urgent need to understand the mechanisms of resistance for combating these bacteria. In the current study, comparative proteomic methodologies were applied to identify Escherichia coli K-12 outer membrane (OM) proteins related to kanamycin resistance. Mass spectrometry and western blotting results revealed that OM proteins TolC, Tsx and OstA were up-regulated, whereas MipA, OmpA, FadL and OmpW were down-regulated in kanamycin-resistant E. coli K-12 strain. Genetic deletion of tolC (ΔtolC-Km) led to a 2-fold decrease in the minimum inhibitory concentration (MIC) of kanamycin and deletion of mipA (ΔmipA-Km) resulted in a 4-fold increase in the MIC of kanamycin. Changes in the MICs for genetically modified strains could be completely recovered by gene complementation. Compared with the wild-type strain, the survival capability of ΔompA-Km was significantly increased and that of Δtsx-Km was significantly decreased. We further evaluated the role and expression of MipA in response to four other antibiotics including nalidixic acid, streptomycin, chloramphenicol and aureomycin, which suggested that MipA was a novel OM protein related to antibiotic resistance. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  8. Sulfonamide-Based Inhibitors of Aminoglycoside Acetyltransferase Eis Abolish Resistance to Kanamycin in Mycobacterium tuberculosis

    Energy Technology Data Exchange (ETDEWEB)

    Garzan, Atefeh; Willby, Melisa J.; Green, Keith D.; Gajadeera, Chathurada S.; Hou, Caixia; Tsodikov, Oleg V.; Posey, James E.; Garneau-Tsodikova, Sylvie

    2016-12-08

    A two-drug combination therapy where one drug targets an offending cell and the other targets a resistance mechanism to the first drug is a time-tested, yet underexploited approach to combat or prevent drug resistance. By high-throughput screening, we identified a sulfonamide scaffold that served as a pharmacophore to generate inhibitors of Mycobacterium tuberculosis acetyltransferase Eis, whose upregulation causes resistance to the aminoglycoside (AG) antibiotic kanamycin A (KAN) in Mycobacterium tuberculosis. Rational systematic derivatization of this scaffold to maximize Eis inhibition and abolish the Eis-mediated KAN resistance of M. tuberculosis yielded several highly potent agents. A crystal structure of Eis in complex with one of the most potent inhibitors revealed that the inhibitor bound Eis in the AG-binding pocket held by a conformationally malleable region of Eis (residues 28–37) bearing key hydrophobic residues. These Eis inhibitors are promising leads for preclinical development of innovative AG combination therapies against resistant TB.

  9. Influence of tra genes of IncP and F plasmids on the mobilization of small Kanamycin resistance ColE1-Like plasmids in bacterial biofilms

    Science.gov (United States)

    Background: Horizontal gene transfer is a mechanism for movement of antibiotic resistance genes among bacteria. Some small kanamycin resistance (KanR) ColE1-like plasmids isolated from different serotypes of Salmonella enterica were shown to carry mobilization genes; although not self-transmissibl...

  10. Comparing amikacin and kanamycin-induced hearing loss in multidrug-resistant tuberculosis treatment under programmatic conditions in a Namibian retrospective cohort

    NARCIS (Netherlands)

    Sagwa, Evans L; Ruswa, Nunurai; Mavhunga, Farai; Rennie, Timothy; Leufkens, Hubert G M; Mantel-Teeuwisse, Aukje K

    2015-01-01

    BACKGROUND: Amikacin and kanamycin are mainly used for treating multidrug-resistant tuberculosis (MDR-TB), especially in developing countries where the burden of MDR-TB is highest. Their protracted use in MDR-TB treatment is known to cause dose-dependent irreversible hearing loss, requiring hearing

  11. Cloning and over expression of non-coding RNA rprA in E.coli and its resistance to Kanamycin without osmotic shock.

    Science.gov (United States)

    Sahni, Azita; Hajjari, Mohammadreza; Raheb, Jamshid; Foroughmand, Ali Mohammad; Asgari, Morteza

    2017-01-01

    Recent reports have indicated that small RNAs have key roles in the response of the E.coli to stress and also in the regulating of virulence factors. It seems that some small non-coding RNAs are involved in multidrug resistance. Previous studies have indicated that rprA can increase the tolerance to Kanamycin in RcsB-deficient Escherichia coli K-12 following osmotic shock. The current study aims to clone and over-express the non-coding RNA rprA in E.coli and investigate its effect on the bacterial resistance to Kanamycin without any osmotic shock. For this purpose, rprA gene was amplified by the PCR and then cloned into the PET-28a (+) vector. The recombinant plasmid was transformed into wild type E.coli BL21 (DE3). The over expression was induced by IPTG and confirmed by qRT-PCR. The resistance to the kanamycin was then measured in different times by spectrophotometry. The statistical analysis showed that the rprA can increase the resistance to Kanamycin in Ecoli K12. The interaction between rprA and rpoS was reviewed and analyzed by in silico methods. The results showed that the bacteria with over-expressed rprA were more resistant to Kanamycin. The present study is an important step to prove the role of non-coding RNA rprA in bacterial resistance. The data can be the basis for future works and can also help to develop and deliver next-generation antibiotics.

  12. Controversy Associated With the Common Component of Most Transgenic Plants – Kanamycin Resistance Marker Gene

    OpenAIRE

    Jelenić, Srećko

    2003-01-01

    Plant genetic engineering is a powerful tool for producing crops resistant to pests, diseases and abiotic stress or crops with improved nutritional value or better quality products. Currently over 70 genetically modified (GM) crops have been approved for use in different countries. These cover a wide range of plant species with significant number of different modified traits. However, beside the technology used for their improvement, the common component of most GM crops is the neomycin phosp...

  13. Metabolic pathway for poly(3-hydroxybutyrate-co-3-hydroxyvalerate) formation in Nocardia corallina: inactivation of mutB by chromosomal integration of a kanamycin resistance gene.

    Science.gov (United States)

    Valentin, H F; Dennis, D

    1996-02-01

    The gene encoding the large subunit of the methylmalonyl-coenzyme A (CoA) mutase in Nocardia corallina (mutBNc) was cloned. A 4.3-kbp BamHI fragment containing almost the entire mutBNc was identified by Southern hybridization experiments employing a digoxigenin-labeled probe deduced from mutB of Streptomyces cinnamonensis, mutBNc was interrupted by insertion of a kanamycin resistance gene block (mutB::kan or mutB::neo) and introduced into N. corallina to obtain mutB-negative strains by homologous recombination. Four of sixteen kanamycin-resistant clones occurred via double-crossover events and harbored only the interrupted mutBNc. These exhibited no growth on odd-chain fatty acids in the presence of kanamycin but exhibited wild-type growth on even-chain fatty acids, glucose, and succinate. Whereas the wild type of N. corallina accumulates a copolyester of 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV) containing more than 60 mol% 3HV from most carbon sources, mutB-negative strains accumulated poly(3HB-co-3HV) containing only 2 to 6 mol% 3HV. Methylmalonyl-CoA mutase activity was not found in these clones. Therefore, this study provides strong evidence that the majority of 3HV units in poly(3HB-co-3HV) accumulated by N. corallina are synthesized via the methylmalonyl-CoA pathway.

  14. CONFERENCE REPORT ANTIRETROVIRAL RESISTANCE

    African Journals Online (AJOL)

    2004-08-02

    Aug 2, 2004 ... had achieved full virological suppression (viral load. < 50 copies/ml). Thirty-two per cent of women had nevirapine-resistant virus. A novel strategy to avoid maternal exposure to nevirapine might be administering the drug to the newborn as post- exposure prophylaxis (PEP). Researchers from South Africa3.

  15. Immunoassay Analysis of Kanamycin in Serum Using the Tobramycin Kit

    NARCIS (Netherlands)

    Dijkstra, J. A.; Voerman, A. J.; Greijdanus, B.; Touw, D. J.; Alffenaar, J. W. C.

    Kanamycin is one of the aminoglycosides used in the treatment of multidrug-resistant tuberculosis. Blood concentrations of kanamycin are predictive for the treatment efficacy and the occurrence of side effects, and dose adjustments can be needed to optimize therapy. However, an immunoassay method

  16. Improved methods in Agrobacterium-mediated transformation of almond using positive (mannose/pmi) or negative (kanamycin resistance) selection-based protocols.

    Science.gov (United States)

    Ramesh, Sunita A; Kaiser, Brent N; Franks, Tricia; Collins, Graham; Sedgley, Margaret

    2006-08-01

    A protocol for Agrobacterium-mediated transformation with either kanamycin or mannose selection was developed for leaf explants of the cultivar Prunus dulcis cv. Ne Plus Ultra. Regenerating shoots were selected on medium containing 15 muM kanamycin (negative selection), while in the positive selection strategy, shoots were selected on 2.5 g/l mannose supplemented with 15 g/l sucrose. Transformation efficiencies based on PCR analysis of individual putative transformed shoots from independent lines relative to the initial numbers of leaf explants tested were 5.6% for kanamycin/nptII and 6.8% for mannose/pmi selection, respectively. Southern blot analysis on six randomly chosen PCR-positive shoots confirmed the presence of the nptII transgene in each, and five randomly chosen lines identified to contain the pmi transgene by PCR showed positive hybridisation to a pmi DNA probe. The positive (mannose/pmi) and the negative (kanamycin) selection protocols used in this study have greatly improved transformation efficiency in almond, which were confirmed with PCR and Southern blot. This study also demonstrates that in almond the mannose/pmi selection protocol is appropriate and can result in higher transformation efficiencies over that of kanamycin/nptII selection protocols.

  17. 35S Promoter Methylation in Kanamycin-Resistant Kalanchoe (Kalanchoe pinnata L.) Plants Expressing the Antimicrobial Peptide Cecropin P1 Transgene.

    Science.gov (United States)

    Shevchuk, T V; Zakharchenko, N S; Tarlachkov, S V; Furs, O V; Dyachenko, O V; Buryanov, Y I

    2016-09-01

    Transgenic kalanchoe plants (Kalanchoe pinnata L.) expressing the antimicrobial peptide cecropin P1 gene (cecP1) under the control of the 35S cauliflower mosaic virus 35S RNA promoter and the selective neomycin phosphotransferase II (nptII) gene under the control of the nopaline synthase gene promoter were studied. The 35S promoter methylation and the cecropin P1 biosynthesis levels were compared in plants growing on media with and without kanamycin. The low level of active 35S promoter methylation further decreases upon cultivation on kanamycin-containing medium, while cecropin P1 synthesis increases.

  18. Synthesis and antibacterial activity of 1-N-(1,3-dihydroxy-2-propyl)kanamycin B (UK-31,214).

    Science.gov (United States)

    Richardson, K; Brammer, K W; Jevons, S; Plews, R M; Wright, J R

    1979-10-01

    1-N-(1,3-Dihydroxy-2-propyl)kanamycin B was prepared and its in vitro activity against aminoglycoside-sensitive and aminoglycoside-resistant organisms was compared with that of kanamycin B and gentamicin. This kanamycin B derivative (code No. UK-31,214) demonstrated potent activity in all of these tests and gave good protection in experimental infections in mice.

  19. RNA-seq analysis of the effect of kanamycin and the ABC transporter AtWBC19 on Arabidopsis thaliana seedlings reveals changes in metal content.

    Directory of Open Access Journals (Sweden)

    Ayalew Mentewab

    Full Text Available Plants are exposed to antibiotics produced by soil microorganisms, but little is known about their responses at the transcriptional level. Likewise, few endogenous mechanisms of antibiotic resistance have been reported. The Arabidopsis thaliana ATP Binding Cassette (ABC transporter AtWBC19 (ABCG19 is known to confer kanamycin resistance, but the exact mechanism of resistance is not well understood. Here we examined the transcriptomes of control seedlings and wbc19 mutant seedlings using RNA-seq analysis. Exposure to kanamycin indicated changes in the organization of the photosynthetic apparatus, metabolic fluxes and metal uptake. Elemental analysis showed a 60% and 80% reduction of iron uptake in control and wbc19 mutant seedlings respectively, upon exposure to kanamycin. The drop in iron content was accompanied by the upregulation of the gene encoding for FERRIC REDUCTION OXIDASE 6 (FRO6 in mutant seedlings but not by the differential expression of other transport genes known to be induced by iron deficiency. In addition, wbc19 mutants displayed a distinct expression profile in the absence of kanamycin. Most notably the expression of several zinc ion binding proteins, including ZINC TRANSPORTER 1 PRECURSOR (ZIP1 was increased, suggesting abnormal zinc uptake. Elemental analysis confirmed a 50% decrease of zinc content in wbc19 mutants. Thus, the antibiotic resistance gene WBC19 appears to also have a role in zinc uptake.

  20. Synthesis and antibacterial activities of 1-N [(S)-omega-amino-2-hydroxyalkyl] kanamycin A derivatives.

    Science.gov (United States)

    Richardson, K; Jevons, S; Moore, J W; Ross, B C; Wright, J R

    1977-10-01

    Four 1-N-aminohydroxy-alkyl derivatives of kanamycin A were prepared and their in vitro activities against aminoglycoside-sensitive and aminoglycoside-resistant organisms were compared with amikacin. 1-N-[(S)-4-Amino-2-hydroxybutyl] kanamycin A (Fig. 1, compound 2, code no. UK-18,892) was equipotent to amikacin in all these tests and in mouse protection studies.

  1. Intrinsic resistance to aminoglycosides in Enterococcus faecium is conferred by the 16S rRNA m5C1404-specific methyltransferase EfmM

    DEFF Research Database (Denmark)

    Galimand, Marc; Schmitt, Emmanuelle; Panvert, Michel

    2011-01-01

    methyltransferase, as well as by the previously characterized aac(6')-Ii that encodes a 6'-N-aminoglycoside acetyltransferase. Inactivation of efmM in E. faecium increases susceptibility to the aminoglycosides kanamycin and tobramycin, and, conversely, expression of a recombinant version of efmM in Escherichia coli...... confers resistance to these drugs. The EfmM protein shows significant sequence similarity to E. coli RsmF (previously called YebU), which is a 5-methylcytidine (m(5)C) methyltransferase modifying 16S rRNA nucleotide C1407. The target for EfmM is shown by mass spectrometry to be a neighboring 16S r...

  2. Influences of the disease resistance conferred by the individual ...

    African Journals Online (AJOL)

    To research possible influences of the disease resistance conferred by different trans-resistance genes on the transgenic rice plants in their yields and grain quality, three transgenic rice lines, including two with the resistance genes Pi-d2 and Pi-d3, respectively, for rice blast, and one with the resistance gene Xa21 for rice ...

  3. Agrobacterium mediated transformation of brassica juncea (l.) czern with chitinase gene conferring resistance against fungal infections

    International Nuclear Information System (INIS)

    Ahmad, B.; Ambreen, S.; Khan, I.

    2015-01-01

    Brassica juncea (Czern and Coss., L.) is an important oilseed crop. Since it is attacked by several bacterial and fungal diseases, therefore, we developed an easy and simple protocol for the regeneration and transformation of B. juncea variety RAYA ANMOL to give rise to transgenic plants conferring resistance against various fungal diseases. The transformation was carried out using Agrobacterium with Chitinase gene. This gene was isolated from Streptomyces griseus HUT6037. We used two types of explants for transformation i.e. hypocotyls and cotyledons. Only hypocotyls explants showed good results regarding callus initiation. Different hormonal concentrations were applied i.e. BAP 2, 4 and 6 mgL-1 and NAA 0.1, 0.2 and 0.3 mgL-1. However, high transformation efficiency was observed by supplementing the medium with combination of 2 mgL-1 BAP and 0.2 mgL-1 for initiation of callus. Similarly 10 mgL-1 kanamycin and 200 mgL-1 cefotaxime also proved successful for the selection of transformed callus. In order to confirm the presence of transgenic callus Polymerase chain reaction was performed using specific primers for Chitinase gene. (author)

  4. Co-spray drying of hygroscopic kanamycin with the hydrophobic drug rifampicin to improve the aerosolization of kanamycin powder for treating respiratory infections.

    Science.gov (United States)

    Momin, Mohammad A M; Tucker, Ian G; Doyle, Colin S; Denman, John A; Sinha, Shubhra; Das, Shyamal C

    2018-04-25

    High dose delivery of drugs to the lung using a dry powder inhaler (DPI) is an emerging approach to combat drug-resistant local infections. To achieve this, highly aerosolizable powders are required. We hypothesized that co-spray-drying kanamycin, a hydrophilic hygroscopic antibiotic, with rifampicin, a hydrophobic antibiotic, would produce inhalable particles with surfaces enriched in rifampicin. Such particles would have higher aerosolization than kanamycin alone, and minimise the mass of powder for inhalation avoiding use of non-active excipients. Kanamycin was co-spray-dried with rifampicin using a Buchi Mini Spray-dryer. All powders were inhalable in size (1.1-5.9 µm) and noncrystalline. X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS) showed the surface of the combination powder was enriched with rifampicin. In vitro aerosolization (fine particle fraction) determined by next generation impactor (NGI), dramatically improved from 29.5 ± 0.2% (kanamycin-only) to 78.2 ± 1.3% (kanamycin-rifampicin combination). The combination powder was flake-shaped in morphology, stable at 15% and 53% RH and 25 ± 2 °C during one-month storage in an open Petri dish, and non-toxic (up to 50 µg/mL) to human alveolar and bronchial cell-lines. Surface enrichment of kanamycin by hydrophobic rifampicin improves aerosolization, which may help to combat drug-resistant local infections by facilitating high dose delivery to deep lung. Copyright © 2018 Elsevier B.V. All rights reserved.

  5. 21 CFR 520.1204 - Kanamycin, bismuth subcarbonate, activated attapulgite.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Kanamycin, bismuth subcarbonate, activated... § 520.1204 Kanamycin, bismuth subcarbonate, activated attapulgite. (a) Specifications—(1) Each 5 milliliters (mL) of suspension contains 100 milligrams (mg) kanamycin (as the sulfate), 250 mg bismuth...

  6. Completion of the nucleotide sequence of the central region of Tn5 confirms the presence of three resistance genes.

    OpenAIRE

    Mazodier, P; Cossart, P; Giraud, E; Gasser, F

    1985-01-01

    The DNA sequence of the region located downstream from the kanamycin resistance gene of Tn5 up to the right inverted repeat IS50R has been determined. This completes the determination of the sequence of Tn5 which is 5818 bp long. The 2.7 Kb central region contains three resistance genes: the kanamycin-neomycin resistance gene, a gene coding for resistance to CL990 an antimitotic-antibiotic compound of the bleomycin family and a third gene that confers streptomycin resistance in some bacterial...

  7. High Prevalence and Predominance of the aph(2″)-If Gene Conferring Aminoglycoside Resistance in Campylobacter.

    Science.gov (United States)

    Yao, Hong; Liu, Dejun; Wang, Yang; Zhang, Qijing; Shen, Zhangqi

    2017-05-01

    Campylobacter is a major foodborne pathogen, and previous studies revealed that Campylobacter isolates from food-producing animals are increasingly resistant to gentamicin in China. The molecular epidemiology and genetic mechanisms responsible for gentamicin resistance in China have not been well understood. In this study, 607 Campylobacter isolates of chicken and swine origins collected in 2014 were analyzed, revealing that 15.6% (25/160) of the Campylobacter jejuni isolates and 79.9% (357/447) of the Campylobacter coli isolates were resistant to gentamicin. PCR detection of the gentamicin resistance genes indicated that aph(2″)-If was more prevalent than the previously identified aacA/aphD gene and has become the dominant gentamicin resistance determinant in Campylobacter Transformation and whole-genome sequencing as well as long-range PCR discovered that aph(2″)-If was located on a chromosomal segment inserted between two conserved genes, Cj0299 and panB Cloning of aph(2″)-If into gentamicin-susceptible C. jejuni NCTC 11168 confirmed its function in conferring high-level resistance to gentamicin and kanamycin. Molecular typing by pulsed-field gel electrophoresis suggested that both regional expansion of a particular clone and horizontal transmission were involved in the dissemination of the aph(2″)-If gene in Campylobacter To our knowledge, this is the first report describing the high prevalence of a chromosomally encoded aph(2″)-If gene in Campylobacter The high prevalence and predominance of this gene might be driven by the use of aminoglycoside antibiotics in food animal production in China and potentially compromise the usefulness of gentamicin as a therapeutic agent for Campylobacter -associated systemic infection. Copyright © 2017 American Society for Microbiology.

  8. A novel gene of Kalanchoe daigremontiana confers plant drought resistance.

    Science.gov (United States)

    Wang, Li; Zhu, Chen; Jin, Lin; Xiao, Aihua; Duan, Jie; Ma, Luyi

    2018-02-07

    Kalanchoe (K.) daigremontiana is important for studying asexual reproduction under different environmental conditions. Here, we describe a novel KdNOVEL41 (KdN41) gene that may confer drought resistance and could thereby affect K. daigremontiana development. The detected subcellular localization of a KdN41/Yellow Fluorescent Protein (YFP) fusion protein was in the nucleus and cell membrane. Drought, salt, and heat stress treatment in tobacco plants containing the KdN41 gene promoter driving β-glucuronidase (GUS) gene transcription revealed that only drought stress triggered strong GUS staining in the vascular tissues. Overexpression (OE) of the KdN41 gene conferred improved drought resistance in tobacco plants compared to wild-type and transformed with empty vector plants by inducing higher antioxidant enzyme activities, decreasing cell membrane damage, increasing abscisic acid (ABA) content, causing reinforced drought resistance related gene expression profiles. The 3,3'-diaminobenzidine (DAB) and nitroblue tetrazolium (NBT) staining results also showed less relative oxygen species (ROS) content in KdN41-overexpressing tobacco leaf during drought stress. Surprisingly, by re-watering after drought stress, KdN41-overexpressing tobacco showed earlier flowering. Overall, the KdN41 gene plays roles in ROS scavenging and osmotic damage reduction to improve tobacco drought resistance, which may increase our understanding of the molecular network involved in developmental manipulation under drought stress in K. daigremontiana.

  9. Combined treatment with the antibiotics kanamycin and streptomycin promotes the conjugation of Escherichia coli.

    Science.gov (United States)

    Zhang, Peng-Yi; Xu, Pei-Pei; Xia, Zhi-Jie; Wang, Jing; Xiong, Juan; Li, Yue-Zhong

    2013-11-01

    It is widely accepted that antibiotics provide a critical selective pressure for the horizontal transfer of antibiotic resistance between bacterial species. This study demonstrated that a combination of low doses of kanamycin and streptomycin, which inhibited the growth of recipient and donor cells, respectively, had positive effects on the transmission of the conjugation plasmids pRK2013, pSU2007, and RP4 from Escherichia coli DH5α to HB101 at their minimum inhibitory concentrations (MICs). Administration of either antibiotic alone as well as other antibiotics in combination or alone did not have this effect. Two-dimensional electrophoresis revealed that 60 proteins were downregulated and 14 proteins were upregulated in the conjugation of E. coli DH5α (pRK2013) and HB101 in the presence of kanamycin and streptomycin. Of these proteins, 64 were subsequently identified by mass spectrometry. Two antibiotic-induced genes encoding oligopeptide-binding protein (OppA) and ribose-binding protein (RbsB) were further confirmed by quantitative real-time PCR. When these genes were deleted, the number of transconjugants decreased in the same fashion as when the cells were treated with kanamycin and streptomycin. These results indicate that the process of E. coli conjugation may be promoted by combination treatment with kanamycin and streptomycin and that two proteins potentially participated in this process. © 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

  10. 21 CFR 524.1200b - Kanamycin ophthalmic aqueous solution.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Kanamycin ophthalmic aqueous solution. 524.1200b... § 524.1200b Kanamycin ophthalmic aqueous solution. (a) Specifications. The drug, which is in an aqueous solution including suitable and harmless preservatives and buffer substances, contains 10 milligrams of...

  11. Spectrum of Resistance Conferred by ml-o Powdery Mildew Resistance Genes in Barley

    DEFF Research Database (Denmark)

    Jørgensen, Jørgen Helms

    1977-01-01

    /(4) in all tests. They were also resistant to field populations of the pathogen when scored in disease nurseries at more than 78 locations in 29 countries in Europe, the Near East, North and South America. New Zealand, and Japan. This indicates that the 11 genes confer the same, world-wide spectrum...

  12. Food supply confers calcifiers resistance to ocean acidification

    KAUST Repository

    Ramajo, Laura

    2016-01-18

    Invasion of ocean surface waters by anthropogenic CO2 emitted to the atmosphere is expected to reduce surface seawater pH to 7.8 by the end of this century compromising marine calcifiers. A broad range of biological and mineralogical mechanisms allow marine calcifiers to cope with ocean acidification, however these mechanisms are energetically demanding which affect other biological processes (trade-offs) with important implications for the resilience of the organisms against stressful conditions. Hence, food availability may play a critical role in determining the resistance of calcifiers to OA. Here we show, based on a meta-analysis of existing experimental results assessing the role of food supply in the response of organisms to OA, that food supply consistently confers calcifiers resistance to ocean acidification.

  13. aadA Confers Streptomycin Resistance in Borrelia burgdorferi

    Science.gov (United States)

    Frank, Kristi L.; Bundle, Sharyl F.; Kresge, Michele E.; Eggers, Christian H.; Samuels, D. Scott

    2003-01-01

    To enhance genetic manipulation of the Lyme disease spirochete Borrelia burgdorferi, we assayed the aadA gene for the ability to confer resistance to the antibiotics spectinomycin and streptomycin. Using the previously described pBSV2 as a backbone, a shuttle vector, termed pKFSS1, which carries the aadA open reading frame fused to the B. burgdorferi flgB promoter was constructed. The hybrid flgB promoter-aadA cassette confers resistance to spectinomycin and streptomycin in both B. burgdorferi and Escherichia coli. pKFSS1 has a replication origin derived from the 9-kb circular plasmid and can be comaintained in B. burgdorferi with extant shuttle vector pCE320, which has a replication origin derived from a 32-kb circular plasmid, or pBSV2, despite the fact that pKFSS1 and pBSV2 have the same replication origin. Our results demonstrate the availability of a new selectable marker and shuttle vector for genetically dissecting B. burgdorferi at the molecular level. PMID:14594849

  14. A Mutation in the Decoding Center of Thermus thermophilus 16S rRNA Suggests a Novel Mechanism of Streptomycin Resistance

    Science.gov (United States)

    Gregory, Steven T.; Carr, Jennifer F.; Dahlberg, Albert E.

    2005-01-01

    A spontaneous kanamycin resistance and capreomycin resistance mutation, A1408G, in the decoding center of 16S rRNA, was identified in the extreme thermophile Thermus thermophilus. Unexpectedly, this mutation also confers resistance to streptomycin. We propose a novel mechanism of streptomycin resistance by which A1408G influences conformational changes in 16S rRNA during tRNA selection. PMID:15743969

  15. Physicochemical characteristics of the kanamycin-lyophilized kit

    International Nuclear Information System (INIS)

    Eva Maria Widyasari; Misyetti; Teguh Hafiz Ambar W; Witri Nuraeni

    2013-01-01

    Kanamycin is a broad-spectrum antibiotic and usually used for the treatment of infections when antibiotics like penicillin are less powerful and can not be given. This research was performed to obtain several physicochemical character of 99m Tc-kanamycin which were made in the form of lyophilized kanamycin kit to ensure the later application in humans. Kanamycin diagnostic kit were provided in lyophilized kit comprising kanamycin as ligand compound, pyrophosphate as co-ligand and SnCl 2 as reducing agent. The radiochemical purity was determined by instant thin layer chromatography (ITLC-SG) using 0.5 N NaOH as the mobile phase and ascending paper chromatography using Whatman paper 3 with acetone as the mobile phase. The plasma binding protein of 99m Tc-kanamycin was investigated in vitro by precipitation method using 5% of trichloro acetic acid (TCA) solution, whereas the lipophilicity (log P) was obtained by determination the partition coefficient in organic solvent-water system. Studies on the effect of the amount of radioactivity and the volume of Na 99m TcO 4 solution to the radiochemical purity of 99m Tc-kanamycin were also performed. From this experiment it was obtained that kanamycin lyophilized-kit was hydrophilic, 59.54% of solutions bound to plasma, radiochemical purity was more than 95%, and the final volume of 2 ml dosage was stable up to 2 hours after the addition of 99m Tc with a radioactivity of less then 3 mCi. (author)

  16. Reaching consensus on drug resistance conferring mutations (Part 1

    Directory of Open Access Journals (Sweden)

    Daniela M Cirillo

    2016-01-01

    A user-friendly interface designed for nonexpert or expert operability.A standardized and validated analysis pipeline for variant analyses of M. tuberculosis next-generation sequencing (NGS data.Access to data beyond the published literature with dynamic and iterative updates of new data generated by global surveillance and clinical trials.A well-developed legal structure to ensure intellectual property rights and data ownership remain with contributors.A structured data-sharing architecture to restrict access to sensitive or unpublished data sets.Metadata standardization using CDISC: supports global, platform-independent data standards that enable information system interoperability.An emphasis on data quality and rigorous, expert curation with multiple quality control checks for whole-genome sequencing and other metadata.Validation of NGS analysis output by an expert committee with grading of resistance conferring mutations based on rigorous statistical standards.Regulatory-compliant analysis pipeline and database architecture. Successful execution of such an extensive database platform requires substantial collaboration from scientists investigating the genetic basis for drug resistance worldwide, and from developers with expertise in database design and implementation.

  17. Mutations in the Plasmodium falciparum Cyclic Amine Resistance Locus (PfCARL Confer Multidrug Resistance

    Directory of Open Access Journals (Sweden)

    Gregory LaMonte

    2016-07-01

    Full Text Available Mutations in the Plasmodium falciparum cyclic amine resistance locus (PfCARL are associated with parasite resistance to the imidazolopiperazines, a potent class of novel antimalarial compounds that display both prophylactic and transmission-blocking activity, in addition to activity against blood-stage parasites. Here, we show that pfcarl encodes a protein, with a predicted molecular weight of 153 kDa, that localizes to the cis-Golgi apparatus of the parasite in both asexual and sexual blood stages. Utilizing clustered regularly interspaced short palindromic repeat (CRISPR-mediated gene introduction of 5 variants (L830V, S1076N/I, V1103L, and I1139K, we demonstrate that mutations in pfcarl are sufficient to generate resistance against the imidazolopiperazines in both asexual and sexual blood-stage parasites. We further determined that the mutant PfCARL protein confers resistance to several structurally unrelated compounds. These data suggest that PfCARL modulates the levels of small-molecule inhibitors that affect Golgi-related processes, such as protein sorting or membrane trafficking, and is therefore an important mechanism of resistance in malaria parasites.

  18. Functional screening of antibiotic resistance genes from human gut microbiota reveals a novel gene fusion.

    Science.gov (United States)

    Cheng, Gong; Hu, Yongfei; Yin, Yeshi; Yang, Xi; Xiang, Chunsheng; Wang, Baohong; Chen, Yanfei; Yang, Fengling; Lei, Fang; Wu, Na; Lu, Na; Li, Jing; Chen, Quanze; Li, Lanjuan; Zhu, Baoli

    2012-11-01

    The human gut microbiota has a high density of bacteria that are considered a reservoir for antibiotic resistance genes (ARGs). In this study, one fosmid metagenomic library generated from the gut microbiota of four healthy humans was used to screen for ARGs against seven antibiotics. Eight new ARGs were obtained: one against amoxicillin, six against d-cycloserine, and one against kanamycin. The new amoxicillin resistance gene encodes a protein with 53% identity to a class D β-lactamase from Riemerella anatipestifer RA-GD. The six new d-cycloserine resistance genes encode proteins with 73-81% identity to known d-alanine-d-alanine ligases. The new kanamycin resistance gene encodes a protein of 274 amino acids with an N-terminus (amino acids 1-189) that has 42% identity to the 6'-aminoglycoside acetyltransferase [AAC(6')] from Enterococcus hirae and a C-terminus (amino acids 190-274) with 35% identity to a hypothetical protein from Clostridiales sp. SSC/2. A functional study on the novel kanamycin resistance gene showed that only the N-terminus conferred kanamycin resistance. Our results showed that functional metagenomics is a useful tool for the identification of new ARGs. © 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

  19. Coresistance to Neomycin and Kanamycin by Mutations in an Escherichia coli Locus that Affects Ribosomes

    Science.gov (United States)

    Apirion, D.; Schlessinger, D.

    1968-01-01

    Mutant strains resistant to neomycin or to kanamycin sulfate were isolated from Escherichia coli K-12. Nine mutants were analyzed; all were resistant to both antibiotics (about 150 and 100 μg/ml, respectively), and were designated nek. In the mutant strains, the ribosomes are changed from those of the parental strain; for when they were used in assays for polypeptide formation directed by polyadenylic acid or polycytidylic acid, coding fidelity in presence of the drugs was increased and inhibition of synthesis by the drugs was lessened. Mating experiments and transduction tests showed that all of the nine nek mutants are either closely linked or allelic, and the nek locus is closely linked to two genes—str (streptomycin) and spc (spectinomycin)—known to affect the 30S ribosome. The two nek mutants tested were recessive to the sensitive, wild-type allele. When the nek mutants were compared to the parental strain, pleiotropic effects of the nek mutations were observed. Resistance to low levels of streptomycin and spectinomycin was increased, whereas resistance to chloramphenicol was decreased. Also, the mutants were less able to adapt to high concentrations of lincomycin, and could no longer show phenotypic suppression of an arginine requirement by neomycin or kanamycin. Such pleiotropic effects are suggested to be the rule for mutations in genes that participate in the biosynthesis of a cellular organelle. PMID:4895052

  20. Drug resistance. K13-propeller mutations confer artemisinin resistance in Plasmodium falciparum clinical isolates.

    Science.gov (United States)

    Straimer, Judith; Gnädig, Nina F; Witkowski, Benoit; Amaratunga, Chanaki; Duru, Valentine; Ramadani, Arba Pramundita; Dacheux, Mélanie; Khim, Nimol; Zhang, Lei; Lam, Stephen; Gregory, Philip D; Urnov, Fyodor D; Mercereau-Puijalon, Odile; Benoit-Vical, Françoise; Fairhurst, Rick M; Ménard, Didier; Fidock, David A

    2015-01-23

    The emergence of artemisinin resistance in Southeast Asia imperils efforts to reduce the global malaria burden. We genetically modified the Plasmodium falciparum K13 locus using zinc-finger nucleases and measured ring-stage survival rates after drug exposure in vitro; these rates correlate with parasite clearance half-lives in artemisinin-treated patients. With isolates from Cambodia, where resistance first emerged, survival rates decreased from 13 to 49% to 0.3 to 2.4% after the removal of K13 mutations. Conversely, survival rates in wild-type parasites increased from ≤0.6% to 2 to 29% after the insertion of K13 mutations. These mutations conferred elevated resistance to recent Cambodian isolates compared with that of reference lines, suggesting a contemporary contribution of additional genetic factors. Our data provide a conclusive rationale for worldwide K13-propeller sequencing to identify and eliminate artemisinin-resistant parasites. Copyright © 2015, American Association for the Advancement of Science.

  1. A novel resistance gene, lnu(H), conferring resistance to lincosamides in Riemerella anatipestifer CH-2.

    Science.gov (United States)

    Luo, Hong-Yan; Liu, Ma-Feng; Wang, Ming-Shu; Zhao, Xin-Xin; Jia, Ren-Yong; Chen, Shun; Sun, Kun-Feng; Yang, Qiao; Wu, Ying; Chen, Xiao-Yue; Biville, Francis; Zou, Yuan-Feng; Jing, Bo; Cheng, An-Chun; Zhu, De-Kang

    2018-01-01

    The Gram-negative bacterium Riemerella anatipestifer CH-2 is resistant to lincosamides, having a lincomycin (LCM) minimum inhibitory concentration (MIC) of 128 µg/mL. The G148_1775 gene of R. anatipestifer CH-2, designated lnu(H), encodes a 260-amino acid protein with ≤41% identity to other reported lincosamide nucleotidylyltransferases. Escherichia coli Rosetta TM (DE3) containing the pBAD24-lnu(H) plasmid showed four- and two-fold increases in the MICs of LCM and clindamycin (CLI), respectively. A kinetic assay of the purified Lnu(H) enzyme for LCM and CLI showed that the protein could inactive lincosamides. Mass spectrometry analysis demonstrated that the Lnu(H) enzyme catalysed adenylylation of lincosamides. In addition, an lnu(H) gene deletion strain exhibited 512- and 32-fold decreases in LCM and CLI MICs, respectively. The wild-type level of lincosamide resistance could be restored by complementation with a shuttle plasmid carrying the lnu(H) gene. The transformant R. anatipestifer ATCC 11845 [lnu(H)] acquired by natural transformation also exhibited high-level lincosamide resistance. Moreover, among 175 R. anatipestifer field isolates, 56 (32.0%) were positive for the lnu(H) gene by PCR. In conclusion, Lnu(H) is a novel lincosamide nucleotidylyltransferase that inactivates LCM and CLI by nucleotidylylation, thus conferring high-level lincosamide resistance to R. anatipestifer CH-2. Copyright © 2017. Published by Elsevier B.V.

  2. Influences of the disease resistance conferred by the individual ...

    African Journals Online (AJOL)

    STORAGESEVER

    2009-10-05

    Oct 5, 2009 ... If the resistance in the transgenic rice plants with a disease resistance gene is similar to the RPM1 resis- tance in Arabidopsis, that is, negatively correlate with yield or grain quality in rice, we should not only consider the disease resistance of the transgenic rice plants but ignore their yields and grain quality, ...

  3. Regeneration of multiple shoots from transgenic potato events facilitates the recovery of phenotypically normal lines: assessing a cry9Aa2 gene conferring insect resistance

    Directory of Open Access Journals (Sweden)

    Jacobs Jeanne ME

    2011-10-01

    Full Text Available Abstract Background The recovery of high performing transgenic lines in clonal crops is limited by the occurrence of somaclonal variation during the tissue culture phase of transformation. This is usually circumvented by developing large populations of transgenic lines, each derived from the first shoot to regenerate from each transformation event. This study investigates a new strategy of assessing multiple shoots independently regenerated from different transformed cell colonies of potato (Solanum tuberosum L.. Results A modified cry9Aa2 gene, under the transcriptional control of the CaMV 35S promoter, was transformed into four potato cultivars using Agrobacterium-mediated gene transfer using a nptII gene conferring kanamycin resistance as a selectable marker gene. Following gene transfer, 291 transgenic lines were grown in greenhouse experiments to assess somaclonal variation and resistance to potato tuber moth (PTM, Phthorimaea operculella (Zeller. Independently regenerated lines were recovered from many transformed cell colonies and Southern analysis confirmed whether they were derived from the same transformed cell. Multiple lines regenerated from the same transformed cell exhibited a similar response to PTM, but frequently exhibited a markedly different spectrum of somaclonal variation. Conclusions A new strategy for the genetic improvement of clonal crops involves the regeneration and evaluation of multiple shoots from each transformation event to facilitate the recovery of phenotypically normal transgenic lines. Most importantly, regenerated lines exhibiting the phenotypic appearance most similar to the parental cultivar are not necessarily derived from the first shoot regenerated from a transformed cell colony, but can frequently be a later regeneration event.

  4. A pigeonpea gene confers resistance to Asian soybean rust in soybean.

    Science.gov (United States)

    Kawashima, Cintia G; Guimarães, Gustavo Augusto; Nogueira, Sônia Regina; MacLean, Dan; Cook, Doug R; Steuernagel, Burkhard; Baek, Jongmin; Bouyioukos, Costas; Melo, Bernardo do V A; Tristão, Gustavo; de Oliveira, Jamile Camargos; Rauscher, Gilda; Mittal, Shipra; Panichelli, Lisa; Bacot, Karen; Johnson, Ebony; Iyer, Geeta; Tabor, Girma; Wulff, Brande B H; Ward, Eric; Rairdan, Gregory J; Broglie, Karen E; Wu, Gusui; van Esse, H Peter; Jones, Jonathan D G; Brommonschenkel, Sérgio H

    2016-06-01

    Asian soybean rust (ASR), caused by the fungus Phakopsora pachyrhizi, is one of the most economically important crop diseases, but is only treatable with fungicides, which are becoming less effective owing to the emergence of fungicide resistance. There are no commercial soybean cultivars with durable resistance to P. pachyrhizi, and although soybean resistance loci have been mapped, no resistance genes have been cloned. We report the cloning of a P. pachyrhizi resistance gene CcRpp1 (Cajanus cajan Resistance against Phakopsora pachyrhizi 1) from pigeonpea (Cajanus cajan) and show that CcRpp1 confers full resistance to P. pachyrhizi in soybean. Our findings show that legume species related to soybean such as pigeonpea, cowpea, common bean and others could provide a valuable and diverse pool of resistance traits for crop improvement.

  5. Hfq mutation confers increased cephalosporin resistance in Klebsiella pneumoniae

    Directory of Open Access Journals (Sweden)

    Li Xinran

    2017-01-01

    Full Text Available Klebsiella pneumoniae (K. pneumoniae, is an opportunistic pathogen raising significant public health concerns owing to its multi-drug resistance. Hfq, one of the main RNA-binding proteins, is a key post-transcriptional regulator. This protein is closely related to virulence and resistance in various pathogenic bacteria. Although the role of hfq in K pneumoniae virulence has been explored, its influence on resistance remains largely unknown. The aim of this study was to investigate the role of hfq in the resistance of K. pneumoniae to cephalosporins. An hfq mutant was constructed, and its resistance to cephalosporins was investigated. The hfq mutant exhibited over 16-fold higher cephalosporin resistance than that exhibited by the wild type. Time-kill curve analysis showed that the hfq mutant could survive under higher concentrations of cephalosporins than the wild-type strain could. Quantitative RT-PCR showed that expression levels for 8 out of the 9 penicillin-binding proteins, which are the targets of cephalosporins, were downregulated in the hfq mutant. Taken together, contrary to its role in many other bacteria, hfq is involved in a negative regulation of K. pneumoniae resistance to cephalosporins by downregulating the expression of penicillin-binding proteins.

  6. Mitochondrial Adaptations to Oxidative Stress Confer Resistance to Apoptosis in Lymphoma Cells

    Directory of Open Access Journals (Sweden)

    Margaret M. Briehl

    2012-08-01

    Full Text Available Acquired resistance to drugs commonly used for lymphoma treatment poses a significant barrier to improving lymphoma patient survival. Previous work with a lymphoma tissue culture model indicates that selection for resistance to oxidative stress confers resistance to chemotherapy-induced apoptosis. This suggests that adaptation to chronic oxidative stress can contribute to chemoresistance seen in lymphoma patients. Oxidative stress-resistant WEHI7.2 cell variants in a lymphoma tissue culture model exhibit a range of apoptosis sensitivities. We exploited this phenotype to test for mitochondrial changes affecting sensitivity to apoptosis in cells made resistant to oxidative stress. We identified impaired release of cytochrome c, and the intermembrane proteins adenylate kinase 2 and Smac/DIABLO, indicating inhibition of the pathway leading to permeabilization of the outer mitochondrial membrane. Blunting of a glucocorticoid-induced signal and intrinsic mitochondrial resistance to cytochrome c release contributed to both points of resistance. The level of Bcl-2 family members or a difference in Bim induction were not contributing factors. The extent of cardiolipin oxidation following dexamethasone treatment, however, did correlate with apoptosis resistance. The differences found in the variants were all proportionate to the degree of resistance to glucocorticoid treatment. We conclude that tolerance to oxidative stress leads to mitochondrial changes that confer resistance to apoptosis.

  7. A secondary RET mutation in the activation loop conferring resistance to vandetanib.

    Science.gov (United States)

    Nakaoku, Takashi; Kohno, Takashi; Araki, Mitsugu; Niho, Seiji; Chauhan, Rakhee; Knowles, Phillip P; Tsuchihara, Katsuya; Matsumoto, Shingo; Shimada, Yoko; Mimaki, Sachiyo; Ishii, Genichiro; Ichikawa, Hitoshi; Nagatoishi, Satoru; Tsumoto, Kouhei; Okuno, Yasushi; Yoh, Kiyotaka; McDonald, Neil Q; Goto, Koichi

    2018-02-12

    Resistance to vandetanib, a type I RET kinase inhibitor, developed in a patient with metastatic lung adenocarcinoma harboring a CCDC6-RET fusion that initially exhibited a response to treatment. The resistant tumor acquired a secondary mutation resulting in a serine-to-phenylalanine substitution at codon 904 in the activation loop of the RET kinase domain. The S904F mutation confers resistance to vandetanib by increasing the ATP affinity and autophosphorylation activity of RET kinase. A reduced interaction with the drug is also observed in vitro for the S904F mutant by thermal shift assay. A crystal structure of the S904F mutant reveals a small hydrophobic core around F904 likely to enhance basal kinase activity by stabilizing an active conformer. Our findings indicate that missense mutations in the activation loop of the kinase domain are able to increase kinase activity and confer drug resistance through allosteric effects.

  8. Proceedings of the CERI 2005 electricity conference : markets, integration, resistance

    International Nuclear Information System (INIS)

    2005-01-01

    This conference was attended by power industry decision makers who face continuing challenges regarding changes in electricity market mechanisms, pricing options, and power generation and transmission alternatives. It provided an opportunity to review energy markets in North American with particular reference to supply and demand and opportunities for traditional or new generation technologies based on renewable energy sources including wind powered generation. The presentations focused on transmission issues, market design and capacity issues as well as market power and pricing. The integration of wind energy into the power grid as a measure to diversity the power generation portfolio in North America was also discussed along with hydrothermal synergies and interconnections. The role of wind, coal and nuclear power in future North American energy markets was also discussed along with their environmental consequences. tabs., figs

  9. Distribution of HIV-1 resistance-conferring polymorphic alleles SDF ...

    Indian Academy of Sciences (India)

    Unknown

    2Department of Genetics, Owaisi Medical and Research Centre, Deccan College of Medical Sciences and ... data, are comparable to those from Africa and Southeast Asia, where AIDS is known to be widespread. ... HIV-1 resistance polymorphism; chemokine receptor; stromal-derived factor-1; Andhra Pradesh; AIDS.

  10. Pyocyanin production by Pseudomonas aeruginosa confers resistance to ionic silver.

    Science.gov (United States)

    Muller, Michael; Merrett, Neil D

    2014-09-01

    Silver in its ionic form (Ag+), but not the bulk metal (Ag0), is toxic to microbial life forms and has been used for many years in the treatment of wound infections. The prevalence of bacterial resistance to silver is considered low due to the nonspecific nature of its toxicity. However, the recent increased use of silver as an antimicrobial agent for medical, consumer, and industrial products has raised concern that widespread silver resistance may emerge. Pseudomonas aeruginosa is a common pathogen that produces pyocyanin, a redox toxin and a reductant for molecular oxygen and ferric (Fe3+) ions. The objective of this study was to determine whether pyocyanin reduces Ag+ to Ag0, which may contribute to silver resistance due to lower bioavailability of the cation. Using surface plasmon resonance spectroscopy and scanning electron microscopy, pyocyanin was confirmed to be a reductant for Ag+, forming Ag0 nanoparticles and reducing the bioavailability of free Ag+ by >95% within minutes. Similarly, a pyocyanin-producing strain of P. aeruginosa (PA14) reduced Ag+ but not a pyocyanin-deficient (ΔphzM) strain of the bacterium. Challenge of each strain with Ag+ (as AgNO3) gave MICs of 20 and 5 μg/ml for the PA14 and ΔphzM strains, respectively. Removal of pyocyanin from the medium strain PA14 was grown in or its addition to the medium that ΔphzM mutant was grown in gave MICs of 5 and 20 μg/ml, respectively. Clinical isolates demonstrated similar pyocyanin-dependent resistance to Ag+. We conclude that pseudomonal silver resistance exists independently of previously recognized intracellular mechanisms and may be more prevalent than previously considered. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  11. Mechanisms of Streptomycin Resistance: Selection of Mutations in the 16S rRNA Gene Conferring Resistance

    Science.gov (United States)

    Springer, Burkhard; Kidan, Yishak G.; Prammananan, Therdsak; Ellrott, Kerstin; Böttger, Erik C.; Sander, Peter

    2001-01-01

    Chromosomally acquired streptomycin resistance is frequently due to mutations in the gene encoding the ribosomal protein S12, rpsL. The presence of several rRNA operons (rrn) and a single rpsL gene in most bacterial genomes prohibits the isolation of streptomycin-resistant mutants in which resistance is mediated by mutations in the 16S rRNA gene (rrs). Three strains were constructed in this investigation: Mycobacterium smegmatis rrnB, M. smegmatis rpsL3+, and M. smegmatis rrnB rpsL3+. M. smegmatis rrnB carries a single functional rrn operon, i.e., rrnA (comprised of 16S, 23S, and 5S rRNA genes) and a single rpsL+ gene; M. smegmatis rpsL3+ is characterized by the presence of two rrn operons (rrnA and rrnB) and three rpsL+ genes; and M. smegmatis rrnB rpsL3+ carries a single functional rrn operon (rrnA) and three rpsL+ genes. By genetically altering the number of rpsL and rrs alleles in the bacterial genome, mutations in rrs conferring streptomycin resistance could be selected, as revealed by analysis of streptomycin-resistant derivatives of M. smegmatis rrnB rpsL3+. Besides mutations well known to confer streptomycin resistance, novel streptomycin resistance conferring mutations were isolated. Most of the mutations were found to map to a functional pseudoknot structure within the 530 loop region of the 16S rRNA. One of the mutations observed, i.e., 524G→C, severely distorts the interaction between nucleotides 524G and 507C, a Watson-Crick interaction which has been thought to be essential for ribosome function. The use of the single rRNA allelic M. smegmatis strain should help to elucidate the principles of ribosome-drug interactions. PMID:11557484

  12. Identification of dfrA14 in two distinct plasmids conferring trimethoprim resistance in Actinobacillus pleuropneumoniae.

    Science.gov (United States)

    Bossé, Janine T; Li, Yanwen; Walker, Stephanie; Atherton, Tom; Fernandez Crespo, Roberto; Williamson, Susanna M; Rogers, Jon; Chaudhuri, Roy R; Weinert, Lucy A; Oshota, Olusegun; Holden, Matt T G; Maskell, Duncan J; Tucker, Alexander W; Wren, Brendan W; Rycroft, Andrew N; Langford, Paul R

    2015-08-01

    The objective of this study was to determine the distribution and genetic basis of trimethoprim resistance in Actinobacillus pleuropneumoniae isolates from pigs in England. Clinical isolates collected between 1998 and 2011 were tested for resistance to trimethoprim and sulphonamide. The genetic basis of trimethoprim resistance was determined by shotgun WGS analysis and the subsequent isolation and sequencing of plasmids. A total of 16 (out of 106) A. pleuropneumoniae isolates were resistant to both trimethoprim (MIC >32 mg/L) and sulfisoxazole (MIC ≥256 mg/L), and a further 32 were resistant only to sulfisoxazole (MIC ≥256 mg/L). Genome sequence data for the trimethoprim-resistant isolates revealed the presence of the dfrA14 dihydrofolate reductase gene. The distribution of plasmid sequences in multiple contigs suggested the presence of two distinct dfrA14-containing plasmids in different isolates, which was confirmed by plasmid isolation and sequencing. Both plasmids encoded mobilization genes, the sulphonamide resistance gene sul2, as well as dfrA14 inserted into strA, a streptomycin-resistance-associated gene, although the gene order differed between the two plasmids. One of the plasmids further encoded the strB streptomycin-resistance-associated gene. This is the first description of mobilizable plasmids conferring trimethoprim resistance in A. pleuropneumoniae and, to our knowledge, the first report of dfrA14 in any member of the Pasteurellaceae. The identification of dfrA14 conferring trimethoprim resistance in A. pleuropneumoniae isolates will facilitate PCR screens for resistance to this important antimicrobial. © The Author 2015. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy.

  13. Defining the structural requirements for a helix in 23 S ribosomal RNA that confers erythromycin resistance

    DEFF Research Database (Denmark)

    Douthwaite, S; Powers, T; Lee, J Y

    1989-01-01

    The helix spanning nucleotides 1198 to 1247 (helix 1200-1250) in Escherichia coli 23 S ribosomal RNA (rRNA) is functionally important in protein synthesis, and deletions in this region confer erythromycin resistance. In order to define the structural requirements for resistance, we have dissected....... However, removal of either these or non-conserved nucleotides from helix 1200-1250 measurably reduces the efficiency of 23 S RNA in forming functional ribosomes. We have used chemical probing and a modified primer extension method to investigate erythromycin binding to wild-type and resistant ribosomes...

  14. Mutations in gidB confer low-level streptomycin resistance in Mycobacterium tuberculosis.

    Science.gov (United States)

    Wong, Sharon Y; Lee, Jong Seok; Kwak, Hyun Kyung; Via, Laura E; Boshoff, Helena I M; Barry, Clifton E

    2011-06-01

    The global threat posed by drug-resistant strains of Mycobacterium tuberculosis demands a greater understanding of the genetic basis and molecular mechanisms that govern how such strains develop resistance against various antituberculous drugs. In this report, we examine a new genetic basis for resistance to one of the oldest and most widely used second-line drugs employed in tuberculosis therapy, streptomycin (SM). This marker for SM resistance was first discovered on the basis of genomic data obtained from drug-resistant M. tuberculosis strains collected in Japan, wherein an association was observed between SM resistance and a mutation in gidB, a putative 16S rRNA methyltransferase. By evaluating an isogenic ΔgidB mutant strain constructed from strain H37Rv, we demonstrate the causal role of gidB in conferring a low-level SM-resistant phenotype in M. tuberculosis with a 16-fold increase in the MIC over the parent strain. Among clinical isolates, the modest increase in SM resistance conferred by a gidB mutation leads to an MIC distribution of gidB mutation-containing strains that spans the recommended SM breakpoint concentration currently used in drug susceptibility testing protocols. As such, some gidB mutation-containing isolates are found to be SM sensitive, while others are SM resistant. On the basis of a pharmacodynamic analysis and Monte Carlo simulation, those isolates that are found to be SM sensitive should still respond favorably to SM treatment, while nearly half of those found to be SM resistant will likely respond poorly. This report provides the first microbiological evidence for the contribution of gidB in streptomycin resistance and examines the clinical implications of mutations in the gidB gene.

  15. Mutations in gidB Confer Low-Level Streptomycin Resistance in Mycobacterium tuberculosis▿†

    Science.gov (United States)

    Wong, Sharon Y.; Lee, Jong Seok; Kwak, Hyun Kyung; Via, Laura E.; Boshoff, Helena I. M.; Barry, Clifton E.

    2011-01-01

    The global threat posed by drug-resistant strains of Mycobacterium tuberculosis demands a greater understanding of the genetic basis and molecular mechanisms that govern how such strains develop resistance against various antituberculous drugs. In this report, we examine a new genetic basis for resistance to one of the oldest and most widely used second-line drugs employed in tuberculosis therapy, streptomycin (SM). This marker for SM resistance was first discovered on the basis of genomic data obtained from drug-resistant M. tuberculosis strains collected in Japan, wherein an association was observed between SM resistance and a mutation in gidB, a putative 16S rRNA methyltransferase. By evaluating an isogenic ΔgidB mutant strain constructed from strain H37Rv, we demonstrate the causal role of gidB in conferring a low-level SM-resistant phenotype in M. tuberculosis with a 16-fold increase in the MIC over the parent strain. Among clinical isolates, the modest increase in SM resistance conferred by a gidB mutation leads to an MIC distribution of gidB mutation-containing strains that spans the recommended SM breakpoint concentration currently used in drug susceptibility testing protocols. As such, some gidB mutation-containing isolates are found to be SM sensitive, while others are SM resistant. On the basis of a pharmacodynamic analysis and Monte Carlo simulation, those isolates that are found to be SM sensitive should still respond favorably to SM treatment, while nearly half of those found to be SM resistant will likely respond poorly. This report provides the first microbiological evidence for the contribution of gidB in streptomycin resistance and examines the clinical implications of mutations in the gidB gene. PMID:21444711

  16. AcrB drug-binding pocket substitution confers clinically relevant resistance and altered substrate specificity.

    Science.gov (United States)

    Blair, Jessica M A; Bavro, Vassiliy N; Ricci, Vito; Modi, Niraj; Cacciotto, Pierpaolo; Kleinekathӧfer, Ulrich; Ruggerone, Paolo; Vargiu, Attilio V; Baylay, Alison J; Smith, Helen E; Brandon, Yvonne; Galloway, David; Piddock, Laura J V

    2015-03-17

    The incidence of multidrug-resistant bacterial infections is increasing globally and the need to understand the underlying mechanisms is paramount to discover new therapeutics. The efflux pumps of Gram-negative bacteria have a broad substrate range and transport antibiotics out of the bacterium, conferring intrinsic multidrug resistance (MDR). The genomes of pre- and posttherapy MDR clinical isolates of Salmonella Typhimurium from a patient that failed antibacterial therapy and died were sequenced. In the posttherapy isolate we identified a novel G288D substitution in AcrB, the resistance-nodulation division transporter in the AcrAB-TolC tripartite MDR efflux pump system. Computational structural analysis suggested that G288D in AcrB heavily affects the structure, dynamics, and hydration properties of the distal binding pocket altering specificity for antibacterial drugs. Consistent with this hypothesis, recreation of the mutation in standard Escherichia coli and Salmonella strains showed that G288D AcrB altered substrate specificity, conferring decreased susceptibility to the fluoroquinolone antibiotic ciprofloxacin by increased efflux. At the same time, the substitution increased susceptibility to other drugs by decreased efflux. Information about drug transport is vital for the discovery of new antibacterials; the finding that one amino acid change can cause resistance to some drugs, while conferring increased susceptibility to others, could provide a basis for new drug development and treatment strategies.

  17. Ancient symbiosis confers desiccation resistance to stored grain pest beetles.

    Science.gov (United States)

    Engl, Tobias; Eberl, Nadia; Gorse, Carla; Krüger, Theresa; Schmidt, Thorsten H P; Plarre, Rudy; Adler, Cornel; Kaltenpoth, Martin

    2017-11-08

    Microbial symbionts of insects provide a range of ecological traits to their hosts that are beneficial in the context of biotic interactions. However, little is known about insect symbiont-mediated adaptation to the abiotic environment, for example, temperature and humidity. Here, we report on an ancient clade of intracellular, bacteriome-located Bacteroidetes symbionts that are associated with grain and wood pest beetles of the phylogenetically distant families Silvanidae and Bostrichidae. In the saw-toothed grain beetle Oryzaephilus surinamensis, we demonstrate that the symbionts affect cuticle thickness, melanization and hydrocarbon profile, enhancing desiccation resistance and thereby strongly improving fitness under dry conditions. Together with earlier observations on symbiont contributions to cuticle biosynthesis in weevils, our findings indicate that convergent acquisitions of bacterial mutualists represented key adaptations enabling diverse pest beetle groups to survive and proliferate under the low ambient humidity that characterizes dry grain storage facilities. © 2017 John Wiley & Sons Ltd.

  18. Physical Localization of a Locus from Agropyron cristatum Conferring Resistance to Stripe Rust in Common Wheat.

    Science.gov (United States)

    Zhang, Zhi; Song, Liqiang; Han, Haiming; Zhou, Shenghui; Zhang, Jinpeng; Yang, Xinming; Li, Xiuquan; Liu, Weihua; Li, Lihui

    2017-11-13

    Stripe rust, caused by Puccinia striiformis f. sp. tritici ( Pst ), is one of the most destructive diseases of wheat ( Triticum aestivum L.) worldwide. Agropyron cristatum (L.) Gaertn. (2 n = 28, PPPP), one of the wild relatives of wheat, exhibits resistance to stripe rust. In this study, wheat- A . cristatum 6P disomic addition line 4844-12 also exhibited resistance to stripe rust. To identify the stripe rust resistance locus from A . cristatum 6P, ten translocation lines, five deletion lines and the BC₂F₂ and BC₃F₂ populations of two wheat- A . cristatum 6P whole-arm translocation lines were tested with a mixture of two races of Pst in two sites during 2015-2016 and 2016-2017, being genotyped with genomic in situ hybridization (GISH) and molecular markers. The result indicated that the locus conferring stripe rust resistance was located on the terminal 20% of 6P short arm's length. Twenty-nine 6P-specific sequence-tagged-site (STS) markers mapped on the resistance locus have been acquired, which will be helpful for the fine mapping of the stripe rust resistance locus. The stripe rust-resistant translocation lines were found to carry some favorable agronomic traits, which could facilitate their use in wheat improvement. Collectively, the stripe rust resistance locus from A . cristatum 6P could be a novel resistance source and the screened stripe rust-resistant materials will be valuable for wheat disease breeding.

  19. Molecular analysis of pyrethroid resistance conferred by target insensitivity and increased metabolic detoxification in Plutella xylostella.

    Science.gov (United States)

    Sonoda, Shoji

    2010-05-01

    The pyrethroid resistance of the diamondback moth Plutella xylostella (L.) is conferred by increased gene expression of cytochrome P450 to detoxify the insecticide and/or through gene mutation of the sodium channel, which makes the individual insensitive to pyrethroids. However, no information is available about the correlation between the increased metabolic detoxification and the target insensitivity in pyrethroid resistance. Frequencies of pyrethroid-resistant alleles (L1014F, T929I and M918I) and two resistance-related mutations (A1101T and P1879S) at the sodium channel and expression levels of the cytochrome P450 gene CYP6BG1 were examined individually using laboratory and field strains of P. xylostella. Real-time quantitative PCR analysis using the laboratory strains revealed that levels of larval expression of the resistant strain, homozygous for the pyrethroid-resistant alleles other than the M918I, are significantly higher than those of the susceptible strain. In the field strains, the expression levels in insects having the same resistant alleles as those of the resistant strains varied greatly among individuals. The expression levels were not significantly higher than those in the heterozygotes. Significant correlation between the target insensitivity and the increased metabolic detoxification in pyrethroid resistance of P. xylostella was observed in the laboratory but not in the field.

  20. Transposon Tn5 encodes streptomycin resistance in nonenteric bacteria.

    OpenAIRE

    O'Neill, E A; Kiely, G M; Bender, R A

    1984-01-01

    Strains of Caulobacter crescentus, Pseudomonas putida, Acinetobacter calcoaceticus, Rhizobium meliloti, and Rhodopseudomonas sphaeroides carrying the kanamycin resistance-encoding transposon Tn5 were 15 to 500 times more resistant to streptomycin than transposon-free strains. The streptomycin resistance determinant, which is separable from the kanamycin resistance determinant of Tn5, was not expressed in Escherichia coli or Klebsiella aerogenes.

  1. Analysis of the Kanamycin in Raw Milk Using the Suspension Array

    Directory of Open Access Journals (Sweden)

    Yanfei Wang

    2013-01-01

    Full Text Available With the monoclonal antibody against kanamycin being prepared successfully, a bead-based indirect competitive fluorescent immunoassay was developed to detect kanamycin in milk. The fact that there was no significant cross-reaction with other aminoglycoside antibiotics implied that the monoclonal antibody was highly specific for kanamycin. The limit of detection (LOD and the 50% inhibition concentration (IC50 in raw milk were 3.2 ng/mL and 52.5 ng/mL, respectively. Using the method developed in this study, the kanamycin concentrations were monitored in raw milk after the intramuscular administration of kanamycin in sick cows. Compared to the conventional enzyme-linked immunosorbent assay (ELISA, the method using the suspension array system was more sensitive. The results obtained in the present study showed a good correlation with that of the ELISA.

  2. Conservation and Dispersion of Genes Conferring Resistance to Tomato Begomoviruses between Tomato and Pepper Genomes

    Directory of Open Access Journals (Sweden)

    Manisha Mangal

    2017-11-01

    Full Text Available In the present climate change scenario, controlling plant disease through exploitation of host plant resistance could contribute toward the sustainable crop production and global food security. In this respect, the identification of new sources of resistance and utilization of genetic diversity within the species may help in the generation of cultivars with improved disease resistance. Begomoviruses namely, Tomato yellow leaf curl virus (TYLCV and Chilli leaf curl virus (ChLCV are known to cause major yield losses in several economically important crop plants of the family Solanaceae. Though co-occurrence, association and synergistic interactions among these viruses in the host plants is reported, whether orthologous genetic loci in related host plants could be responsible for conferring resistance to these viruses has not been investigated yet. Several loci including Ty1, Ty2, Ty3, Ty4, and ty5 have been reported to confer resistance to leaf curl viruses in tomato. Here, we examined the pepper orthologous markers, corresponding to these QTL regions, for polymorphism between ChLCV susceptible and resistant genotypes of pepper. Further, to examine if the polymorphic markers are segregating with the disease resistance, Bulk Segregant Analysis (BSA was performed on F2 population derived from crosses between resistant and susceptible lines. However, none of the markers showed polymorphism in BSA suggesting that the tested markers are not linked to genes/QTLs responsible for conferring resistance to ChLCV in the selected genotypes. In silico analysis was performed to study the synteny and collinearity of genes located within these QTL regions in tomato and pepper genomes, which revealed that more than 60% genes located in Ty2 and Ty4, 13.71% genes in Ty1, 23.07% in Ty3, and 44.77% genes located within ty5 QTL region in tomato are conserved in pepper genome. However, despite such a high conservation in gene content, the linkage relationship in these

  3. Novel α-Tubulin Mutations Conferring Resistance to Dinitroaniline Herbicides in Lolium rigidum

    Directory of Open Access Journals (Sweden)

    Zhizhan Chu

    2018-02-01

    Full Text Available The dinitroaniline herbicides (particularly trifluralin have been globally used in many crops for selective grass weed control. Consequently, trifluralin resistance has been documented in several important crop weed species and has recently reached a level of concern in Australian Lolium rigidum populations. Here, we report novel mutations in the L. rigidum α-tubulin gene which confer resistance to trifluralin and other dinitroaniline herbicides. Nucleotide mutations at the highly conserved codon Arg-243 resulted in amino acid substitutions of Met or Lys. Rice calli transformed with the mutant 243-Met or 243-Lys α-tubulin genes were 4- to 8-fold more resistant to trifluralin and other dinitroaniline herbicides (e.g., ethalfluralin and pendimethalin compared to calli transformed with the wild type α-tubulin gene from L. rigidum. Comprehensive modeling of molecular docking predicts that Arg-243 is close to the trifluralin binding site on the α-tubulin surface and that replacement of Arg-243 by Met/Lys-243 results in a spatial shift of the trifluralin binding domain, reduction of trifluralin-tubulin contacts, and unfavorable interactions. The major effect of these substitutions is a significant rise of free interaction energy between α-tubulin and trifluralin, as well as between trifluralin and its whole molecular environment. These results demonstrate that the Arg-243 residue in α-tubulin is a determinant for trifluralin sensitivity, and the novel Arg-243-Met/Lys mutations may confer trifluralin resistance in L. rigidum.

  4. Analysis of acetohydroxyacid synthase1 gene in chickpea conferring resistance to imazamox herbicide.

    Science.gov (United States)

    Jain, Parul; Tar'an, Bunyamin

    2014-11-01

    Chickpea (Cicer arietinum L.) production in the Canadian prairies is challenging due to a lack of effective weed management mainly because of poor competition ability of the crop and limited registered herbicide options. Chickpea genotype with resistance to imidazolinone (IMI) herbicides has been identified. A point mutation in the acetohydroxyacid synthase1 (AHAS1) gene at C581 to T581, resulting in an amino acid substitution from Ala194 to Val194 (position 205, standardized to arabidopsis), confers the resistance to imazamox in chickpea. However, the molecular mechanism leading to the resistance is not fully understood. In many plant species, contrasting transcription levels of AHAS gene has been implicated in the resistant and susceptible genotypes in response to IMI. The objectives of this research were to compare the AHAS gene expression and AHAS enzyme activity in resistant and susceptible chickpea cultivars in response to imazamox herbicide treatment. Results from RT-qPCR indicated that there is no significant change in the transcript levels of AHAS1 between the susceptible and the resistant genotypes in response to imazamox treatment. Protein hydrophobic cluster analysis, protein-ligand docking analysis, and AHAS enzyme activity assay all indicated that the resistance to imazamox in chickpea is due to the alteration of interaction of the AHAS1 enzyme with the imazamox herbicide.

  5. Molecular mapping of two loci that confer resistance to Asian rust in soybean.

    Science.gov (United States)

    Silva, Danielle C G; Yamanaka, Naoki; Brogin, Rodrigo L; Arias, Carlos A A; Nepomuceno, Alexandre L; Di Mauro, Antônio O; Pereira, Selma S; Nogueira, Livia M; Passianotto, André L L; Abdelnoor, Ricardo V

    2008-06-01

    Asian soybean rust (ASR) is caused by the fungal pathogen Phakopsora pachyrhizi Sydow & Sydow. It was first identified in Brazil in 2001 and quickly infected soybean areas in several countries in South America. Primary efforts to combat this disease must involve the development of resistant cultivars. Four distinct genes that confer resistance against ASR have been reported: Rpp1, Rpp2, Rpp3, and Rpp4. However, no cultivar carrying any of those resistance loci has been released. The main objective of this study was to genetically map Rpp2 and Rpp4 resistance genes. Two F(2:3) populations, derived from the crosses between the resistant lines PI 230970 (Rpp2), PI 459025 (Rpp4) and the susceptible cultivar BRS 184, were used in this study. The mapping populations and parental lines were inoculated with a field isolate of P. pachyrhizi and evaluated for lesion type as resistant (RB lesions) or susceptible (TAN lesions). The mapping populations were screened with SSR markers, using the bulk segregant analysis (BSA) to expedite the identification of linked markers. Both resistance genes showed an expected segregation ratio for a dominant trait. This study allowed mapping Rpp2 and Rpp4 loci on the linkage groups J and G, respectively. The associated markers will be of great value on marker assisted selection for this trait.

  6. 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

  7. Introgression of resistance-conferring ALS mutations in herbicide-resistant weedy rice

    Science.gov (United States)

    Weedy red rice (Oryza sativa) competes aggressively with rice, reducing yields and grain quality. Clearfield™ rice, a nontransgenic, herbicide-resistant (HR) rice introduced in 2002 to control weedy rice, has resulted in some ALS-resistant weedy rice apparently due to gene flow. Studies were conduct...

  8. Molecular characterization of Als1, an acetohydroxyacid synthase mutation conferring resistance to sulfonylurea herbicides in soybean.

    Science.gov (United States)

    Ghio, Cecilia; Ramos, María Laura; Altieri, Emiliano; Bulos, Mariano; Sala, Carlos A

    2013-12-01

    The AHAS gene family in soybean was characterized. The locus Als1 for sulfonylurea resistance was mapped and the resistant allele was characterized at the molecular level. Sulfonylurea (SU) resistance in soybean is controlled by Als1, a semi-dominant allele obtained by EMS mutagenesis over the cultivar Williams 82. The overall objective of this research was to map Als1 in the soybean genome and to determine the nucleotidic changes conferring resistance to SU. Four nucleotide sequences (GmAhas1-4) showing high homology with the Arabidopsis thaliana acetohydroxyacid synthase (AHAS, EC 4.1.3.18) gene sequence were identified by in silico analysis, PCR-amplified from the SU-resistant line BTK323STS and sequenced. Expression analysis showed that GmAhas1, located on chromosome 4 by in silico analysis, is the most expressed sequence in true leaves. F2:3 families derived from the cross between susceptible and resistant lines were evaluated for SU resistance. Mapping results indicate that the locus als1 is located on chromosome 4. Sequence comparison of GmAhas1 between BTK323STS and Williams 82 showed a single nucleotide change from cytosine to thymine at position 532. This transversion generates an amino acid change from proline to serine at position 197 (A. thaliana nomenclature) of the AHAS catalytic subunit. An allele-specific marker developed for the GmAhas1 mutant sequence cosegregated with SU resistance in the F2 population. Taking together, the mapping, expression and sequencing results indicate that the GmAhas1 sequence corresponds to the Als1 gene sequence controlling SU resistance in soybean. The molecular breeding tools described herein create the basis to speed up the identification of new mutations in soybean AHAS leading to enhanced levels of resistance to SU or to other families of AHAS inhibitor herbicides.

  9. Identification of mutations conferring streptomycin resistance in multidrug-resistant tuberculosis of China.

    Science.gov (United States)

    Zhao, Li-Li; Liu, Hai-Can; Sun, Qing; Xiao, Tong-Yang; Zhao, Xiu-Qin; Li, Gui-Lian; Zeng, Chun-Yan; Wan, Kang-Lin

    2015-10-01

    We investigated the spectrum and frequency of mutations in rpsL, rrs, and gidB among 140 multidrug-resistant tuberculosis (MDR-TB) clinical isolates from China. The association between mutations and different genotypes was also analyzed. Our data revealed that 65.7% of MDR-TB were resistant to streptomycin (STR), and 90.2% of STR-resistant isolates were Beijing strains. STR resistance was correlated with Beijing family (P=0.00). Compared with phenotypic data, detection of mutations for the combination of these 3 genes exhibited 94.6% sensitivity, 91.7% specificity, and 93.6% accuracy. The most common mutations in STR-resistant isolates were rpsL128, 262, and rrs514, of which rpsL128 showed association with Beijing lineage (P=0.00). A combination of these 3 mutations can serve as the reliable predictors for STR resistance, showing the sensitivity, specificity, and accuracy of 85.9%, 97.9%, and 90.0%, respectively. Furthermore, gidBA276C, not A615G, was Beijing lineage specific. These findings are useful to develop rapid molecular diagnostic methods for STR resistance in China. Copyright © 2015 Elsevier Inc. All rights reserved.

  10. Over-expression of Oryza sativa Xrn4 confers plant resistance to virus infection.

    Science.gov (United States)

    Jiang, Shanshan; Jiang, Liangliang; Yang, Jian; Peng, Jiejun; Lu, Yuwen; Zheng, Hongying; Lin, Lin; Chen, Jianping; Yan, Fei

    2018-01-10

    Plant Xrn4 is a cytoplasmic 5' to 3' exoribonuclease that is reported to play an antiviral role during viral infection as demonstrated by experiments using the Xrn4s of Nicotiana benthamiana and Arabidopsis thaliana. Meanwhile, little is known about the anti-viral activity of Xrn4 from other plants. Here, we cloned the cytoplasmic Xrn4 gene of Oryza sativa (OsXrn4), and demonstrated that its over-expression elevated the 5'-3' exoribonuclease activity in rice plants and conferred resistance to rice stripe virus, a negative-sense RNA virus causing serious losses in East Asia. The accumulation of viral RNAs was also decreased. Moreover, the ectopic expression of OsXrn4 in N. benthamiana also conferred plant resistance to tobacco mosaic virus infection. These results show that the monocotyledonous plant cytoplasmic Xrn4 also has an antiviral role and thus provides a strategy for producing transgenic plants resistant to viral infection. Copyright © 2017. Published by Elsevier B.V.

  11. Molecular and biochemical characterization of an induced mutation conferring imidazolinone resistance in sunflower.

    Science.gov (United States)

    Sala, Carlos A; Bulos, Mariano; Echarte, Mariel; Whitt, Sherry R; Ascenzi, Robert

    2008-12-01

    A partially dominant nuclear gene conferring resistance to the imidazolinone herbicides was previously identified in the cultivated sunflower (Helianthus annuus L.) line CLHA-Plus developed by seed mutagenesis. The objective of this study was to characterize this resistant gene at the phenotypic, biochemical and molecular levels. CLHA-Plus showed a complete susceptibility to sulfonylureas (metsulfuron, tribenuron and chlorsulfuron) but, on the other hand, it showed a complete resistance to imidazolinones (imazamox, imazapyr and imazapic) at two rates of herbicide application. This pattern was in close association with the AHAS-inhibition kinetics of protein extracts of CLHA-Plus challenged with different doses of imazamox and chlorsulfuron. Nucleotide and deduced amino acid sequence comparisons between resistant and susceptible lines indicated that the imidazolinone-resistant AHAS of CLHA-Plus has a threonine codon (ACG) at position 122 (relative to the Arabidopsis thaliana AHAS sequence), whereas the herbicide-susceptible enzyme from BTK47 has an alanine residue (GCG) at this position. Since the resistance genes to AHAS-inhibiting herbicides so far characterized in sunflower code for the catalytic (large) subunit of AHAS, we propose to redesignate the wild type allele as ahasl1 and the incomplete dominant resistant alleles as Ahasl1-1 (previously Imr1 or Ar ( pur )), Ahasl1-2 (previously Ar ( kan )) and Ahasl1-3 (for the allele present in CLHA-Plus). The higher tolerance level to imidazolinones and the lack of cross-resistance to other AHAS-inhibiting herbicides of Ahasl1-3 indicate that this induced mutation can be used to develop commercial hybrids with superior levels of tolerance and, at the same time, to assist weed management where control of weedy common sunflower is necessary.

  12. Pathogens of bovine respiratory disease in North American feedlots conferring multidrug resistance via integrative conjugative elements.

    Science.gov (United States)

    Klima, Cassidy L; Zaheer, Rahat; Cook, Shaun R; Booker, Calvin W; Hendrick, Steve; Alexander, Trevor W; McAllister, Tim A

    2014-02-01

    In this study, we determined the prevalence of bovine respiratory disease (BRD)-associated viral and bacterial pathogens in cattle and characterized the genetic profiles, antimicrobial susceptibilities, and nature of antimicrobial resistance determinants in collected bacteria. Nasopharyngeal swab and lung tissue samples from 68 BRD mortalities in Alberta, Canada (n = 42), Texas (n = 6), and Nebraska (n = 20) were screened using PCR for bovine viral diarrhea virus (BVDV), bovine respiratory syncytial virus, bovine herpesvirus 1, parainfluenza type 3 virus, Mycoplasma bovis, Mannheimia haemolytica, Pasteurella multocida, and Histophilus somni. Excepting bovine herpesvirus 1, all agents were detected. M. haemolytica (91%) and BVDV (69%) were the most prevalent, with cooccurrence in 63% of the cattle. Isolates of M. haemolytica (n = 55), P. multocida (n = 8), and H. somni (n = 10) from lungs were also collected. Among M. haemolytica isolates, a clonal subpopulation (n = 8) was obtained from a Nebraskan feedlot. All three bacterial pathogens exhibited a high rate of antimicrobial resistance, with 45% exhibiting resistance to three or more antimicrobials. M. haemolytica (n = 18), P. multocida (n = 3), and H. somni (n = 3) from Texas and Nebraska possessed integrative conjugative elements (ICE) that conferred resistance for up to seven different antimicrobial classes. ICE were shown to be transferred via conjugation from P. multocida to Escherichia coli and from M. haemolytica and H. somni to P. multocida. ICE-mediated multidrug-resistant profiles of bacterial BRD pathogens could be a major detriment to many of the therapeutic antimicrobial strategies currently used to control BRD.

  13. Identification of QTLs conferring resistance to downy mildew in legacy cultivars of lettuce

    Science.gov (United States)

    Simko, Ivan; Atallah, Amy J.; Ochoa, Oswaldo E.; Antonise, Rudie; Galeano, Carlos H.; Truco, Maria Jose; Michelmore, Richard W.

    2013-01-01

    Many cultivars of lettuce (Lactuca sativa L.), the most popular leafy vegetable, are susceptible to downy mildew disease caused by Bremia lactucae. Cultivars Iceberg and Grand Rapids that were released in the 18th and 19th centuries, respectively, have high levels of quantitative resistance to downy mildew. We developed a population of recombinant inbred lines (RILs) originating from a cross between these two legacy cultivars, constructed a linkage map, and identified two QTLs for resistance on linkage groups 2 (qDM2.1) and 5 (qDM5.1) that determined resistance under field conditions in California and the Netherlands. The same QTLs determined delayed sporulation at the seedling stage in laboratory experiments. Alleles conferring elevated resistance at both QTLs originate from cultivar Iceberg. An additional QTL on linkage group 9 (qDM9.1) was detected through simultaneous analysis of all experiments with mixed-model approach. Alleles for elevated resistance at this locus originate from cultivar Grand Rapids. PMID:24096732

  14. Macrolide resistance conferred by rRNA mutations in field isolates of Mannheimia haemolytica and Pasteurella multocida

    DEFF Research Database (Denmark)

    Olsen, Anders S; Warrass, Ralf; Douthwaite, Stephen Roger

    2014-01-01

    OBJECTIVES: To determine how resistance to macrolides is conferred in field isolates of Pasteurella multocida and Mannheimia haemolytica that lack previously identified resistance determinants for rRNA methylation, efflux and macrolide-modifying enzymes. METHODS: Isolates of P. multocida and M...... by genome sequencing and primer extension on the rRNAs. RESULTS: Macrolide resistance in one M. haemolytica isolate was conferred by the 23S rRNA mutation A2058G; resistance in three P. multocida isolates were caused by mutations at the neighbouring nucleotide A2059G. In each strain, all six copies...... of the rrn operons encoded the respective mutations. There were no mutations in the ribosomal protein genes rplD or rplV, and no other macrolide resistance mechanism was evident. CONCLUSIONS: High-level macrolide resistance can arise from 23S rRNA mutations in P. multocida and M. haemolytica despite...

  15. 2,3-Dihydroxybenzoic acid attenuates kanamycin-induced volume reduction in mouse utricular type I hair cells

    DEFF Research Database (Denmark)

    Severinsen, Stig Åvall; Kirkegaard, Mette; Nyengaard, Jens Randel

    2006-01-01

    interest. This study estimates cellular quantitative changes in the utricular macula of mice following systemic treatment with kanamycin alone or in combination with DHB. The animals were injected with either saline, kanamycin or kanamycin+DHB for 15 days and perfusion fixed three weeks after last...... macula, hair cell type I and supporting cells decreased significantly in animals injected with kanamycin but not in animals co-treated with DHB. Hair and supporting cell numbers remained unchanged in all three groups. In conclusion, the kanamycin-induced volume reduction of type I hair cells...

  16. Overexpression of VOZ2 confers biotic stress tolerance but decreases abiotic stress resistance in Arabidopsis.

    Science.gov (United States)

    Nakai, Yusuke; Fujiwara, Sumire; Kubo, Yasuyuki; Sato, Masa H

    2013-03-01

    VOZ (vascular plant one zinc-finger protein) is a plant specific one-zinc finger type transcriptional activator, which is highly conserved through land plant evolution. We have previously shown that loss-of-function mutations in VOZ1 and VOZ2 showed increased cold and drought stress tolerances whereas decreased biotic stress resistance in Arabidopsis. Here, we demonstrate that transgenic plants overexpressing VOZ2 impairs freezing and drought stress tolerances but increases resistance to a fungal pathogen, Colletoricum higginsianum. Consistent with changes in the tolerance to biotic and abiotic stresses, the expression of marker genes for these stresses is significantly altered compared with those of the wild-type plant. These results indicate that a overexpression of VOZ2 confers biotic stress tolerance but impairs abiotic stress tolerances in Arabidopsis.

  17. Genetic engineering in insects: Cloning and transformation of genes conferring resistance to insecticides

    International Nuclear Information System (INIS)

    Mouches, C.

    1988-01-01

    Genetic engineering and transformation offer the possibility of modifying the genetic material of insects. These techniques will make it possible, for example, to transfer genes conferring resistance to insecticides into the genome of beneficial species, or to develop new methods of combating insect pests and disease carrying insects. We cloned two genes which contain the code for proteins that detoxify insecticides. The first, esterase B1 from Culex quinquefasciatus, is amplified approximately 250 times in Californian mosquitoes resistant to organic phosphate insecticides. A second esterase gene was cloned from bacteria which break down various organic phosphates. Experiments are in progress to transfer these genes to Drosophila and beneficial insects. These same genes could also serve as selection markers for the purpose of developing transformation techniques for different insects whose genome one wishes to modify using genetic engineering techniques. (author). 5 refs

  18. Multiple origins of resistance-conferring mutations in Plasmodium vivax dihydrofolate reductase

    Directory of Open Access Journals (Sweden)

    O'Neil Michael T

    2008-04-01

    Full Text Available Abstract Background In order to maximize the useful therapeutic life of antimalarial drugs, it is crucial to understand the mechanisms by which parasites resistant to antimalarial drugs are selected and spread in natural populations. Recent work has demonstrated that pyrimethamine-resistance conferring mutations in Plasmodium falciparum dihydrofolate reductase (dhfr have arisen rarely de novo, but spread widely in Asia and Africa. The origin and spread of mutations in Plasmodium vivax dhfr were assessed by constructing haplotypes based on sequencing dhfr and its flanking regions. Methods The P. vivax dhfr coding region, 792 bp upstream and 683 bp downstream were amplified and sequenced from 137 contemporary patient isolates from Colombia, India, Indonesia, Papua New Guinea, Sri Lanka, Thailand, and Vanuatu. A repeat motif located 2.6 kb upstream of dhfr was also sequenced from 75 of 137 patient isolates, and mutational relationships among the haplotypes were visualized using the programme Network. Results Synonymous and non-synonymous single nucleotide polymorphisms (SNPs within the dhfr coding region were identified, as was the well-documented in-frame insertion/deletion (indel. SNPs were also identified upstream and downstream of dhfr, with an indel and a highly polymorphic repeat region identified upstream of dhfr. The regions flanking dhfr were highly variable. The double mutant (58R/117N dhfr allele has evolved from several origins, because the 58R is encoded by at least 3 different codons. The triple (58R/61M/117T and quadruple (57L/61M/117T/173F, 57I/58R/61M/117T and 57L/58R/61M/117T mutant alleles had at least three independent origins in Thailand, Indonesia, and Papua New Guinea/Vanuatu. Conclusion It was found that the P. vivax dhfr coding region and its flanking intergenic regions are highly polymorphic and that mutations in P. vivax dhfr that confer antifolate resistance have arisen several times in the Asian region. This contrasts

  19. Resistance gene homologues in melon are linked to genetic loci conferring disease and pest resistance.

    Science.gov (United States)

    Brotman, Y.; Silberstein, L.; Kovalski, I.; Perin, C.; Dogimont, C.; Pitrat, M.; Klingler, J.; Thompson, A.; Perl-Treves, R.

    2002-05-01

    Genomic and cDNA fragments with homology to known disease resistance genes (RGH fragments) were cloned from Cucumis melo using degenerate-primer PCR. Fifteen homologues of the NBS-LRR gene family have been isolated. The NBS-LRR homologues show high divergence and, based on the partial NBS-fragment sequences, appear to include members of the two major subfamilies that have been described in dicot plants, one that possesses a TIR-protein element and one that lacks such a domain. Genomic organization of these sequences was explored by DNA gel-blot analysis, and conservation among other Cucurbitaceae was assessed. Two mapping populations that segregate for several disease and pest resistance loci were used to map the RGH probes onto the melon genetic map. Several NBS-LRR related sequences mapped to the vicinity of genetic loci that control resistance to papaya ringspot virus, Fusarium oxysporum race 1, F. oxysporum race 2 and to the insect pest Aphis gossypii. The utility of such markers for breeding resistant melon cultivars and for cloning the respective R-genes is discussed.

  20. Diversity of Molecular Mechanisms Conferring Carbapenem Resistance to Pseudomonas aeruginosa Isolates from Saudi Arabia

    Directory of Open Access Journals (Sweden)

    Mohamed H. Al-Agamy

    2016-01-01

    Full Text Available Background. This study described various molecular and epidemiological characters determining antibiotic resistance patterns in Pseudomonas aeruginosa isolates. Methods. A total of 34 carbapenem-resistant P. aeruginosa clinical isolates were isolated from samples collected at a tertiary hospital in Riyadh, Saudi Arabia, from January to December 2011. Susceptibility testing, serotyping, molecular characterization of carbapenem resistance, and pulsed-field gel electrophoresis (PFGE were performed. Results. All isolates were resistant to ceftazidime, and more than half were highly resistant (minimum inhibitory concentration (MIC > 256 mg/L. Fifteen isolates had MIC values ≥64 mg/L for any of the carbapenems examined. Vietnamese extended-spectrum β-lactamase (VEB-1 (n=16/34 and oxacillinase (OXA-10 (n=14/34 were the most prevalent extended-spectrum β-lactamase and penicillinase, respectively. Verona imipenemase (VIM-1, VIM-2, VIM-4, VIM-11, and VIM-28 and imipenemase (IMP-7 variants were found in metallo-β-lactamase producers. A decrease in outer membrane porin gene (oprD expression was seen in nine isolates, and an increase in efflux pump gene (MexAB expression was detected in five isolates. Six serotypes (O:1, O:4, O:7, O:10, O:11, and O:15 were found among the 34 isolates. The predominant serotype was O:11 (16 isolates, followed by O:15 (nine isolates. PFGE analysis of the 34 carbapenem-resistant P. aeruginosa isolates revealed 14 different pulsotypes. Conclusions. These results revealed diverse mechanisms conferring carbapenem resistance to P. aeruginosa isolates from Saudi Arabia.

  1. Apoptosis-related genes confer resistance to Fusarium wilt in transgenic 'Lady Finger' bananas.

    Science.gov (United States)

    Paul, Jean-Yves; Becker, Douglas K; Dickman, Martin B; Harding, Robert M; Khanna, Harjeet K; Dale, James L

    2011-12-01

    Fusarium wilt, caused by Fusarium oxysporum f. sp. cubense (Foc), is one of the most devastating diseases of banana (Musa spp.). Apart from resistant cultivars, there are no effective control measures for the disease. We investigated whether the transgenic expression of apoptosis-inhibition-related genes in banana could be used to confer disease resistance. Embryogenic cell suspensions of the banana cultivar, 'Lady Finger', were stably transformed with animal genes that negatively regulate apoptosis, namely Bcl-xL, Ced-9 and Bcl-2 3' UTR, and independently transformed plant lines were regenerated for testing. Following a 12-week exposure to Foc race 1 in small-plant glasshouse bioassays, seven transgenic lines (2 × Bcl-xL, 3 × Ced-9 and 2 × Bcl-2 3' UTR) showed significantly less internal and external disease symptoms than the wild-type susceptible 'Lady Finger' banana plants used as positive controls. Of these, one Bcl-2 3' UTR line showed resistance that was equivalent to that of wild-type Cavendish bananas that were included as resistant negative controls. Further, the resistance of this line continued for 23-week postinoculation at which time the experiment was terminated. Using TUNEL assays, Foc race 1 was shown to induce apoptosis-like features in the roots of wild-type 'Lady Finger' plants consistent with a necrotrophic phase in the life cycle of this pathogen. This was further supported by the observed reduction in these effects in the roots of the resistant Bcl-2 3' UTR-transgenic line. This is the first report on the generation of transgenic banana plants with resistance to Fusarium wilt. © 2011 The Authors. Plant Biotechnology Journal © 2011 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd.

  2. Deletion of OSH3 gene confers resistance against ISP-1 in Saccharomyces cerevisiae.

    Science.gov (United States)

    Yano, Tatsuya; Inukai, Masatoshi; Isono, Fujio

    2004-02-27

    Sphingolipids have been reported to regulate the growth and death of mammalian and yeast cells, but their precise mechanisms are unknown. In this paper, it was shown that the deletion of the oxysterol binding protein homologue 3 (OSH3) gene confers hyper resistance against ISP-1, an inhibitor of sphingolipid biosynthesis, in the yeast Saccharomyces cerevisiae. Furthermore, the overexpression of the ROK1 gene, which directly binds to Osh3p, conferred resistance against ISP-1, and the deletion of the KEM1 gene, which regulates microtubule functions, exhibited ISP-1 hypersensitivity. And yet, an ISP-1 treatment caused an abnormal mitotic spindle formation, and the ISP-1-induced cell cycle arrest was rescued by the deletion of the OSH3 gene. Taken together, it is suggested that the expression levels of the OSH3 gene influence the ISP-1 sensitivity of S. cerevisiae, and the sphingolipids are necessary for normal mitotic spindle formation in which the Osh3p may play a pivotal role.

  3. Detection of Multidrug-Resistant Salmonella enterica Serotype typhimurium DT104 Based on a Gene Which Confers Cross-Resistance to Florfenicol and Chloramphenicol

    Science.gov (United States)

    Bolton, Lance F.; Kelley, Lynda C.; Lee, Margie D.; Fedorka-Cray, Paula J.; Maurer, John J.

    1999-01-01

    Salmonella enterica serotype typhimurium (S. typhimurium) DT104 (DT104) first emerged as a major pathogen in Europe and is characterized by its pentadrug-resistant pattern. It has also been associated with outbreaks in the United States. The organism typically carries resistance to ampicillin, chloramphenicol, streptomycin, sulfonamides, and tetracycline. The mechanism of chloramphenicol resistance in DT104 was determined by producing antibiotic-resistant Escherichia coli host strain clones from DT104 DNA. DNA from chloramphenicol-resistant clones was sequenced, and probes specific for the genes floS. typhimurium (floSt), int, invA, and spvC were produced for colony blot hybridizations. One hundred nine Salmonella isolates, including 44 multidrug-resistant DT104 isolates, were tested to evaluate the specificities of the probes. The gene floSt, reported in this study, confers chloramphenicol and florfenicol resistance on S. typhimurium DT104. Florfenicol resistance is unique to S. typhimurium DT104 and multidrug-resistant S. typhimurium isolates with the same drug resistance profile among all isolates evaluated. Of 44 DT104 isolates tested, 98% were detected based on phenotypic florfenicol resistance and 100% had the floSt-positive genotype. Resistances to florfenicol and chloramphenicol are conferred by the gene floSt, described in this paper. Presumptive identification of S. typhimurium DT104 can be made rapidly based on the presence of the floSt gene or its resulting phenotype. PMID:10203484

  4. Role of H- and D- MATE-type transporters from multidrug resistant clinical isolates of Vibrio fluvialis in conferring fluoroquinolone resistance.

    Directory of Open Access Journals (Sweden)

    Priyabrata Mohanty

    Full Text Available BACKGROUND: The study seeks to understand the role of efflux pumps in multidrug resistance displayed by the clinical isolates of Vibrio fluvialis, a pathogen known to cause cholera-like diarrhoea. METHODOLOGY: Two putative MATE family efflux pumps (H- and D-type were PCR amplified from clinical isolates of V. fluvialis obtained from Kolkata, India, in 2006 and sequenced. Bioinformatic analysis of these proteins was done to predict protein structures. Subsequently, the genes were cloned and expressed in a drug hypersusceptible Escherichia coli strain KAM32 using the vector pBR322. The recombinant clones were tested for the functionality of the efflux pump proteins by MIC determination and drug transport assays using fluorimeter. RESULTS: The sequences of the genes were found to be around 99% identical to their counterparts in V. cholerae. Protein structure predicting servers TMHMM and I-TASSER depicted ten-twelve membrane helical structures for both type of pumps. Real time PCR showed that these genes were expressed in the native V. fluvialis isolates. In the drug transport assays, the V. fluvialis clinical isolates as well as recombinant E. coli harbouring the efflux pump genes showed the energy-dependent and sodium ion-dependent drug transport activity. KAM32 cells harbouring the recombinant plasmids showed elevated MIC to the fluoroquinolones, norfloxacin and ciprofloxacin but H-type pumps VCH and VFH from V. cholerae and V. fluvialis respectively, showed decreased MIC to aminoglycosides like gentamicin, kanamycin and streptomycin. Decrease in MIC was also observed for acriflavin, ethidium bromide, safranin and nalidixic acid. SIGNIFICANCE: Increased resistance towards fluoroquinolones exhibited due to these efflux pumps from multidrug resistant clinical isolates of V. fluvialis implies that treatment procedure may become more elaborate for this simple but highly infectious disease. To the best of our knowledge, this is the first report of

  5. IDENTIFICATION OF A MAJOR QUANTITATIVE TRAIT LOCUS CONFERRING RICE BLAST RESISTANCE USING RECOMBINANT INBRED LINES

    Directory of Open Access Journals (Sweden)

    Sobrizal Sobrizal

    2013-05-01

    Full Text Available Blast disease caused by Pyricularia oryzae is one of the limiting factors for rice production world wide. The use of resistant varieties for managing blast disease is considered as the most eco-friendly approaches. However, their resistances may be broken down within a few years due to the appearance of new virulent blast races in the field. The objective of the present study was to identify the quantitative trait locus (QTL conferring resistance to blast disease using 126 recombinant inbred (RI lines originated from a crossing of a durably resistant upland rice genotype (Laka and a highly susceptible rice accession cultivar (Kencana Bali. The RI population was developed through a single seed descent method from 1997 to 2004. Resistance of the RI lines was evaluated for blast in an endemic area of Sukabumi, West Java, in 2005. Disease intensity of the blast was examined following the standard evaluation system developed by the International Rice Research Institute (IRRI. At the same year the RI lines were analyzed with 134 DNA markers. Results of the study showed that one major QTL was found to be associated with blast resistance, and this QTL was located near RM2136 marker on the long arm of chromosome 11. This QTL explained 87% of the phenotypic variation with 37% additive effect. The map position of this QTL differed from that of a partial resistant gene, Pi34, identified previously on chromosome 11 in the Japanese durably resistant variety, Chubu 32. The QTL, however, was almost at the same position as that of the multiple allele-resistant gene, Pik. Therefore, an allelic test should be conducted to clarify the allelic relationship between QTL identified in this study and the Pik. The RI lines are the permanent segregating population that could be very useful for analysing phenotypic variations of important agronomic traits possibly owned by the RI lines. The major QTL identified in this study could be used as a genetic resource in

  6. Screening for streptomycin resistance-conferring mutations in Mycobacterium tuberculosis clinical isolates from Poland.

    Science.gov (United States)

    Jagielski, Tomasz; Ignatowska, Helena; Bakuła, Zofia; Dziewit, Łukasz; Napiórkowska, Agnieszka; Augustynowicz-Kopeć, Ewa; Zwolska, Zofia; Bielecki, Jacek

    2014-01-01

    Currently, mutations in three genes, namely rrs, rpsL, and gidB, encoding 16S rRNA, ribosomal protein S12, and 16S rRNA-specific methyltransferase, respectively, are considered to be involved in conferring resistance to streptomycin (STR) in Mycobacterium tuberculosis. The aim of this study was to investigate the spectrum and frequency of these mutations in M. tuberculosis clinical isolates, both resistant and susceptible to STR. Sixty-four M. tuberculosis isolates recovered from as many TB patients from Poland in 2004 were included in the study. Within the sample were 50 multidrug-resistant (32 STR-resistant and 18 STR-susceptible) and 14 pan-susceptible isolates. Preliminary testing for STR resistance was performed with the 1% proportion method. The MICs of STR were determined by the Etest method. Mutation profiling was carried out by amplifying and sequencing the entire rrs, rpsL, and gidB genes. Non-synonymous mutations in either rrs or rpsL gene were detected in 23 (71.9%) of the STR-resistant and none of the STR-susceptible isolates. Mutations in the gidB gene were distributed among 12 (37.5%) STR-resistant and 13 (40.6%) STR-susceptible isolates. Four (12.5%) STR-resistant isolates were wild-type at all three loci examined. None of the rrs, rpsL or gidB mutations could be linked to low, intermediate or high level of STR resistance. In accordance with previous findings, the gidB 47T→G (L16R) mutation was associated with the Latin American-Mediterranean genotype family, whereas 276A→C (E92D) and 615A→G (A205A) mutations of the gidB gene were associated with the Beijing lineage. The study underlines the usefulness of rrs and rpsL mutations as molecular markers for STR resistance yet not indicative of its level. The gidB polymorphisms can serve as phylogenetic markers.

  7. Dominant mutations confer resistance to the immunosuppressant, rapamycin, in variants of a T cell lymphoma.

    Science.gov (United States)

    Dumont, F J; Staruch, M J; Grammer, T; Blenis, J; Kastner, C A; Rupprecht, K M

    1995-06-01

    Rapamycin (RAP) disrupts signaling events implicated in cytokine-dependent proliferation of lymphocytes and other cells. This action is known to involve the formation of molecular complexes between the drug and intracellular binding proteins, termed FKBPs. However, the biochemical target(s) for the effector RAP-FKBP complexes remain uncharacterized. As an approach to explore the mechanism of action of RAP, we have isolated three independent sets of somatic mutants of the YAC-1 murine T cell line with markedly reduced sensitivity to the drug's inhibitory effects on proliferation and on IL-1-induced IFN-gamma production. These mutants were still fully sensitive to FK-506, an immunosuppressant structurally related to RAP whose mode of action also involves an interaction with FKBPs. Furthermore, the 12-kDa FKBP, FKBP12, was detectable in immunoblots from cytosolic extracts and eluates from RAP-affinity matrix in the mutants as in wild-type cells, suggesting that the resistance to RAP in the mutants is not due to a lack of FKBP12 expression. Cell fusion experiments were conducted to further define the nature of the alterations imparting RAP resistance in these mutants. Clones deficient in either thymidine kinase or hypoxanthine-guanine phosphoribosyltransferase, suitable as fusion partners for aminopterin-based selection of hybrids were generated from the wild-type or mutant lines. In most instances, the hybrids derived from the fusion between RAP-sensitive clones and RAP-resistant clones exhibited a RAP-resistant phenotype. Similar results were obtained with hybrids between RAP-resistant YAC-1 clones and the RAP-sensitive EL-4 cell line. Therefore, the mutations that confer resistance to RAP in the present system are dominant. Altogether, our observations are consistent with a model where pharmacologically relevant targets for the RAP-FKBP complex, rather than FKBP, might be altered in the mutants such that the inactivation of these targets by the effector complex is

  8. Identification of a novel mutation at the primary dimer interface of GyrA conferring fluoroquinolone resistance in Clostridium difficile.

    Science.gov (United States)

    Mac Aogáin, Micheál; Kilkenny, Shauna; Walsh, Claire; Lindsay, Sinéad; Moloney, Geraldine; Morris, Trefor; Jones, Sophie; Rogers, Thomas R

    2015-12-01

    The aim of this study was to determine whether alternative resistance mechanisms, other than mutation in the quinolone resistance-determining region (QRDR) of DNA gyrase, could confer fluoroquinolone resistance in Clostridium difficile. An in vitro-generated C. difficile mutant exhibiting increased fluoroquinolone resistance was isolated through antibiotic selection on ciprofloxacin. The QRDR of this mutant was investigated by chain-termination sequencing and was found to be devoid of mutation. To determine the nature of the non-QRDR resistance mechanism in this strain, the genomes of the mutant and wild-type strains were sequenced. The gyrBA region from a collection of clinical isolates exhibiting variable fluoroquinolone resistance levels was also sequenced and was compared with that present in 918 publicly available C. difficile genomic data sets. Whole-genome sequence analysis of the fluoroquinolone-resistant mutant revealed a single non-synonymous substitution (Ala384Asp) at the predicted primary dimer interface of GyrA, far beyond the classically defined QRDR. This novel mutation caused increased resistance to ciprofloxacin, ofloxacin, levofloxacin and moxifloxacin while conferring hypersusceptibility to novobiocin. Several novel extra-QRDR polymorphisms in C. difficile DNA gyrase were identified among clinical isolates, whilst observed fluoroquinolone resistance in strains devoid of gyrBA mutations confirmed the existence of DNA gyrase-independent resistance mechanisms in this species. In conclusion, we report the first non-QRDR mutation to confer fluoroquinolone resistance in C. difficile. Although the Ala384Asp substitution was not detected in clinical isolates, this study revealed a diversity of alternative extra-QRDR polymorphisms in DNA gyrase whose association with fluoroquinolone resistance warrants further investigation. Copyright © 2015 International Society for Chemotherapy of Infection and Cancer. Published by Elsevier Ltd. All rights reserved.

  9. Ultra-sensitive detection of kanamycin for food safety using a reduced graphene oxide-based fluorescent aptasensor

    Science.gov (United States)

    Ha, Na-Reum; Jung, In-Pil; La, Im-Joung; Jung, Ho-Sup; Yoon, Moon-Young

    2017-01-01

    Overuse of antibiotics has caused serious problems, such as appearance of super bacteria, whose accumulation in the human body through the food chain is a concern. Kanamycin is a common antibiotic used to treat diverse infections; however, residual kanamycin can cause many side effects in humans. Thus, development of an ultra-sensitive, precise, and simple detection system for residual kanamycin in food products is urgently needed for food safety. In this study, we identified kanamycin-binding aptamers via a new screening method, and truncated variants were analyzed for optimization of the minimal sequence required for target binding. We found various aptamers with high binding affinity from 34.7 to 669 nanomolar Kdapp values with good specificity against kanamycin. Furthermore, we developed a reduced graphene oxide (RGO)-based fluorescent aptasensor for kanamycin detection. In this system, kanamycin was detected at a concentration as low as 1 pM (582.6 fg/mL). In addition, this method could detect kanamycin accurately in kanamycin-spiked blood serum and milk samples. Consequently, this simple, rapid, and sensitive kanamycin detection system with newly structural and functional analysis aptamer exhibits outstanding detection compared to previous methods and provides a new possibility for point of care testing and food safety.

  10. Conferred resistance to Botrytis cinerea in Lilium by overexpression of the RCH10 chitinase gene.

    Science.gov (United States)

    Núñez de Cáceres González, Francisco F; Davey, Michael R; Cancho Sanchez, Ester; Wilson, Zoe A

    2015-07-01

    Transgenic Lilium lines have been generated by Agrobacterium -mediated transformation that have enhanced resistance to Botrytis cinerea as a consequence of ectopic expression of a rice chitinase gene. The production of ornamentals is an important global industry, with Lilium being one of the six major bulb crops in the world. The international trade in ornamentals is in the order of £60-75 billion and is expected to increase worldwide by 2-4% per annum. The continued success of the floriculture industry depends on the introduction of new species/cultivars with major alterations in key agronomic characteristics, such as resistance to pathogens. Fungal diseases are the cause of reduced yields and marketable quality of cultivated plants, including ornamental species. The fungal pathogen Botrytis causes extreme economic losses to a wide range of crop species, including ornamentals such as Lilium. Agrobacterium-mediated transformation was used to develop Lilium oriental cv. 'Star Gazer' plants that ectopically overexpress the Rice Chitinase 10 gene (RCH10), under control of the CaMV35S promoter. Levels of conferred resistance linked to chitinase expression were evaluated by infection with Botrytis cinerea; sporulation was reduced in an in vitro assay and the relative expression of the RCH10 gene was determined by quantitative reverse transcriptase-PCR. The extent of resistance to Botrytis, compared to that of the wild type plants, showed a direct correlation with the level of chitinase gene expression. Transgenic plants grown to flowering showed no detrimental phenotypic effects associated with transgene expression. This is the first report of Lilium plants with resistance to Botrytis cinerea generated by a transgenic approach.

  11. Lr67/Yr46 confers adult plant resistance to stem rust and powdery mildew in wheat.

    Science.gov (United States)

    Herrera-Foessel, Sybil A; Singh, Ravi P; Lillemo, Morten; Huerta-Espino, Julio; Bhavani, Sridhar; Singh, Sukhwinder; Lan, Caixia; Calvo-Salazar, Violeta; Lagudah, Evans S

    2014-04-01

    We demonstrate that Lr67/Yr46 has pleiotropic effect on stem rust and powdery mildew resistance and is associated with leaf tip necrosis. Genes are designated as Sr55, Pm46 and Ltn3 , respectively. Wheat (Triticum aestivum) accession RL6077, known to carry the pleiotropic slow rusting leaf and yellow rust resistance genes Lr67/Yr46 in Thatcher background, displayed significantly lower stem rust (P. graminis tritici; Pgt) and powdery mildew (Blumeria graminis tritici; Bgt) severities in Kenya and in Norway, respectively, compared to its recurrent parent Thatcher. We investigated the resistance of RL6077 to stem rust and powdery mildew using Avocet × RL6077 F6 recombinant inbred lines (RILs) derived from two photoperiod-insensitive F3 families segregating for Lr67/Yr46. Greenhouse seedling tests were conducted with Mexican Pgt race RTR. Field evaluations were conducted under artificially initiated stem rust epidemics with Pgt races RTR and TTKST (Ug99 + Sr24) at Ciudad Obregon (Mexico) and Njoro (Kenya) during 2010-2011; and under natural powdery mildew epiphytotic in Norway at Ås and Hamar during 2011 and 2012. In Mexico, a mean reduction of 41 % on stem rust severity was obtained for RILs carrying Lr67/Yr46, compared to RILs that lacked the gene, whereas in Kenya the difference was smaller (16 %) but significant. In Norway, leaf tip necrosis was associated with Lr67/Yr46 and RILs carrying Lr67/Yr46 showed a 20 % reduction in mean powdery mildew severity at both sites across the 2 years of evaluation. Our study demonstrates that Lr67/Yr46 confers partial resistance to stem rust and powdery mildew and is associated with leaf tip necrosis. The corresponding pleiotropic, or tightly linked, genes, designated as Sr55, Pm46, and Ltn3, can be utilized to provide broad-spectrum durable disease resistance in wheat.

  12. Alcohol dehydrogenase 1 (ADH1) confers both abiotic and biotic stress resistance in Arabidopsis.

    Science.gov (United States)

    Shi, Haitao; Liu, Wen; Yao, Yue; Wei, Yunxie; Chan, Zhulong

    2017-09-01

    Although the transcriptional regulation and upstream transcription factors of AtADH1 in response to abiotic stress are widely revealed, the in vivo roles of AtADH1 remain unknown. In this study, we found that the expression of AtADH1 was largely induced after salt, drought, cold and pathogen infection. Further studies found that AtADH1 overexpressing plants were more sensitive to abscisic acid (ABA) in comparison to wide type (WT), while AtADH1 knockout mutants showed no significant difference compared with WT in ABA sensitivity. Consistently, AtADH1 overexpressing plants showed improved stress resistance to salt, drought, cold and pathogen infection than WT, but the AtADH1 knockout mutants had no significant difference in abiotic and biotic stress resistance. Moreover, overexpression of AtADH1 expression increased the transcript levels of multiple stress-related genes, accumulation of soluble sugars and callose depositions. All these results indicate that AtADH1 confers enhanced resistance to both abiotic and biotic stresses. Copyright © 2017 Elsevier B.V. All rights reserved.

  13. Identification of wheat gene Sr35 that confers resistance to Ug99 stem rust race group.

    Science.gov (United States)

    Saintenac, Cyrille; Zhang, Wenjun; Salcedo, Andres; Rouse, Matthew N; Trick, Harold N; Akhunov, Eduard; Dubcovsky, Jorge

    2013-08-16

    Wheat stem rust, caused by Puccinia graminis f. sp. tritici (Pgt), is a devastating disease that can cause severe yield losses. A previously uncharacterized Pgt race, designated Ug99, has overcome most of the widely used resistance genes and is threatening major wheat production areas. Here, we demonstrate that the Sr35 gene from Triticum monococcum is a coiled-coil, nucleotide-binding, leucine-rich repeat gene that confers near immunity to Ug99 and related races. This gene is absent in the A-genome diploid donor and in polyploid wheat but is effective when transferred from T. monococcum to polyploid wheat. The cloning of Sr35 opens the door to the use of biotechnological approaches to control this devastating disease and to analyses of the molecular interactions that define the wheat-rust pathosystem.

  14. High mobility group A1 enhances tumorigenicity of human cholangiocarcinoma and confers resistance to therapy.

    Science.gov (United States)

    Quintavalle, Cristina; Burmeister, Katharina; Piscuoglio, Salvatore; Quagliata, Luca; Karamitopoulou, Eva; Sepe, Romina; Fusco, Alfredo; Terracciano, Luigi M; Andersen, Jesper B; Pallante, Pierlorenzo; Matter, Matthias S

    2017-09-01

    High mobility group A1 (HMGA1) protein has been described to play an important role in numerous types of human carcinoma. By the modulation of several target genes HMGA1 promotes proliferation and epithelial-mesenchymal transition of tumor cells. However, its role in cholangiocarcinoma (CCA) has not been addressed yet. Therefore, we determined HMGA1 mRNA expression in CCA samples in a transcriptome array (n = 104) and a smaller cohort (n = 13) by qRT-PCR. Protein expression was evaluated by immunohistochemistry in a tissue microarray (n = 67). In addition, we analyzed changes in cell proliferation, colony formation, response to gemcitabine treatment, and target gene expression after modulation of HMGA1 expression in CCA cell lines. mRNA levels of HMGA1 were found to be upregulated in 15-62% depending on the cohort analyzed. Immunohistochemistry showed HMGA1 overexpression in 51% of CCA specimens. Integration with clinico-pathological data revealed that high HMGA1 expression was associated with reduced time to recurrence and a positive lymph node status in extrahepatic cholangiocellular carcinoma. In vitro experiments showed that overexpression of HMGA1 in CCA cell lines promoted cell proliferation, whereas its suppression reduced growth rate. HMGA1 further promoted colony formation in an anchorage independent growth and conferred resistance to gemcitabine treatment. Finally, HMGA1 modulated the expression of two genes involved in CCA carcinogenesis, iNOS and ERBB2. In conclusion, our findings indicate that HMGA1 expression is increased in a substantial number of CCA specimens. HMGA1 further promotes CCA tumorigenicity and confers resistance to chemotherapy. © 2017 Wiley Periodicals, Inc.

  15. Molecular mapping and characterization of two genes conferring resistance to Phytophthora sojae in a soybean landrace PI 567139B

    Science.gov (United States)

    Phytophthora root and stem rot (PRR), caused by the soil-borne oomycete pathogen Phytophthora sojae, is one of the most destructive diseases of soybean. PRR can be effectively controlled by race-specific genes conferring resistance to P. sojae (Rps). However, the Rps genes are usually non-durable, a...

  16. satG, Conferring Resistance to Streptogramin A, Is Widely Distributed in Enterococcus faecium Strains but Not in Staphylococci

    Science.gov (United States)

    Haroche, Julien; Allignet, Jeanine; Aubert, Sylvie; Van Den Bogaard, Anthony E.; El Solh, Névine

    2000-01-01

    A gene almost identical to satG was isolated from an Enterococcus faecium strain. This gene was transferred to a Staphylococcus aureus recipient strain where it conferred resistance to streptogramin A. satG was found to be widely distributed among E. faecium strains but not detected among staphylococci. PMID:10602747

  17. Magnitude of gene mutations conferring drug resistance in mycobacterium tuberculosis isolates from lymph node aspirates in ethiopia.

    Science.gov (United States)

    Biadglegne, Fantahun; Tessema, Belay; Rodloff, Arne C; Sack, Ulrich

    2013-01-01

    Resistance to drugs is due to particular genomic mutations in the specific genes of Mycobacterium tuberculosis. Timely genetic characterization will allow identification of resistance mutations that will optimize an effective antibiotic treatment regimen. We determine the magnitude of gene mutations conferring resistance to isoniazid (INH), rifampicin (RMP) and ethambutol (EMB) among tuberculosis (TB) lymphadenitis patients. A cross sectional prospective study was conducted among 226 M.tuberculosis isolates from culture positive lymph node aspirates collected from TB lymphadenitis patients between April 2012 and May 2012. Detection of mutations conferring resistance to drugs was carried out using GenoType(®) MTBDRplus and GenoType® MTBDRsl assay. Out of the 226 strains, mutations conferring resistance to INH, RMP, multidrug resistance tuberculosis (MDR-TB) and EMB were 8, 3, 2 and 2 isolates, respectively. There was no isolated strain that showed mutation in the inhA promoter region gene. All INH resistant strains had mutations in the katG gene at codon 315 with amino acid change of S315T1. Among rifampicin resistant strains, two isolates displayed mutations at codon 531 in the rpoB gene with amino acid change of S531L and one isolate was by omission of wild type probes at Q513L. According to mutations associated with ethambutol resistance, all of the isolates had mutations in the embB gene with aminoacid change of M306I. All isolates resistant to INH, RMP and MDR using BacT/AlerT 3D system were correctly identified by GenoType® MTBDRplus assay. We observed mutations conferring resistance to INH at S315T1 of the katG gene, RMP at S531L and Q513L in the rpoB genes and EMB at M306I of the embB gene. In the absence of conventional drug susceptibility testing, the effort to develop easy, rapid and cost effective molecular assays for drug resistance TB monitoring is definitely desirable and the GenoType® MTBDRplus assay was found to be a useful method for diagnosis

  18. Macrolide resistance conferred by rRNA mutations in field isolates of Mannheimia haemolytica and Pasteurella multocida.

    Science.gov (United States)

    Olsen, Anders S; Warrass, Ralf; Douthwaite, Stephen

    2015-02-01

    To determine how resistance to macrolides is conferred in field isolates of Pasteurella multocida and Mannheimia haemolytica that lack previously identified resistance determinants for rRNA methylation, efflux and macrolide-modifying enzymes. Isolates of P. multocida and M. haemolytica identified as being highly resistant (MICs >64 mg/L) to the macrolides erythromycin, gamithromycin, tilmicosin, tildipirosin and tulathromycin were screened by multiplex PCR for the previously identified resistance genes erm(42), msr(E) and mph(E). Strains lacking these determinants were analysed by genome sequencing and primer extension on the rRNAs. Macrolide resistance in one M. haemolytica isolate was conferred by the 23S rRNA mutation A2058G; resistance in three P. multocida isolates were caused by mutations at the neighbouring nucleotide A2059G. In each strain, all six copies of the rrn operons encoded the respective mutations. There were no mutations in the ribosomal protein genes rplD or rplV, and no other macrolide resistance mechanism was evident. High-level macrolide resistance can arise from 23S rRNA mutations in P. multocida and M. haemolytica despite their multiple copies of rrn. Selective pressures from exposure to different macrolide or lincosamide drugs presumably resulted in consolidation of either the A2058G or the A2059G mutation. © The Author 2014. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

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

    Science.gov (United States)

    Nakano, Shigeru; Fukaya, Masahiro

    2008-06-30

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

  20. Cellular expression of gH confers resistance to herpes simplex virus type-1 entry

    International Nuclear Information System (INIS)

    Scanlan, Perry M.; Tiwari, Vaibhav; Bommireddy, Susmita; Shukla, Deepak

    2003-01-01

    Entry of herpes simplex virus-1 (HSV-1) into cells requires a concerted action of four viral glycoproteins gB, gD, and gH-gL. Previously, cell surface expression of gD had been shown to confer resistance to HSV-1 entry. To investigate any similar effects caused by other entry glycoproteins, gB and gH-gL were coexpressed with Nectin-1 in Chinese hamster ovary (CHO) cells. Interestingly, cellular expression of gB had no effect on HSV-1(KOS) entry. In contrast, entry was significantly reduced in cells expressing gH-gL. This effect was further analyzed by expressing gH and gL separately. Cells expressing gL were normally susceptible, whereas gH-expressing cells were significantly resistant. Further experiments suggested that the gH-mediated interference phenomenon was not specific to any particular gD receptor and was also observed in gH-expressing HeLa cells. Moreover, contrary to a previous report, gL-independent cell surface expression of gH was detected in stably transfected CHO cells, possibly implicating cell surface gH in the interference phenomenon. Thus, taken together these findings indicate that cellular expression of gH interferes with HSV-1 entry

  1. Disruptive environmental chemicals and cellular mechanisms that confer resistance to cell death.

    Science.gov (United States)

    Narayanan, Kannan Badri; Ali, Manaf; Barclay, Barry J; Cheng, Qiang Shawn; D'Abronzo, Leandro; Dornetshuber-Fleiss, Rita; Ghosh, Paramita M; Gonzalez Guzman, Michael J; Lee, Tae-Jin; Leung, Po Sing; Li, Lin; Luanpitpong, Suidjit; Ratovitski, Edward; Rojanasakul, Yon; Romano, Maria Fiammetta; Romano, Simona; Sinha, Ranjeet K; Yedjou, Clement; Al-Mulla, Fahd; Al-Temaimi, Rabeah; Amedei, Amedeo; Brown, Dustin G; Ryan, Elizabeth P; Colacci, Annamaria; Hamid, Roslida A; Mondello, Chiara; Raju, Jayadev; Salem, Hosni K; Woodrick, Jordan; Scovassi, A Ivana; Singh, Neetu; Vaccari, Monica; Roy, Rabindra; Forte, Stefano; Memeo, Lorenzo; Kim, Seo Yun; Bisson, William H; Lowe, Leroy; Park, Hyun Ho

    2015-06-01

    Cell death is a process of dying within biological cells that are ceasing to function. This process is essential in regulating organism development, tissue homeostasis, and to eliminate cells in the body that are irreparably damaged. In general, dysfunction in normal cellular death is tightly linked to cancer progression. Specifically, the up-regulation of pro-survival factors, including oncogenic factors and antiapoptotic signaling pathways, and the down-regulation of pro-apoptotic factors, including tumor suppressive factors, confers resistance to cell death in tumor cells, which supports the emergence of a fully immortalized cellular phenotype. This review considers the potential relevance of ubiquitous environmental chemical exposures that have been shown to disrupt key pathways and mechanisms associated with this sort of dysfunction. Specifically, bisphenol A, chlorothalonil, dibutyl phthalate, dichlorvos, lindane, linuron, methoxychlor and oxyfluorfen are discussed as prototypical chemical disruptors; as their effects relate to resistance to cell death, as constituents within environmental mixtures and as potential contributors to environmental carcinogenesis. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  2. Cerebroside elicitor confers resistance to fusarium disease in various plant species.

    Science.gov (United States)

    Umemura, Kenji; Tanino, Shigeki; Nagatsuka, Tadako; Koga, Jinichiro; Iwata, Michiaki; Nagashima, Kenji; Amemiya, Yoshimiki

    2004-08-01

    ABSTRACT In the rice blast fungus pathosystem, cerebroside, a compound categorized as a sphingolipid, was found in our previous study to be a non-racespecific elicitor, which elicits defense responses in rice. Here we describe that cerebroside C is produced in diverse strains of Fusarium oxysporum, a common soilborne agent of wilt disease affecting a wide range of plant species. In addition, some type of cerebroside elicitor involving cerebroside A, B, or C was detected in other soilborne phytopathogens, such as Pythium and Botrytis. Treatment of lettuce (Lactuca sativa), tomato (Lycopersicon esculentum), melon (Cucumis melo), and sweet potato (Ipomoea batatas) with cerebroside B resulted in resistance to infection by each pathogenic strain of F. oxysporum. Induction of pathogenesis-related genes and H(2)O(2) production by treatment with cerebroside B were observed in tomato root tissues. The cerebroside elicitor showed no antifungal activity against F. oxysporum in vitro, indicating that the cerebroside elicitor activates defense mechanisms to confer resistance to Fusarium disease. These results suggest that cerebroside functions as a non-race-specific elicitor in a wide range of plant-phytopathogenic fungus interactions. Additionally, cerebroside elicitor serves as a potential biologically derived control agent.

  3. Label-free detection of kanamycin based on a G-quadruplex DNA aptamer-based fluorescent intercalator displacement assay

    Science.gov (United States)

    Xing, Yun-Peng; Liu, Chun; Zhou, Xiao-Hong; Shi, Han-Chang

    2015-01-01

    This work was the first to report that the kanamycin-binding DNA aptamer (5'-TGG GGG TTG AGG CTA AGC CGA-3') can form stable parallel G-quadruplex DNA (G4-DNA) structures by themselves and that this phenomenon can be verified by nondenaturing polyacrylamide gel electrophoresis and circular dichroism spectroscopy. Based on these findings, we developed a novel label-free strategy for kanamycin detection based on the G4-DNA aptamer-based fluorescent intercalator displacement assay with thiazole orange (TO) as the fluorescence probe. In the proposed strategy, TO became strongly fluorescent upon binding to kanamycin-binding G4-DNA. However, the addition of kanamycin caused the displacement of TO from the G4-DNA-TO conjugate, thereby resulting in decreased fluorescent signal, which was inversely related to the kanamycin concentration. The detection limit of the proposed assay decreased to 59 nM with a linear working range of 0.1 μM to 20 μM for kanamycin. The cross-reactivity against six other antibiotics was negligible compared with the response to kanamycin. A satisfactory recovery of kanamycin in milk samples ranged from 80.1% to 98.0%, confirming the potential of this bioassay in the measurement of kanamycin in various applications. Our results also served as a good reference for developing similar fluorescent G4-DNA-based bioassays in the future.

  4. Resistance to Downy Mildew in Lettuce 'La Brillante' is Conferred by Dm50 Gene and Multiple QTL.

    Science.gov (United States)

    Simko, Ivan; Ochoa, Oswaldo E; Pel, Mathieu A; Tsuchida, Cayla; Font I Forcada, Carolina; Hayes, Ryan J; Truco, Maria-Jose; Antonise, Rudie; Galeano, Carlos H; Michelmore, Richard W

    2015-09-01

    Many cultivars of lettuce (Lactuca sativa L.) are susceptible to downy mildew, a nearly globally ubiquitous disease caused by Bremia lactucae. We previously determined that Batavia type cultivar 'La Brillante' has a high level of field resistance to the disease in California. Testing of a mapping population developed from a cross between 'Salinas 88' and La Brillante in multiple field and laboratory experiments revealed that at least five loci conferred resistance in La Brillante. The presence of a new dominant resistance gene (designated Dm50) that confers complete resistance to specific isolates was detected in laboratory tests of seedlings inoculated with multiple diverse isolates. Dm50 is located in the major resistance cluster on linkage group 2 that contains at least eight major, dominant Dm genes conferring resistance to downy mildew. However, this Dm gene is ineffective against the isolates of B. lactucae prevalent in the field in California and the Netherlands. A quantitative trait locus (QTL) located at the Dm50 chromosomal region (qDM2.2) was detected, though, when the amount of disease was evaluated a month before plants reached harvest maturity. Four additional QTL for resistance to B. lactucae were identified on linkage groups 4 (qDM4.1 and qDM4.2), 7 (qDM7.1), and 9 (qDM9.2). The largest effect was associated with qDM7.1 (up to 32.9% of the total phenotypic variance) that determined resistance in multiple field experiments. Markers identified in the present study will facilitate introduction of these resistance loci into commercial cultivars of lettuce.

  5. Substitutions in PBP3 confer resistance to both ampicillin and extended-spectrum cephalosporins in Haemophilus parainfluenzae as revealed by site-directed mutagenesis and gene recombinants

    DEFF Research Database (Denmark)

    Wienholtz, Nanna H; Ciechanowski, Aynur Barut; Nørskov-Lauritsen, Niels

    2017-01-01

    I from clinical strains encoding four substitutions in the transpeptidase region of PBP3 conferred resistance to ampicillin, but not to cephalosporins. Introduction of ftsI from a clinical strain encoding eight substitutions conferred resistance to ampicillin, cefotaxime and ceftriaxone. MICs....../H/S in combination with V511A were resistant to ampicillin. Substitution S385T increased the MICs of third-generation cephalosporins if V511A was also present. Conclusions: Substitutions in PBP3 are sufficient to confer resistance to both ampicillin and third-generation cephalosporins in H. parainfluenzae...... . A combination of substitutions at positions Val-511 and Asn-526 confers resistance to ampicillin. Resistance to third-generation cephalosporins probably requires more than four substitutions in PBP3....

  6. Fine mapping and candidate gene analysis of two loci conferring resistance to Phytophthora sojae in soybean.

    Science.gov (United States)

    Li, Linghong; Lin, Feng; Wang, Weidong; Ping, Jieqing; Fitzgerald, Joshua C; Zhao, Meixia; Li, Shuai; Sun, Lianjun; Cai, Chunmei; Ma, Jianxin

    2016-12-01

    RpsUN1 and RpsUN2 were fine mapped to two genomic regions harboring disease resistance-like genes. The haplotypes and instability of the regions and candidate genes for the two resistance loci were characterized. Phytophthora root and stem rot caused by Phytophthora sojae, is one of the most destructive diseases of soybean. Deploying soybean cultivars carrying race-specific resistance conferred by Rps genes is the most practical approach to managing this disease. Previously, two Rps genes, RpsUN1 and RpsUN2 were identified in a landrace PI 567139B and mapped to a 6.5 cM region on chromosome 3 and a 3.0 cM region on chromosome 16, corresponding to 1387 and 423 kb of the soybean reference genome sequences. By analyzing recombinants defined by genotypic and phenotypic screening of the 826 F 2:3 families derived from two reciprocal crosses between the two parental lines, RpsUN1 and RpsUN2, were further narrowed to a 151 kb region that harbors five genes including three disease resistance (R)-like genes, and a 36 kb region that contains four genes including five R-like genes, respectively, according to the reference genome. Expressional changes of these nine genes before and after inoculation with the pathogen, as revealed by RNA-seq, suggest that Glyma.03g034600 in the RpsUN1 region and Glyma.16g215200 and Glyma.16g214900 in the RpsUN2 region of PI 567139B may be associated with the resistance to P. sojae. It is also suggested that unequal recombination between/among R-like genes may have occurred, resulting in the formation of two recombinants with inconsistent genotypic and phenotypic observations. The haplotype variation of genomic regions where RpsUN1 and RpsUN2 reside in the entire soybean germplasm deposited in the US soybean germplasm collection suggests that RpsUN1 and RpsUN2 are most likely novel genes.

  7. Error-prone PCR mutation of Ls-EPSPS gene from Liriope spicata conferring to its enhanced glyphosate-resistance.

    Science.gov (United States)

    Mao, Chanjuan; Xie, Hongjie; Chen, Shiguo; Valverde, Bernal E; Qiang, Sheng

    2017-09-01

    Liriope spicata (Thunb.) Lour has a unique LsEPSPS structure contributing to the highest-ever-recognized natural glyphosate tolerance. The transformed LsEPSPS confers increased glyphosate resistance to E. coli and A. thaliana. However, the increased glyphosate-resistance level is not high enough to be of commercial value. Therefore, LsEPSPS was subjected to error-prone PCR to screen mutant EPSPS genes capable of endowing higher resistance levels. A mutant designated as ELs-EPSPS having five mutated amino acids (37Val, 67Asn, 277Ser, 351Gly and 422Gly) was selected for its ability to confer improved resistance to glyphosate. Expression of ELs-EPSPS in recombinant E. coli BL21 (DE3) strains enhanced resistance to glyphosate in comparison to both the LsEPSPS-transformed and -untransformed controls. Furthermore, transgenic ELs-EPSPS A. thaliana was about 5.4 fold and 2-fold resistance to glyphosate compared with the wild-type and the Ls-EPSPS-transgenic plants, respectively. Therefore, the mutated ELs-EPSPS gene has potential value for has potential for the development of glyphosate-resistant crops. Copyright © 2016 Elsevier Inc. All rights reserved.

  8. tcrB, a gene conferring transferable copper resistance in Enterococcus faecium: occurrence, transferability, and linkage to macrolide and glycopeptide resistance

    DEFF Research Database (Denmark)

    Hasman, Henrik; Aarestrup, Frank Møller

    2002-01-01

    B protein from Enterococcus hirae. The tcrB gene was found in E. faecium isolated from pigs (75%), broilers (34%), calves (16%), and humans (10%) but not in isolates from sheep. Resistant isolates, containing the tcrB gene, grew on brain heart infusion agar plates containing up to 28 mM CuSO4 compared......A newly discovered gene, designated tcrB, which is located on a conjugative plasmid conferring acquired copper resistance in Enterococcus faecium, was identified in an isolate from a pig. The tcrB gene encodes a putative protein belonging to the CPx-type ATPase family with homology (46%) to the Cop...... for resistance to these two antimicrobial agents. The frequent occurrence of this new copper resistance gene in isolates from pigs, where copper sulfate is being used in large amounts as feed additive, suggests that the use of copper has selected for resistance....

  9. Genomic Analysis of Hospital Plumbing Reveals Diverse Reservoir of Bacterial Plasmids Conferring Carbapenem Resistance.

    Science.gov (United States)

    Weingarten, Rebecca A; Johnson, Ryan C; Conlan, Sean; Ramsburg, Amanda M; Dekker, John P; Lau, Anna F; Khil, Pavel; Odom, Robin T; Deming, Clay; Park, Morgan; Thomas, Pamela J; Henderson, David K; Palmore, Tara N; Segre, Julia A; Frank, Karen M

    2018-02-06

    The hospital environment is a potential reservoir of bacteria with plasmids conferring carbapenem resistance. Our Hospital Epidemiology Service routinely performs extensive sampling of high-touch surfaces, sinks, and other locations in the hospital. Over a 2-year period, additional sampling was conducted at a broader range of locations, including housekeeping closets, wastewater from hospital internal pipes, and external manholes. We compared these data with previously collected information from 5 years of patient clinical and surveillance isolates. Whole-genome sequencing and analysis of 108 isolates provided comprehensive characterization of bla KPC / bla NDM -positive isolates, enabling an in-depth genetic comparison. Strikingly, despite a very low prevalence of patient infections with bla KPC -positive organisms, all samples from the intensive care unit pipe wastewater and external manholes contained carbapenemase-producing organisms (CPOs), suggesting a vast, resilient reservoir. We observed a diverse set of species and plasmids, and we noted species and susceptibility profile differences between environmental and patient populations of CPOs. However, there were plasmid backbones common to both populations, highlighting a potential environmental reservoir of mobile elements that may contribute to the spread of resistance genes. Clear associations between patient and environmental isolates were uncommon based on sequence analysis and epidemiology, suggesting reasonable infection control compliance at our institution. Nonetheless, a probable nosocomial transmission of Leclercia sp. from the housekeeping environment to a patient was detected by this extensive surveillance. These data and analyses further our understanding of CPOs in the hospital environment and are broadly relevant to the design of infection control strategies in many infrastructure settings. IMPORTANCE Carbapenemase-producing organisms (CPOs) are a global concern because of the morbidity and

  10. A new point mutation in the iron-sulfur subunit of succinate dehydrogenase confers resistance to boscalid in Sclerotinia sclerotiorum.

    Science.gov (United States)

    Wang, Yong; Duan, Yabing; Wang, Jianxin; Zhou, Mingguo

    2015-09-01

    Research has established that mutations in highly conserved amino acids of the succinate dehydrogenase (SDH) complex in various fungi confer SDH inhibitor (SDHI) resistance. For Sclerotinia sclerotiorum (Lib.) de Bary, a necrotrophic fungus with a broad host range and a worldwide distribution, boscalid resistance has been attributed to the mutation H132R in the highly conserved SdhD subunit protein of the SDH complex. In our previous study, however, only one point mutation, A11V in SdhB (GCA to GTA change in SdhB), was detected in S. sclerotiorum boscalid-resistant (BR) mutants. In the current study, replacement of the SdhB gene in a boscalid-sensitive (BS) S. sclerotiorum strain with the mutant SdhB gene conferred resistance. Compared with wild-type strains, BR and GSM (SdhB gene in the wild-type strain replaced by the mutant SdhB gene) mutants were more sensitive to osmotic stress, lacked the ability to produce sclerotia and exhibited lower expression of the pac1 gene. Importantly, the point mutation was not located in the highly conserved sequence of the iron-sulfur subunit of SDH. These results suggest that resistance based on non-conserved vs. conserved protein domains differs in mechanism. In addition to increasing our understanding of boscalid resistance in S. sclerotiorum, the new information will be useful for the development of alternative antifungal drugs. © 2014 BSPP AND JOHN WILEY & SONS LTD.

  11. Genomic Analysis of Hospital Plumbing Reveals Diverse Reservoir of Bacterial Plasmids Conferring Carbapenem Resistance

    Science.gov (United States)

    2018-01-01

    ABSTRACT The hospital environment is a potential reservoir of bacteria with plasmids conferring carbapenem resistance. Our Hospital Epidemiology Service routinely performs extensive sampling of high-touch surfaces, sinks, and other locations in the hospital. Over a 2-year period, additional sampling was conducted at a broader range of locations, including housekeeping closets, wastewater from hospital internal pipes, and external manholes. We compared these data with previously collected information from 5 years of patient clinical and surveillance isolates. Whole-genome sequencing and analysis of 108 isolates provided comprehensive characterization of blaKPC/blaNDM-positive isolates, enabling an in-depth genetic comparison. Strikingly, despite a very low prevalence of patient infections with blaKPC-positive organisms, all samples from the intensive care unit pipe wastewater and external manholes contained carbapenemase-producing organisms (CPOs), suggesting a vast, resilient reservoir. We observed a diverse set of species and plasmids, and we noted species and susceptibility profile differences between environmental and patient populations of CPOs. However, there were plasmid backbones common to both populations, highlighting a potential environmental reservoir of mobile elements that may contribute to the spread of resistance genes. Clear associations between patient and environmental isolates were uncommon based on sequence analysis and epidemiology, suggesting reasonable infection control compliance at our institution. Nonetheless, a probable nosocomial transmission of Leclercia sp. from the housekeeping environment to a patient was detected by this extensive surveillance. These data and analyses further our understanding of CPOs in the hospital environment and are broadly relevant to the design of infection control strategies in many infrastructure settings. PMID:29437920

  12. Genomic Analysis of Hospital Plumbing Reveals Diverse Reservoir of Bacterial Plasmids Conferring Carbapenem Resistance

    Directory of Open Access Journals (Sweden)

    Rebecca A. Weingarten

    2018-02-01

    Full Text Available The hospital environment is a potential reservoir of bacteria with plasmids conferring carbapenem resistance. Our Hospital Epidemiology Service routinely performs extensive sampling of high-touch surfaces, sinks, and other locations in the hospital. Over a 2-year period, additional sampling was conducted at a broader range of locations, including housekeeping closets, wastewater from hospital internal pipes, and external manholes. We compared these data with previously collected information from 5 years of patient clinical and surveillance isolates. Whole-genome sequencing and analysis of 108 isolates provided comprehensive characterization of blaKPC/blaNDM-positive isolates, enabling an in-depth genetic comparison. Strikingly, despite a very low prevalence of patient infections with blaKPC-positive organisms, all samples from the intensive care unit pipe wastewater and external manholes contained carbapenemase-producing organisms (CPOs, suggesting a vast, resilient reservoir. We observed a diverse set of species and plasmids, and we noted species and susceptibility profile differences between environmental and patient populations of CPOs. However, there were plasmid backbones common to both populations, highlighting a potential environmental reservoir of mobile elements that may contribute to the spread of resistance genes. Clear associations between patient and environmental isolates were uncommon based on sequence analysis and epidemiology, suggesting reasonable infection control compliance at our institution. Nonetheless, a probable nosocomial transmission of Leclercia sp. from the housekeeping environment to a patient was detected by this extensive surveillance. These data and analyses further our understanding of CPOs in the hospital environment and are broadly relevant to the design of infection control strategies in many infrastructure settings.

  13. Coordination between Apoplastic and Symplastic Detoxification Confers Plant Aluminum Resistance1[C][W][OPEN

    Science.gov (United States)

    Zhu, Xiao Fang; Lei, Gui Jie; Wang, Zhi Wei; Shi, Yuan Zhi; Braam, Janet; Li, Gui Xin; Zheng, Shao Jian

    2013-01-01

    Whether aluminum toxicity is an apoplastic or symplastic phenomenon is still a matter of debate. Here, we found that three auxin overproducing mutants, yucca, the recessive mutant superroot2, and superroot1 had increased aluminum sensitivity, while a transfer DNA insertion mutant, xyloglucan endotransglucosylase/hydrolases15 (xth15), showed enhanced aluminum resistance, accompanied by low endogenous indole-3-acetic acid levels, implying that auxin may be involved in plant responses to aluminum stress. We used yucca and xth15 mutants for further study. The two mutants accumulated similar total aluminum in roots and had significantly reduced cell wall aluminum and increased symplastic aluminum content relative to the wild-type ecotype Columbia, indicating that altered aluminum levels in the symplast or cell wall cannot fully explain the differential aluminum resistance of these two mutants. The expression of Al sensitive1 (ALS1), a gene that functions in aluminum redistribution between the cytoplasm and vacuole and contributes to symplastic aluminum detoxification, was less abundant in yucca and more abundant in xth15 than the wild type, consistent with possible ALS1 function conferring altered aluminum sensitivity in the two mutants. Consistent with the idea that xth15 can tolerate more symplastic aluminum because of possible ALS1 targeting to the vacuole, morin staining of yucca root tip sections showed more aluminum accumulation in the cytosol than in the wild type, and xth15 showed reduced morin staining of cytosolic aluminum, even though yucca and xth15 had similar overall symplastic aluminum content. Exogenous application of an active auxin analog, naphthylacetic acid, to the wild type mimicked the aluminum sensitivity and distribution phenotypes of yucca, verifying that auxin may regulate aluminum distribution in cells. Together, these data demonstrate that auxin negatively regulates aluminum tolerance through altering ALS1 expression and aluminum distribution

  14. Antibiotic stress-induced modulation of the endoribonucleolytic activity of RNase III and RNase G confers resistance to aminoglycoside antibiotics in Escherichia coli

    Science.gov (United States)

    Song, Wooseok; Kim, Yong-Hak; Sim, Se-Hoon; Hwang, Soonhye; Lee, Jung-Hyun; Lee, Younghoon; Bae, Jeehyeon; Hwang, Jihwan; Lee, Kangseok

    2014-01-01

    Here, we report a resistance mechanism that is induced through the modulation of 16S ribosomal RNA (rRNA) processing on the exposure of Escherichia coli cells to aminoglycoside antibiotics. We observed decreased expression levels of RNase G associated with increased RNase III activity on rng mRNA in a subgroup of E. coli isolates that transiently acquired resistance to low levels of kanamycin or streptomycin. Analyses of 16S rRNA from the aminoglycoside-resistant E. coli cells, in addition to mutagenesis studies, demonstrated that the accumulation of 16S rRNA precursors containing 3–8 extra nucleotides at the 5’ terminus, which results from incomplete processing by RNase G, is responsible for the observed aminoglycoside resistance. Chemical protection, mass spectrometry analysis and cell-free translation assays revealed that the ribosomes from rng-deleted E. coli have decreased binding capacity for, and diminished sensitivity to, streptomycin and neomycin, compared with wild-type cells. It was observed that the deletion of rng had similar effects in Salmonella enterica serovar Typhimurium strain SL1344. Our findings suggest that modulation of the endoribonucleolytic activity of RNase III and RNase G constitutes a previously uncharacterized regulatory pathway for adaptive resistance in E. coli and related gram-negative bacteria to aminoglycoside antibiotics. PMID:24489121

  15. Antibiotic stress-induced modulation of the endoribonucleolytic activity of RNase III and RNase G confers resistance to aminoglycoside antibiotics in Escherichia coli.

    Science.gov (United States)

    Song, Wooseok; Kim, Yong-Hak; Sim, Se-Hoon; Hwang, Soonhye; Lee, Jung-Hyun; Lee, Younghoon; Bae, Jeehyeon; Hwang, Jihwan; Lee, Kangseok

    2014-04-01

    Here, we report a resistance mechanism that is induced through the modulation of 16S ribosomal RNA (rRNA) processing on the exposure of Escherichia coli cells to aminoglycoside antibiotics. We observed decreased expression levels of RNase G associated with increased RNase III activity on rng mRNA in a subgroup of E. coli isolates that transiently acquired resistance to low levels of kanamycin or streptomycin. Analyses of 16S rRNA from the aminoglycoside-resistant E. coli cells, in addition to mutagenesis studies, demonstrated that the accumulation of 16S rRNA precursors containing 3-8 extra nucleotides at the 5' terminus, which results from incomplete processing by RNase G, is responsible for the observed aminoglycoside resistance. Chemical protection, mass spectrometry analysis and cell-free translation assays revealed that the ribosomes from rng-deleted E. coli have decreased binding capacity for, and diminished sensitivity to, streptomycin and neomycin, compared with wild-type cells. It was observed that the deletion of rng had similar effects in Salmonella enterica serovar Typhimurium strain SL1344. Our findings suggest that modulation of the endoribonucleolytic activity of RNase III and RNase G constitutes a previously uncharacterized regulatory pathway for adaptive resistance in E. coli and related gram-negative bacteria to aminoglycoside antibiotics.

  16. Bmi1 confers resistance to oxidative stress on hematopoietic stem cells.

    Directory of Open Access Journals (Sweden)

    Shunsuke Nakamura

    Full Text Available The polycomb-group (PcG proteins function as general regulators of stem cells. We previously reported that retrovirus-mediated overexpression of Bmi1, a gene encoding a core component of polycomb repressive complex (PRC 1, maintained self-renewing hematopoietic stem cells (HSCs during long-term culture. However, the effects of overexpression of Bmi1 on HSCs in vivo remained to be precisely addressed.In this study, we generated a mouse line where Bmi1 can be conditionally overexpressed under the control of the endogenous Rosa26 promoter in a hematopoietic cell-specific fashion (Tie2-Cre;R26Stop(FLBmi1. Although overexpression of Bmi1 did not significantly affect steady state hematopoiesis, it promoted expansion of functional HSCs during ex vivo culture and efficiently protected HSCs against loss of self-renewal capacity during serial transplantation. Overexpression of Bmi1 had no effect on DNA damage response triggered by ionizing radiation. In contrast, Tie2-Cre;R26Stop(FLBmi1 HSCs under oxidative stress maintained a multipotent state and generally tolerated oxidative stress better than the control. Unexpectedly, overexpression of Bmi1 had no impact on the level of intracellular reactive oxygen species (ROS.Our findings demonstrate that overexpression of Bmi1 confers resistance to stresses, particularly oxidative stress, onto HSCs. This thereby enhances their regenerative capacity and suggests that Bmi1 is located downstream of ROS signaling and negatively regulated by it.

  17. Crystallization and preliminary diffraction studies of SFC-1, a carbapenemase conferring antibiotic resistance

    International Nuclear Information System (INIS)

    Hong, Myoung-Ki; Lee, Jae Jin; Wu, Xing; Kim, Jin-Kwang; Jeong, Byeong Chul; Pham, Tan-Viet; Kim, Seung-Hwan; Lee, Sang Hee; Kang, Lin-Woo

    2012-01-01

    The SFC-1 gene from S. fonticola was cloned and SFC-1 was expressed, purified and crystallized. X-ray diffraction data were collected from an SFC-1 crystal to 1.6 Å resolution. SFC-1, a class A carbapenemase that confers antibiotic resistance, hydrolyzes the β-lactam rings of β-lactam antibiotics (carbapenems, cephalosporins, penicillins and aztreonam). SFC-1 presents an enormous challenge to infection control, particularly in the eradication of Gram-negative pathogens. As SFC-1 exhibits a remarkably broad substrate range, including β-lactams of all classes, the enzyme is a potential target for the development of antimicrobial agents against pathogens producing carbapenemases. In this study, SFC-1 was cloned, overexpressed, purified and crystallized. The SFC-1 crystal diffracted to 1.6 Å resolution and belonged to the orthorhombic space group P2 1 2 1 2 1 , with unit-cell parameters a = 65.8, b = 68.3, c = 88.8 Å. Two molecules are present in the asymmetric unit, with a corresponding V M of 1.99 Å 3 Da −1 and a solvent content of 38.1%

  18. Resistance to Bacillus thuringiensis Toxin Cry2Ab in Trichoplusia ni Is Conferred by a Novel Genetic Mechanism

    Science.gov (United States)

    Song, Xiaozhao; Kain, Wendy; Cassidy, Douglas

    2015-01-01

    The resistance to the Bacillus thuringiensis (Bt) toxin Cry2Ab in a greenhouse-originated Trichoplusia ni strain resistant to both Bt toxins Cry1Ac and Cry2Ab was characterized. Biological assays determined that the Cry2Ab resistance in the T. ni strain was a monogenic recessive trait independent of Cry1Ac resistance, and there existed no significant cross-resistance between Cry1Ac and Cry2Ab in T. ni. From the dual-toxin-resistant T. ni strain, a strain resistant to Cry2Ab only was isolated, and the Cry2Ab resistance trait was introgressed into a susceptible laboratory strain to facilitate comparative analysis of the Cry2Ab resistance with the susceptible T. ni strain. Results from biochemical analysis showed no significant difference between the Cry2Ab-resistant and -susceptible T. ni larvae in midgut proteases, including caseinolytic proteolytic activity and zymogram profile and serine protease activities, in midgut aminopeptidase and alkaline phosphatase activity, and in midgut esterases and hemolymph plasma melanization activity. For analysis of genetic linkage of Cry2Ab resistance with potential Cry toxin receptor genes, molecular markers for the midgut cadherin, alkaline phosphatase (ALP), and aminopeptidase N (APN) genes were identified between the original greenhouse-derived dual-toxin-resistant and the susceptible laboratory T. ni strains. Genetic linkage analysis showed that the Cry2Ab resistance in T. ni was not genetically associated with the midgut genes coding for the cadherin, ALP, and 6 APNs (APN1 to APN6) nor associated with the ABC transporter gene ABCC2. Therefore, the Cry2Ab resistance in T. ni is conferred by a novel but unknown genetic mechanism. PMID:26025894

  19. Durable broad-spectrum powdery mildew resistance in pea er1 plants is conferred by natural loss-of-function mutations in PsMLO1

    NARCIS (Netherlands)

    Humphry, M.; Reinstädler, A.; Ivanov, S.; Bisseling, T.; Panstruga, R.

    2011-01-01

    Loss-of-function alleles of plant-specific MLO (Mildew Resistance Locus O) genes confer broad-spectrum powdery mildew resistance in monocot (barley) and dicot (Arabidopsis thaliana, tomato) plants. Recessively inherited powdery mildew resistance in pea (Pisum sativum) er1 plants is, in many aspects,

  20. Target-site mutations conferring resistance to glyphosate in feathertop Rhodes grass (Chloris virgata) populations in Australia.

    Science.gov (United States)

    Ngo, The D; Krishnan, Mahima; Boutsalis, Peter; Gill, Gurjeet; Preston, Christopher

    2018-05-01

    Chloris virgata is a warm-season, C 4 , annual grass weed affecting field crops in northern Australia that has become an emerging weed in southern Australia. Four populations with suspected resistance to glyphosate were collected in South Australia, Queensland and New South Wales, Australia, and compared with one susceptible (S) population to confirm glyphosate resistance and elucidate possible mechanisms of resistance. Based on the rate of glyphosate required to kill 50% of treated plants (LD 50 ), glyphosate resistance (GR) was confirmed in four populations of C. virgata (V12, V14.2, V14.16 and V15). GR plants were 2-9.7-fold more resistant and accumulated less shikimate after glyphosate treatment than S plants. GR and S plants did not differ in glyphosate absorption and translocation. Target-site EPSPS mutations corresponding to Pro-106-Leu (V14.2) and Pro-106-Ser (V15, V14.16 and V12) substitutions were found in GR populations. The population with Pro-106-Leu substitution was 2.9-4.9-fold more resistant than the three other populations with Pro-106-Ser substitution. This report confirms glyphosate resistance in C. virgata and shows that target-site EPSPS mutations confer resistance to glyphosate in this species. The evolution of glyphosate resistance in C. virgata highlights the need to identify alternative control tactics. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

  1. A Novel FexA Variant from a Canine Staphylococcus pseudintermedius Isolate That Does Not Confer Florfenicol Resistance

    Science.gov (United States)

    Gómez-Sanz, Elena; Kadlec, Kristina; Feßler, Andrea T.; Zarazaga, Myriam; Schwarz, Stefan

    2013-01-01

    Transposon Tn558 integrated in the chromosomal radC gene was detected for the first time in Staphylococus pseudintermedius. It carried a novel fexA variant (fexAv) that confers only chloramphenicol resistance. The exporter FexAv exhibited two amino acid substitutions, Gly33Ala and Ala37Val, both of which seem to be important for substrate recognition. Site-directed mutagenesis that reverted the mutated base pairs to those present in the original fexA gene restored the chloramphenicol-plus-florfenicol resistance phenotype. PMID:23979755

  2. Distinct mechanisms govern the dosage-dependent and developmentally regulated resistance conferred by the maize Hm2 gene.

    Science.gov (United States)

    Chintamanani, Satya; Multani, Dilbag S; Ruess, Holly; Johal, Gurmukh S

    2008-01-01

    The maize Hm2 gene provides protection against the leaf spot and ear mold disease caused by Cochliobolus carbonum race 1 (CCR1). In this regard, it is similar to Hm1, the better-known disease resistance gene of the maize-CCR1 pathosystem. However, in contrast to Hm1, which provides completely dominant resistance at all stages of plant development, Hm2-conferred resistance is only partially dominant and becomes fully effective only at maturity. To investigate why Hm2 behaves in this manner, we cloned it on the basis of its homology to Hm1. As expected, Hm2 is a duplicate of Hm1, although the protein it encodes is grossly truncated compared with HM1. The efficacy of Hm2 in conferring resistance improves gradually over time, changing from having little or no impact in seedling tissues to providing complete immunity at anthesis. The developmentally specified phenotype of Hm2 is not dictated transcriptionally, because the expression level of the gene, whether occurring constitutively or undergoing substantial and transient induction in response to infection, does not change with plant age. In contrast, however, the Hm2 transcript is much more abundant in plants homozygous for this gene compared with plants that contain only one copy of the gene, suggesting a transcriptional basis for the dosage-dependent nature of Hm2. Thus, different mechanisms seem to underlie the developmentally programmed versus the partially dominant resistance phenotype of Hm2.

  3. Phage display-selected single-chain antibodies confer high levels of resistance against Tomato spotted wilt virus.

    Science.gov (United States)

    Prins, Marcel; Lohuis, Dick; Schots, Arjen; Goldbach, Rob

    2005-07-01

    Rational design of antibodies targeting essential viral proteins can complement the palette of antiviral resistance strategies. Here, stable and high expression of single-chain monoclonal antibodies targeting the nucleoprotein of the economically important plant virus Tomato spotted wilt virus, a protein that is involved in multiple steps in the viral infection cycle, is reported. High cytoplasmic expression levels of three selected phage display-derived anti-viral single-chain antibodies were established. Of these antibodies, two led to high levels of resistance against this plant virus. Protoplast experiments provided evidence that the two resistance-conferring antibodies may have a different mode of action and could be combined for higher durability of resistance in the field.

  4. Inheritance and molecular mapping of a gene conferring seedling resistance against Puccinia hordei in the barley cultivar Ricardo.

    Science.gov (United States)

    Sandhu, K S; Forrest, K L; Kong, S; Bansal, U K; Singh, D; Hayden, M J; Park, R F

    2012-11-01

    Genetic studies were undertaken to determine the inheritance and genomic location of uncharacterised seedling resistance to leaf rust, caused by Puccinia hordei, in the barley cultivar Ricardo. The resistance was shown to be conferred by a single dominant gene, which was tentatively designated RphRic. Bulk segregant analysis (BSA) and genetic mapping of an F(3) mapping population using multiplex-ready SSR genotyping and Illumina GoldenGate SNP assay located RphRic in chromosome 4H. Given that this is the first gene for leaf rust resistance mapped on chromosome 4H, it was designated Rph21. The presence of an additional gene, Rph2, in Ricardo, was confirmed by the test of allelism. The seedling gene Rph21 has shown effectiveness against all Australian pathotypes of P. hordei tested since at least 1992 and hence represents a new and useful source of resistance to this pathogen.

  5. A New Ala-122-Asn Amino Acid Change Confers Decreased Fitness to ALS-Resistant Echinochloa crus-galli.

    Science.gov (United States)

    Panozzo, Silvia; Scarabel, Laura; Rosan, Valentina; Sattin, Maurizio

    2017-01-01

    Gene mutations conferring herbicide resistance may cause pleiotropic effects on plant fitness. Knowledge of these effects is important for managing the evolution of herbicide-resistant weeds. An Echinochloa crus-galli population resistant to acetolactate synthase (ALS) herbicides was collected in a maize field in north-eastern Italy and the cross-resistance pattern, resistance mechanism and fitness costs associated to mutant-resistant plants under field conditions in the presence or absence of intra-specific competition were determined. The study reports for the first time the Ala-122-Asn amino-acid change in the ALS gene that confers high levels of cross-resistance to all ALS inhibitors tested. Results of 3-year growth analysis showed that mutant resistant E. crus-galli plants had a delayed development in comparison with susceptible plants and this was registered in both competitive (3, 7, and 20 plants m -2 ) and non-competitive (spaced plants) situations. The number of panicles produced by resistant plants was also lower (about 40% fewer panicles) than susceptible plants under no-intraspecific competition. Instead, with the increasing competition level, the difference in panicle production at harvest time decreased until it became negligible at 20 plants m -2 . Evaluation of total dry biomass as well as biomass allocation in vegetative parts did not highlight any difference between resistant and susceptible plants. Instead, panicle dry weight was higher in susceptible plants indicating that they allocated more biomass than resistant ones to the reproductive organs, especially in no-competition and in competition situations at lower plant densities. The different fitness between resistant and susceptible phenotypes suggests that keeping the infestation density as low as possible can increase the reproduction success of the susceptible phenotype and therefore contribute to lowering the ratio between resistant and susceptible alleles. If adequately embedded in a

  6. A New Ala-122-Asn Amino Acid Change Confers Decreased Fitness to ALS-Resistant Echinochloa crus-galli

    Directory of Open Access Journals (Sweden)

    Silvia Panozzo

    2017-11-01

    Full Text Available Gene mutations conferring herbicide resistance may cause pleiotropic effects on plant fitness. Knowledge of these effects is important for managing the evolution of herbicide-resistant weeds. An Echinochloa crus-galli population resistant to acetolactate synthase (ALS herbicides was collected in a maize field in north-eastern Italy and the cross-resistance pattern, resistance mechanism and fitness costs associated to mutant-resistant plants under field conditions in the presence or absence of intra-specific competition were determined. The study reports for the first time the Ala-122-Asn amino-acid change in the ALS gene that confers high levels of cross-resistance to all ALS inhibitors tested. Results of 3-year growth analysis showed that mutant resistant E. crus-galli plants had a delayed development in comparison with susceptible plants and this was registered in both competitive (3, 7, and 20 plants m-2 and non-competitive (spaced plants situations. The number of panicles produced by resistant plants was also lower (about 40% fewer panicles than susceptible plants under no-intraspecific competition. Instead, with the increasing competition level, the difference in panicle production at harvest time decreased until it became negligible at 20 plants m-2. Evaluation of total dry biomass as well as biomass allocation in vegetative parts did not highlight any difference between resistant and susceptible plants. Instead, panicle dry weight was higher in susceptible plants indicating that they allocated more biomass than resistant ones to the reproductive organs, especially in no-competition and in competition situations at lower plant densities. The different fitness between resistant and susceptible phenotypes suggests that keeping the infestation density as low as possible can increase the reproduction success of the susceptible phenotype and therefore contribute to lowering the ratio between resistant and susceptible alleles. If adequately

  7. Overexpression of Salmonella enterica serovar Typhi recA gene confers fluoroquinolone resistance in Escherichia coli DH5α

    Directory of Open Access Journals (Sweden)

    M.A.M. Yassien

    2015-11-01

    Full Text Available A spontaneous fluoroquinolone-resistant mutant (STM1 was isolated from its parent Salmonella enterica serovar Typhi (S. Typhi clinical isolate. Unlike its parent isolate, this mutant has selective resistance to fluoroquinolones without any change in its sensitivity to various other antibiotics. DNA gyrase assays revealed that the fluoroquinolone resistance phenotype of the STM1 mutant did not result from alteration of the fluoroquinolone sensitivity of the DNA gyrase isolated from it. To study the mechanism of fluoroquinolone resistance, a genomic library from the STM1 mutant was constructed in Escherichia coli DH5α and two recombinant plasmids were obtained. Only one of these plasmids (STM1-A conferred the selective fluoroquinolone resistance phenotype to E. coli DH5α. The chromosomal insert from STM1-A, digested with EcoRI and HindIII restriction endonucleases, produced two DNA fragments and these were cloned separately into pUC19 thereby generating two new plasmids, STM1-A1 and STM1-A2. Only STM1-A1 conferred the selective fluoroquinolone resistance phenotype to E. coli DH5α. Sequence and subcloning analyses of STM1-A1 showed the presence of an intact RecA open reading frame. Unlike that of the wild-type E. coli DH5α, protein analysis of a crude STM1-A1 extract showed overexpression of a 40 kDa protein. Western blotting confirmed the 40 kDa protein band to be RecA. When a RecA PCR product was cloned into pGEM-T and introduced into E. coli DH5α, the STM1-A11 subclone retained fluoroquinolone resistance. These results suggest that overexpression of RecA causes selective fluoroquinolone resistance in E. coli DH5α.

  8. Overexpression of Salmonella enterica serovar Typhi recA gene confers fluoroquinolone resistance in Escherichia coli DH5α.

    Science.gov (United States)

    Yassien, M A M; Elfaky, M A

    2015-11-01

    A spontaneous fluoroquinolone-resistant mutant (STM1) was isolated from its parent Salmonella enterica serovar Typhi (S. Typhi) clinical isolate. Unlike its parent isolate, this mutant has selective resistance to fluoroquinolones without any change in its sensitivity to various other antibiotics. DNA gyrase assays revealed that the fluoroquinolone resistance phenotype of the STM1 mutant did not result from alteration of the fluoroquinolone sensitivity of the DNA gyrase isolated from it. To study the mechanism of fluoroquinolone resistance, a genomic library from the STM1 mutant was constructed in Escherichia coli DH5α and two recombinant plasmids were obtained. Only one of these plasmids (STM1-A) conferred the selective fluoroquinolone resistance phenotype to E. coli DH5α. The chromosomal insert from STM1-A, digested with EcoRI and HindIII restriction endonucleases, produced two DNA fragments and these were cloned separately into pUC19 thereby generating two new plasmids, STM1-A1 and STM1-A2. Only STM1-A1 conferred the selective fluoroquinolone resistance phenotype to E. coli DH5α. Sequence and subcloning analyses of STM1-A1 showed the presence of an intact RecA open reading frame. Unlike that of the wild-type E. coli DH5α, protein analysis of a crude STM1-A1 extract showed overexpression of a 40 kDa protein. Western blotting confirmed the 40 kDa protein band to be RecA. When a RecA PCR product was cloned into pGEM-T and introduced into E. coli DH5α, the STM1-A11 subclone retained fluoroquinolone resistance. These results suggest that overexpression of RecA causes selective fluoroquinolone resistance in E. coli DH5α.

  9. Non-injurious neonatal hypoxia confers resistance to brain senescence in aged male rats.

    Directory of Open Access Journals (Sweden)

    Nicolas Martin

    Full Text Available Whereas brief acute or intermittent episodes of hypoxia have been shown to exert a protective role in the central nervous system and to stimulate neurogenesis, other studies suggest that early hypoxia may constitute a risk factor that influences the future development of mental disorders. We therefore investigated the effects of a neonatal "conditioning-like" hypoxia (100% N₂, 5 min on the brain and the cognitive outcomes of rats until 720 days of age (physiologic senescence. We confirmed that such a short hypoxia led to brain neurogenesis within the ensuing weeks, along with reduced apoptosis in the hippocampus involving activation of Erk1/2 and repression of p38 and death-associated protein (DAP kinase. At 21 days of age, increased thicknesses and cell densities were recorded in various subregions, with strong synapsin activation. During aging, previous exposure to neonatal hypoxia was associated with enhanced memory retrieval scores specifically in males, better preservation of their brain integrity than controls, reduced age-related apoptosis, larger hippocampal cell layers, and higher expression of glutamatergic and GABAergic markers. These changes were accompanied with a marked expression of synapsin proteins, mainly of their phosphorylated active forms which constitute major players of synapse function and plasticity, and with increases of their key regulators, i.e. Erk1/2, the transcription factor EGR-1/Zif-268 and Src kinase. Moreover, the significantly higher interactions between PSD-95 scaffolding protein and NMDA receptors measured in the hippocampus of 720-day-old male animals strengthen the conclusion of increased synaptic functional activity and plasticity associated with neonatal hypoxia. Thus, early non-injurious hypoxia may trigger beneficial long term effects conferring higher resistance to senescence in aged male rats, with a better preservation of cognitive functions.

  10. Non-injurious neonatal hypoxia confers resistance to brain senescence in aged male rats.

    Science.gov (United States)

    Martin, Nicolas; Bossenmeyer-Pourié, Carine; Koziel, Violette; Jazi, Rozat; Audonnet, Sandra; Vert, Paul; Guéant, Jean-Louis; Daval, Jean-Luc; Pourié, Grégory

    2012-01-01

    Whereas brief acute or intermittent episodes of hypoxia have been shown to exert a protective role in the central nervous system and to stimulate neurogenesis, other studies suggest that early hypoxia may constitute a risk factor that influences the future development of mental disorders. We therefore investigated the effects of a neonatal "conditioning-like" hypoxia (100% N₂, 5 min) on the brain and the cognitive outcomes of rats until 720 days of age (physiologic senescence). We confirmed that such a short hypoxia led to brain neurogenesis within the ensuing weeks, along with reduced apoptosis in the hippocampus involving activation of Erk1/2 and repression of p38 and death-associated protein (DAP) kinase. At 21 days of age, increased thicknesses and cell densities were recorded in various subregions, with strong synapsin activation. During aging, previous exposure to neonatal hypoxia was associated with enhanced memory retrieval scores specifically in males, better preservation of their brain integrity than controls, reduced age-related apoptosis, larger hippocampal cell layers, and higher expression of glutamatergic and GABAergic markers. These changes were accompanied with a marked expression of synapsin proteins, mainly of their phosphorylated active forms which constitute major players of synapse function and plasticity, and with increases of their key regulators, i.e. Erk1/2, the transcription factor EGR-1/Zif-268 and Src kinase. Moreover, the significantly higher interactions between PSD-95 scaffolding protein and NMDA receptors measured in the hippocampus of 720-day-old male animals strengthen the conclusion of increased synaptic functional activity and plasticity associated with neonatal hypoxia. Thus, early non-injurious hypoxia may trigger beneficial long term effects conferring higher resistance to senescence in aged male rats, with a better preservation of cognitive functions.

  11. Transgenic cotton expressing Cry10Aa toxin confers high resistance to the cotton boll weevil.

    Science.gov (United States)

    Ribeiro, Thuanne Pires; Arraes, Fabricio Barbosa Monteiro; Lourenço-Tessutti, Isabela Tristan; Silva, Marilia Santos; Lisei-de-Sá, Maria Eugênia; Lucena, Wagner Alexandre; Macedo, Leonardo Lima Pepino; Lima, Janaina Nascimento; Santos Amorim, Regina Maria; Artico, Sinara; Alves-Ferreira, Márcio; Mattar Silva, Maria Cristina; Grossi-de-Sa, Maria Fatima

    2017-08-01

    Genetically modified (GM) cotton plants that effectively control cotton boll weevil (CBW), which is the most destructive cotton insect pest in South America, are reported here for the first time. This work presents the successful development of a new GM cotton with high resistance to CBW conferred by Cry10Aa toxin, a protein encoded by entomopathogenic Bacillus thuringiensis (Bt) gene. The plant transformation vector harbouring cry10Aa gene driven by the cotton ubiquitination-related promoter uceA1.7 was introduced into a Brazilian cotton cultivar by biolistic transformation. Quantitative PCR (qPCR) assays revealed high transcription levels of cry10Aa in both T 0 GM cotton leaf and flower bud tissues. Southern blot and qPCR-based 2 -ΔΔCt analyses revealed that T 0 GM plants had either one or two transgene copies. Quantitative and qualitative analyses of Cry10Aa protein expression showed variable protein expression levels in both flower buds and leaves tissues of T 0 GM cotton plants, ranging from approximately 3.0 to 14.0 μg g -1 fresh tissue. CBW susceptibility bioassays, performed by feeding adults and larvae with T 0 GM cotton leaves and flower buds, respectively, demonstrated a significant entomotoxic effect and a high level of CBW mortality (up to 100%). Molecular analysis revealed that transgene stability and entomotoxic effect to CBW were maintained in T 1 generation as the Cry10Aa toxin expression levels remained high in both tissues, ranging from 4.05 to 19.57 μg g -1 fresh tissue, and the CBW mortality rate remained around 100%. In conclusion, these Cry10Aa GM cotton plants represent a great advance in the control of the devastating CBW insect pest and can substantially impact cotton agribusiness. © 2017 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

  12. The Batten disease gene CLN3 confers resistance to endoplasmic reticulum stress induced by tunicamycin

    Energy Technology Data Exchange (ETDEWEB)

    Wu, Dan, E-mail: danw@bjmu.edu.cn [Department of Medical Genetics, Peking University Health Science Center, No 38 Xueyuan Road, Haidian district, Beijing 100191 (China); Liu, Jing; Wu, Baiyan [Department of Medical Genetics, Peking University Health Science Center, No 38 Xueyuan Road, Haidian district, Beijing 100191 (China); Tu, Bo; Zhu, Weiguo [Department of Biochemistry and Molecular Biology, Peking University Health Science Center, No 38 Xueyuan Road, Haidian district, Beijing 100191 (China); Luo, Jianyuan, E-mail: jluo@som.umaryland.edu [Department of Medical Genetics, Peking University Health Science Center, No 38 Xueyuan Road, Haidian district, Beijing 100191 (China); Department of Medical and Research Technology, School of Medicine, University of Maryland, Baltimore 21201 (United States)

    2014-04-25

    Highlights: • The work reveals a protective properties of CLN3 towards TM-induced apoptosis. • CLN3 regulates expression of the GRP78 and the CHOP in response to the ER stress. • CLN3 plays a specific role in the ERS response. - Abstract: Mutations in CLN3 gene cause juvenile neuronal ceroid lipofuscinosis (JNCL or Batten disease), an early-onset neurodegenerative disorder that is characterized by the accumulation of ceroid lipofuscin within lysosomes. The function of the CLN3 protein remains unclear and is presumed to be related to Endoplasmic reticulum (ER) stress. To investigate the function of CLN3 in the ER stress signaling pathway, we measured proliferation and apoptosis in cells transfected with normal and mutant CLN3 after treatment with the ER stress inducer tunicamycin (TM). We found that overexpression of CLN3 was sufficient in conferring increased resistance to ER stress. Wild-type CLN3 protected cells from TM-induced apoptosis and increased cell proliferation. Overexpression of wild-type CLN3 enhanced expression of the ER chaperone protein, glucose-regulated protein 78 (GRP78), and reduced expression of the proapoptotic protein CCAAT/-enhancer-binding protein homologous protein (CHOP). In contrast, overexpression of mutant CLN3 or siRNA knockdown of CLN3 produced the opposite effect. Together, our data suggest that the lack of CLN3 function in cells leads to a failure of management in the response to ER stress and this may be the key deficit in JNCL that causes neuronal degeneration.

  13. Elevated Cell Wall Chitin in Candida albicans Confers Echinocandin Resistance In Vivo

    Science.gov (United States)

    Lee, Keunsook K.; MacCallum, Donna M.; Jacobsen, Mette D.; Walker, Louise A.; Odds, Frank C.

    2012-01-01

    Candida albicans cells with increased cell wall chitin have reduced echinocandin susceptibility in vitro. The aim of this study was to investigate whether C. albicans cells with elevated chitin levels have reduced echinocandin susceptibility in vivo. BALB/c mice were infected with C. albicans cells with normal chitin levels and compared to mice infected with high-chitin cells. Caspofungin therapy was initiated at 24 h postinfection. Mice infected with chitin-normal cells were successfully treated with caspofungin, as indicated by reduced kidney fungal burdens, reduced weight loss, and decreased C. albicans density in kidney lesions. In contrast, mice infected with high-chitin C. albicans cells were less susceptible to caspofungin, as they had higher kidney fungal burdens and greater weight loss during early infection. Cells recovered from mouse kidneys at 24 h postinfection with high-chitin cells had 1.6-fold higher chitin levels than cells from mice infected with chitin-normal cells and maintained a significantly reduced susceptibility to caspofungin when tested in vitro. At 48 h postinfection, caspofungin treatment induced a further increase in chitin content of C. albicans cells harvested from kidneys compared to saline treatment. Some of the recovered clones had acquired, at a low frequency, a point mutation in FKS1 resulting in a S645Y amino acid substitution, a mutation known to confer echinocandin resistance. This occurred even in cells that had not been exposed to caspofungin. Our results suggest that the efficacy of caspofungin against C. albicans was reduced in vivo due to either elevation of chitin levels in the cell wall or acquisition of FKS1 point mutations. PMID:21986821

  14. Identification and molecular mapping of Rps11, a novel gene conferring resistance to Phytophthora sojae in soybean.

    Science.gov (United States)

    Ping, Jieqing; Fitzgerald, Joshua C; Zhang, Chunbao; Lin, Feng; Bai, Yonghe; Wang, Dechun; Aggarwal, Rajat; Rehman, Maqsood; Crasta, Oswald; Ma, Jianxin

    2016-02-01

    Rps11 confers excellent resistance to predominant Phytophthora sojae isolates capable of defeating major Rps genes deployed into soybean production, representing a novel source of resistance for soybean cultivar enhancement. Phytophthora root and stem rot (PRSR), caused by the soil-borne pathogen Phytophthora sojae, is a devastating disease of soybean [Glycine max (L.) Merr.] throughout the world. Deploying resistant soybean cultivars is the most effective and environmentally friendly approach to managing this disease. The soybean landrace PI 594527 was found to carry excellent resistance to all P. sojae isolates examined, some of which were capable of overcoming the major Rps genesp, such as Rps1-k, Rps1-c, and Rps3-a, predominantly used for soybean protection in the past decades. A mapping population consisting of 58 F2 individuals and 209 F2:3 families derived from a cross between PI 594527 and the susceptible cultivar 'Williams' was used to characterize the inheritance pattern of the resistance to P. soja (Rps) in PI 594527. It was found that the resistance was conferred by a single Rps gene, designated Rps11, which was initially defined as an ~5 Mb genomic region at the beginning of chromosome 7 by bulked segregant analysis (BSA) with a nucleotide polymorphism (SNP) chip comprising 7039 SNP markers. Subsequently, simple sequence repeat (SSR) markers in the defined region were used to genotype the F2:3 mapping population to map Rps11 to a 225.3 kb genomic region flanked by SSR markers BARCSOYSSR_07_0286 and BARCSOYSSR_07_0300, according to the soybean reference genome sequence. Particularly, an SSR marker (i.e., BARCSOYSSR_07_0295) was found to tightly co-segregate with Rps11 in the mapping population and can be effectively used for marker-assisted selection of this gene for development of resistant soybean cultivars.

  15. Lr67 and Lr34 rust resistance genes have much in common – they confer broad spectrum resistance to multiple pathogens in wheat

    Science.gov (United States)

    2013-01-01

    Background Adult plant rust resistance genes Lr67 and Lr34 confer race non-specific resistance to multiple fungal pathogens of wheat. Induced, susceptible mutants were characterised for both genes. Results Three categories of Lr34 mutants were identified that were either partial susceptible, fully susceptible or hyper-susceptible to stripe rust and leaf rust. The likely impact of the mutational change on the predicted Lr34 protein correlated with differences in response to rust infection. Four independent Lr67 mutants were recovered that were susceptible to stripe rust, leaf rust and stem rust pathogens, including one possible hyper-susceptible Lr67 mutant. Conclusions Detailed study of Lr34 mutants revealed that subtle changes in resistance response to multiple pathogens were correlated with mutational changes in the predicted protein. Recovery of independent Lr67 mutants indicates that as for Lr34, a single gene at the Lr67 locus is likely to confer resistance to multiple pathogens. The infection phenotypes of Lr67 mutants closely resembled that of Lr34 mutants. PMID:23819608

  16. The Vat locus encodes for a CC-NBS-LRR protein that confers resistance to Aphis gossypii infestation and A. gossypii-mediated virus resistance.

    Science.gov (United States)

    Dogimont, Catherine; Chovelon, Veronique; Pauquet, Jerome; Boualem, Adnane; Bendahmane, Abdelhafid

    2014-12-01

    Aphis gossypii is a polyphagous sucking aphid and a vector for many viruses. In Cucumis melo, a dominant locus, Vat, confers a high level of resistance to Aphis gossypii infestation and to viruses transmitted by this vector. To investigate the mechanism underlying this double resistance, we first genetically dissected the Vat locus. We delimited the double resistance to a single gene that encodes for a coiled-coil-nucleotide-binding-site-leucine-rich repeat (CC-NBS-LRR) protein type. To validate the genetic data, transgenic lines expressing the Vat gene were generated and assessed for the double resistance. In this analysis, Vat-transgenic plants were resistant to A. gossypii infestation as well as A. gossypii-mediated virus transmission. When the plants were infected mechanically, virus infection occurred on both transgenic and non-transgenic control plants. These results confirmed that the cloned CC-NBS-LRR gene mediates both resistance to aphid infestation and virus infection using A. gossypii as a vector. This resistance also invokes a separate recognition and response phases in which the recognition phase involves the interaction of an elicitor molecule from the aphid and Vat from the plant. The response phase is not specific and blocks both aphid infestation and virus infection. Sequence analysis of Vat alleles suggests a major role of an unusual conserved LRR repeat in the recognition of A. gossypii. © 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd.

  17. Linked, if not the same, Mi-1 homologues confer resistance to tomato powdery mildew and root-knot nematodes.

    Science.gov (United States)

    Seifi, Alireza; Kaloshian, Isgouhi; Vossen, Jack; Che, Daidi; Bhattarai, Kishor K; Fan, Junmei; Naher, Zabun; Goverse, Aska; Tjallingii, W Freddy; Lindhout, Pim; Visser, Richard G F; Bai, Yuling

    2011-04-01

    On the short arm of tomato chromosome 6, a cluster of disease resistance (R) genes have evolved harboring the Mi-1 and Cf genes. The Mi-1 gene confers resistance to root-knot nematodes, aphids, and whiteflies. Previously, we mapped two genes, Ol-4 and Ol-6, for resistance to tomato powdery mildew in this cluster. The aim of this study was to investigate whether Ol-4 and Ol-6 are homologues of the R genes located in this cluster. We show that near-isogenic lines (NIL) harboring Ol-4 (NIL-Ol-4) and Ol-6 (NIL-Ol-6) are also resistant to nematodes and aphids. Genetically, the resistance to nematodes cosegregates with Ol-4 and Ol-6, which are further fine-mapped to the Mi-1 cluster. We provide evidence that the composition of Mi-1 homologues in NIL-Ol-4 and NIL-Ol-6 is different from other nematode-resistant tomato lines, Motelle and VFNT, harboring the Mi-1 gene. Furthermore, we demonstrate that the resistance to both nematodes and tomato powdery mildew in these two NIL is governed by linked (if not the same) Mi-1 homologues in the Mi-1 gene cluster. Finally, we discuss how Solanum crops exploit Mi-1 homologues to defend themselves against distinct pathogens.

  18. Identification of regulated genes conferring resistance to high concentrations of glyphosate in a new strain of Enterobacter.

    Science.gov (United States)

    Fei, Yun-Yan; Gai, Jun-Yi; Zhao, Tuan-Jie

    2013-12-01

    Glyphosate is a widely used herbicide that inhibits 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) activity. Most plants and microbes are sensitive to glyphosate. However, transgenic-resistant crops that contain a modified epsps obtained from the resistant microbes have been commercially successful and therefore, new resistance genes and their adaptive regulatory mechanisms are of great interest. In this study, a soil-borne, glyphosate-resistant bacterium was selected and identified as Enterobacter. The EPSPS in this strain was found to have been altered to a resistant one. A total of 42 differentially expressed genes (DEGs) in the glyphosate were screened using microarray techniques. Under treatment, argF, sdhA, ivbL, rrfA-H were downregulated, whereas the transcripts of speA, osmY, pflB, ahpC, fusA, deoA, uxaC, rpoD and a few ribosomal protein genes were upregulated. Data were verified by quantitative real-time PCR on selected genes. All transcriptional changes appeared to protect the bacteria from glyphosate and associated osmotic, acidic and oxidative stresses. Many DEGs may have the potential to confer resistance to glyphosate alone, and some may be closely related to the shikimate pathway, reflecting the complex gene interaction network for glyphosate resistance. © 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

  19. The Fd-GOGAT1 mutant gene lc7 confers resistance to Xanthomonas oryzae pv. Oryzae in rice.

    Science.gov (United States)

    Chen, Honglin; Li, Chunrong; Liu, Liping; Zhao, Jiying; Cheng, Xuzhen; Jiang, Guanghuai; Zhai, Wenxue

    2016-05-23

    Disease resistance is an important goal of crop improvement. The molecular mechanism of resistance requires further study. Here, we report the identification of a rice leaf color mutant, lc7, which is defective in chlorophyll synthesis and photosynthesis but confers resistance to Xanthomonas oryzae pv. Oryzae (Xoo). Map-based cloning revealed that lc7 encodes a mutant ferredoxin-dependent glutamate synthase1 (Fd-GOGAT1). Fd-GOGAT1 has been proposed to have great potential for improving nitrogen-use efficiency, but its function in bacterial resistance has not been reported. The lc7 mutant accumulates excessive levels of ROS (reactive oxygen species) in the leaves, causing the leaf color to become yellow after the four-leaf stage. Compared to the wild type, lc7 mutants have a broad-spectrum high resistance to seven Xoo strains. Differentially expressed genes (DEGs) and qRT-PCR analysis indicate that many defense pathways that are involved in this broad-spectrum resistance are activated in the lc7 mutant. These results suggest that Fd-GOGAT1 plays an important role in broad-spectrum bacterial blight resistance, in addition to modulating nitrogen assimilation and chloroplast development.

  20. TetAB46, a predicted heterodimeric ABC transporter conferring tetracycline resistance in Streptococcus australis isolated from the oral cavity.

    Science.gov (United States)

    Warburton, Philip J; Ciric, Lena; Lerner, Avigdor; Seville, Lorna A; Roberts, Adam P; Mullany, Peter; Allan, Elaine

    2013-01-01

    To identify the genes responsible for tetracycline resistance in a strain of Streptococcus australis isolated from pooled saliva from healthy volunteers in France. S. australis is a viridans Streptococcus, originally isolated from the oral cavity of children in Australia, and subsequently reported in the lungs of cystic fibrosis patients and as a cause of invasive disease in an elderly patient. Agar containing 2 mg/L tetracycline was used for the isolation of tetracycline-resistant organisms. A genomic library in Escherichia coli was used to isolate the tetracycline resistance determinant. In-frame deletions and chromosomal repair were used to confirm function. Antibiotic susceptibility was determined by agar dilution and disc diffusion assay. The tetracycline resistance determinant from S. australis FRStet12 was isolated from a genomic library in E. coli and DNA sequencing showed two open reading frames predicted to encode proteins with similarity to multidrug resistance-type ABC transporters. Both genes were required for tetracycline resistance (to both the naturally occurring and semi-synthetic tetracyclines) and they were designated tetAB(46). This is the first report of a predicted ABC transporter conferring tetracycline resistance in a member of the oral microbiota.

  1. Evaluation of dihydrofolate reductase and dihydropteroate synthetase genotypes that confer resistance to sulphadoxine-pyrimethamine in Plasmodium falciparum in Haiti

    Directory of Open Access Journals (Sweden)

    Carter Tamar E

    2012-08-01

    Full Text Available Abstract Background Malaria caused by Plasmodium falciparum infects roughly 30,000 individuals in Haiti each year. Haiti has used chloroquine (CQ as a first-line treatment for malaria for many years and as a result there are concerns that malaria parasites may develop resistance to CQ over time. Therefore it is important to prepare for alternative malaria treatment options should CQ resistance develop. In many other malaria-endemic regions, antifolates, particularly pyrimethamine (PYR and sulphadoxine (SDX treatment combination (SP, have been used as an alternative when CQ resistance has developed. This study evaluated mutations in the dihydrofolate reductase (dhfr and dihydropteroate synthetase (dhps genes that confer PYR and SDX resistance, respectively, in P. falciparum to provide baseline data in Haiti. This study is the first comprehensive study to examine PYR and SDX resistance genotypes in P. falciparum in Haiti. Methods DNA was extracted from dried blood spots and genotyped for PYR and SDX resistance mutations in P. falciparum using PCR and DNA sequencing methods. Sixty-one samples were genotyped for PYR resistance in codons 51, 59, 108 and 164 of the dhfr gene and 58 samples were genotyped for SDX resistance codons 436, 437, 540 of the dhps gene in P. falciparum. Results Thirty-three percent (20/61 of the samples carried a mutation at codon 108 (S108N of the dhfr gene. No mutations in dhfr at codons 51, 59, 164 were observed in any of the samples. In addition, no mutations were observed in dhps at the three codons (436, 437, 540 examined. No significant difference was observed between samples collected in urban vs rural sites (Welch’s T-test p-value = 0.53 and permutations p-value = 0.59. Conclusion This study has shown the presence of the S108N mutation in P. falciparum that confers low-level PYR resistance in Haiti. However, the absence of SDX resistance mutations suggests that SP resistance may not be present in Haiti. These

  2. Evaluation of dihydrofolate reductase and dihydropteroate synthetase genotypes that confer resistance to sulphadoxine-pyrimethamine in Plasmodium falciparum in Haiti.

    Science.gov (United States)

    Carter, Tamar E; Warner, Megan; Mulligan, Connie J; Existe, Alexander; Victor, Yves S; Memnon, Gladys; Boncy, Jacques; Oscar, Roland; Fukuda, Mark M; Okech, Bernard A

    2012-08-13

    Malaria caused by Plasmodium falciparum infects roughly 30,000 individuals in Haiti each year. Haiti has used chloroquine (CQ) as a first-line treatment for malaria for many years and as a result there are concerns that malaria parasites may develop resistance to CQ over time. Therefore it is important to prepare for alternative malaria treatment options should CQ resistance develop. In many other malaria-endemic regions, antifolates, particularly pyrimethamine (PYR) and sulphadoxine (SDX) treatment combination (SP), have been used as an alternative when CQ resistance has developed. This study evaluated mutations in the dihydrofolate reductase (dhfr) and dihydropteroate synthetase (dhps) genes that confer PYR and SDX resistance, respectively, in P. falciparum to provide baseline data in Haiti. This study is the first comprehensive study to examine PYR and SDX resistance genotypes in P. falciparum in Haiti. DNA was extracted from dried blood spots and genotyped for PYR and SDX resistance mutations in P. falciparum using PCR and DNA sequencing methods. Sixty-one samples were genotyped for PYR resistance in codons 51, 59, 108 and 164 of the dhfr gene and 58 samples were genotyped for SDX resistance codons 436, 437, 540 of the dhps gene in P. falciparum. Thirty-three percent (20/61) of the samples carried a mutation at codon 108 (S108N) of the dhfr gene. No mutations in dhfr at codons 51, 59, 164 were observed in any of the samples. In addition, no mutations were observed in dhps at the three codons (436, 437, 540) examined. No significant difference was observed between samples collected in urban vs rural sites (Welch's T-test p-value = 0.53 and permutations p-value = 0.59). This study has shown the presence of the S108N mutation in P. falciparum that confers low-level PYR resistance in Haiti. However, the absence of SDX resistance mutations suggests that SP resistance may not be present in Haiti. These results have important implications for ongoing discussions on

  3. A horizontally gene transferred copper resistance locus confers hyper-resistance to antibacterial copper toxicity and enables survival of community acquired methicillin resistant Staphylococcus aureus USA300 in macrophages.

    Science.gov (United States)

    Purves, Joanne; Thomas, Jamie; Riboldi, Gustavo P; Zapotoczna, Marta; Tarrant, Emma; Andrew, Peter W; Londoño, Alejandra; Planet, Paul J; Geoghegan, Joan A; Waldron, Kevin J; Morrissey, Julie A

    2018-03-09

    Excess copper is highly toxic and forms part of the host innate immune system's antibacterial arsenal, accumulating at sites of infection and acting within macrophages to kill engulfed pathogens. We show for the first time that a novel, horizontally gene transferred copper resistance locus (copXL), uniquely associated with the SCCmec elements of the highly virulent, epidemic, community acquired methicillin resistant Staphylococcus aureus (CA-MRSA) USA300, confers copper hyper-resistance. These genes are additional to existing core genome copper resistance mechanisms, and are not found in typical S. aureus lineages, but are increasingly identified in emerging pathogenic isolates. Our data show that CopX, a putative P 1B-3 -ATPase efflux transporter, and CopL, a novel lipoprotein, confer copper hyper-resistance compared to typical S. aureus strains. The copXL genes form an operon that is tightly repressed in low copper environments by the copper regulator CsoR. Significantly, CopX and CopL are important for S. aureus USA300 intracellular survival within macrophages. Therefore, the emergence of new S. aureus clones with the copXL locus has significant implications for public health because these genes confer increased resistance to antibacterial copper toxicity, enhancing bacterial fitness by altering S. aureus interaction with innate immunity. © 2018 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd.

  4. MAPLE fabrication of thin films based on kanamycin functionalized magnetite nanoparticles with anti-pathogenic properties

    Science.gov (United States)

    Grumezescu, Valentina; Andronescu, Ecaterina; Holban, Alina Maria; Mogoantă, Laurenţiu; Mogoşanu, George Dan; Grumezescu, Alexandru Mihai; Stănculescu, Anca; Socol, Gabriel; Iordache, Florin; Maniu, Horia; Chifiriuc, Mariana Carmen

    2015-05-01

    In this study we aimed to evaluate the biocompatibility and antimicrobial activity of kanamycin functionalized 5 nm-magnetite (Fe3O4@KAN) nanoparticles thin films deposited by Matrix Assisted Pulsed Laser Evaporation (MAPLE) technique. A laser deposition regime was established in order to stoichiometrically transfer Fe3O4@KAN thin films on silicone and glass substrates. Morphological and physico-chemical properties of powders and coatings were characterized by XRD, TEM, SEM, AFM and IR microscopy (IRM). Our nanostructured thin films have proved efficiency in the prevention of microbial adhesion and mature biofilms development as a result of antibiotic release in its active form. Furthermore, kanamycin functionalized nanostructures exhibit a good biocompatibility, both in vivo and in vitro, demonstrating their potential for implants application. This is the first study reporting the assessment of the in vivo biocompatibility of a magnetite-antimicrobial thin films produced by MAPLE technique.

  5. Investigation of porous silicon nanopowders functionalized by antibiotic Kanamycin, fluorophore Indocyanine Green

    Science.gov (United States)

    Bespalova, K.; Somov, P. A.; Spivak, Yu M.

    2017-11-01

    Porous silicon nanopowders for target drug delivery were obtained by electrochemical anodic etching in a hydrofluoric acid solution using the monocrystalline silicon n-type conductivity. Porous silicon powders were obtained by sonification of porous silicon layers. The powders were functionalized by antibiotic Kanamycin and fluorophore Indocyanine Green by the passive adsorption method. The peculiarities of absorption spectra in 190-600 nm region were revealed for functionalized porous silicon powders dispersions in water.

  6. RENAL CLEARANCE AND URINARY EXCRETION OF KANAMYCIN IN DOMESTIC RUMINANT SPECIES

    Directory of Open Access Journals (Sweden)

    I. JAVED, Z. U. RAHMAN, F. H. KHAN, F. MUHAMMAD, Z. IQBAL AND B. ASLAM

    2006-01-01

    Full Text Available Species dependent geonetical differences in renal clearance and urinary excretion of kanamycin were investigated in adult female buffaloes, cows, sheep and goats. The drug was administered as a single intravenous dose (5 mg/kg b.wt. Blood and urine samples were collected at various time intervals after drug administration. The plasma and urine concentrations of the drug were determined using the microbiological assay. The mean (± SE values for endogenous creatinine clearance (an index of glomerular filtration rate were 0.77 ± 0.05, 0.49 ± 0.07, 0.81 ± 0.07 and 0.98 ± 0.13 ml/min.kg in buffaloes, cows, sheep and goats, respectively. Experiments regarding kidney handling of kanamycin in these ruminant species revealed respective values of renal clearance as 0.08 ± 0.01, 0.07 ± 0.01, 0.19 ± 0.02 and 0.23 ± 0.04 ml/min.kg. Besides glomerular filtration, kanamycin was reabsorbed from the renal tubules of all ruminant species and actively secreted into the renal tubules of buffaloes and goats. The cumulative percentages of intravenous dose of kanamycin excreted through urine during 12 hours in buffaloes, cows, sheep and goats were 4.31 ± 0.37, 2.53 ± 0.30, 11.0 ± 1.04 and 15.8 ± 2.22, respectively. This species variation in the percentage of urinary excretion in these domestic ruminants coincides with their respective glomerular filtration rates, being the highest in goats, lowest in cows and intermediate in sheep and buffaloes.

  7. Aminoglycoside resistance profile and structural architecture of the aminoglycoside acetyltransferase AAC(6')-Im.

    Science.gov (United States)

    Smith, Clyde A; Bhattacharya, Monolekha; Toth, Marta; Stewart, Nichole K; Vakulenko, Sergei B

    2017-11-09

    Aminoglycoside 6'-acetyltransferase-Im (AAC(6')-Im) is the closest monofunctional homolog of the AAC(6')-Ie acetyltransferase of the bifunctional enzyme AAC(6')-Ie/APH(2")-Ia. The AAC(6')-Im acetyltransferase confers 4- to 64-fold higher MICs to 4,6-disubstituted aminoglycosides and the 4,5-disubstituted aminoglycoside neomycin than AAC(6')-Ie, yet unlike AAC(6')-Ie, the AAC(6')-Im enzyme does not confer resistance to the atypical aminoglycoside fortimicin. The structure of the kanamycin A complex of AAC(6')-Im shows that the substrate binds in a shallow positively-charged pocket, with the N6' amino group positioned appropriately for an efficient nucleophilic attack on an acetyl-CoA cofactor. The AAC(6')-Ie enzyme binds kanamycin A in a sufficiently different manner to position the N6' group less efficiently, thereby reducing the activity of this enzyme towards the 4,6-disubstituted aminoglycosides. Conversely, docking studies with fortimicin in both acetyltransferases suggest that the atypical aminoglycoside might bind less productively in AAC(6')-Im, thus explaining the lack of resistance to this molecule.

  8. Development of pyramidal lines with two major QTLs conferring resistance to sheath blight in rice (Oryza sativa L.)

    Science.gov (United States)

    Hossain, Md Kamal; Jena, Kshirod; Bhuiyan, Md Atiqur Rahman; Ratnam, Wickneswari

    2014-09-01

    Sheath blight is an emerging threat in rice cultivation. It is animportant disease caused by the soil-borne necrotrophic pathogenic fungus, Rhizoctonia solani Kühn. However, to date neither known major genes for quantitative resistance, nor any rice lines immune to this disease has been identified. The disease resistance is quantitative in nature. Numerous genes are involved in this resistance process. There are few quantitative trait loci (QTLs) detected conferring improved resistance against the disease. Teqing and Tetepshowimproved resistance having QTLs, qSB-9 and qSBR11-1, respectively. Since, these QTLs demonstrates additive effects, pyramiding of these QTLs might be an option to increase the sheath blight resistance in rice. Nine rice cultivars were screened at greenhouse conditions. Results showed that Tetep and Teqing had the lowest disease ratings. UKMRC2a new high yielding cultivar was as recipient parent. Crosses between UKMRC2 and Teqing, and UKMRC2 and Tetep were made and confirmed. Subsequently 4-way crosses between the two F1s were performed to develop pyramidal lines.

  9. Differentiation of encephalitogenic T cells confers resistance to an inhibitory anti-CD4 monoclonal antibody.

    Science.gov (United States)

    Mannie, M D; Morrison-Plummer, J; McConnell, T J

    1993-12-15

    The anti-CD4 mAb W3/25 inhibits experimental autoimmune encephalomyelitis (EAE) in Lewis rats by blocking Th cell responses to encephalitogenic determinants of myelin basic protein (MBP). However, it has yet to be resolved how W3/25 modulates CD4 to inhibit EAE-associated T cell responses. This study revealed that W3/25 profoundly inhibited MBP-stimulated proliferation by sensitized lymph node cells but only partially inhibited the respective response of uncloned and cloned lines of MBP-specific T cells. That is, low concentrations of W3/25 blocked 30 to 60% of MBP-stimulated proliferation, but 100-fold higher concentrations did not result in additional inhibition. W3/25 also inhibited MBP-induced acquisition of EAE transfer activity, but only in cultures of freshly isolated lymph node cells and not in cultures of continuously propagated T cells. Studies focusing on the GP2.E5 T cell line revealed that the lack of sensitivity to W3/25 in encephalitogenic and proliferative assays was nevertheless associated with an effective blockage of MBP-stimulated IL-2 production. Importantly, W3/25 specifically inhibited antigenic but not mitogenic stimulation of IL-2 production. Reverse transcriptase/polymerase chain reaction analyses revealed that MBP-activated GP2.E5 T cells produced mRNA for both IL-2 and IL-4, and that W3/25 selectively inhibited accumulation of IL-2 as compared to IL-4 mRNA. Thus, GP2.E5 T cells apparently express a IL-4-dependent pathway that confers resistance to the inhibitory activity of W3/25. Studies focusing on two CD4+ T cell hybridomas revealed that W3/25 profoundly inhibited MBP-stimulated IL-2 production but did not affect the alternative response of MBP-induced growth inhibition. Several other hybrids also mediated MBP-stimulated IL-2 production but did not express CD4 and were not affected by W3/25. These results indicate that: 1) interactions of W3/25 with CD4 do not necessarily block class II MHC-restricted recognition of MBP; and 2

  10. Dissection of two soybean QTL conferring partial resistance to Phytophthora sojae through sequence and gene expression analysis

    Directory of Open Access Journals (Sweden)

    Wang Hehe

    2012-08-01

    Full Text Available Abstract Background Phytophthora sojae is the primary pathogen of soybeans that are grown on poorly drained soils. Race-specific resistance to P. sojae in soybean is gene-for-gene, although in many areas of the US and worldwide there are populations that have adapted to the most commonly deployed resistance to P. sojae ( Rps genes. Hence, this system has received increased attention towards identifying mechanisms and molecular markers associated with partial resistance to this pathogen. Several quantitative trait loci (QTL have been identified in the soybean cultivar ‘Conrad’ that contributes to the expression of partial resistance to multiple P. sojae isolates. Results In this study, two of the Conrad QTL on chromosome 19 were dissected through sequence and expression analysis of genes in both resistant (Conrad and susceptible (‘Sloan’ genotypes. There were 1025 single nucleotide polymorphisms (SNPs in 87 of 153 genes sequenced from Conrad and Sloan. There were 304 SNPs in 54 genes sequenced from Conrad compared to those from both Sloan and Williams 82, of which 11 genes had SNPs unique to Conrad. Eleven of 19 genes in these regions analyzed with qRT-PCR had significant differences in fold change of transcript abundance in response to infection with P. sojae in lines with QTL haplotype from the resistant parent compared to those with the susceptible parent haplotype. From these, 8 of the 11 genes had SNPs in the upstream, untranslated region, exon, intron, and/or downstream region. These 11 candidate genes encode proteins potentially involved in signal transduction, hormone-mediated pathways, plant cell structural modification, ubiquitination, and basal resistance. Conclusions These findings may indicate a complex defense network with multiple mechanisms underlying these two soybean QTL conferring resistance to P. sojae. SNP markers derived from these candidate genes can contribute to fine mapping of QTL and marker assisted breeding for

  11. Deficiency of insulin-like growth factor-1 receptor confers resistance to oxidative stress in C2C12 myoblasts.

    Directory of Open Access Journals (Sweden)

    Sachin Thakur

    Full Text Available IGF-1 receptor (IGF-1R signaling regulates cell growth, transformation and survival. Haploinsufficiency of the IGF-1R is reported to paradoxically confer resistance to oxidative stress in vivo and in cells cultured from Igf1r(+/- mice. In order to determine whether IGF-1R deficiency directly confers resistance to oxidative stress in specific cell types, an siRNA-mediated approach was applied to reduce IGF-1R in C2C12 myoblasts, NIH3T3 fibroblasts and MC3T3-E1 osteoblasts. Treating the IGF-1R deficient myoblasts with H2O2 resulted in significantly higher phosphorylation of Akt as compared to cells having normal expression of IGF-1R. Similar results were obtained with UV treatment, another inducer of oxidative stress. This enhanced activation of Akt was associated with reduced level of cleaved caspase-3 and PARP. Moreover, in the IGF-1R knockdown myoblasts, phosphorylation of the Akt substrate Bad was enhanced after peroxide treatment. However, in NIH-3T3 fibroblasts and MC3T3-E1 osteoblasts, the loss of IGF-1R by siRNA directed knockdown was associated with reduced levels of phosphorylated Akt on treatment with H2O2 or UV as compared to control cells and these cells showed more apoptosis. These results suggest a novel mechanism of cell type specific differential regulation of resistance to oxidative stress induced apoptosis by reduced levels of IGF-1R.

  12. A new amino acid substitution (Ala-205-Phe) in acetolactate synthase (ALS) confers broad spectrum resistance to ALS-inhibiting herbicides.

    Science.gov (United States)

    Brosnan, James T; Vargas, Jose J; Breeden, Gregory K; Grier, Logan; Aponte, Raphael A; Tresch, Stefan; Laforest, Martin

    2016-01-01

    This is a first report of an Ala-205-Phe substitution in acetolactate synthase conferring resistance to imidazolinone, sulfonylurea, triazolopyrimidines, sulfonylamino-carbonyl-triazolinones, and pyrimidinyl (thio) benzoate herbicides. Resistance to acetolactate synthase (ALS) and photosystem II inhibiting herbicides was confirmed in a population of allotetraploid annual bluegrass (Poa annua L.; POAAN-R3) selected from golf course turf in Tennessee. Genetic sequencing revealed that seven of eight POAAN-R3 plants had a point mutation in the psbA gene resulting in a known Ser-264-Gly substitution on the D1 protein. Whole plant testing confirmed that this substitution conferred resistance to simazine in POAAN-R3. Two homeologous forms of the ALS gene (ALSa and ALSb) were detected and expressed in all POAAN-R3 plants sequenced. The seven plants possessing the Ser-264-Gly mutation conferring resistance to simazine also had a homozygous Ala-205-Phe substitution on ALSb, caused by two nucleic acid substitutions in one codon. In vitro ALS activity assays with recombinant protein and whole plant testing confirmed that this Ala-205-Phe substitution conferred resistance to imidazolinone, sulfonylurea, triazolopyrimidines, sulfonylamino-carbonyl- triazolinones, and pyrimidinyl (thio) benzoate herbicides. This is the first report of Ala-205-Phe mutation conferring wide spectrum resistance to ALS inhibiting herbicides.

  13. Identification and characterization of Sr13, a tetraploid wheat gene that confers resistance to the Ug99 stem rust race group

    Science.gov (United States)

    The Puccinia graminis f. sp. tritici (Pgt) Ug99 race group is virulent to most stem rust resistance genes currently deployed in wheat and poses a serious threat to global wheat production. The durum wheat (Triticum turgidum ssp. durum) gene Sr13 confers resistance to Ug99 in addition to virulent rac...

  14. 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

  15. An AFLP marker linked to the Pm-1 gene that confers resistance to Podosphaera xanthii race 1 in Cucumis melo

    Directory of Open Access Journals (Sweden)

    Ana Paula Matoso Teixeira

    2008-01-01

    Full Text Available Brazil produced 330,000 metric tons of melons in 2005, principally in the Northeast region where one of the most important melon pathogens is the powdery mildew fungus Podosphaera xanthii. The disease is controlled mainly by incorporating single dominant resistance genes into commercial hybrids. We report on linkage analysis of the Pm-1 resistance gene, introgressed from the AF125Pm-1 Cantalupensis Charentais-type breeding line into the yellow-fleshed melon (Group Inodorus breeding line AF426-S by backcrossing to produce the resistant line AF426-R, and the amplified fragment length polymorphism (AFLP marker M75/H35_155 reported to be polymorphic between AF426-S and AF426-R. Segregation analysis of M75/H35_155 using a backcross population of 143 plants derived from [AF426-R x AF426-S] x AF426-S and screened for resistance to P. xanthii race 1 produced a recombination frequency of 4.9%, indicating close linkage between M75/H35_155 and Pm-1. Using the same segregating population, the M75/H35_155 marker had previously been reported to be distantly linked to Prv¹, a gene conferring resistance to papaya ringspot virus-type W. Since M75/H35_155 is linked to Prv¹ at a distance of 40.9 cM it is possible that Pm-1 and Prv¹ are also linked.

  16. Conformational Response of 30S-bound IF3 to A-Site Binders Streptomycin and Kanamycin.

    Science.gov (United States)

    Chulluncuy, Roberto; Espiche, Carlos; Nakamoto, Jose Alberto; Fabbretti, Attilio; Milón, Pohl

    2016-12-13

    Aminoglycoside antibiotics are widely used to treat infectious diseases. Among them, streptomycin and kanamycin (and derivatives) are of importance to battle multidrug-resistant (MDR) Mycobacterium tuberculosis . Both drugs bind the small ribosomal subunit (30S) and inhibit protein synthesis. Genetic, structural, and biochemical studies indicate that local and long-range conformational rearrangements of the 30S subunit account for this inhibition. Here, we use intramolecular FRET between the C- and N-terminus domains of the flexible IF3 to monitor real-time perturbations of their binding sites on the 30S platform. Steady and pre-steady state binding experiments show that both aminoglycosides bring IF3 domains apart, promoting an elongated state of the factor. Binding of Initiation Factor IF1 triggers closure of IF3 bound to the 30S complex, while both aminoglycosides revert the IF1-dependent conformation. Our results uncover dynamic perturbations across the 30S subunit, from the A-site to the platform, and suggest that both aminoglycosides could interfere with prokaryotic translation initiation by modulating the interaction between IF3 domains with the 30S platform.

  17. Conformational Response of 30S-bound IF3 to A-Site Binders Streptomycin and Kanamycin

    Directory of Open Access Journals (Sweden)

    Roberto Chulluncuy

    2016-12-01

    Full Text Available Aminoglycoside antibiotics are widely used to treat infectious diseases. Among them, streptomycin and kanamycin (and derivatives are of importance to battle multidrug-resistant (MDR Mycobacterium tuberculosis. Both drugs bind the small ribosomal subunit (30S and inhibit protein synthesis. Genetic, structural, and biochemical studies indicate that local and long-range conformational rearrangements of the 30S subunit account for this inhibition. Here, we use intramolecular FRET between the C- and N-terminus domains of the flexible IF3 to monitor real-time perturbations of their binding sites on the 30S platform. Steady and pre-steady state binding experiments show that both aminoglycosides bring IF3 domains apart, promoting an elongated state of the factor. Binding of Initiation Factor IF1 triggers closure of IF3 bound to the 30S complex, while both aminoglycosides revert the IF1-dependent conformation. Our results uncover dynamic perturbations across the 30S subunit, from the A-site to the platform, and suggest that both aminoglycosides could interfere with prokaryotic translation initiation by modulating the interaction between IF3 domains with the 30S platform.

  18. Hepatocyte-specific deletion of the keap1 gene activates Nrf2 and confers potent resistance against acute drug toxicity

    International Nuclear Information System (INIS)

    Okawa, Hiromi; Motohashi, Hozumi; Kobayashi, Akira; Aburatani, Hiroyuki; Kensler, Thomas W.; Yamamoto, Masayuki

    2006-01-01

    Nrf2 is a key regulator of many detoxifying enzyme genes, and cytoplasmic protein Keap1 represses the Nrf2 activity under quiescent conditions. Germ line deletion of the keap1 gene results in constitutive activation of Nrf2, but the pups unexpectedly died before weaning. To investigate how constitutive activation of Nrf2 influences the detoxification system in adult mice, we generated mice bearing a hepatocyte-specific disruption of the keap1 gene. Homozygous mice were viable and their livers displayed no apparent abnormalities, but nuclear accumulation of Nrf2 is elevated. Microarray analysis revealed that, while many detoxifying enzyme genes are highly expressed, some of the typical Nrf2-dependent genes are only marginally increased in the Keap1-deficient liver. The mutant mice were significantly more resistant to toxic doses of acetaminophen than control animals. These results demonstrate that chronic activation of Nrf2 confers animals with resistance to xenobiotics without affecting the morphological and physiological integrity of hepatocytes

  19. Bacterial Enzymes and Antibiotic Resistance- Oral Presentation

    Energy Technology Data Exchange (ETDEWEB)

    Maltz, Lauren [SLAC National Accelerator Lab., Menlo Park, CA (United States)

    2015-08-25

    By using protein crystallography and X-ray diffraction, structures of bacterial enzymes were solved to gain a better understanding of how enzymatic modification acts as an antibacterial resistance mechanism. Aminoglycoside phosphotransferases (APHs) are one of three aminoglycoside modifying enzymes that confer resistance to the aminoglycoside antibiotics via enzymatic modification, rendering many drugs obsolete. Specifically, the APH(2”) family vary in their substrate specificities and also in their preference for the phosphate donor (ADP versus GDP). By solving the structures of members of the APH(2”) family of enzymes, we can see how domain movements are important to their substrate specificity. Our structure of the ternary complex of APH(2”)-IIIa with GDP and kanamycin, when compared to the known structures of APH(2”)-IVa, reveals that there are real physical differences between these two enzymes, a structural finding that explains why the two enzymes differ in their preferences for certain aminoglycosides. Another important group of bacterial resistance enzymes are the Class D β-lactamases. Oxacillinase carbapenemases (OXAs) are part of this enzyme class and have begun to confer resistance to ‘last resort’ drugs, most notably carbapenems. Our structure of OXA-143 shows that the conformational flexibility of a conserved hydrophobic residue in the active site (Val130) serves to control the entry of a transient water molecule responsible for a key step in the enzyme’s mechanism. Our results provide insight into the structural mechanisms of these two different enzymes.

  20. HLA-DRB1-DQB1 haplotypes confer susceptibility and resistance to multiple sclerosis in Sardinia.

    Directory of Open Access Journals (Sweden)

    Eleonora Cocco

    :02, *06:01 alleles. CONCLUSIONS: These findings show that the association of specific, independent DRB1*-DQB1* haplotypes confers susceptibility or resistance to MS in the MS-prone Sardinian population. The data also supports a functional role for specific residues of the DRB1 and DQB1 proteins in predisposing patients to MS.

  1. A novel amino acid substitution Trp574Arg in acetolactate synthase (ALS) confers broad resistance to ALS-inhibiting herbicides in crabgrass (Digitaria sanguinalis).

    Science.gov (United States)

    Li, Jian; Li, Mei; Gao, Xingxiang; Fang, Feng

    2017-12-01

    Crabgrass (Digitaria sanguinalis) is an annual monocotyledonous weed. In recent years, field applications of nicosulfuron have been ineffective in controlling crabgrass populations in Shandong Province, China. To investigate the mechanisms of resistance to nicosulfuron in crabgrass populations, the acetolactate synthase (ALS) gene fragment covering known resistance-confering mutation sites was amplified and sequenced. Dose-response experiments suggested that the resistant population SD13 (R) was highly resistant to nicosulfuron (resistance index R/S = 43.7) compared with the sensitive population SD22 (S). ALS gene sequencing revealed a Trp574Arg substitution in the SD13 population, and no other known resistance-conferring mutations were found. In vitro ALS enzyme assays further confirmed that the SD13 population was resistant to all tested ALS-inhibiting herbicides. The resistance pattern experiments revealed that, compared with SD22, the SD13 population exhibited broad-spectrum resistance to nicosulfuron (43.7-fold), imazethapyr (11.4-fold) and flumetsulam (16.1-fold); however, it did not develop resistance to atrazine, mesotrione and topramezone. This study demonstrated that Trp574Arg substitution was the main reason for crabgrass resistance to ALS-inhibiting herbicides. To our knowledge, this is the first report of Trp574Arg substitution in a weed species, and is the first report of target-site mechanisms of herbicide resistance for crabgrass. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

  2. Mapping quantitative trait loci conferring resistance to rice black-streaked virus in maize (Zea mays L.).

    Science.gov (United States)

    Luan, Junwen; Wang, Fei; Li, Yujie; Zhang, Bin; Zhang, Juren

    2012-08-01

    Maize rough dwarf disease (MRDD) is one of the most serious virus diseases of maize worldwide, and it causes great reduction of maize production. In China, the pathogen was shown to be rice black-streaked virus (RBSDV). Currently, MRDD has spread broadly and leads to significant loss in China. However, there has been little research devoted to this disease. Our aims were to identify the markers and loci underlying resistance to this virus disease. In this study, segregation populations were constructed from two maize elite lines '90110', which is highly resistant to MRDD and 'Ye478', which is highly susceptible to MRDD. The F(2) and BC(1) populations were used for bulk sergeant analysis (BSA) to identify resistance-related markers. One hundred and twenty F(7:9) RILs were used for quantitative trait loci (QTL) mapping through the experiment of multiple environments over 3 years. Natural occurrence and artificial inoculation were both used and combined to determine the phenotype of plants. Five QTL, qMRD2, qMRD6, qMRD7, qMRD8 and qMRD10 were measured in the experiments. The qMRD8 on chromosome 8 was proved to be one major QTL conferring resistance to RBSDV disease in almost all traits and environments, which explained 12.0-28.9 % of the phenotypic variance for disease severity in this present study.

  3. Mutations in 23S rRNA confer resistance against azithromycin in Pseudomonas aeruginosa.

    Science.gov (United States)

    Marvig, Rasmus Lykke; Søndergaard, Mette S R; Damkiær, Søren; Høiby, Niels; Johansen, Helle Krogh; Molin, Søren; Jelsbak, Lars

    2012-08-01

    The emergence of antibiotic-resistant Pseudomonas aeruginosa is an important concern in the treatment of long-term airway infections in cystic fibrosis patients. In this study, we report the occurrence of azithromycin resistance among clinical P. aeruginosa DK2 isolates. We demonstrate that resistance is associated with specific mutations (A2058G, A2059G, and C2611T in Escherichia coli numbering) in domain V of 23S rRNA and that introduction of A2058G and C2611T into strain PAO1 results in azithromycin resistance.

  4. Mapping of loci from Solanum lycopersicoides conferring resistance or susceptibility to Botrytis cinerea in tomato.

    Science.gov (United States)

    Davis, Joel; Yu, Daozhan; Evans, Wendy; Gokirmak, Tufan; Chetelat, Roger T; Stotz, Henrik U

    2009-07-01

    Cultivated tomato (Solanum lycopersicum, syn. Lycopersicon esculentum) is susceptible to the necrotrophic ascomycete and causal agent of gray mold, Botrytis cinerea. Resistance to this fungal pathogen is elevated in wild relatives of tomato, including Solanum lycopersicoides. An introgression line population (IL) containing chromosomal segments of S. lycopersicoides within the background of tomato cv. VF36 was used to screen the genome for foliar resistance and susceptibility to B. cinerea. Based on this screen, putative quantitative trait loci (QTL) were identified, five for resistance and two for susceptibility. Four resistance QTL decreased infection frequency while the fifth reduced lesion diameter. One susceptibility QTL increased infection frequency whereas the other increased lesion diameter. Overlapping chromosomal segments provided strong evidence for partial resistance on chromosomes 1 and 9 and for elevated susceptibility on chromosome 11. Segregation analysis confirmed the major resistance QTL on the long arm of chromosome 1 and susceptibility on chromosome 11. Linkage of partial resistance to chromosome 9 could not be confirmed. The usefulness of these data for resistance breeding and for map-based cloning of foliar resistance to B. cinerea is discussed.

  5. EPSPS gene amplification conferring resistance to glyphosate in windmill grass (Chloris truncata) in Australia.

    Science.gov (United States)

    Ngo, The D; Malone, Jenna M; Boutsalis, Peter; Gill, Gurjeet; Preston, Christopher

    2018-05-01

    Five glyphosate-resistant populations of Chloris truncata originally collected from New South Wales were compared with one susceptible (S) population from South Australia to confirm glyphosate resistance and elucidate possible mechanisms of resistance. Based on the amounts of glyphosate required to kill 50% of treated plants (LD 50 ), glyphosate resistance (GR) was confirmed in five populations of C. truncata (A536, A528, T27, A534 and A535.1). GR plants were 2.4-8.7-fold more resistant and accumulated less shikimate after glyphosate treatment than S plants. There was no difference in glyphosate absorption and translocation between GR and S plants. The EPSPS gene did not contain any point mutation that had previously been associated with resistance to glyphosate. The resistant plants (A528 and A536) contained up to 32-48 more copies of the EPSPS gene than the susceptible plants. This study has identified EPSPS gene amplification contributing to glyphosate resistance in C. truncata. In addition, a Glu-91-Ala mutation within EPSPS was identified that may contribute to glyphosate resistance in this species. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

  6. Mutations in 23S rRNA Confer Resistance against Azithromycin in Pseudomonas aeruginosa

    DEFF Research Database (Denmark)

    Marvig, Rasmus Lykke; Søndergaard, Mette S. R.; Pedersen, Søren Damkiær

    2012-01-01

    The emergence of antibiotic-resistant Pseudomonas aeruginosa is an important concern in the treatment of long-term airway infections in cystic fibrosis patients. In this study, we report the occurrence of azithromycin resistance among clinical P. aeruginosa DK2 isolates. We demonstrate...... that resistance is associated with specific mutations (A2058G, A2059G, and C2611T in Escherichia coli numbering) in domain V of 23S rRNA and that introduction of A2058G and C2611T into strain PAO1 results in azithromycin resistance....

  7. Screening for streptomycin resistance conferring mutations in Mycobacterium tuberculosis isolates from Iran.

    Science.gov (United States)

    Rezaei, Faranak; Haeili, Mehri; Imani Fooladi, Abbasali; Azari Garmjan, Gholam Ali; Feizabadi, Mohammad Mehdi

    2017-02-01

    Point mutations in the rpsL and rrs genes can lead to development of streptomycin (STR) resistance in Mycobacterium tuberculosis. The aims of this study were to determine the frequency of mutations in STR resistant M. tuberculosis isolates in Iran and to analyze the possible relationship between bacterial genotype and STR resistance. Twenty-three M. tuberculosis samples comprising 9 multidrug-resistant (MDR) and 14 non-MDR isolates, recovered from TB patients in four regions: Tehran (n = 14), Isfahan (n = 2), Zahedan (n = 2), and Khorasan (n = 5), were analysed. Mutational profiling was performed by sequencing of the rrs and rpsL genes and spoligotyping method was used for genotyping. Nineteen isolates were resistant to STR, among them 7 exhibited mutations in the rpsL gene and 7 had mutations in the rrs gene. The remaining 5 STR resistant as well as all susceptible isolates lacked any mutation in both genes. Beijing genotype was associated with both MDR and STR resistance in which all mutations occurred at codon 43 of the rpsL gene. There was an association between mutations in the rpsL and rrs genes and STR resistance. We also found a correlation between Beijing genotype and STR resistance.

  8. Towards positional isolation of three quantitative trait loci conferring resistance to powdery mildew in two Spanish barley landraces.

    Directory of Open Access Journals (Sweden)

    Cristina Silvar

    Full Text Available Three quantitative trait loci (QTL conferring broad spectrum resistance to powdery mildew, caused by the fungus Blumeria graminis f. sp. hordei, were previously identified on chromosomes 7HS, 7HL and 6HL in the Spanish barley landrace-derived lines SBCC097 and SBCC145. In the present work, a genome-wide putative linear gene index of barley (Genome Zipper and the first draft of the physical, genetic and functional sequence of the barley genome were used to go one step further in the shortening and explicit demarcation on the barley genome of these regions conferring resistance to powdery mildew as well as in the identification of candidate genes. First, a comparative analysis of the target regions to the barley Genome Zippers of chromosomes 7H and 6H allowed the development of 25 new gene-based molecular markers, which slightly better delimit the QTL intervals. These new markers provided the framework for anchoring of genetic and physical maps, figuring out the outline of the barley genome at the target regions in SBCC097 and SBCC145. The outermost flanking markers of QTLs on 7HS, 7HL and 6HL defined a physical area of 4 Mb, 3.7 Mb and 3.2 Mb, respectively. In total, 21, 10 and 16 genes on 7HS, 7HL and 6HL, respectively, could be interpreted as potential candidates to explain the resistance to powdery mildew, as they encode proteins of related functions with respect to the known pathogen defense-related processes. The majority of these were annotated as belonging to the NBS-LRR class or protein kinase family.

  9. Benzoxazinone kanamycin A conjugate. A new fluorescent probe suitable to detect mycoplasmas in cell culture.

    Science.gov (United States)

    Monsigny, M; Midoux, P; Depierreux, C; Bebear, C; Le Bris, M T; Valeur, B

    1990-01-01

    The synthesis of a new benzoxazinone derivative suitable to detect early infection of cultured cells with mycoplasmas is described. p-[beta-(7-dimethylamino 1,4-benzoxazin 2-one 3yl)-vinyl]- phenylpropenoic acid was coupled to kanamycin A, an aminoglycoside leading to a cationic fluorescent probe which fluoresces at 600 nm upon excitation at 490 nm. This fluorescent probe is shown to heavily label the glycocallix of all the mycoplasma strains tested which are found to be associated with contaminated cultured cells and to allow an easy and rapid detection of contamination by fluorescence microscopy and flow cytometry.

  10. Loci and candidate genes conferring resistance to soybean cyst nematode HG type 2.5.7.

    Science.gov (United States)

    Zhao, Xue; Teng, Weili; Li, Yinghui; Liu, Dongyuan; Cao, Guanglu; Li, Dongmei; Qiu, Lijuan; Zheng, Hongkun; Han, Yingpeng; Li, Wenbin

    2017-06-14

    Soybean (Glycine max L. Merr.) cyst nematode (SCN, Heterodera glycines I,) is a major pest of soybean worldwide. The most effective strategy to control this pest involves the use of resistant cultivars. The aim of the present study was to investigate the genome-wide genetic architecture of resistance to SCN HG Type 2.5.7 (race 1) in landrace and elite cultivated soybeans. A total of 200 diverse soybean accessions were screened for resistance to SCN HG Type 2.5.7 and genotyped through sequencing using the Specific Locus Amplified Fragment Sequencing (SLAF-seq) approach with a 6.14-fold average sequencing depth. A total of 33,194 SNPs were identified with minor allele frequencies (MAF) over 4%, covering 97% of all the genotypes. Genome-wide association mapping (GWAS) revealed thirteen SNPs associated with resistance to SCN HG Type 2.5.7. These SNPs were distributed on five chromosomes (Chr), including Chr7, 8, 14, 15 and 18. Four SNPs were novel resistance loci and nine SNPs were located near known QTL. A total of 30 genes were identified as candidate genes underlying SCN resistance. A total of sixteen novel soybean accessions were identified with significant resistance to HG Type 2.5.7. The beneficial alleles and candidate genes identified by GWAS might be valuable for improving marker-assisted breeding efficiency and exploring the molecular mechanisms underlying SCN resistance.

  11. Modified cellulose synthase gene from Arabidopsis thaliana confers herbicide resistance to plants

    Science.gov (United States)

    Somerville, Chris R [Portola Valley, CA; Scheible, Wolf [Golm, DE

    2007-07-10

    Cellulose synthase ("CS"), a key enzyme in the biosynthesis of cellulose in plants is inhibited by herbicides comprising thiazolidinones such as 5-tert-butyl-carbamoyloxy-3-(3-trifluromethyl)phenyl-4-thiazolidinone (TZ), isoxaben and 2,6-dichlorobenzonitrile (DCB). Two mutant genes encoding isoxaben and TZ-resistant cellulose synthase have been isolated from isoxaben and TZ-resistant Arabidopsis thaliana mutants. When compared with the gene coding for isoxaben or TZ-sensitive cellulose synthase, one of the resistant CS genes contains a point mutation, wherein glycine residue 998 is replaced by an aspartic acid. The other resistant mutation is due to a threonine to isoleucine change at amino acid residue 942. The mutant CS gene can be used to impart herbicide resistance to a plant; thereby permitting the utilization of the herbicide as a single application at a concentration which ensures the complete or substantially complete killing of weeds, while leaving the transgenic crop plant essentially undamaged.

  12. Induction of Xa10-like Genes in Rice Cultivar Nipponbare Confers Disease Resistance to Rice Bacterial Blight.

    Science.gov (United States)

    Wang, Jun; Tian, Dongsheng; Gu, Keyu; Yang, Xiaobei; Wang, Lanlan; Zeng, Xuan; Yin, Zhongchao

    2017-06-01

    Bacterial blight of rice, caused by Xanthomonas oryzae pv. oryzae, is one of the most destructive bacterial diseases throughout the major rice-growing regions in the world. The rice disease resistance (R) gene Xa10 confers race-specific disease resistance to X. oryzae pv. oryzae strains that deliver the corresponding transcription activator-like (TAL) effector AvrXa10. Upon bacterial infection, AvrXa10 binds specifically to the effector binding element in the promoter of the R gene and activates its expression. Xa10 encodes an executor R protein that triggers hypersensitive response and activates disease resistance. 'Nipponbare' rice carries two Xa10-like genes in its genome, of which one is the susceptible allele of the Xa23 gene, a Xa10-like TAL effector-dependent executor R gene isolated recently from 'CBB23' rice. However, the function of the two Xa10-like genes in disease resistance to X. oryzae pv. oryzae strains has not been investigated. Here, we designated the two Xa10-like genes as Xa10-Ni and Xa23-Ni and characterized their function for disease resistance to rice bacterial blight. Both Xa10-Ni and Xa23-Ni provided disease resistance to X. oryzae pv. oryzae strains that deliver the matching artificially designed TAL effectors (dTALE). Transgenic rice plants containing Xa10-Ni and Xa23-Ni under the Xa10 promoter provided specific disease resistance to X. oryzae pv. oryzae strains that deliver AvrXa10. Xa10-Ni and Xa23-Ni knock-out mutants abolished dTALE-dependent disease resistance to X. oryzae pv. oryzae. Heterologous expression of Xa10-Ni and Xa23-Ni in Nicotiana benthamiana triggered cell death. The 19-amino-acid residues at the N-terminal regions of XA10 or XA10-Ni are dispensable for their function in inducing cell death in N. benthamiana and the C-terminal regions of XA10, XA10-Ni, and XA23-Ni are interchangeable among each other without affecting their function. Like XA10, both XA10-Ni and XA23-Ni locate to the endoplasmic reticulum (ER) membrane

  13. Transgenic Cotton Plants Expressing Cry1Ia12 Toxin Confer Resistance to Fall Armyworm (Spodoptera frugiperda) and Cotton Boll Weevil (Anthonomus grandis).

    Science.gov (United States)

    de Oliveira, Raquel S; Oliveira-Neto, Osmundo B; Moura, Hudson F N; de Macedo, Leonardo L P; Arraes, Fabrício B M; Lucena, Wagner A; Lourenço-Tessutti, Isabela T; de Deus Barbosa, Aulus A; da Silva, Maria C M; Grossi-de-Sa, Maria F

    2016-01-01

    Gossypium hirsutum (commercial cooton) is one of the most economically important fibers sources and a commodity crop highly affected by insect pests and pathogens. Several transgenic approaches have been developed to improve cotton resistance to insect pests, through the transgenic expression of different factors, including Cry toxins, proteinase inhibitors, and toxic peptides, among others. In the present study, we developed transgenic cotton plants by fertilized floral buds injection (through the pollen-tube pathway technique) using an DNA expression cassette harboring the cry1Ia12 gene, driven by CaMV35S promoter. The T0 transgenic cotton plants were initially selected with kanamycin and posteriorly characterized by PCR and Southern blot experiments to confirm the genetic transformation. Western blot and ELISA assays indicated the transgenic cotton plants with higher Cry1Ia12 protein expression levels to be further tested in the control of two major G. hirsutum insect pests. Bioassays with T1 plants revealed the Cry1Ia12 protein toxicity on Spodoptera frugiperda larvae, as evidenced by mortality up to 40% and a significant delay in the development of the target insects compared to untransformed controls (up to 30-fold). Also, an important reduction of Anthonomus grandis emerging adults (up to 60%) was observed when the insect larvae were fed on T1 floral buds. All the larvae and adult insect survivors on the transgenic lines were weaker and significantly smaller compared to the non-transformed plants. Therefore, this study provides GM cotton plant with simultaneous resistance against the Lepidopteran (S. frugiperda), and the Coleopteran (A. grandis) insect orders, and all data suggested that the Cry1Ia12 toxin could effectively enhance the cotton transgenic plants resistance to both insect pests.

  14. Suppression of resistance to Erysiphe graminis f.sp. hordei conferred by the mlo5 barley powdery mildew resistance gene

    DEFF Research Database (Denmark)

    Lyngkjær, M.F.; Carver, T.L.W.; Zeyen, R.J.

    1997-01-01

    . Additional suppression of mlo5 penetration resistance against the avirulent E. graminis isolate was achieved by using DDG, mannose, or glucose in combination with the phenylalanine ammonia lyase inhibitor alpha-aminooxy-beta-phenylpropionic acid (AOPP). A mlo virulent isolate of E. graminis was also tested...

  15. The N550K/H Mutations in FGFR2 Confer Differential Resistance to PD173074, Dovitinib, and Ponatinib ATP-Competitive Inhibitors

    Directory of Open Access Journals (Sweden)

    Sara A Byron

    2013-08-01

    Full Text Available We sought to identify fibroblast growth factor receptor 2 (FGFR2 kinase domain mutations that confer resistance to the pan-FGFR inhibitor, dovitinib, and explore the mechanism of action of the drug-resistant mutations. We cultured BaF3 cells overexpressing FGFR2 in high concentrations of dovitinib and identified 14 dovitinib-resistant mutations, including the N550K mutation observed in 25% of FGFR2mutant endometrial cancers (ECs. Structural and biochemical in vitro kinase analyses, together with BaF3 proliferation assays, showed that the resistance mutations elevate the intrinsic kinase activity of FGFR2. BaF3 lines were used to assess the ability of each mutation to confer cross-resistance to PD173074 and ponatinib. Unlike PD173074, ponatinib effectively inhibited all the dovitinib-resistant FGFR2 mutants except the V565I gatekeeper mutation, suggesting ponatinib but not dovitinib targets the active conformation of FGFR2 kinase. EC cell lines expressing wild-type FGFR2 were relatively resistant to all inhibitors, whereas EC cell lines expressing mutated FGFR2 showed differential sensitivity. Within the FGFR2mutant cell lines, three of seven showed marked resistance to PD173074 and relative resistance to dovitinib and ponatinib. This suggests that alternative mechanisms distinct from kinase domain mutations are responsible for intrinsic resistance in these three EC lines. Finally, overexpression of FGFR2N550K in JHUEM-2 cells (FGFR2C383R conferred resistance (about five-fold to PD173074, providing independent data that FGFR2N550K can be associated with drug resistance. Biochemical in vitro kinase analyses also show that ponatinib is more effective than dovitinib at inhibiting FGFR2N550K. We propose that tumors harboring mutationally activated FGFRs should be treated with FGFR inhibitors that specifically bind the active kinase.

  16. Effect of multidrug resistance-conferring mutations on the fitness and virulence of Pseudomonas aeruginosa.

    Science.gov (United States)

    Abdelraouf, Kamilia; Kabbara, Samer; Ledesma, Kimberly R; Poole, Keith; Tam, Vincent H

    2011-06-01

    Multidrug resistance has become a quandary in the treatment of bacterial infections. The effect of resistance mutations and the fitness cost on the pathogenicity of Pseudomonas aeruginosa is not well established. The objective of this study was to examine the impact of multidrug resistance on the fitness and virulence of P. aeruginosa. Fourteen P. aeruginosa strains with various resistance mechanisms were used. In vitro growth of these isolates was investigated in full-strength and 0.25-strength Mueller-Hinton broth (MHB). Exponential growth rates were estimated from serial bacterial burden over 24 h. In vitro growth of two multidrug-resistant strains (PAO1ΔmexRΔoprD and PA9019) was studied when each was grown in co-culture with wild-type strain PAO1. In vivo growth was compared between PAO1 and PAO1ΔmexRΔopD using a murine pneumonia model; virulence over 10 days was studied in six isolates. Significant reduction in growth rate was observed in selected mutants (P resistance mutations were associated with fitness cost in P. aeruginosa, and accumulation of such mutations was associated with a reduction in virulence. However, it was difficult to predict the impact in clinical isolates. Knowledge of multidrug resistance mechanisms and compensatory mutations would likely be helpful.

  17. Stacking of antimicrobial genes in potato transgenic plants confers increased resistance to bacterial and fungal pathogens.

    Science.gov (United States)

    Rivero, Mercedes; Furman, Nicolás; Mencacci, Nicolás; Picca, Pablo; Toum, Laila; Lentz, Ezequiel; Bravo-Almonacid, Fernando; Mentaberry, Alejandro

    2012-01-20

    Solanum tuberosum plants were transformed with three genetic constructions expressing the Nicotiana tabacum AP24 osmotine, Phyllomedusa sauvagii dermaseptin and Gallus gallus lysozyme, and with a double-transgene construction expressing the AP24 and lysozyme sequences. Re-transformation of dermaseptin-transformed plants with the AP24/lysozyme construction allowed selection of plants simultaneously expressing the three transgenes. Potato lines expressing individual transgenes or double- and triple-transgene combinations were assayed for resistance to Erwinia carotovora using whole-plant and tuber infection assays. Resistance levels for both infection tests compared consistently for most potato lines and allowed selection of highly resistant phenotypes. Higher resistance levels were found in lines carrying the dermaseptin and lysozyme sequences, indicating that theses proteins are the major contributors to antibacterial activity. Similar results were obtained in tuber infection tests conducted with Streptomyces scabies. Plant lines showing the higher resistance to bacterial infections were challenged with Phytophthora infestans, Rhizoctonia solani and Fusarium solani. Considerable levels of resistance to each of these pathogens were evidenced employing semi-quantitative tests based in detached-leaf inoculation, fungal growth inhibition and in vitro plant inoculation. On the basis of these results, we propose that stacking of these transgenes is a promising approach to achieve resistance to both bacterial and fungal pathogens. Copyright © 2011 Elsevier B.V. All rights reserved.

  18. Non-recessive Bt toxin resistance conferred by an intracellular cadherin mutation in field-selected populations of cotton bollworm.

    Directory of Open Access Journals (Sweden)

    Haonan Zhang

    Full Text Available Transgenic crops producing Bacillus thuringiensis (Bt toxins have been planted widely to control insect pests, yet evolution of resistance by the pests can reduce the benefits of this approach. Recessive mutations in the extracellular domain of toxin-binding cadherin proteins that confer resistance to Bt toxin Cry1Ac by disrupting toxin binding have been reported previously in three major lepidopteran pests, including the cotton bollworm, Helicoverpa armigera. Here we report a novel allele from cotton bollworm with a deletion in the intracellular domain of cadherin that is genetically linked with non-recessive resistance to Cry1Ac. We discovered this allele in each of three field-selected populations we screened from northern China where Bt cotton producing Cry1Ac has been grown intensively. We expressed four types of cadherin alleles in heterologous cell cultures: susceptible, resistant with the intracellular domain mutation, and two complementary chimeric alleles with and without the mutation. Cells transfected with each of the four cadherin alleles bound Cry1Ac and were killed by Cry1Ac. However, relative to cells transfected with either the susceptible allele or the chimeric allele lacking the intracellular domain mutation, cells transfected with the resistant allele or the chimeric allele containing the intracellular domain mutation were less susceptible to Cry1Ac. These results suggest that the intracellular domain of cadherin is involved in post-binding events that affect toxicity of Cry1Ac. This evidence is consistent with the vital role of the intracellular region of cadherin proposed by the cell signaling model of the mode of action of Bt toxins. Considered together with previously reported data, the results suggest that both pore formation and cell signaling pathways contribute to the efficacy of Bt toxins.

  19. Carbonic anhydrase II confers resistance to deltamethrin in Culex pipiens pallens.

    Science.gov (United States)

    Ye, Wenyun; Zhang, Cheng; Xu, Na; Sun, Yan; Ma, Lei; Shen, Bo; Zhou, Dan; Zhu, Changliang

    2017-12-01

    Carbonic anhydrases (CAs) are metabolic enzymes that regulate the physiological equilibrium in a variety of organisms. In this study, we investigated the function of CA II in Culex pipiens pallens using real-time quantitative polymerase chain reaction and double-stranded RNA injection. CA II transcripts were more abundant in the deltamethrin-susceptible strain than the deltamethrin-resistant strain. The activities of metabolic enzymes increased when the CA II expression was silenced. These findings suggest CA II regulates deltamethrin resistance by altering metabolic enzyme activity, and could serve as a potential genetic marker for monitoring deltamethrin resistance in mosquitoes. © 2017 Wiley Periodicals, Inc.

  20. EmtA, a rRNA methyltransferase conferring high-level evernimicin resistance

    DEFF Research Database (Denmark)

    Mann, P. A.; Xiong, L.; Mankin, A. S.

    2001-01-01

    Enterococcus faecium strain 9631355 was isolated from animal sources on the basis of its resistance to the growth promotant avilamycin. The strain also exhibited high-level resistance to evernimicin, a drug undergoing evaluation as a therapeutic agent in humans. Ribosomes from strain 9631355...... exhibited a dramatic reduction in evernimicin binding, shown by both cell-free translation assays and direct-binding assays. The resistance determinant was cloned from strain 9631355; sequence alignments suggested it was a methyltransferase and therefore it was designated emtA for evernimicin...

  1. Cloning of disease-resistance homologues in end sequences of BAC clones linked to Fom-2, a gene conferring resistance to Fusarium wilt in melon (Cucumis melo L.).

    Science.gov (United States)

    Wang, Yi-Hong; Choi, Woobong; Thomas, Claude E; Dean, Ralph A

    2002-06-01

    Disease resistance has not yet been characterized at the molecular level in cucurbits, a group of high-value, nutritious, horticultural plants. Previously, we genetically mapped the Fom-2 gene that confers resistance to Fusarium wilt races 0 and I of melon. In this paper, two cosegregating codominant markers (AM, AFLP marker; FM, Fusarium marker) were used to screen a melon bacterial artificial chromosome (BAC) library. Identified clones were fingerprinted and end sequenced. Fingerprinting analysis showed that clones identified by each marker assembled into two separate contigs at high stringency. GenBank searches produced matches to leucine-rich repeats (LRRs) of resistance genes (R genes); to retroelements and to cellulose synthase in clones identified by FM; and to nucleotide-binding sites (NBSs) of R genes, retroelements, and cytochrome P-450 in clones identified by AM. A 6.5-kb fragment containing both NBS and LRR sequences was found to share high homology to TIR (Toll-interleukin-1 receptor)-NBS-LRR R genes, such as N, with 42% identity and 58% similarity in the TIR-NBS and LRR regions. The sequence information may be useful for identifying NBS-LRR class of R genes in other cucurbits.

  2. Microbiological/clinical characteristics and validation of topical therapy with kanamycin in aerobic vaginitis: a pilot study.

    Science.gov (United States)

    Tempera, G; Bonfiglio, G; Cammarata, E; Corsello, S; Cianci, A

    2004-07-01

    The term 'aerobic vaginitis' defines a 'new' vaginal pathology that is neither classifiable as specific vaginitis nor as bacterial vaginosis. We studied a sample of 30 women with a clinical and microbiological diagnosis of aerobic vaginitis and compared the efficacy and tolerability of kanamycin and meclocycline, two products commercially available in Italy in the form of vaginal pessaries. In chronological order of enrollment, the patients were alternately treated with kanamycin or meclocycline; the dose of administration in both groups was of one pessary per day for 6 days. The evaluation of the therapeutic efficacy was carried out both at the first check-up (7th-8th day) and at a second check-up (13th-16th day). At the first follow-up carried out immediately at the end of therapy, the percentage of normalisation of clinical signs and symptoms was increased independently of the type of treatment in the case of moderate grade aerobic vaginitis, while kanamycin was produced a better effect in the group with severe aerobic vaginitis. Furthermore, at the second follow-up, a direct correlation with recovery of vaginal homeostasis was demonstrated by the normalisation of the vaginal pH and by the presence of lactobacilli, only in kanamycin treated group. In conclusion, our results showed the validity of the treatment with kanamycin intravaginally in this recently recognised disease.

  3. Phosphoprotein enriched in diabetes (PED/PEA15) promotes migration in hepatocellular carcinoma and confers resistance to sorafenib.

    Science.gov (United States)

    Quintavalle, Cristina; Hindupur, Sravanth Kumar; Quagliata, Luca; Pallante, Pierlorenzo; Nigro, Cecilia; Condorelli, Gerolama; Andersen, Jesper Bøje; Tagscherer, Katrin Elisabeth; Roth, Wilfried; Beguinot, Francesco; Heim, Markus Hermann; Ng, Charlotte Kiu Yan; Piscuoglio, Salvatore; Matter, Matthias Sebastian

    2017-10-26

    Hepatocellular carcinoma (HCC) is the third-leading cause of cancer-related death with limited treatment options and frequent resistance to sorafenib, the only drug currently approved for first-line therapy. Therefore, better understanding of HCC tumor biology and its resistance to treatment is urgently needed. Here, we analyzed the role of phosphoprotein enriched in diabetes (PED) in HCC. PED has been shown to regulate cell proliferation, apoptosis and migration in several types of cancer. However, its function in HCC has not been addressed yet. Our study revealed that both transcript and protein levels of PED were significantly high in HCC compared with non-tumoral tissue. Clinico-pathological correlation revealed that PED high HCCs showed an enrichment of gene signatures associated with metastasis and poor prognosis. Further, we observed that PED overexpression elevated the migration potential and PED silencing the decreased migration potential in liver cancer cell lines without effecting cell proliferation. Interestingly, we found that PED expression was regulated by a hepatocyte specific nuclear factor, HNF4α. A reduction of HNF4α induced an increase in PED expression and consequently, promoted cell migration in vitro. Finally, PED reduced the antitumoral effect of sorafenib by inhibiting caspase-3/7 activity. In conclusion, our data suggest that PED has a prominent role in HCC biology. It acts particularly on promoting cell migration and confers resistance to sorafenib treatment. PED may be a novel target for HCC therapy and serve as a predictive marker for treatment response against sorafenib.

  4. SU-C-303-01: Activation-Induced Cytidine Deaminase Confers Cancer Resistance to Radiation Therapy

    International Nuclear Information System (INIS)

    Yi, S; La Count, S; Liu, J; Bai, X; Lu, L

    2015-01-01

    Purpose: To study the role of activation-induced cytidine deaminase (AID) in malignant cell resistance to radiation therapy. Methods: We first developed several small devices that could be used to adopt radiation beams from clinical high dose rate brachy therapy (HDR) or linac-based megavoltage machines to perform pre-clinical cell and mouse experiments. Then we used these devices to deliver radiation to AID-positive and AID-silenced cancer cells or tumors formed by these cells in mice. Cells and mice bearing tumors received the same dose under the same experimental conditions. For cells, we observed the apoptosis and the cell survival rate over time. For mice bearing tumors, we measured and recorded the tumor sizes every other day for 4 weeks. Results: For cell experiments, we found that the AID-positive cells underwent much less apoptosis compared with AID-silenced cells upon radiation. And for mouse experiments, we found that AID-positive tumors grew significantly faster than the AID-silenced tumors despite of receiving the same doses of radiation. Conclusion: Our study suggests that AID may confer cancer resistance to radiation therapy, and AID may be a significant biomarker predicting cancer resistance to radiation therapy for certain cancer types

  5. Metagenomic Analysis of Apple Orchard Soil Reveals Antibiotic Resistance Genes Encoding Predicted Bifunctional Proteins▿

    Science.gov (United States)

    Donato, Justin J.; Moe, Luke A.; Converse, Brandon J.; Smart, Keith D.; Berklein, Flora C.; McManus, Patricia S.; Handelsman, Jo

    2010-01-01

    To gain insight into the diversity and origins of antibiotic resistance genes, we identified resistance genes in the soil in an apple orchard using functional metagenomics, which involves inserting large fragments of foreign DNA into Escherichia coli and assaying the resulting clones for expressed functions. Among 13 antibiotic-resistant clones, we found two genes that encode bifunctional proteins. One predicted bifunctional protein confers resistance to ceftazidime and contains a natural fusion between a predicted transcriptional regulator and a β-lactamase. Sequence analysis of the entire metagenomic clone encoding the predicted bifunctional β-lactamase revealed a gene potentially involved in chloramphenicol resistance as well as a predicted transposase. A second clone that encodes a predicted bifunctional protein confers resistance to kanamycin and contains an aminoglycoside acetyltransferase domain fused to a second acetyltransferase domain that, based on nucleotide sequence, was predicted not to be involved in antibiotic resistance. This is the first report of a transcriptional regulator fused to a β-lactamase and of an aminoglycoside acetyltransferase fused to an acetyltransferase not involved in antibiotic resistance. PMID:20453147

  6. Bacteriophage-resistant Staphylococcus aureus mutant confers broad immunity against staphylococcal infection in mice.

    Directory of Open Access Journals (Sweden)

    Rosanna Capparelli

    Full Text Available In the presence of a bacteriophage (a bacteria-attacking virus resistance is clearly beneficial to the bacteria. As expected in such conditions, resistant bacteria emerge rapidly. However, in the absence of the phage, resistant bacteria often display reduced fitness, compared to their sensitive counterparts. The present study explored the fitness cost associated with phage-resistance as an opportunity to isolate an attenuated strain of S. aureus. The phage-resistant strain A172 was isolated from the phage-sensitive strain A170 in the presence of the M(Sa phage. Acquisition of phage-resistance altered several properties of A172, causing reduced growth rate, under-expression of numerous genes and production of capsular polysaccharide. In vivo, A172 modulated the transcription of the TNF-alpha, IFN-gamma and Il-1beta genes and, given intramuscularly, protected mice from a lethal dose of A170 (18/20. The heat-killed vaccine also afforded protection from heterologous methicillin-resistant S. aureus (MRSA (8/10 mice or vancomycin-intermediate S. aureus (VISA (9/10 mice. The same vaccine was also effective when administered as an aerosol. Anti-A172 mouse antibodies, in the dose of 10 microl/mouse, protected the animals (10/10, in two independent experiments from a lethal dose of A170. Consisting predominantly of the sugars glucose and galactose, the capsular polysaccharide of A172, given in the dose of 25 microg/mouse, also protected the mice (20/20 from a lethal dose of A170. The above results demonstrate that selection for phage-resistance can facilitate bacterial vaccine preparation.

  7. IncA/C plasmids conferring high azithromycin resistance in vibrio cholerae.

    Science.gov (United States)

    Wang, Ruibai; Liu, Haican; Zhao, Xiuqin; Li, Jie; Wan, Kanglin

    2018-01-01

    Azithromycin (AZM) is a clinically important antibiotic against Vibrio cholerae, especially for inhibiting V. cholerae colonisation of the intestine and for the treatment of severe cholera in children and pregnant women. An IncA/C plasmid was isolated from two high minimum inhibitory concentration (MIC) AZM-resistant V. cholerae strains of the two mainly pathogenic serogroups (O1 and O139) isolated in China. In the 172 predicted open reading frames (ORFs), 16 genes were related to antibiotic resistance, of which 5 were well-defined genes associated with macrolide resistance. The five macrolide resistance genes distributed in two clusters, mphR-mrx-mph(K) and mel-mph2, flanked by insertion sequence elements and involving two kinds of resistance mechanism. Deletion of the complete region of the two clusters deceased the AZM MIC from ≥64 µg/mL to ≤0.5 µg/mL. This IncA/C plasmid shows great ability to accumulate antibiotic resistance genes. In addition to 11 resistance genes to other antibiotics, 5 macrolide resistance genes with different function were gathered repeatedly through transposition on one plasmid. This genotype could not be simply explained by antibiotic stress applied on the host from the environment or treatment. These phosphorylases and transmembrane transporters might be involved in the transport and metabolism of other non-antibiotic substances, enabling this kind of plasmid to propagate better in the host. Copyright © 2017 Elsevier B.V. and International Society of Chemotherapy. All rights reserved.

  8. Hairpin RNA Targeting Multiple Viral Genes Confers Strong Resistance to Rice Black-Streaked Dwarf Virus

    Directory of Open Access Journals (Sweden)

    Fangquan Wang

    2016-05-01

    Full Text Available Rice black-streaked dwarf virus (RBSDV belongs to the genus Fijivirus in the family of Reoviridae and causes severe yield loss in rice-producing areas in Asia. RNA silencing, as a natural defence mechanism against plant viruses, has been successfully exploited for engineering virus resistance in plants, including rice. In this study, we generated transgenic rice lines harbouring a hairpin RNA (hpRNA construct targeting four RBSDV genes, S1, S2, S6 and S10, encoding the RNA-dependent RNA polymerase, the putative core protein, the RNA silencing suppressor and the outer capsid protein, respectively. Both field nursery and artificial inoculation assays of three generations of the transgenic lines showed that they had strong resistance to RBSDV infection. The RBSDV resistance in the segregating transgenic populations correlated perfectly with the presence of the hpRNA transgene. Furthermore, the hpRNA transgene was expressed in the highly resistant transgenic lines, giving rise to abundant levels of 21–24 nt small interfering RNA (siRNA. By small RNA deep sequencing, the RBSDV-resistant transgenic lines detected siRNAs from all four viral gene sequences in the hpRNA transgene, indicating that the whole chimeric fusion sequence can be efficiently processed by Dicer into siRNAs. Taken together, our results suggest that long hpRNA targeting multiple viral genes can be used to generate stable and durable virus resistance in rice, as well as other plant species.

  9. Cypermethrin resistance conferred by increased target insensitivity and metabolic detoxification in Culex pipiens pallens Coq.

    Science.gov (United States)

    Xu, Wenping; Liu, Songlin; Zhang, Yang; Gao, Jufang; Yang, Mingjun; Liu, Xiao; Tao, Liming

    2017-10-01

    In order to elucidate the molecular mechanisms of cypermethrin resistance in Culex pipiens pallens Coq, the susceptible strain (SS strain) and cypermethrin resistant strain (CR strain) of Cx. p. pallens were investigated in this paper. The cypermethrin resistance ratio of CR strain to SS strain was measured by biological assays method, the cDNA sequence of sodium channel was cloned and analyzed. Real-time quantitative RT-PCR was used to detect the expression levels of the detoxification-related genes across between CR strain and SS strain of Cx. p. pallens. Bioassays indicated that CR strain was 283.06 and 80.68-fold resistance to cypermethrin and permethrin as compared to the susceptible strain, respectively. The sequence variability analysis of sodium channel gene between SS strain and CR strain shows that 4 point mutations (R954Q, L1023F, S1775G and A1989E) appear on the amino acid sequence of sodium channel of CR strain. The transcriptional levels of CYP6Z10, CYP9M10, CPGSTd1 and CPGSTd2 in the resistant strain are significantly higher than it is in the susceptible. The transcripts of CYP4H34 and E4 esterase have no significant difference between the CR strain and SS strain. The results indicated that sodium channel mutations, combined with elevated levels of P450s and GSTs, are associated with cypermethrin resistance in CR strain. Copyright © 2017 Elsevier B.V. All rights reserved.

  10. The potyviral suppressor of RNA silencing confers enhanced resistance to multiple pathogens

    International Nuclear Information System (INIS)

    Pruss, Gail J.; Lawrence, Christopher B.; Bass, Troy; Li Qingshun Q.; Bowman, Lewis H.; Vance, Vicki

    2004-01-01

    Helper component-protease (HC-Pro) is a plant viral suppressor of RNA silencing, and transgenic tobacco expressing HC-Pro has increased susceptibility to a broad range of viral pathogens. Here we report that these plants also exhibit enhanced resistance to unrelated heterologous pathogens. Tobacco mosaic virus (TMV) infection of HC-Pro-expressing plants carrying the N resistance gene results in fewer and smaller lesions compared to controls without HC-Pro. The resistance to TMV is compromised but not eliminated by expression of nahG, which prevents accumulation of salicylic acid (SA), an important defense signaling molecule. HC-Pro-expressing plants are also more resistant to tomato black ring nepovirus (TBRV) and to the oomycete Peronospora tabacina. Enhanced TBRV resistance is SA-independent, whereas the response to P. tabacina is associated with early induction of markers characteristic of SA-dependent defense. Thus, a plant viral suppressor of RNA silencing enhances resistance to multiple pathogens via both SA-dependent and SA-independent mechanisms

  11. Identification of 8-methyladenosine as the modification catalyzed by the radical SAM methyltransferase Cfr that confers antibiotic resistance in bacteria

    DEFF Research Database (Denmark)

    Giessing, Anders; Jensen, Søren Skov; Rasmussen, Anette

    2009-01-01

    with those of a chemically synthesized nucleoside standard shows that Cfr catalyzes formation of 8-methyladenosine. In addition, analysis of RNA derived from E. coli strains lacking the m(2)A2503 methyltransferase reveals that Cfr also has the ability to catalyze methylation at position 2 to form 2......The Cfr methyltransferase confers combined resistance to five different classes of antibiotics that bind to the peptidyl transferase center of bacterial ribosomes. The Cfr-mediated modification has previously been shown to occur on nucleotide A2503 of 23S rRNA and has a mass corresponding...... to an additional methyl group, but its specific identity and position remained to be elucidated. A novel tandem mass spectrometry approach has been developed to further characterize the Cfr-catalyzed modification. Comparison of nucleoside fragmentation patterns of A2503 from Escherichia coli cfr+ and cfr- strains...

  12. Identification of QTLs Conferring Resistance to Deltamethrin in Culex pipiens pallens.

    Science.gov (United States)

    Zou, Feifei; Chen, Chen; Zhong, Daibin; Shen, Bo; Zhang, Donghui; Guo, Qin; Wang, Weijie; Yu, Jing; Lv, Yuan; Lei, Zhentao; Ma, Kai; Ma, Lei; Zhu, Changliang; Yan, Guiyun

    2015-01-01

    Culex pipiens pallens is the most abundant Culex mosquito species in northern China and is an important vector of bancroftian filariasis and West Nile virus. Deltamethrin is an insecticide that is widely used for mosquito control, however resistance to this and other insecticides has become a major challenge in the control of vector-borne diseases that appear to be inherited quantitatively. Furthermore, the genetic basis of insecticide resistance remains poorly understood. In this study, quantitative trait loci (QTL) mapping of resistance to deltamethrin was conducted in F2 intercross segregation populations using bulked segregation analysis (BSA) and amplified fragment length polymorphism markers (AFLP) in Culex pipiens pallens. A genetic linkage map covering 381 cM was constructed and a total of seven QTL responsible for resistance to deltamethrin were detected by composite interval mapping (CIM), which explained 95% of the phenotypic variance. The major QTL in linkage group 2 accounted for 62% of the variance and is worthy of further study. 12 AFLP markers in the map were cloned and the genomic locations of these marker sequences were determined by applying the Basic Local Alignment Search Tool (BLAST) tool to the genome sequence of the closely related Culex quinquefasciatus. Our results suggest that resistance to deltamethrin is a quantitative trait under the control of a major QTL in Culex pipiens pallens. Cloning of related AFLP markers confirm the potential utility for anchoring the genetic map to the physical map. The results provide insight into the genetic architecture of the trait.

  13. Identification of QTLs Conferring Resistance to Deltamethrin in Culex pipiens pallens.

    Directory of Open Access Journals (Sweden)

    Feifei Zou

    Full Text Available Culex pipiens pallens is the most abundant Culex mosquito species in northern China and is an important vector of bancroftian filariasis and West Nile virus. Deltamethrin is an insecticide that is widely used for mosquito control, however resistance to this and other insecticides has become a major challenge in the control of vector-borne diseases that appear to be inherited quantitatively. Furthermore, the genetic basis of insecticide resistance remains poorly understood. In this study, quantitative trait loci (QTL mapping of resistance to deltamethrin was conducted in F2 intercross segregation populations using bulked segregation analysis (BSA and amplified fragment length polymorphism markers (AFLP in Culex pipiens pallens. A genetic linkage map covering 381 cM was constructed and a total of seven QTL responsible for resistance to deltamethrin were detected by composite interval mapping (CIM, which explained 95% of the phenotypic variance. The major QTL in linkage group 2 accounted for 62% of the variance and is worthy of further study. 12 AFLP markers in the map were cloned and the genomic locations of these marker sequences were determined by applying the Basic Local Alignment Search Tool (BLAST tool to the genome sequence of the closely related Culex quinquefasciatus. Our results suggest that resistance to deltamethrin is a quantitative trait under the control of a major QTL in Culex pipiens pallens. Cloning of related AFLP markers confirm the potential utility for anchoring the genetic map to the physical map. The results provide insight into the genetic architecture of the trait.

  14. Transgenic Rice Plants Harboring Genomic DNA from Zizania latifolia Confer Bacterial Blight Resistance

    Directory of Open Access Journals (Sweden)

    Wei-wei SHEN

    2011-03-01

    Full Text Available Based on the sequence of a resistance gene analog FZ14 derived from Zizania latifolia (Griseb., a pair of specific PCR primers FZ14P1/FZ14P2 was designed to isolate candidate disease resistance gene. The pooled-PCR approach was adopted using the primer pair to screen a genomic transformation-competent artificial chromosome (TAC library derived from Z. latifolia. A positive TAC clone (ZR1 was obtained and confirmed by sequence analysis. The results indicated that ZR1 consisted of conserved motifs similar to P-loop (kinase 1a, kinase 2, kinase 3a and GLPL (Gly-Leu-Pro-Leu, suggesting that it could be a portion of NBS-LRR type of resistance gene. Using Agrobacterium-mediated transformation of Nipponbare mature embryo, a total of 48 independent transgenic T0 plants were obtained. Among them, 36 plants were highly resistant to the virulent bacterial blight strain PXO71. The results indicate that ZR1 contains at least one functional bacterial blight resistance gene.

  15. Modified cellulose synthase gene from 'Arabidopsis thaliana' confers herbicide resistance to plants

    Energy Technology Data Exchange (ETDEWEB)

    Somerville, Chris R.; Scieble, Wolf

    2000-10-11

    Cellulose synthase ('CS'), a key enzyme in the biosynthesis of cellulose in plants is inhibited by herbicides comprising thiazolidinones such as 5-tert-butyl-carbamoyloxy-3-(3-trifluromethyl) phenyl-4-thiazolidinone (TZ), isoxaben and 2,6-dichlorobenzonitrile (DCB). Two mutant genes encoding isoxaben and TZ-resistant cellulose synthase have been isolated from isoxaben and TZ-resistant Arabidopsis thaliana mutants. When compared with the gene coding for isoxaben or TZ-sensitive cellulose synthase, one of the resistant CS genes contains a point mutation, wherein glycine residue 998 is replaced by an aspartic acid. The other resistant mutation is due to a threonine to isoleucine change at amino acid residue 942. The mutant CS gene can be used to impart herbicide resistance to a plant; thereby permitting the utilization of the herbicide as a single application at a concentration which ensures the complete or substantially complete killing of weeds, while leaving the transgenic crop plant essentially undamaged.

  16. Mutations in the RAM network confer resistance to the thiol oxidant 4,4'-dipyridyl disulfide

    DEFF Research Database (Denmark)

    López-Mirabal, H Reynaldo; Winther, Jakob R; Thorsen, Michael

    2008-01-01

    -specific oxidant dipyridyl disulfide (DPS) yielded tao3-516, which is impaired in the function of the RAM signaling network protein Tao3/Pag1p. We suggest that the DPS-resistance of the tao3-516 mutant might be due to deficient cell-cycle-regulated production of the chitinase Cts1p, which functions in post......-mitotic cell separation and depends on Tao3p and the RAM network for regulated expression. Consistent with this, deletion of other RAM genes or CTS1 also resulted in increased resistance to DPS. Exposure to DPS caused extensive depolarization of the actin cytoskeleton. We found that tao3-516 is resistant...... to latrunculin, a specific inhibitor of actin polymerization, and that ram, Deltaace2, and Deltacts1 mutants are resistant to benomyl, a microtubule-destabilizing drug. Since septum build-up depends on the organization of cytoskeletal proteins, the resistance to cytoskeletal stress of Cts1p-deficient mutants...

  17. Candidate gene analysis and identification of TRAP and SSR markers linked to the Or5 gene, which confers sunflower resistance to race E of broomrape (Orobanche cumana Wallr.)

    Science.gov (United States)

    Sunflower broomrape (Orobanche cumana Wallr.) is a root holoparasitic angiosperm considered as being one of the major constraints for sunflower production in Mediterranean areas. Breeding for resistance has been crucial for protecting sunflowers from broomrape damage. The Or5 gene, which confers re...

  18. BNYVV-derived dsRNA confers resistance to rhizomania disease of sugar beet as evidenced by a novel transgenic hairy root approach

    NARCIS (Netherlands)

    Pavli, R.; Panopoulos, N.J.; Goldbach, R.W.; Skaracis, G.N.

    2010-01-01

    Agrobacterium rhizogenes-transformed sugar beet hairy roots, expressing dsRNA from the Beet necrotic yellow vein virus replicase gene, were used as a novel approach to assess the efficacy of three intron-hairpin constructs at conferring resistance to rhizomania disease. Genetically engineered roots

  19. 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,

  20. Obesogenic memory can confer long-term increases in adipose tissue but not liver inflammation and insulin resistance after weight loss

    Directory of Open Access Journals (Sweden)

    J. Schmitz

    2016-05-01

    Conclusions: These results demonstrate that although sustained weight loss improves systemic glucose homeostasis, primarily through improved inflammation and insulin action in liver, a remarkable obesogenic memory can confer long-term increases in adipose tissue inflammation and insulin resistance in mice as well as in a significant subpopulation of obese patients.

  1. Distinction between the Cfr Methyltransferase Conferring Antibiotic Resistance and the Housekeeping RlmN Methyltransferase

    DEFF Research Database (Denmark)

    Atkinson, Gemma C; Hansen, Lykke H; Tenson, Tanel

    2013-01-01

    The cfr gene encodes the Cfr methyltransferase that primarily methylates C-8 in A2503 of 23S rRNA in the peptidyl transferase region of bacterial ribosomes. The methylation provides resistance to six classes of antibiotics of clinical and veterinary importance. The rlmN gene encodes the Rlm......N methyltransferase that methylates C-2 in A2503 in 23S rRNA and A37 in tRNA, but RlmN does not significantly influence antibiotic resistance. The enzymes are homologous and use the same mechanism involving radical S-adenosyl methionine to methylate RNA via an intermediate involving a methylated cysteine....... The differentiation between the two classes is supported by previous and new experimental evidence from antibiotic resistance, primer extensions, and mass spectrometry. Finally, evolutionary aspects of the distribution of Cfr- and RlmN-like enzymes are discussed....

  2. Structural analysis of base substitutions in Thermus thermophilus 16S rRNA conferring streptomycin resistance.

    Science.gov (United States)

    Demirci, Hasan; Murphy, Frank V; Murphy, Eileen L; Connetti, Jacqueline L; Dahlberg, Albert E; Jogl, Gerwald; Gregory, Steven T

    2014-08-01

    Streptomycin is a bactericidal antibiotic that induces translational errors. It binds to the 30S ribosomal subunit, interacting with ribosomal protein S12 and with 16S rRNA through contacts with the phosphodiester backbone. To explore the structural basis for streptomycin resistance, we determined the X-ray crystal structures of 30S ribosomal subunits from six streptomycin-resistant mutants of Thermus thermophilus both in the apo form and in complex with streptomycin. Base substitutions at highly conserved residues in the central pseudoknot of 16S rRNA produce novel hydrogen-bonding and base-stacking interactions. These rearrangements in secondary structure produce only minor adjustments in the three-dimensional fold of the pseudoknot. These results illustrate how antibiotic resistance can occur as a result of small changes in binding site conformation. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  3. 2nd International Conference on Historic Earthquake-Resistant Timber Frames in the Mediterranean Area

    CERN Document Server

    Machado, José; Costa, Alfredo; Candeias, Paulo; Ruggieri, Nicola; Catarino, José

    2016-01-01

    This book presents a selection of the best papers from the HEaRT 2015 conference, held in Lisbon, Portugal, which provided a valuable forum for engineers and architects, researchers and educators to exchange views and findings concerning the technological history, construction features and seismic behavior of historical timber-framed walls in the Mediterranean countries. The topics covered are wide ranging and include historical aspects and examples of the use of timber-framed construction systems in response to earthquakes, such as the gaiola system in Portugal and the Bourbon system in southern Italy; interpretation of the response of timber-framed walls to seismic actions based on calculations and experimental tests; assessment of the effectiveness of repair and strengthening techniques, e.g., using aramid fiber wires or sheets; and modelling analyses. In addition, on the basis of case studies, a methodology is presented that is applicable to diagnosis, strengthening and improvement of seismic performance ...

  4. Transgenic expression of the Aedes aegypti CYP9J28 confers pyrethroid resistance in Drosophila melanogaster

    NARCIS (Netherlands)

    Pavlidi, N.; Monastirioti, M.; Daborn, P.; Van Leeuwen, T.; Vontas, J.

    2012-01-01

    The emergence and spread of insecticide resistance in mosquitoes, such as the major vector of dengue and yellow fever Aedes aegypti, is a major public health problem. A number of studies have been conducted to-date aiming to identify specific molecular changes that are associated with the phenotype,

  5. A genetic marker allele conferring resistance to Ascaris suum in pigs

    DEFF Research Database (Denmark)

    Skallerup, Per; Thamsborg, Stig M.; Jørgensen, Claus B.

    2013-01-01

    Mapping of quantitative trait loci (QTLs) has helped dissecting the genetics underlying the variation in resistance to helminth infections. In pigs, two single nucleotide polymorphisms (SNP TXNIP and SNP ARNT), both on chromosome 4, have been reported to be associated with Ascaris suum worm burden...

  6. A truncating mutation of HDAC2 in human cancers confers resistance to histone deacetylase inhibition

    DEFF Research Database (Denmark)

    Ropero, S; Fraga, MF; Ballestar, E

    2006-01-01

    arising in individuals with hereditary nonpolyposis colorectal cancer syndrome. The presence of the HDAC2 frameshift mutation causes a loss of HDAC2 protein expression and enzymatic activity and renders these cells more resistant to the usual antiproliferative and proapoptotic effects of histone...

  7. Ectopic accumulation of linalool confers resistance to Xanthomonas citri subsp. citri in transgenic sweet orange plants.

    Science.gov (United States)

    Shimada, Takehiko; Endo, Tomoko; Rodríguez, Ana; Fujii, Hiroshi; Goto, Shingo; Matsuura, Takakazu; Hojo, Yuko; Ikeda, Yoko; Mori, Izumi C; Fujikawa, Takashi; Peña, Leandro; Omura, Mitsuo

    2017-05-01

    In order to clarify whether high linalool content in citrus leaves alone induces strong field resistance to citrus canker caused by Xanthomonas citri subsp. citri (Xcc), and to assess whether this trait can be transferred to a citrus type highly sensitive to the bacterium, transgenic 'Hamlin' sweet orange (Citrus sinensis L. Osbeck) plants over-expressing a linalool synthase gene (CuSTS3-1) were generated. Transgenic lines (LIL) with the highest linalool content showed strong resistance to citrus canker when spray inoculated with the bacterium. In LIL plants inoculated by wounding (multiple-needle inoculation), the linalool level was correlated with the repression of the bacterial titer and up-regulation of defense-related genes. The exogenous application of salicylic acid, methyl jasmonate or linalool triggered responses similar to those constitutively induced in LIL plants. The linalool content in Ponkan mandarin leaves was significantly higher than that of leaves from six other representative citrus genotypes with different susceptibilities to Xcc. We propose that linalool-mediated resistance might be unique to citrus tissues accumulating large amounts of volatile organic compounds in oil cells. Linalool might act not only as a direct antibacterial agent, but also as a signal molecule involved in triggering a non-host resistance response against Xcc. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  8. Label-free longitudinal monitoring of melanogenesis in the evolution of melanoma treatment resistance (Conference Presentation)

    Science.gov (United States)

    Osseiran, Sam; Wang, Hequn; Dutton-Regester, Ken; Garraway, Levi A.; Evans, Conor L.

    2017-02-01

    While melanoma is not the most common form of skin cancer, it represents the vast majority of skin cancer-related deaths. Indeed, while combination therapies such as Dabrafenib and Trametinib have shown great promise in clinical trials for treating metastatic disease, some melanoma subtypes nevertheless develop resistances to front-line treatments. Under in vitro conditions, some metastatic human melanoma cell lines have been observed to evolve resistance to treatment while simultaneously changing color under brightfield microscopy, hinting at perturbations in pigment synthesis. The process known as melanogenesis gives rise to the two forms of melanin found in mammals: eumelanin, a dark brown/black pigment, and pheomelanin, a much more pale red/blond pigment. Interestingly, pheomelanin has been shown to contribute to the onset and development of melanoma in an ultraviolet-radiation-independent manner through a mechanism of oxidative stress. Eumelanin, on the other hand, is a known antioxidant whose chemical properties seem to shield cells against oxidative damage. To study these pigments in closer detail, nonlinear optical microscopy including coherent anti-Stokes Raman scattering (CARS) was used for the specific visualization and quantification of the relative abundance of pheomelanin and eumelanin within these treatment resistant cell lines. These microscopy toolkits provide a means to monitor changes in pigmentation in a noninvasive and non-destructive manner without the use of exogenous dyes to better understand the molecular basis of treatment resistance.

  9. Identification of QTLs conferring resistance to downy mildew in legacy cultivars of lettuce

    Science.gov (United States)

    Many cultivars of lettuce (Lactuca sativa L.), the most popular leafy vegetable, are susceptible to downy mildew disease caused by Bremia lactucae. Cultivars Iceberg and Grand Rapids that were released in 18th and 19th century, respectively, have high levels of quantitative resistance to downy milde...

  10. Transgenic resistance confers effective field level control of bacterial spot disease in tomato.

    Directory of Open Access Journals (Sweden)

    Diana M Horvath

    Full Text Available We investigated whether lines of transgenic tomato (Solanum lycopersicum expressing the Bs2 resistance gene from pepper, a close relative of tomato, demonstrate improved resistance to bacterial spot disease caused by Xanthomonas species in replicated multi-year field trials under commercial type growing conditions. We report that the presence of the Bs2 gene in the highly susceptible VF 36 background reduced disease to extremely low levels, and VF 36-Bs2 plants displayed the lowest disease severity amongst all tomato varieties tested, including commercial and breeding lines with host resistance. Yields of marketable fruit from transgenic lines were typically 2.5 times that of the non-transformed parent line, but varied between 1.5 and 11.5 fold depending on weather conditions and disease pressure. Trials were conducted without application of any copper-based bactericides, presently in wide use despite negative impacts on the environment. This is the first demonstration of effective field resistance in a transgenic genotype based on a plant R gene and provides an opportunity for control of a devastating pathogen while eliminating ineffective copper pesticides.

  11. Overexpression of a soybean salicylic acid methlyltransferase gene confers resistance to soybean cyst nematode

    Science.gov (United States)

    Soybean cyst nematode (Heterodera glycines Ichinohe, SCN) is the most pervasive pest of soybean [Glycine max (L.) Merr.] in the USA and worldwide. SCN reduced soybean yields worldwide by an estimated billion dollars annually. These losses remained stable with the use of resistant cultivars but over ...

  12. Potentially Deceptive Health Nutrition-Related Advertising Claims: The Role of Inoculation in Conferring Resistance

    Science.gov (United States)

    Mason, Alicia M.; Miller, Claude H.

    2016-01-01

    Objective: This study sought to examine the efficacy of inoculation message treatments to facilitate resistance to health nutrition-related (HNR) commercial food advertising claims. Design: Data were collected across three phases extending across a 5-week period conducted over two semesters at a Midwest US university. A 2 × 3 between-subjects…

  13. Transient elevation of glycolysis confers radio-resistance by facilitating DNA repair in cells

    International Nuclear Information System (INIS)

    Bhatt, Anant Narayan; Chauhan, Ankit; Khanna, Suchit; Rai, Yogesh; Singh, Saurabh; Soni, Ravi; Kalra, Namita; Dwarakanath, Bilikere S

    2015-01-01

    Cancer cells exhibit increased glycolysis for ATP production (the Warburg effect) and macromolecular biosynthesis; it is also linked with therapeutic resistance that is generally associated with compromised respiratory metabolism. Molecular mechanisms underlying radio-resistance linked to elevated glycolysis remain incompletely understood. We stimulated glycolysis using mitochondrial respiratory modifiers (MRMs viz. di-nitro phenol, DNP; Photosan-3, PS3; Methylene blue, MB) in established human cell lines (HEK293, BMG-1 and OCT-1). Glucose utilization and lactate production, levels of glucose transporters and glycolytic enzymes were investigated as indices of glycolysis. Clonogenic survival, DNA repair and cytogenetic damage were studied as parameters of radiation response. MRMs induced the glycolysis by enhancing the levels of two important regulators of glucose metabolism GLUT-1 and HK-II and resulted in 2 fold increase in glucose consumption and lactate production. This increase in glycolysis resulted in resistance against radiation-induced cell death (clonogenic survival) in different cell lines at an absorbed dose of 5 Gy. Inhibition of glucose uptake and glycolysis (using fasentin, 2-deoxy-D-glucose and 3-bromopyruvate) in DNP treated cells failed to increase the clonogenic survival of irradiated cells, suggesting that radio-resistance linked to inhibition of mitochondrial respiration is glycolysis dependent. Elevated glycolysis also facilitated rejoining of radiation-induced DNA strand breaks by activating both non-homologous end joining (NHEJ) and homologous recombination (HR) pathways of DNA double strand break repair leading to a reduction in radiation-induced cytogenetic damage (micronuclei formation) in these cells. These findings suggest that enhanced glycolysis generally observed in cancer cells may be responsible for the radio-resistance, partly by enhancing the repair of DNA damage

  14. Salicylic acid confers enhanced resistance to Glomerella leaf spot in apple.

    Science.gov (United States)

    Zhang, Ying; Shi, Xiangpeng; Li, Baohua; Zhang, Qingming; Liang, Wenxing; Wang, Caixia

    2016-09-01

    Glomerella leaf spot (GLS) caused by Glomerella cingulata is a newly emergent disease that results in severe defoliation and fruit spots in apple. Currently, there are no effective means to control this disease except for the traditional fungicide sprays. Induced resistance by elicitors against pathogens infection is a widely accepted eco-friendly strategy. In the present study, we investigated whether exogenous application of salicylic acid (SA) could improve resistance to GLS in a highly susceptible apple cultivar (Malus domestica Borkh. cv. 'Gala') and the underlying mechanisms. The results showed that pretreatment with SA, at 0.1-1.0 mM, induced strong resistance against GLS in 'Gala' apple leaves, with SA treated leaves showing significant reduction in lesion numbers and disease index. Concurrent with the enhanced disease resistance, SA treatment markedly increased the total antioxidant capacity (T-AOC) and defence-related enzyme activities, including catalase (CAT), superoxide dismutase (SOD), peroxidase (POD), phenylalanine ammonia-lyase (PAL) and polyphenol oxidase (PPO). As expected, SA treatment also induced the expression levels of five pathogenesis-related (PR) genes including PR1, PR5, PR8, Chitinase and β-1,3-glucanase. Furthermore, the most pronounced and/or rapid increase was observed in leaves treated with SA and subsequently inoculated with G. cingulata compared to the treatment with SA or inoculation with the pathogen. Together, these results suggest that exogenous SA triggered increase in reactive oxygen species levels and the antioxidant system might be responsible for enhanced resistance against G. cingulata in 'Gala' apple leaves. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  15. Transient elevation of glycolysis confers radio-resistance by facilitating DNA repair in cells.

    Science.gov (United States)

    Bhatt, Anant Narayan; Chauhan, Ankit; Khanna, Suchit; Rai, Yogesh; Singh, Saurabh; Soni, Ravi; Kalra, Namita; Dwarakanath, Bilikere S

    2015-05-01

    Cancer cells exhibit increased glycolysis for ATP production (the Warburg effect) and macromolecular biosynthesis; it is also linked with therapeutic resistance that is generally associated with compromised respiratory metabolism. Molecular mechanisms underlying radio-resistance linked to elevated glycolysis remain incompletely understood. We stimulated glycolysis using mitochondrial respiratory modifiers (MRMs viz. di-nitro phenol, DNP; Photosan-3, PS3; Methylene blue, MB) in established human cell lines (HEK293, BMG-1 and OCT-1). Glucose utilization and lactate production, levels of glucose transporters and glycolytic enzymes were investigated as indices of glycolysis. Clonogenic survival, DNA repair and cytogenetic damage were studied as parameters of radiation response. MRMs induced the glycolysis by enhancing the levels of two important regulators of glucose metabolism GLUT-1 and HK-II and resulted in 2 fold increase in glucose consumption and lactate production. This increase in glycolysis resulted in resistance against radiation-induced cell death (clonogenic survival) in different cell lines at an absorbed dose of 5 Gy. Inhibition of glucose uptake and glycolysis (using fasentin, 2-deoxy-D-glucose and 3-bromopyruvate) in DNP treated cells failed to increase the clonogenic survival of irradiated cells, suggesting that radio-resistance linked to inhibition of mitochondrial respiration is glycolysis dependent. Elevated glycolysis also facilitated rejoining of radiation-induced DNA strand breaks by activating both non-homologous end joining (NHEJ) and homologous recombination (HR) pathways of DNA double strand break repair leading to a reduction in radiation-induced cytogenetic damage (micronuclei formation) in these cells. These findings suggest that enhanced glycolysis generally observed in cancer cells may be responsible for the radio-resistance, partly by enhancing the repair of DNA damage.

  16. Genetic Variation in Plant CYP51s Confers Resistance against Voriconazole, a Novel Inhibitor of Brassinosteroid-Dependent Sterol Biosynthesis

    Science.gov (United States)

    Rozhon, Wilfried; Husar, Sigrid; Kalaivanan, Florian; Khan, Mamoona; Idlhammer, Markus; Shumilina, Daria; Lange, Theo; Hoffmann, Thomas; Schwab, Wilfried; Fujioka, Shozo; Poppenberger, Brigitte

    2013-01-01

    Brassinosteroids (BRs) are plant steroid hormones with structural similarity to mammalian sex steroids and ecdysteroids from insects. The BRs are synthesized from sterols and are essential regulators of cell division, cell elongation and cell differentiation. In this work we show that voriconazole, an antifungal therapeutic drug used in human and veterinary medicine, severely impairs plant growth by inhibiting sterol-14α-demethylation and thereby interfering with BR production. The plant growth regulatory properties of voriconazole and related triazoles were identified in a screen for compounds with the ability to alter BR homeostasis. Voriconazole suppressed growth of the model plant Arabidopsis thaliana and of a wide range of both monocotyledonous and dicotyledonous plants. We uncover that voriconazole toxicity in plants is a result of a deficiency in BRs that stems from an inhibition of the cytochrome P450 CYP51, which catalyzes a step of BR-dependent sterol biosynthesis. Interestingly, we found that the woodland strawberry Fragaria vesca, a member of the Rosaceae, is naturally voriconazole resistant and that this resistance is conferred by the specific CYP51 variant of F. vesca. The potential of voriconazole as a novel tool for plant research is discussed. PMID:23335967

  17. A novel NADPH-dependent aldehyde reductase gene from Vigna radiata confers resistance to the grapevine fungal toxin eutypine.

    Science.gov (United States)

    Guillén, P; Guis, M; Martínez-Reina, G; Colrat, S; Dalmayrac, S; Deswarte, C; Bouzayen, M; Roustan, J P; Fallot, J; Pech, J C; Latché, A

    1998-11-01

    Eutypine, 4-hydroxy-3-(3-methyl-3-butene-1-ynyl) benzyl aldehyde, is a toxin produced by Eutypa lata, the causal agent of eutypa dieback of grapevines. It has previously been demonstrated that tolerance of some cultivars to this disease was correlated with their capacity to convert eutypine to the corresponding alcohol, eutypinol, which lacks phytotoxicity. We have thus purified to homogeneity a protein from Vigna radiata that exhibited eutypine-reducing activity and have isolated the corresponding cDNA. This encodes an NADPH-dependent reductase of 36 kDa that we have named Vigna radiata eutypine-reducing enzyme (VR-ERE), based on the capacity of a recombinant form of the protein to reduce eutypine into eutypinol. The strongest homologies (86.8%) of VR-ERE at the amino acid level were found with CPRD14, a drought-inducible gene of unknown function, isolated from Vigna unguiculata and with an aromatic alcohol dehydrogenase (71.7%) from Eucalyptus gunnii. Biochemical characterization of VR-ERE revealed that a variety of compounds containing an aldehyde group can act as substrates. However, the highest affinity was observed with 3-substituted benzaldehydes. Expression of a VR-ERE transgene in Vitis vinifera cells cultured in vitro conferred resistance to the toxin. This discovery opens up new biotechnological approaches for the generation of grapevines resistant to eutypa dieback.

  18. An improved method for transformation of lettuce by Agrobacterium tumefaciens with a gene that confers freezing resistance

    Directory of Open Access Journals (Sweden)

    Pileggi Marcos

    2001-01-01

    Full Text Available An efficient method for constructing transgenic lettuce cultivars by Agrobacterium tumefaciens was described by Torres et al., 1993. In the present work, an improvement of the above procedure is described and applied to transform the cultivar Grand Rapids with a mutated P5CS gene. The major modifications were concerned with turning more practical the transformation and regeneration protocols. Also we tried to improve transformation steps by increasing injured area in explants and prolonging co-cultivation with Agrobacteria (in larger concentration. A more significant selective pressure was used against non-transformed plants and bacteria. In these work we were concerned to obtain T1 and T2 seeds. The P5CS gene codes for a delta¹-pyrroline-5-carboxylate synthetase, a bifunctional enzyme that catalyzes two steps of proline biosynthesis in plants (Zhang et al., 1995; Peng et al., 1996, while the mutated gene is insensitive to feedback inhibition by proline. The potential benefit of this gene is to confer water stress resistance (drought, salt, cold due to increased intracellular levels of proline that works like an osmoprotectant. In this work could obtain and characterize transgenic lettuce lineages which are resistant to freezing temperature.

  19. Genetic variation in plant CYP51s confers resistance against voriconazole, a novel inhibitor of brassinosteroid-dependent sterol biosynthesis.

    Science.gov (United States)

    Rozhon, Wilfried; Husar, Sigrid; Kalaivanan, Florian; Khan, Mamoona; Idlhammer, Markus; Shumilina, Daria; Lange, Theo; Hoffmann, Thomas; Schwab, Wilfried; Fujioka, Shozo; Poppenberger, Brigitte

    2013-01-01

    Brassinosteroids (BRs) are plant steroid hormones with structural similarity to mammalian sex steroids and ecdysteroids from insects. The BRs are synthesized from sterols and are essential regulators of cell division, cell elongation and cell differentiation. In this work we show that voriconazole, an antifungal therapeutic drug used in human and veterinary medicine, severely impairs plant growth by inhibiting sterol-14α-demethylation and thereby interfering with BR production. The plant growth regulatory properties of voriconazole and related triazoles were identified in a screen for compounds with the ability to alter BR homeostasis. Voriconazole suppressed growth of the model plant Arabidopsis thaliana and of a wide range of both monocotyledonous and dicotyledonous plants. We uncover that voriconazole toxicity in plants is a result of a deficiency in BRs that stems from an inhibition of the cytochrome P450 CYP51, which catalyzes a step of BR-dependent sterol biosynthesis. Interestingly, we found that the woodland strawberry Fragaria vesca, a member of the Rosaceae, is naturally voriconazole resistant and that this resistance is conferred by the specific CYP51 variant of F. vesca. The potential of voriconazole as a novel tool for plant research is discussed.

  20. Genetic variation in plant CYP51s confers resistance against voriconazole, a novel inhibitor of brassinosteroid-dependent sterol biosynthesis.

    Directory of Open Access Journals (Sweden)

    Wilfried Rozhon

    Full Text Available Brassinosteroids (BRs are plant steroid hormones with structural similarity to mammalian sex steroids and ecdysteroids from insects. The BRs are synthesized from sterols and are essential regulators of cell division, cell elongation and cell differentiation. In this work we show that voriconazole, an antifungal therapeutic drug used in human and veterinary medicine, severely impairs plant growth by inhibiting sterol-14α-demethylation and thereby interfering with BR production. The plant growth regulatory properties of voriconazole and related triazoles were identified in a screen for compounds with the ability to alter BR homeostasis. Voriconazole suppressed growth of the model plant Arabidopsis thaliana and of a wide range of both monocotyledonous and dicotyledonous plants. We uncover that voriconazole toxicity in plants is a result of a deficiency in BRs that stems from an inhibition of the cytochrome P450 CYP51, which catalyzes a step of BR-dependent sterol biosynthesis. Interestingly, we found that the woodland strawberry Fragaria vesca, a member of the Rosaceae, is naturally voriconazole resistant and that this resistance is conferred by the specific CYP51 variant of F. vesca. The potential of voriconazole as a novel tool for plant research is discussed.

  1. Characterisation of a mobilisable plasmid conferring florfenicol and chloramphenicol resistance in Actinobacillus pleuropneumoniae

    Science.gov (United States)

    Bossé, Janine T; Li, Yanwen; Atherton, Tom G; Walker, Stephanie; Williamson, Susanna M; Rogers, Jon; Chaudhuri, Roy R; Weinert, Lucy A; Holden, Matthew TG; Maskell, Duncan J; Tucker, Alexander W; Wren, Brendan W; Rycroft, Andrew N; Langford, Paul R

    2015-01-01

    The complete nucleotide sequence of a 7.7 kb mobilisable plasmid (pM3446F), isolated from a florfenicol resistant isolate of Actinobacillus pleuropneumoniae, showed extended similarity to plasmids found in other members of the Pasteurellaceae containing the floR gene as well as replication and mobilisation genes. Mobilisation into other Pasteurellaceae species confirmed that this plasmid can be transferred horizontally. PMID:26049592

  2. Overexpression of NPR1 in Brassica juncea Confers Broad Spectrum Resistance to Fungal Pathogens

    Directory of Open Access Journals (Sweden)

    Sajad Ali

    2017-10-01

    Full Text Available Brassica juncea (Indian mustard is a commercially important oil seed crop, which is highly affected by many biotic stresses. Among them, Alternaria leaf blight and powdery mildew are the most devastating diseases leading to huge yield losses in B. juncea around the world. In this regard, genetic engineering is a promising tool that may possibly allow us to enhance the B. juncea disease resistance against these pathogens. NPR1 (non-expressor of pathogen-related gene 1 is a bonafide receptor of salicylic acid (SA which modulates multiple immune responses in plants especially activation of induced and systemic acquired resistance (SAR. Here, we report the isolation and characterization of new NPR1 homolog (BjNPR1 from B. juncea. The phylogenetic tree constructed based on the deduced sequence of BjNPR1 with homologs from other species revealed that BjNPR1 grouped together with other known NPR1 proteins of Cruciferae family, and was nearest to B. napus. Furthermore, expression analysis showed that BjNPR1 was upregulated after SA treatment and fungal infection but not by jasmonic acid or abscisic acid. To understand the defensive role of this gene, we generated B. juncea transgenic lines overexpressing BjNPR1, and further confirmed by PCR and Southern blotting. The transgenic lines showed no phenotypic abnormalities, and constitutive expression of BjNPR1 activates defense signaling pathways by priming the expression of antifungal PR genes. Moreover, BjNPR1 transgenic lines showed enhanced resistance to Alternaria brassicae and Erysiphe cruciferarum as there was delay in symptoms and reduced disease severity than non-transgenic plants. In addition, the rate of disease spreading to uninfected or distal parts was also delayed in transgenic plants thus suggesting the activation of SAR. Altogether, the present study suggests that BjNPR1 is involved in broad spectrum of disease resistance against fungal pathogens.

  3. Point mutations in the sodium channel gene conferring tau-fluvalinate resistance in Varroa destructor.

    Science.gov (United States)

    Hubert, Jan; Nesvorna, Marta; Kamler, Martin; Kopecky, Jan; Tyl, Jan; Titera, Dalibor; Stara, Jitka

    2014-06-01

    Sodium channels (SCs) in mites and insects are target sites for pesticides, including pyrethroids. Point mutations in the SC gene have been reported to change the structural conformation of the protein and its sensitivity to pesticides. To find mutations in the SC gene of the mite Varroa destructor (VmNa), the authors analysed the VmNa gene sequences available in GenBank and prepared specific primers for the amplification of two fragments containing the regions coding for (i) the domain II S4-S6 region (bp 2805-3337) and (ii) the domain III S4-3' terminus region (bp 4737-6500), as determined according to the VmNa cDNA sequence AY259834. Sensitive and resistant mite populations did not differ in the amino acid sequences of the III S4-3' terminus VmNa region. However, differences were found in the IIS4-IIS6 fragment. In the resistant population, the mutation C(3004) → G resulted in the substitution L(1002) → V (codon ctg → gtg) at the position equivalent to that of the housefly L925 in the domain II S5 helix. Additionally, the mutation F(1052) → L (codon ttc → ctc) at the position equivalent to that of the housefly F975 in the domain II P-loop connecting segments S5 and S6 was detected in both the resistant and sensitive populations. All individuals that survived the tau-fluvalinate treatment in the bioassay harboured the L(1002) → V mutation combined with the F(1052), while dead individuals from both the sensitive and resistant populations harboured mostly the L(1002) residue and either of the two residues at position 1052. © 2013 Society of Chemical Industry.

  4. Castration resistance in human prostate cancer is conferred by a frequently occurring androgen receptor splice variant

    Science.gov (United States)

    Sun, Shihua; Sprenger, Cynthia C.T.; Vessella, Robert L.; Haugk, Kathleen; Soriano, Kathryn; Mostaghel, Elahe A.; Page, Stephanie T.; Coleman, Ilsa M.; Nguyen, Holly M.; Sun, Huiying; Nelson, Peter S.; Plymate, Stephen R.

    2010-01-01

    Progression of prostate cancer following castration is associated with increased androgen receptor (AR) expression and signaling despite AR blockade. Recent studies suggest that these activities are due to the generation of constitutively active AR splice variants, but the mechanisms by which these splice variants could mediate such effects are not fully understood. Here we have identified what we believe to be a novel human AR splice variant in which exons 5, 6, and 7 are deleted (ARv567es) and demonstrated that this variant can contribute to cancer progression in human prostate cancer xenograft models in mice following castration. We determined that, in human prostate cancer cell lines, ARv567es functioned as a constitutively active receptor, increased expression of full-length AR (ARfl), and enhanced the transcriptional activity of AR. In human xenografts, human prostate cancer cells transfected with ARv567es cDNA formed tumors that were resistant to castration. Furthermore, the ratio of ARv567es to ARfl expression within the xenografts positively correlated with resistance to castration. Importantly, we also detected ARv567es frequently in human prostate cancer metastases. In summary, these data indicate that constitutively active AR splice variants can contribute to the development of castration-resistant prostate cancers and may serve as biomarkers for patients who are likely to suffer from early recurrence and are candidates for therapies directly targeting the AR rather than ligand. PMID:20644256

  5. Production of transgenic brassica juncea with the synthetic chitinase gene (nic) conferring resistance to alternaria brassicicola

    International Nuclear Information System (INIS)

    Munir, I.; Hussan, W.; Kazi, M.; Mian, A.

    2016-01-01

    Brassica juncea is an important oil seed crop throughout the world. The demand and cultivation of oil seed crops has gained importance due to rapid increase in world population and industrialization. Fungal diseases pose a great threat to Brassica productivity worldwide. Absence of resistance genes against fungal infection within crossable germplasms of this crop necessitates deployment of genetic engineering approaches to produce transgenic plants with resistance against fungal infections. In the current study, hypocotyls and cotyledons of Brassica juncea, used as explants, were transformed with Agrobacterium tumefacien strain EHA101 harboring binary vector pEKB/NIC containing synthetic chitinase gene (NIC), an antifungal gene under the control of cauliflower mosaic virus promoter (CaMV35S). Bar genes and nptII gene were used as selectable markers. Presence of chitinase gene in trangenic lines was confirmed by PCR and southern blotting analysis. Effect of the extracted proteins from non-transgenic and transgenic lines was observed on the growth of Alternaria brassicicola, a common disease causing pathogen in brassica crop. In comparison to non-transgenic control lines, the leaf tissue extracts of the transgenic lines showed considerable resistance and antifungal activity against A. brassicicola. The antifungal activity in transgenic lines was observed as corresponding to the transgene copy number. (author)

  6. FLCN and AMPK Confer Resistance to Hyperosmotic Stress via Remodeling of Glycogen Stores.

    Directory of Open Access Journals (Sweden)

    Elite Possik

    2015-10-01

    Full Text Available Mechanisms of adaptation to environmental changes in osmolarity are fundamental for cellular and organismal survival. Here we identify a novel osmotic stress resistance pathway in Caenorhabditis elegans (C. elegans, which is dependent on the metabolic master regulator 5'-AMP-activated protein kinase (AMPK and its negative regulator Folliculin (FLCN. FLCN-1 is the nematode ortholog of the tumor suppressor FLCN, responsible for the Birt-Hogg-Dubé (BHD tumor syndrome. We show that flcn-1 mutants exhibit increased resistance to hyperosmotic stress via constitutive AMPK-dependent accumulation of glycogen reserves. Upon hyperosmotic stress exposure, glycogen stores are rapidly degraded, leading to a significant accumulation of the organic osmolyte glycerol through transcriptional upregulation of glycerol-3-phosphate dehydrogenase enzymes (gpdh-1 and gpdh-2. Importantly, the hyperosmotic stress resistance in flcn-1 mutant and wild-type animals is strongly suppressed by loss of AMPK, glycogen synthase, glycogen phosphorylase, or simultaneous loss of gpdh-1 and gpdh-2 enzymes. Our studies show for the first time that animals normally exhibit AMPK-dependent glycogen stores, which can be utilized for rapid adaptation to either energy stress or hyperosmotic stress. Importantly, we show that glycogen accumulates in kidneys from mice lacking FLCN and in renal tumors from a BHD patient. Our findings suggest a dual role for glycogen, acting as a reservoir for energy supply and osmolyte production, and both processes might be supporting tumorigenesis.

  7. O-specific polysaccharide confers lysozyme resistance to extraintestinal pathogenic Escherichia coli.

    Science.gov (United States)

    Bao, Yinli; Zhang, Haobo; Huang, Xinxin; Ma, Jiale; Logue, Catherine M; Nolan, Lisa K; Li, Ganwu

    2018-12-31

    Extraintestinal pathogenic Escherichia coli (ExPEC) is the leading cause of bloodstream and other extraintestinal infections in human and animals. The greatest challenge encountered by ExPEC during an infection is posed by the host defense mechanisms, including lysozyme. ExPEC have developed diverse strategies to overcome this challenge. The aim of this study was to characterize the molecular mechanism of ExPEC resistance to lysozyme. For this, 15,000 transposon mutants of a lysozyme-resistant ExPEC strain NMEC38 were screened; 20 genes were identified as involved in ExPEC resistance to lysozyme-of which five were located in the gene cluster between galF and gnd, and were further confirmed to be involved in O-specific polysaccharide biosynthesis. The O-specific polysaccharide was able to inhibit the hydrolytic activity of lysozyme; it was also required by the complete lipopolysaccharide (LPS)-mediated protection of ExPEC against the bactericidal activity of lysozyme. The O-specific polysaccharide was further shown to be able to directly interact with lysozyme. Furthermore, LPS from ExPEC strains of different O serotypes was also able to inhibit the hydrolytic activity of lysozyme. Because of their cell surface localization and wide distribution in Gram-negative bacteria, O-specific polysaccharides appear to play a long-overlooked role in protecting bacteria against exogenous lysozyme.

  8. Chromosomal locations of the maize (Zea mays L. HtP and rt genes that confer resistance to Exserohilum turcicum

    Directory of Open Access Journals (Sweden)

    Juliana Bernardi Ogliari

    2007-01-01

    Full Text Available We used 125 microsatellite markers to genotype the maize (Zea mays L. near isogenic lines (NIL L30HtPHtPRtRt and L30htphtpRtRt and the L40htphtprtrt line which contrast regarding the presence of the recently described dominant HtP and the recessive rt genes that confer resistance to Exserohilum turcicum. Five microsatellite markers revealed polymorphisms between the NIL and were considered candidate linked markers for the HtP resistance gene. Linkage was confirmed by bulked segregant sample (BSS analysis of 32 susceptible and 34 resistant plants from a BC1F1 population derived from the cross (L30HtPHtPRtRt x L40htphtprtrt x L40htphtprtrt. The bnlg198 and dupssr25 markers, both located on maize chromosome 2L (bin 2.08, were polymorphic between bulks. Linkage distances were estimated based on co-segregation data of the 32 susceptible plants and indicated distances of 28.7 centimorgans (cM between HtP and bnlg198 and 23.5 cM between HtP and dupssr25. The same set of susceptible plants was also genotyped with markers polymorphic between L30HtPHtPRtRt and L40htphtprtrt in order to find markers linked to the rt gene. Marker bnlg197, from chromosome 3L (bin 3.06, was found linked to rt at a distance of 9.7 cM. This is the first report on the chromosomal locations of these newly described genes.

  9. A deletion mutation in nfxB of in vitro-induced moxifloxacin-resistant Pseudomonas aeruginosa confers multidrug resistance.

    Science.gov (United States)

    Thai, Van Chi; Pham, Hoang Vy; Nguyen, Duc Nhat Minh; Lambert, Peter; Nguyen, Thi Thu Hoai

    2017-09-01

    The modulation of efflux pump functions under fluoroquinolone (FQ) exposure is of great concern as it could result in occurrence of multidrug-resistant (MDR) bacterial strains. In this study, MDR mechanism in Pseudomonas aeruginosa induced via moxifloxacin (MOX) pressure was investigated. After serial MOX [concentration of 0.5 × the minimum inhibitory concentration (MIC)] exposure, the fully susceptible P. aeruginosa ATCC 9027 strain has increased its MIC not only toward MOX (1→128 mg/L) but also to other antibiotics. Furthermore, this MOX-exposed strain did not revert to antibiotic-sensitive phenotype when being cultured in antibiotic-free medium for 12 days. No mutation was observed for FQ-target (gyrA and parC) or most investigated efflux regulatory genes (mexT, mexR, and nalC) except nfxB in which a 100-bp deletion was found. This associated with the elevated expression of multidrug efflux pump operon (mexCD-oprJ) which could directly result in MDR phenotype.

  10. Transfer Potential of Plasmids Conferring Extended-Spectrum-Cephalosporin Resistance in Escherichia coli from Poultry.

    Science.gov (United States)

    Mo, Solveig Sølverød; Sunde, Marianne; Ilag, Hanna Karin; Langsrud, Solveig; Heir, Even

    2017-06-15

    Escherichia coli strains resistant to extended-spectrum cephalosporins (ESC) are widely distributed in Norwegian broiler production, and the majority harbor transferable IncK or IncI1 plasmids carrying bla CMY-2 Persistent occurrence in broiler farms may occur through the survival of ESC-resistant E. coli strains in the farm environment, or by transfer and maintenance of resistance plasmids within a population of environmental bacteria with high survival abilities. The aim of this study was to determine the transferability of two successful bla CMY-2 -carrying plasmids belonging to the incompatibility groups IncK and IncI1 into E. coli and Serratia species recipients. Initially, conjugative plasmid transfer from two E. coli donors to potential recipients was tested in an agar assay. Conjugation was further investigated for selected mating pairs in surface and planktonic assays at temperatures from 12°C to 37°C. Transfer of plasmids was observed on agar, in broth, and in biofilm at temperatures down to 25°C. The IncK plasmid was able to transfer into Serratia marcescens , and transconjugants were able to act as secondary plasmid donors to different E. coli and Serratia species recipients. All transconjugants displayed an AmpC phenotype corresponding to the acquisition of bla CMY-2 In summary, the results indicate that the IncK plasmid may transfer between E. coli and Serratia spp. under conditions relevant for broiler production. IMPORTANCE Certain bla CMY-2 -carrying plasmids are successful and disseminated in European broiler production. Traditionally, plasmid transferability has been studied under conditions that are optimal for bacterial growth. Plasmid transfer has previously been reported between E. coli bacteria in biofilms at 37°C and in broth at temperatures ranging from 8 to 37°C. However, intergenus transfer of bla CMY-2 -carrying plasmids from E. coli to environmental bacteria in the food-processing chain has not been previously studied. We

  11. Identification of amino acids conferring high-level resistance to expanded-spectrum cephalosporins in the penA gene from Neisseria gonorrhoeae strain H041.

    Science.gov (United States)

    Tomberg, Joshua; Unemo, Magnus; Ohnishi, Makoto; Davies, Christopher; Nicholas, Robert A

    2013-07-01

    The recent identification of a high-level-ceftriaxone-resistant (MIC = 2 to 4 μg/ml) isolate of Neisseria gonorrhoeae from Japan (H041) portends the loss of ceftriaxone as an effective treatment for gonococcal infections. This is of grave concern because ceftriaxone is the last remaining option for first-line empirical antimicrobial monotherapy. The penA gene from H041 (penA41) is a mosaic penA allele similar to mosaic alleles conferring intermediate-level cephalosporin resistance (Ceph(i)) worldwide but has 13 additional mutations compared to the mosaic penA gene from the previously studied Ceph(i) strain 35/02 (penA35). When transformed into the wild-type strain FA19, the penA41 allele confers 300- and 570-fold increases in the MICs for ceftriaxone and cefixime, respectively. In order to understand the mechanisms involved in high-level ceftriaxone resistance and to improve surveillance and epidemiology during the potential emergence of ceftriaxone resistance, we sought to identify the minimum number of amino acid alterations above those in penA35 that confer high-level resistance to ceftriaxone. Using restriction fragment exchange and site-directed mutagenesis, we identified three mutations, A311V, T316P, and T483S, that, when incorporated into the mosaic penA35 allele, confer essentially all of the increased resistance of penA41. A311V and T316P are close to the active-site nucleophile Ser310 that forms the acyl-enzyme complex, while Thr483 is predicted to interact with the carboxylate of the β-lactam antibiotic. These three mutations have thus far been described only for penA41, but dissemination of these mutations in other mosaic alleles would spell the end of ceftriaxone as an effective treatment for gonococcal infections.

  12. Exosomal transfer of tumor-associated macrophage-derived miR-21 confers cisplatin resistance in gastric cancer cells.

    Science.gov (United States)

    Zheng, Peiming; Chen, Lei; Yuan, Xiangliang; Luo, Qin; Liu, Yi; Xie, Guohua; Ma, Yanhui; Shen, Lisong

    2017-04-13

    Cisplatin-based chemotherapy is frequently used to treat advanced gastric cancer (GC). However, the resistance often occurs with the mechanisms being not well understood. Recently, emerging evidence indicates that tumor-associated macrophages (TAMs) play an important role in chemoresistance of cancer. As the important mediators in intercellular communications, exosomes secreted by host cells mediate the exchange of genetic materials and proteins to be involved in tumor aggressiveness. The aim of the study was to investigate whether exosomes derived from TAMs mediate cisplatin resistance in gastric cancer. M2 polarized macrophages were obtained from mouse bone marrow or human PBMCs stimulated with IL-4 and IL-13. Exosomes isolated from M2 macrophages culture medium were characterized, and miRNA expression profiles of M2 derived exosomes (M2-exos) were analyzed using miRNA microarray. In vitro cell coculture was further conducted to investigate M2-exos mediated crosstalk between TAMs and tumor cells. Moreover, the in vivo experiments were performed using a subcutaneous transplantation tumor model in athymic nude mice. In this study, we showed that M2 polarized macrophages promoted cisplatin (DDP) resistance in gastric cancer cells and exosomes derived from M2 macrophages (M2-exos) are involved in mediating the resistance to DDP. Using miRNA profiles assay, we identify significantly higher levels of microRNA-21 (miR21) isomiRNAs in exosomes and cell lysate isolated from M2 polarized macrophage. Functional studies revealed that exosomal miR-21 can be directly transferred from macrophages to the gastric cancer cells, where it suppresses cell apoptosis and enhances activation of PI3K/AKT signaling pathway by down-regulation of PTEN. Our findings suggest that exosomal transfer of tumor-associated macrophages derived miR-21 confer DDP resistance in gastric cancer, and targeting exosome communication may be a promising new therapeutic strategy for gastric cancer

  13. Upregulation of the PatAB Transporter Confers Fluoroquinolone Resistance to Streptococcus pseudopneumoniae

    Directory of Open Access Journals (Sweden)

    María Alvarado

    2017-10-01

    Full Text Available We characterized the mechanism of fluoroquinolone-resistance in two isolates of Streptococcus pseudopneumoniae having fluoroquinolone-efflux as unique mechanism of resistance. Whole genome sequencing and genetic transformation experiments were performed together with phenotypic determinations of the efflux mechanism. The PatAB pump was identified as responsible for efflux of ciprofloxacin (MIC of 4 μg/ml, ethidium bromide (MICs of 8–16 μg/ml and acriflavine (MICs of 4–8 μg/ml in both isolates. These MICs were at least 8-fold lower in the presence of the efflux inhibitor reserpine. Complete genome sequencing indicated that the sequence located between the promoter of the patAB operon and the initiation codon of patA, which putatively forms an RNA stem-loop structure, may be responsible for the efflux phenotype. RT-qPCR determinations performed on RNAs of cultures treated or not treated with subinhibitory ciprofloxacin concentrations were performed. While no significant changes were observed in wild-type Streptococcus pneumoniae R6 strain, increases in transcription were detected in the ciprofloxacin-efflux transformants obtained with DNA from efflux-positive isolates, in the ranges of 1.4 to 3.4-fold (patA and 2.1 to 2.9-fold (patB. Ciprofloxacin-induction was related with a lower predicted free energy for the stem-loop structure in the RNA of S. pseudopneumoniae isolates (−13.81 and −8.58 than for R6 (−15.32 kcal/mol, which may ease transcription. The presence of these regulatory variations in commensal S. pseudopneumoniae isolates, and the possibility of its transfer to Streptococcus pneumoniae by genetic transformation, could increase fluoroquinolone resistance in this important pathogen.

  14. GTSE1 expression represses apoptotic signaling and confers cisplatin resistance in gastric cancer cells

    International Nuclear Information System (INIS)

    Subhash, Vinod Vijay; Tan, Shi Hui; Tan, Woei Loon; Yeo, Mei Shi; Xie, Chen; Wong, Foong Ying; Kiat, Zee Ying; Lim, Robert; Yong, Wei Peng

    2015-01-01

    Platinum based therapy is commonly used in the treatment of advanced gastric cancer. However, resistance to chemotherapy is a major challenge that causes marked variation in individual response rate and survival rate. In this study, we aimed to identify the expression of GTSE1 and its correlation with cisplatin resistance in gastric cancer cells. Methylation profiling was carried out in tissue samples from gastric cancer patients before undergoing neoadjuvent therapy using docetaxel, cisplatin and 5FU (DCX) and in gastric cancer cell lines. The correlation between GTSE1 expression and methylation in gastric cancer cells was determined by RT-PCR and MSP respectively. GTSE1 expression was knocked-down using shRNA’s and its effects on cisplatin cytotoxicity and cell survival were detected by MTS, proliferation and clonogenic survival assays. Additionally, the effect of GTSE1 knock down in drug induced apoptosis was determined by western blotting and apoptosis assays. GTSE1 exhibited a differential methylation index in gastric cancer patients and in cell lines that correlated with DCX treatment response and cisplatin sensitivity, respectively. In-vitro, GTSE1 expression showed a direct correlation with hypomethylation. Interestingly, Cisplatin treatment induced a dose dependent up regulation as well as nuclear translocation of GTSE1 expression in gastric cancer cells. Knock down of GTSE1 enhanced cisplatin cytotoxity and led to a significant reduction in cell proliferation and clonogenic survival. Also, loss of GTSE1 expression caused a significant increase in P53 mediated apoptosis in cisplatin treated cells. Our study identifies GTSE1 as a biomarker for cisplatin resistance in gastric cancer cells. This study also suggests the repressive role of GTSE1 in cisplatin induced apoptosis and signifies its potential utility as a therapeutic target for better clinical management of gastric cancer patients. The online version of this article (doi:10.1186/s12885

  15. Human Recombinant Antimannan Immunoglobulin G1 Antibody Confers Resistance to Hematogenously Disseminated Candidiasis in Mice

    Science.gov (United States)

    Zhang, Mason X.; Bohlman, M. Charlotte; Itatani, Carol; Burton, Dennis R.; Parren, Paul W. H. I.; St. Jeor, Stephen C.; Kozel, Thomas R.

    2006-01-01

    Mannan is a major cell wall component found in Candida species. Natural antimannan antibody is present in sera from most normal adults, but its role in host resistance to hematogenously disseminated candidiasis is unknown. The purpose of this study was to develop recombinant human antimannan antibody and to study its protective function. A phage Fab display combinatorial library containing Fab genes from bone marrow lymphocytes was screened with Candida albicans yeast cells and chemically purified mannan. One antimannan Fab, termed M1, was converted to a full-length immunoglobulin G1 antibody, M1g1, and M1g1 was produced in CHO cells. The M1g1 epitope was found in C. albicans serotypes A and B, Candida tropicalis, Candida guilliermondii, Candida glabrata, and Candida parapsilosis. Its expression was active at both 23°C and 37°C and uniform over the cell surface. BALB/c mice passively immunized with M1g1 were more resistant than control mice to a lethal hematogenous infection by C. albicans, as evidenced by extension of survival in an M1g1 dose-dependent manner (P, 0.08 to <0.001) and by reduction in number of infection foci and their size in the kidney. In vitro studies found that M1g1 promoted phagocytosis and phagocytic killing of C. albicans yeast cells by mouse peritoneal macrophages and was required for activation of the mouse complement cascade. Thus, human antimannan antibody may have a protective role in host resistance to systemic candidiasis. PMID:16368991

  16. RNAi-mediated oncogene silencing confers resistance to crown gall tumorigenesis

    Science.gov (United States)

    Escobar, Matthew A.; Civerolo, Edwin L.; Summerfelt, Kristin R.; Dandekar, Abhaya M.

    2001-01-01

    Crown gall disease, caused by the soil bacterium Agrobacterium tumefaciens, results in significant economic losses in perennial crops worldwide. A. tumefaciens is one of the few organisms with a well characterized horizontal gene transfer system, possessing a suite of oncogenes that, when integrated into the plant genome, orchestrate de novo auxin and cytokinin biosynthesis to generate tumors. Specifically, the iaaM and ipt oncogenes, which show ≈90% DNA sequence identity across studied A. tumefaciens strains, are required for tumor formation. By expressing two self-complementary RNA constructions designed to initiate RNA interference (RNAi) of iaaM and ipt, we generated transgenic Arabidopsis thaliana and Lycopersicon esculentum plants that are highly resistant to crown gall disease development. In in vitro root inoculation bioassays with two biovar I strains of A. tumefaciens, transgenic Arabidopsis lines averaged 0.0–1.5% tumorigenesis, whereas wild-type controls averaged 97.5% tumorigenesis. Similarly, several transformed tomato lines that were challenged by stem inoculation with three biovar I strains, one biovar II strain, and one biovar III strain of A. tumefaciens displayed between 0.0% and 24.2% tumorigenesis, whereas controls averaged 100% tumorigenesis. This mechanism of resistance, which is based on mRNA sequence homology rather than the highly specific receptor–ligand binding interactions characteristic of traditional plant resistance genes, should be highly durable. If successful and durable under field conditions, RNAi-mediated oncogene silencing may find broad applicability in the improvement of tree crop and ornamental rootstocks. PMID:11687652

  17. Identification of yeast genes that confer resistance to chitosan oligosaccharide (COS using chemogenomics

    Directory of Open Access Journals (Sweden)

    Jaime Maria DLA

    2012-06-01

    Full Text Available Abstract Background Chitosan oligosaccharide (COS, a deacetylated derivative of chitin, is an abundant, and renewable natural polymer. COS has higher antimicrobial properties than chitosan and is presumed to act by disrupting/permeabilizing the cell membranes of bacteria, yeast and fungi. COS is relatively non-toxic to mammals. By identifying the molecular and genetic targets of COS, we hope to gain a better understanding of the antifungal mode of action of COS. Results Three different chemogenomic fitness assays, haploinsufficiency (HIP, homozygous deletion (HOP, and multicopy suppression (MSP profiling were combined with a transcriptomic analysis to gain insight in to the mode of action and mechanisms of resistance to chitosan oligosaccharides. The fitness assays identified 39 yeast deletion strains sensitive to COS and 21 suppressors of COS sensitivity. The genes identified are involved in processes such as RNA biology (transcription, translation and regulatory mechanisms, membrane functions (e.g. signalling, transport and targeting, membrane structural components, cell division, and proteasome processes. The transcriptomes of control wild type and 5 suppressor strains overexpressing ARL1, BCK2, ERG24, MSG5, or RBA50, were analyzed in the presence and absence of COS. Some of the up-regulated transcripts in the suppressor overexpressing strains exposed to COS included genes involved in transcription, cell cycle, stress response and the Ras signal transduction pathway. Down-regulated transcripts included those encoding protein folding components and respiratory chain proteins. The COS-induced transcriptional response is distinct from previously described environmental stress responses (i.e. thermal, salt, osmotic and oxidative stress and pre-treatment with these well characterized environmental stressors provided little or any resistance to COS. Conclusions Overexpression of the ARL1 gene, a member of the Ras superfamily that regulates membrane

  18. Identification of yeast genes that confer resistance to chitosan oligosaccharide (COS) using chemogenomics.

    Science.gov (United States)

    Jaime, Maria D L A; Lopez-Llorca, Luis Vicente; Conesa, Ana; Lee, Anna Y; Proctor, Michael; Heisler, Lawrence E; Gebbia, Marinella; Giaever, Guri; Westwood, J Timothy; Nislow, Corey

    2012-06-22

    Chitosan oligosaccharide (COS), a deacetylated derivative of chitin, is an abundant, and renewable natural polymer. COS has higher antimicrobial properties than chitosan and is presumed to act by disrupting/permeabilizing the cell membranes of bacteria, yeast and fungi. COS is relatively non-toxic to mammals. By identifying the molecular and genetic targets of COS, we hope to gain a better understanding of the antifungal mode of action of COS. Three different chemogenomic fitness assays, haploinsufficiency (HIP), homozygous deletion (HOP), and multicopy suppression (MSP) profiling were combined with a transcriptomic analysis to gain insight in to the mode of action and mechanisms of resistance to chitosan oligosaccharides. The fitness assays identified 39 yeast deletion strains sensitive to COS and 21 suppressors of COS sensitivity. The genes identified are involved in processes such as RNA biology (transcription, translation and regulatory mechanisms), membrane functions (e.g. signalling, transport and targeting), membrane structural components, cell division, and proteasome processes. The transcriptomes of control wild type and 5 suppressor strains overexpressing ARL1, BCK2, ERG24, MSG5, or RBA50, were analyzed in the presence and absence of COS. Some of the up-regulated transcripts in the suppressor overexpressing strains exposed to COS included genes involved in transcription, cell cycle, stress response and the Ras signal transduction pathway. Down-regulated transcripts included those encoding protein folding components and respiratory chain proteins. The COS-induced transcriptional response is distinct from previously described environmental stress responses (i.e. thermal, salt, osmotic and oxidative stress) and pre-treatment with these well characterized environmental stressors provided little or any resistance to COS. Overexpression of the ARL1 gene, a member of the Ras superfamily that regulates membrane trafficking, provides protection against COS

  19. Neamin as an immunogen for the development of a generic ELISA detecting gentamicin, kanamycin and neomycin in milk

    NARCIS (Netherlands)

    Loomans, E.E.M.G.; Wiltenburg, van J.; Koets, M.; Amerongen, van A.

    2003-01-01

    A broad-specific ELISA using one antibody preparation for the detection of gentamicin, kanamycin, and neomycin in milk is reported for the first time. For the immunization of rabbits, neamin was used as the generic hapten on the basis of the facts that it is a two-ring fragment of neomycin and, in

  20. Fluoroquinolone resistance in Helicobacter pylori: role of mutations at position 87 and 91 of GyrA on the level of resistance and identification of a resistance conferring mutation in GyrB.

    Science.gov (United States)

    Rimbara, Emiko; Noguchi, Norihisa; Kawai, Takashi; Sasatsu, Masanori

    2012-02-01

    Fluoroquinolone-containing regimens have been suggested as an alternate to standard triple therapy for the treatment of Helicobacter pylori infections. To determine the relationship between fluoroquinolone resistance and mutations of GyrA and GyrB in H. pylori, we exchanged the mutations at positions 87 and 91 of GyrA among fluoroquinolone-resistant clinical isolates. GyrB of a strain with no mutations in GyrA was also analyzed to identify mechanisms of resistance to norfloxacin. Natural transformation was performed using the amplified fragment of the gyrA and gyrB gene as donor DNA. The amino acid sequences of GyrA and GyrB were determined by DNA sequencing of the gyrA and gyrB genes. Norfloxacin-resistant strains which had mutations at position 87 and 91 became susceptible when the mutations were converted to the wild type. When the mutation from Asp to Asn at position 91 was exchanged to the mutation from Asn to Lys at position 87, the MIC to levofloxacin, gatifloxacin, and sitafloxacin increased. Norfloxacin-resistant strain TS132 with no mutations in GyrA but had a mutation at position 463 in GyrB. Transformants obtained by natural transformation using gyrB DNA of TS132 had a mutation at position 463 of GyrB and revealed resistant to norfloxacin and levofloxacin. Mutation from Asn to Lys at position 87 of GyrA confers higher resistance to levofloxacin and gatifloxacin than does mutation from Asp to Asn at position 91. We propose that mutation at position 463 in GyrB as a novel mechanism of fluoroquinolone resistance in H. pylori. © 2011 Blackwell Publishing Ltd.

  1. Expression of the Galanthus nivalis agglutinin (GNA) gene in transgenic potato plants confers resistance to aphids.

    Science.gov (United States)

    Mi, Xiaoxiao; Liu, Xue; Yan, Haolu; Liang, Lina; Zhou, Xiangyan; Yang, Jiangwei; Si, Huaijun; Zhang, Ning

    2017-01-01

    Aphids, the largest group of sap-sucking pests, cause significant yield losses in agricultural crops worldwide every year. The massive use of pesticides to combat this pest causes severe damage to the environment, putting in risk the human health. In this study, transgenic potato plants expressing Galanthus nivalis agglutinin (GNA) gene were developed using CaMV 35S and ST-LS1 promoters generating six transgenic lines (35S1-35S3 and ST1-ST3 corresponding to the first and second promoter, respectively). Quantitative real-time polymerase chain reaction (qRT-PCR) analysis indicated that the GNA gene was expressed in leaves, stems and roots of transgenic plants under the control of the CaMV 35S promoter, while it was only expressed in leaves and stems under the control of the ST-LS1 promoter. The levels of aphid mortality after 5 days of the inoculation in the assessed transgenic lines ranged from 20 to 53.3%. The range of the aphid population in transgenic plants 15 days after inoculation was between 17.0±1.43 (ST2) and 36.6±0.99 (35S3) aphids per plant, which corresponds to 24.9-53.5% of the aphid population in non-transformed plants. The results of our study suggest that GNA expressed in transgenic potato plants confers a potential tolerance to aphid attack, which appears to be an alternative against the use of pesticides in the future. Copyright © 2016 Académie des sciences. Published by Elsevier SAS. All rights reserved.

  2. The Staphylococcus aureus α-Acetolactate Synthase ALS Confers Resistance to Nitrosative Stress

    Directory of Open Access Journals (Sweden)

    Sandra M. Carvalho

    2017-07-01

    Full Text Available Staphylococcus aureus is a worldwide pathogen that colonizes the human nasal cavity and is a major cause of respiratory and cutaneous infections. In the nasal cavity, S. aureus thrives with high concentrations of nitric oxide (NO produced by the innate immune effectors and has available for growth slow-metabolizing free hexoses, such as galactose. Here, we have used deep sequencing transcriptomic analysis (RNA-Seq and 1H-NMR to uncover how S. aureus grown on galactose, a major carbon source present in the nasopharynx, survives the deleterious action of NO. We observed that, like on glucose, S. aureus withstands high concentrations of NO when using galactose. Data indicate that this resistance is, most likely, achieved through a distinct metabolism that relies on the increased production of amino acids, such as glutamate, threonine, and branched-chain amino acids (BCAAs. Moreover, we found that under NO stress the S. aureus α-acetolactate synthase (ALS enzyme, which converts pyruvate into α-acetolactate, plays an important role. ALS is proposed to prevent intracellular acidification, to promote the production of BCAAs and the activation of the TCA cycle. Additionally, ALS is shown to contribute to the successful infection of murine macrophages. Furthermore, ALS contributes to the resistance of S. aureus to beta-lactam antibiotics such as methicillin and oxacillin.

  3. Translesion polymerase η is upregulated by cancer therapeutics and confers anticancer drug resistance.

    Science.gov (United States)

    Tomicic, Maja T; Aasland, Dorthe; Naumann, Steffen C; Meise, Ruth; Barckhausen, Christina; Kaina, Bernd; Christmann, Markus

    2014-10-01

    DNA repair processes are a key determinant of the sensitivity of cancer cells to DNA-damaging chemotherapeutics, which may induce certain repair genes as a mechanism to promote resistance. Here, we report the results of a screen for repair genes induced in cancer cells treated with DNA crosslinking agents, which identified the translesion polymerase η (PolH) as a p53-regulated target acting as one defense against interstrand crosslink (ICL)-inducing agents. PolH was induced by fotemustine, mafosfamide, and lomustine in breast cancer, glioma, and melanoma cells in vitro and in vivo, with similar inductions observed in normal cells such as lymphocytes and diploid fibroblasts. PolH contributions to the protection against ICL-inducing agents were evaluated by its siRNA-mediated attenuation in cells, which elevated sensitivity to these drugs in all tumor cell models. Conversely, PolH overexpression protected cancer cells against these drugs. PolH attenuation reduced repair of ICL lesions as measured by host cell reactivation assays and enhanced persistence of γH2AX foci. Moreover, we observed a strong accumulation of PolH in the nucleus of drug-treated cells along with direct binding to damaged DNA. Taken together, our findings implicated PolH in ICL repair as a mechanism of cancer drug resistance and normal tissue protection. ©2014 American Association for Cancer Research.

  4. Pyomelanin produced by Vibrio cholerae confers resistance to predation by Acanthamoeba castellanii

    Science.gov (United States)

    Noorian, Parisa; Hu, Jie; Chen, Zhiliang; Kjelleberg, Staffan; Wilkins, Marc R; Sun, Shuyang

    2017-01-01

    Abstract Protozoan predation is one of the main environmental factors constraining bacterial growth in aquatic environments, and thus has led to the evolution of a number of defence mechanisms that protect bacteria from predation. These mechanisms may also function as virulence factors in infection of animal and human hosts. Whole transcriptome shotgun sequencing of Vibrio cholerae biofilms during predation by the amoebae, Acanthamoeba castellanii, revealed that 131 transcripts were significantly differentially regulated when compared to the non-grazed control. Differentially regulated transcripts included those involved in biosynthetic and metabolic pathways. The transcripts of genes involved in tyrosine metabolism were down-regulated in the grazed population, which indicates that the tyrosine metabolic regulon may have a role in the response of V. cholerae biofilms to A. castellanii predation. Homogentisate 1, 2-dioxygenase (HGA) is the main intermediate of the normal L-tyrosine catabolic pathway which is known to auto-oxidize, leading to the formation of the pigment, pyomelanin. Indeed, a pigmented mutant, disrupted in hmgA, was more resistant to amoebae predation than the wild type. Increased grazing resistance was correlated with increased production of pyomelanin and thus reactive oxygen species (ROS), suggesting that ROS production is a defensive mechanism used by bacterial biofilms against predation by amoebae A. castellanii. PMID:29095994

  5. Aberrant lipogenesis is a metabolic marker for azole-resistant candida albicans (Conference Presentation)

    Science.gov (United States)

    Karanja, Caroline; Hong, Weili; Younis, Waleed; Cheng, Ji-Xin; Seleem, Mohamed

    2017-02-01

    Candida is the single most important cause of fungal bloodstream infections worldwide causing significant mortality as high as 50%. This high mortality rate is, in part, due to the inability to rapidly diagnose and simultaneously initiate an effective antifungal therapy early in the disease process. Current culture-based diagnostics are often slow, requiring several days to complete, and are only 50% sensitive in diagnosing candidemia (Candida bloodstream infection). For every 12 hours of delay in starting correct antifungal therapy, the risk of death for a given patient with candidemia increases by 200%. To address this unmet need, we explored the potential of employing stimulated Raman Scattering (SRS) imaging to diagnose candidemia and probe metabolic differences between resistant and susceptible strain at a single cell level. Metabolism is integral to pathogenicity; microorganism have very short life cycles, and therefore only a few hours are needed to observe a full metabolic cycle. SRS imaging at C-H vibration frequency at 2850 cm-1 revealed a substantial difference in lipogenesis between the susceptible and resistant C. albicans. Treating the C. albicans with fluconazole, an antimicrobial drug that targets ergosterol biosynthesis only affected the lipogenesis in the susceptible strain. Our results show that single-cell metabolic imaging under a SRS microscope can be used for diagnose candidemia and early detection of antimicrobial susceptibility.

  6. Nuclear PIM1 confers resistance to rapamycin-impaired endothelial proliferation

    Energy Technology Data Exchange (ETDEWEB)

    Walpen, Thomas; Kalus, Ina [Research Unit, Division Internal Medicine, University Hospital Zuerich, 8091 Zuerich (Switzerland); Schwaller, Juerg [Department of Biomedicine, University of Basel, 4031 Basel (Switzerland); Peier, Martin A. [Research Unit, Division Internal Medicine, University Hospital Zuerich, 8091 Zuerich (Switzerland); Battegay, Edouard J. [Research Unit, Division Internal Medicine, University Hospital Zuerich, 8091 Zuerich (Switzerland); Zurich Center for Integrative Human Physiology (ZIHP), 8057 Zuerich (Switzerland); Humar, Rok, E-mail: Rok.Humar@usz.ch [Research Unit, Division Internal Medicine, University Hospital Zuerich, 8091 Zuerich (Switzerland); Zurich Center for Integrative Human Physiology (ZIHP), 8057 Zuerich (Switzerland)

    2012-12-07

    Highlights: Black-Right-Pointing-Pointer Pim1{sup -/-} endothelial cell proliferation displays increased sensitivity to rapamycin. Black-Right-Pointing-Pointer mTOR inhibition by rapamycin enhances PIM1 cytosolic and nuclear protein levels. Black-Right-Pointing-Pointer Truncation of Pim1 beyond serine 276 results in nuclear localization of the kinase. Black-Right-Pointing-Pointer Nuclear PIM1 increases endothelial proliferation independent of rapamycin. -- Abstract: The PIM serine/threonine kinases and the mTOR/AKT pathway integrate growth factor signaling and promote cell proliferation and survival. They both share phosphorylation targets and have overlapping functions, which can partially substitute for each other. In cancer cells PIM kinases have been reported to produce resistance to mTOR inhibition by rapamycin. Tumor growth depends highly on blood vessel infiltration into the malignant tissue and therefore on endothelial cell proliferation. We therefore investigated how the PIM1 kinase modulates growth inhibitory effects of rapamycin in mouse aortic endothelial cells (MAEC). We found that proliferation of MAEC lacking Pim1 was significantly more sensitive to rapamycin inhibition, compared to wildtype cells. Inhibition of mTOR and AKT in normal MAEC resulted in significantly elevated PIM1 protein levels in the cytosol and in the nucleus. We observed that truncation of the C-terminal part of Pim1 beyond Ser 276 resulted in almost exclusive nuclear localization of the protein. Re-expression of this Pim1 deletion mutant significantly increased the proliferation of Pim1{sup -/-} cells when compared to expression of the wildtype Pim1 cDNA. Finally, overexpression of the nuclear localization mutant and the wildtype Pim1 resulted in complete resistance to growth inhibition by rapamycin. Thus, mTOR inhibition-induced nuclear accumulation of PIM1 or expression of a nuclear C-terminal PIM1 truncation mutant is sufficient to increase endothelial cell proliferation

  7. Suppression of oxidative phosphorylation confers resistance against bevacizumab in experimental glioma.

    Science.gov (United States)

    Eriksson, Jule A; Wanka, Christina; Burger, Michael C; Urban, Hans; Hartel, Ines; von Renesse, Janusz; Harter, Patrick N; Mittelbronn, Michel; Steinbach, Joachim P; Rieger, Johannes

    2017-11-27

    Although bevacizumab initially shows high response rates in gliomas and other tumours, therapy resistance usually develops later. Because anti-angiogenic agents are supposed to induce hypoxia, we asked whether rendering glioma cells independent of oxidative phosphorylation modulates their sensitivity against hypoxia and bevacizumab. LNT-229 glioma cells without functional mitochondria (rho 0 ) and control (rho + ) cells were generated. LNT-229 rho 0 -cells displayed reduced expression of oxidative phosphorylation-related genes and diminished oxygen consumption. Conversely, glycolysis was up-regulated in these cells, as shown by increased lactate production and stronger expression of glucose transporter-1 and lactate dehydrogenase-A. However, hypoxia-induced cell death in vitro was nearly completely abolished in the LNT-229 rho 0 -cells, these cells were more sensitive towards glucose restriction and the treatment with the glycolysis inhibitor 2-deoxy-D-glucose. In an orthotopic mouse xenograft experiment, bevacizumab induced hypoxia as reflected by elevated Hypoxia-inducible factor 1-alpha staining in both, rho + - and rho 0 -tumours. However, it prolonged survival only in the mice bearing rho + -tumours (74 days vs. 105 days, p = 0.024 log-rank test) and had no effect on survival in mice carrying LNT-229 rho 0 -tumours (75 days vs. 70 days, p = 0.52 log-rank test). Interestingly, inhibition of glycolysis in vivo with 2-deoxy-D-glucose re-established sensitivity of rho 0 -tumours against bevacizumab (98 days vs. 80 days, p = 0.0001). In summary, ablation of oxidative phosphorylation in glioma cells leads to a more glycolytic and hypoxia-resistant phenotype and is sufficient to induce bevacizumab-refractory tumours. These results add to increasing evidence that a switch towards glycolysis is one mechanism how tumour cells may evade anti-angiogenic treatments and suggest anti-glycolytic strategies as promising approaches to overcome bevacizumab

  8. Identification of ABC transporter genes conferring combined pleuromutilin-lincosamide-streptogramin A resistance in bovine methicillin-resistant Staphylococcus aureus and coagulase-negative staphylococci.

    Science.gov (United States)

    Wendlandt, Sarah; Kadlec, Kristina; Feßler, Andrea T; Schwarz, Stefan

    2015-06-12

    The aim of this study was to investigate the genetic basis of combined pleuromutilin-lincosamide-streptogramin A resistance in 26 unrelated methicillin-resistant Staphylococcus aureus (MRSA) and coagulase-negative staphylococci (CoNS) from dairy cows suffering from mastitis. The 26 pleuromutilin-resistant staphylococcal isolates were screened for the presence of the genes vga(A), vga(B), vga(C), vga(E), vga(E) variant, sal(A), vmlR, cfr, lsa(A), lsa(B), lsa(C), and lsa(E) by PCR. None of the 26 isolates carried the genes vga(B), vga(C), vga(E), vga(E) variant, vmlR, cfr, lsa(A), lsa(B), or lsa(C). Two Staphylococcus haemolyticus and single Staphylococcus xylosus, Staphylococcus lentus, and Staphylococcus hominis were vga(A)-positive. Twelve S. aureus, two Staphylococcus warneri, as well as single S. lentus and S. xylosus carried the lsa(E) gene. Moreover, single S. aureus, S. haemolyticus, S. xylosus, and Staphylococcus epidermidis were positive for both genes, vga(A) and lsa(E). The sal(A) gene was found in a single Staphylococcus sciuri. All ABC transporter genes were located in the chromosomal DNA, except for a plasmid-borne vga(A) gene in the S. epidermidis isolate. The genetic environment of the lsa(E)-positive isolates was analyzed using previously described PCR assays. Except for the S. warneri and S. xylosus, all lsa(E)-positive isolates harbored a part of the previously described enterococcal multiresistance gene cluster. This is the first report of the novel lsa(E) gene in the aforementioned bovine CoNS species. This is also the first identification of the sal(A) gene in a S. sciuri from a case of bovine mastitis. Moreover, the sal(A) gene was shown to also confer pleuromutilin resistance. Copyright © 2015 Elsevier B.V. All rights reserved.

  9. Structural Insights into HIV Reverse Transcriptase Mutations Q151M and Q151M Complex That Confer Multinucleoside Drug Resistance

    Energy Technology Data Exchange (ETDEWEB)

    Das, Kalyan; Martinez, Sergio E.; Arnold, Eddy

    2017-04-10

    HIV-1 reverse transcriptase (RT) is targeted by multiple drugs. RT mutations that confer resistance to nucleoside RT inhibitors (NRTIs) emerge during clinical use. Q151M and four associated mutations, A62V, V75I, F77L, and F116Y, were detected in patients failing therapies with dideoxynucleosides (didanosine [ddI], zalcitabine [ddC]) and/or zidovudine (AZT). The cluster of the five mutations is referred to as the Q151M complex (Q151Mc), and an RT or virus containing Q151Mc exhibits resistance to multiple NRTIs. To understand the structural basis for Q151M and Q151Mc resistance, we systematically determined the crystal structures of the wild-type RT/double-stranded DNA (dsDNA)/dATP (complex I), wild-type RT/dsDNA/ddATP (complex II), Q151M RT/dsDNA/dATP (complex III), Q151Mc RT/dsDNA/dATP (complex IV), and Q151Mc RT/dsDNA/ddATP (complex V) ternary complexes. The structures revealed that the deoxyribose rings of dATP and ddATP have 3'-endo and 3'-exo conformations, respectively. The single mutation Q151M introduces conformational perturbation at the deoxynucleoside triphosphate (dNTP)-binding pocket, and the mutated pocket may exist in multiple conformations. The compensatory set of mutations in Q151Mc, particularly F116Y, restricts the side chain flexibility of M151 and helps restore the DNA polymerization efficiency of the enzyme. The altered dNTP-binding pocket in Q151Mc RT has the Q151-R72 hydrogen bond removed and has a switched conformation for the key conserved residue R72 compared to that in wild-type RT. On the basis of a modeled structure of hepatitis B virus (HBV) polymerase, the residues R72, Y116, M151, and M184 in Q151Mc HIV-1 RT are conserved in wild-type HBV polymerase as residues R41, Y89, M171, and M204, respectively; functionally, both Q151Mc HIV-1 and wild-type HBV are resistant to dideoxynucleoside analogs.

  10. Overexpression of tobacco osmotin (Tbosm) in soybean conferred resistance to salinity stress and fungal infections.

    Science.gov (United States)

    Subramanyam, Kondeti; Arun, Muthukrishnan; Mariashibu, Thankaraj Salammal; Theboral, Jeevaraj; Rajesh, Manoharan; Singh, Narendra K; Manickavasagam, Markandan; Ganapathi, Andy

    2012-12-01

    Salinity and fungal diseases are the two significant constraints limiting soybean productivity. In order to address these problems, we have transformed soybean cv. Pusa 16 via somatic embryogenesis with salinity induced and apoplastically secreted pathogenesis-related tobacco osmotin (Tbosm) gene using Agrobacterium-mediated genetic transformation. Integration of Tbosm in randomly selected five GUS assay-positive independently transformed soybean plants was confirmed by polymerase chain reaction (PCR) and Southern hybridization. Reverse transcriptase-PCR (RT-PCR) and Western blotting confirmed that the Tbosm was expressed in three of the five transformed soybean plants. Further the Western blotting revealed that the truncated osmotin protein accumulated more in apoplastic fluid. The transformed (T(1)) soybean plants survived up to 200 mM NaCl, whereas non-transformed (NT) plants could withstand till 100 mM and perished at 150 mM NaCl. The biochemical analysis revealed the T(1) soybean plants accumulated higher amount of proline, chlorophyll, APX, CAT, SOD, DHAR, MDHAR, and RWC than NT plants. Leaf gas exchange measurements revealed that T(1) soybean plants maintained higher net photosynthetic rate, CO(2) assimilation, and stomatal conductance than NT plants. The three T(1) soybean plants expressing the osmotin gene also showed resistance against three important fungal pathogens of soybean--Microsphaera diffusa, Septoria glycines and Phakopsora pachyrhizi. The T(1) soybean plants produced 32-35 soybean pods/plant containing 10.3-12.0 g of seeds at 200 mM NaCl, whereas NT plant produced 28.6 soybean pods containing 9.6 g of seeds at 100 mM NaCl. The present investigation clearly shows that expression of Tbosm enhances salinity tolerance and fungal disease resistance in transformed soybean plants.

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

    Science.gov (United States)

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

    2012-02-01

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

  12. Loss of a conserved 7-methylguanosine modification in 16S rRNA confers low-level streptomycin resistance in bacteria.

    Science.gov (United States)

    Okamoto, Susumu; Tamaru, Aki; Nakajima, Chie; Nishimura, Kenji; Tanaka, Yukinori; Tokuyama, Shinji; Suzuki, Yasuhiko; Ochi, Kozo

    2007-02-01

    Streptomycin has been an important drug for the treatment of tuberculosis since its discovery in 1944. But numerous strains of Mycobacterium tuberculosis, the bacterial pathogen that causes tuberculosis, are now streptomycin resistant. Although such resistance is often mediated by mutations within rrs, a 16S rRNA gene or rpsL, which encodes the ribosomal protein S12, these mutations are found in a limited proportion of clinically isolated streptomycin-resistant M. tuberculosis strains. Here we have succeeded in identifying a mutation that confers low-level streptomycin resistance to bacteria, including M. tuberculosis. We found that mutations within the gene gidB confer low-level streptomycin resistance and are an important cause of resistance found in 33% of resistant M. tuberculosis isolates. We further clarified that the gidB gene encodes a conserved 7-methylguanosine (m(7)G) methyltransferase specific for the 16S rRNA, apparently at position G527 located in the so-called 530 loop. Thus, we have identified gidB as a new streptomycin-resistance locus and uncovered a resistance mechanism that is mediated by loss of a conserved m(7)G modification in 16S rRNA. The clinical significance of M. tuberculosis gidB mutation also is noteworthy, as gidB mutations emerge spontaneously at a high frequency of 10(-6) and, once emerged, result in vigorous emergence of high-level streptomycin-resistant mutants at a frequency more than 2000 times greater than that seen in wild-type strains. Further studies on the precise function of GidB may provide a basis for developing strategies to suppress pathogenic bacteria, including M. tuberculosis.

  13. Critical biophysical properties in the Pseudomonas aeruginosa efflux gene regulator MexR are targeted by mutations conferring multidrug resistance.

    Science.gov (United States)

    Andrésen, Cecilia; Jalal, Shah; Aili, Daniel; Wang, Yi; Islam, Sohidul; Jarl, Anngelica; Liedberg, Bo; Wretlind, Bengt; Mårtensson, Lars-Göran; Sunnerhagen, Maria

    2010-04-01

    The self-assembling MexA-MexB-OprM efflux pump system, encoded by the mexO operon, contributes to facile resistance of Pseudomonas aeruginosa by actively extruding multiple antimicrobials. MexR negatively regulates the mexO operon, comprising two adjacent MexR binding sites, and is as such highly targeted by mutations that confer multidrug resistance (MDR). To understand how MDR mutations impair MexR function, we studied MexR-wt as well as a selected set of MDR single mutants distant from the proposed DNA-binding helix. Although DNA affinity and MexA-MexB-OprM repression were both drastically impaired in the selected MexR-MDR mutants, MexR-wt bound its two binding sites in the mexO with high affinity as a dimer. In the MexR-MDR mutants, secondary structure content and oligomerization properties were very similar to MexR-wt despite their lack of DNA binding. Despite this, the MexR-MDR mutants showed highly varying stabilities compared with MexR-wt, suggesting disturbed critical interdomain contacts, because mutations in the DNA-binding domains affected the stability of the dimer region and vice versa. Furthermore, significant ANS binding to MexR-wt in both free and DNA-bound states, together with increased ANS binding in all studied mutants, suggest that a hydrophobic cavity in the dimer region already shown to be involved in regulatory binding is enlarged by MDR mutations. Taken together, we propose that the biophysical MexR properties that are targeted by MDR mutations-stability, domain interactions, and internal hydrophobic surfaces-are also critical for the regulation of MexR DNA binding.

  14. A Naturally Occurring Domestic Cat APOBEC3 Variant Confers Resistance to Feline Immunodeficiency Virus Infection.

    Science.gov (United States)

    Yoshikawa, Rokusuke; Izumi, Taisuke; Yamada, Eri; Nakano, Yusuke; Misawa, Naoko; Ren, Fengrong; Carpenter, Michael A; Ikeda, Terumasa; Münk, Carsten; Harris, Reuben S; Miyazawa, Takayuki; Koyanagi, Yoshio; Sato, Kei

    2016-01-01

    Apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3 (APOBEC3; A3) DNA cytosine deaminases can be incorporated into progeny virions and inhibit lentiviral replication. On the other hand, viral infectivity factor (Vif) of lentiviruses antagonizes A3-mediated antiviral activities by degrading A3 proteins. It is known that domestic cat (Felis catus) APOBEC3Z3 (A3Z3), the ortholog of human APOBEC3H, potently suppresses the infectivity of vif-defective feline immunodeficiency virus (FIV). Although a recent report has shown that domestic cat encodes 7 haplotypes (hap I to hap VII) of A3Z3, the relevance of A3Z3 polymorphism in domestic cats with FIV Vif has not yet been addressed. In this study, we demonstrated that these feline A3Z3 variants suppress vif-defective FIV infectivity. We also revealed that codon 65 of feline A3Z3 is a positively selected site and that A3Z3 hap V is subject to positive selection during evolution. It is particularly noteworthy that feline A3Z3 hap V is resistant to FIV Vif-mediated degradation and still inhibits vif-proficient viral infection. Moreover, the side chain size, but not the hydrophobicity, of the amino acid at position 65 determines the resistance to FIV Vif-mediated degradation. Furthermore, phylogenetic analyses have led to the inference that feline A3Z3 hap V emerged approximately 60,000 years ago. Taken together, these findings suggest that feline A3Z3 hap V may have been selected for escape from an ancestral FIV. This is the first evidence for an evolutionary "arms race" between the domestic cat and its cognate lentivirus. Gene diversity and selective pressure are intriguing topics in the field of evolutionary biology. A direct interaction between a cellular protein and a viral protein can precipitate an evolutionary arms race between host and virus. One example is primate APOBEC3G, which potently restricts the replication of primate lentiviruses (e.g., human immunodeficiency virus type 1 [HIV-1] and simian

  15. Thanatin confers partial resistance against aflatoxigenic fungi in maize (Zea mays).

    Science.gov (United States)

    Schubert, Max; Houdelet, Marcel; Kogel, Karl-Heinz; Fischer, Rainer; Schillberg, Stefan; Nölke, Greta

    2015-10-01

    Aflatoxin-producing fungi can contaminate plants and plant-derived products with carcinogenic secondary metabolites that present a risk to human and animal health. In this study, we investigated the effect of antimicrobial peptides on the major aflatoxigenic fungi Aspergillus flavus and A. parasiticus. In vitro assays with different chemically-synthesized peptides demonstrated that the broad-spectrum peptide thanatin from the spined soldier bug (Podisus maculiventris) had the greatest potential to eliminate aflatoxigenic fungi. The minimal inhibitory concentrations of thanatin against A. flavus and A. parasiticus were 3.13 and 12.5 µM, respectively. A thanatin cDNA was subsequently cloned in a plant expression vector under the control of the ubiquitin-1 promoter allowing the recombinant peptide to be directed to the apoplast in transgenic maize plants. Successful integration of the thanatin expression cassette was confirmed by PCR and expression was demonstrated by semi-quantitative RT-PCR in transgenic maize kernels. Infection assays with maize kernels from T1 transgenic plants showed up to three-fold greater resistance against Aspergillus spp. infections compared to non-transgenic kernels. We demonstrated for the first time that heterologous expression of the antimicrobial peptide thanatin inhibits the growth of Aspergillus spp. in transgenic maize plants offering a solution to protect crops from aflatoxin-producing fungi and the resulting aflatoxin contamination in the field and under storage conditions.

  16. A red and far-red light receptor mutation confers resistance to the herbicide glyphosate

    KAUST Repository

    Sharkhuu, Altanbadralt

    2014-06-01

    Glyphosate is a widely applied broad-spectrum systemic herbicide that inhibits competitively the penultimate enzyme 5-enolpyruvylshikimate 3-phosphate synthase (EPSPS) from the shikimate pathway, thereby causing deleterious effects. A glyphosate-resistant Arabidopsis mutant (gre1) was isolated and genetic analyses indicated that a dysfunctional red (R) and far-red (FR) light receptor, phytochrome B (phyB), caused this phenotype. This finding is consistent with increased glyphosate sensitivity and glyphosate-induced shikimate accumulation in low R:FR light, and the induction of genes encoding enzymes of the shikimate pathway in high R:FR light. Expression of the shikimate pathway genes exhibited diurnal oscillation and this oscillation was altered in the phyB mutant. Furthermore, transcript analysis suggested that this diurnal oscillation was not only dependent on phyB but was also due to circadian regulatory mechanisms. Our data offer an explanation of the well documented observation that glyphosate treatment at various times throughout the day, with their specific composition of light quality and intensity, results in different efficiencies of the herbicide.

  17. A plant Bcl-2-associated athanogene is proteolytically activated to confer fungal resistance

    Directory of Open Access Journals (Sweden)

    Mehdi Kabbage

    2016-04-01

    Full Text Available The Bcl-2-associated athanogene (BAG family is a multifunctional group of proteins involved in numerous cellular functions ranging from apoptosis to tumorigenesis. These proteins are evolutionarily conserved and encode a characteristic region known as the BAG domain. BAGs function as adapter proteins forming complexes with signaling molecules and molecular chaperones. In humans, a role for BAG proteins has been suggested in tumor growth, HIV infection, and neurodegenerative diseases; as a result, the BAGs are attractive targets for therapeutic interventions, and their expression in cells may serve as a predictive tool for disease development. The Arabidopsis genome contains seven homologs of BAG family proteins (Figure 1, including four with a domain organization similar to animal BAGs (BAG1-4. The remaining three members (BAG5-7 contain a predicted calmodulin-binding motif near the BAG domain, a feature unique to plant BAG proteins that possibly reflects divergent mechanisms associated with plant-specific functions. As reported for animal BAGs, plant BAGs also regulate several stress and developmental processes (Figure 2. The recent article by Li et al. focuses on the role of BAG6 in plant innate immunity. This study shows that BAG6 plays a key role in basal plant defense against fungal pathogens. Importantly, this work further shows that BAG6 is proteolytically activated to induce autophagic cell death and resistance in plants. This finding underscores the importance of proteases in the execution of plant cell death, yet little is known about proteases and their substrates in plants.

  18. A red and far-red light receptor mutation confers resistance to the herbicide glyphosate

    Science.gov (United States)

    Sharkhuu, Altanbadralt; Narasimhan, Meena L; Merzaban, Jasmeen S; Bressan, Ray A; Weller, Steve; Gehring, Chris

    2014-01-01

    Glyphosate is a widely applied broad-spectrum systemic herbicide that inhibits competitively the penultimate enzyme 5-enolpyruvylshikimate 3-phosphate synthase (EPSPS) from the shikimate pathway, thereby causing deleterious effects. A glyphosate-resistant Arabidopsis mutant (gre1) was isolated and genetic analyses indicated that a dysfunctional red (R) and far-red (FR) light receptor, phytochrome B (phyB), caused this phenotype. This finding is consistent with increased glyphosate sensitivity and glyphosate-induced shikimate accumulation in low R:FR light, and the induction of genes encoding enzymes of the shikimate pathway in high R:FR light. Expression of the shikimate pathway genes exhibited diurnal oscillation and this oscillation was altered in the phyB mutant. Furthermore, transcript analysis suggested that this diurnal oscillation was not only dependent on phyB but was also due to circadian regulatory mechanisms. Our data offer an explanation of the well documented observation that glyphosate treatment at various times throughout the day, with their specific composition of light quality and intensity, results in different efficiencies of the herbicide. PMID:24654847

  19. Ectopic Expression of JcWRKY Confers Enhanced Resistance in Transgenic Tobacco Against Macrophomina phaseolina.

    Science.gov (United States)

    Agarwal, Parinita; Patel, Khantika; Agarwal, Pradeep K

    2018-04-01

    Plants possess an innate immune system comprising of a complex network of closely regulated defense responses involving differential gene expression mediated by transcription factors (TFs). The WRKYs comprise of an important plant-specific TF family, which is involved in regulation of biotic and abiotic defenses. The overexpression of JcWRKY resulted in improved resistance in transgenic tobacco against Macrophomina phaseolina. The production of reactive oxygen species (ROS) and its detoxification through antioxidative system in the transgenics facilitates defense against Macrophomina. The enhanced catalase activity on Macrophomina infection limits the spread of infection. The transcript expression of antioxidative enzymes gene (CAT and SOD) and salicylic acid (SA) biosynthetic gene ICS1 showed upregulation during Macrophomina infection and combinatorial stress. The enhanced transcript of pathogenesis-related genes PR-1 indicates the accumulation of SA during different stresses. The PR-2 and PR-5 highlight the activation of defense responses comprising of activation of hydrolytic cleavage of glucanases and thaumatin-like proteins causing disruption of fungal cells. The ROS homeostasis in coordination with signaling molecules regulate the defense responses and inhibit fungal growth.

  20. E2F8 confers cisplatin resistance to ER+ breast cancer cells via transcriptionally activating MASTL.

    Science.gov (United States)

    Tian, Jianjun; Lin, Yuting; Yu, Jianhua

    2017-08-01

    MASTL (microtubule-associated serine/threonine kinase-like) is a critical kinase modulating mitotic entry. In this study, we investigated the mechanism of its dysregulation in breast cancer and its involvement in cisplatin resistance in ER+ breast cancer cells. Data mining in Kaplan-Meier Plotter showed that high MASTL expression was associated with worse distant metastasis free survival (DMFS) and relapse free survival (RFS) in ER+ breast cancer patients. In TCGA breast cancer cohort (TCGA-BRCA), MASTL was strongly co-upregulated with E2F8. High E2F8 expression was also strongly associated with unfavorable DMFS and RFS in ER+ breast cancer patients. Promoter scanning in JASPAR Database showed that the MASTL promoter region has a highly possible E2F8 binding site upstream the TSS site. The following western blot, dual luciferase assay and ChIP-qPCR validated this binding site. In MCF-7 cells, E2F8 overexpression alleviated cisplatin induced cell apoptosis by shortening G2/M arrest and promoting mitotic entry, the effect of which was largely canceled by inhibiting MASTL. Therefore, we infer that E2F8 can shorten cisplatin induced G2/M arrest by promoting MASTL mediated mitotic progression in ER+ breast cancer cells. These findings might help to explain why high MASTL or high E2F8 expression is associated with worse RFS in ER+ breast cancer. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  1. FEN1 promotes tumor progression and confers cisplatin resistance in non-small-cell lung cancer.

    Science.gov (United States)

    He, Lingfeng; Luo, Libo; Zhu, Hong; Yang, Huan; Zhang, Yilan; Wu, Huan; Sun, Hongfang; Jiang, Feng; Kathera, Chandra S; Liu, Lingjie; Zhuang, Ziheng; Chen, Haoyan; Pan, Feiyan; Hu, Zhigang; Zhang, Jing; Guo, Zhigang

    2017-06-01

    Lung cancer is one of the leading causes of cancer mortality worldwide. The therapeutic effect of chemotherapy is limited due to the resistance of cancer cells, which remains a challenge in cancer therapeutics. In this work, we found that flap endonuclease 1 (FEN1) is overexpressed in lung cancer cells. FEN1 is a major component of the base excision repair pathway for DNA repair systems and plays important roles in maintaining genomic stability through DNA replication and repair. We showed that FEN1 is critical for the rapid proliferation of lung cancer cells. Suppression of FEN1 resulted in decreased DNA replication and accumulation of DNA damage, which subsequently induced apoptosis. Manipulating the amount of FEN1 altered the response of lung cancer cells to chemotherapeutic drugs. A small-molecule inhibitor (C20) was used to target FEN1 and this enhanced the therapeutic effect of cisplatin. The FEN1 inhibitor significantly suppressed cell proliferation and induced DNA damage in lung cancer cells. In mouse models, the FEN1 inhibitor sensitized lung cancer cells to a DNA damage-inducing agent and efficiently suppressed cancer progression in combination with cisplatin treatment. Our study suggests that targeting FEN1 may be a novel and efficient strategy for a tumor-targeting therapy for lung cancer. © 2017 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.

  2. An antidiabetic polyherbal phytomedicine confers stress resistance and extends lifespan in Caenorhabditis elegans.

    Science.gov (United States)

    Rathor, Laxmi; Pant, Aakanksha; Awasthi, Harshika; Mani, Dayanandan; Pandey, Rakesh

    2017-02-01

    An Ayurvedic polyherbal extract (PHE) comprising six herbs viz. Berberis aristata, Cyperus rotundus, Cedrus deodara, Emblica officinalis, Terminalia chebula and Terminalia bellirica is mentioned as an effective anti-hyperglycemic agent in 'Charaka Samhita', the classical text of Ayurveda. Previously, antidiabetic drug metformin was found to elicit antiaging effects and PHE was also found to exhibit antidiabetic effects in humans. Therefore, we screened it for its in vivo antioxidant antiaging effect on stress and lifespan using human homologous Caenorhabditis elegans model system. The effect on aging is evaluated by studying effect of PHE on mean survival in worms. The stress modulatory potential was assessed by quantification of intracellular ROS level, autofluorescent age pigment lipofuscin, oxidative and thermal stress assays. Additionally, stress response was quantified using gene reporter assays. The 0.01 µg/ml dose of PHE was able to enhance mean lifespan by 16.09% (P elegans. Furthermore, PHE treated worms demonstrated oxidative stress resistance in both wild type and stress hypersensitive mev-1 mutant along with upregulation of stress response genes sod-3 and gst-4. The delayed aging under stress can be attributed to its direct reactive oxygen species-scavenging activity and regulation of some age associated genes like daf-2, daf-16, skn-1, sod-3 and gst-4 in wild-type worms. Additonally, PHE delayed age related paralysis phenotype in CL4176 transgenic worms. Altogether, our results suggest PHE significantly improves the oxidative stress and life span in C. elegans. Overall the present study suggests this polyherbal formulation might play important role in regultaing aging and related complications like diabetes.

  3. Chronic Iron Limitation Confers Transient Resistance to Oxidative Stress in Marine Diatoms1

    Science.gov (United States)

    Graff van Creveld, Shiri; Rosenwasser, Shilo; Vardi, Assaf

    2016-01-01

    Diatoms are single-celled, photosynthetic, bloom-forming algae that are responsible for at least 20% of global primary production. Nevertheless, more than 30% of the oceans are considered “ocean deserts” due to iron limitation. We used the diatom Phaeodactylum tricornutum as a model system to explore diatom’s response to iron limitation and its interplay with susceptibility to oxidative stress. By analyzing physiological parameters and proteome profiling, we defined two distinct phases: short-term (5 d, phase II) iron limitation. While at phase I no significant changes in physiological parameters were observed, molecular markers for iron starvation, such as Iron Starvation Induced Protein and flavodoxin, were highly up-regulated. At phase II, down-regulation of numerous iron-containing proteins was detected in parallel to reduction in growth rate, chlorophyll content, photosynthetic activity, respiration rate, and antioxidant capacity. Intriguingly, while application of oxidative stress to phase I and II iron-limited cells similarly oxidized the reduced glutathione (GSH) pool, phase II iron limitation exhibited transient resistance to oxidative stress, despite the down regulation of many antioxidant proteins. By comparing proteomic profiles of P. tricornutum under iron limitation and metatranscriptomic data of an iron enrichment experiment conducted in the Pacific Ocean, we propose that iron-limited cells in the natural environment resemble the phase II metabolic state. These results provide insights into the trade-off between optimal growth rate and susceptibility to oxidative stress in the response of diatoms to iron quota in the marine environment. PMID:27503604

  4. Chronic Iron Limitation Confers Transient Resistance to Oxidative Stress in Marine Diatoms.

    Science.gov (United States)

    Graff van Creveld, Shiri; Rosenwasser, Shilo; Levin, Yishai; Vardi, Assaf

    2016-10-01

    Diatoms are single-celled, photosynthetic, bloom-forming algae that are responsible for at least 20% of global primary production. Nevertheless, more than 30% of the oceans are considered "ocean deserts" due to iron limitation. We used the diatom Phaeodactylum tricornutum as a model system to explore diatom's response to iron limitation and its interplay with susceptibility to oxidative stress. By analyzing physiological parameters and proteome profiling, we defined two distinct phases: short-term (5 d, phase II) iron limitation. While at phase I no significant changes in physiological parameters were observed, molecular markers for iron starvation, such as Iron Starvation Induced Protein and flavodoxin, were highly up-regulated. At phase II, down-regulation of numerous iron-containing proteins was detected in parallel to reduction in growth rate, chlorophyll content, photosynthetic activity, respiration rate, and antioxidant capacity. Intriguingly, while application of oxidative stress to phase I and II iron-limited cells similarly oxidized the reduced glutathione (GSH) pool, phase II iron limitation exhibited transient resistance to oxidative stress, despite the down regulation of many antioxidant proteins. By comparing proteomic profiles of P. tricornutum under iron limitation and metatranscriptomic data of an iron enrichment experiment conducted in the Pacific Ocean, we propose that iron-limited cells in the natural environment resemble the phase II metabolic state. These results provide insights into the trade-off between optimal growth rate and susceptibility to oxidative stress in the response of diatoms to iron quota in the marine environment. © 2016 American Society of Plant Biologists. All Rights Reserved.

  5. Endogenous ω-3 polyunsaturated fatty acid production confers resistance to obesity, dyslipidemia, and diabetes in mice.

    Science.gov (United States)

    Li, Jie; Li, Fanghong R; Wei, Dong; Jia, Wei; Kang, Jing X; Stefanovic-Racic, Maja; Dai, Yifan; Zhao, Allan Z

    2014-08-01

    Despite the well-documented health benefits of ω-3 polyunsaturated fatty acids (PUFAs), their use in clinical management of hyperglycemia and obesity has shown little success. To better define the mechanisms of ω-3 PUFAs in regulating energy balance and insulin sensitivity, we deployed a transgenic mouse model capable of endogenously producing ω-3 PUFAs while reducing ω-6 PUFAs owing to the expression of a Caenorhabditis elegans fat-1 gene encoding an ω-3 fatty acid desaturase. When challenged with high-fat diets, fat-1 mice strongly resisted obesity, diabetes, hypercholesterolemia, and hepatic steatosis. Endogenous elevation of ω-3 PUFAs and reduction of ω-6 PUFAs did not alter the amount of food intake but led to increased energy expenditure in the fat-1 mice. The requirements for the levels of ω-3 PUFAs as well as the ω-6/ω-3 ratios in controlling blood glucose and obesity are much more stringent than those in lipid metabolism. These metabolic phenotypes were accompanied by attenuation of the inflammatory state because tissue levels of prostaglandin E2, leukotriene B4, monocyte chemoattractant protein-1, and TNF-α were significantly decreased. TNF-α-induced nuclear factor-κB signaling was almost completely abolished. Consistent with the reduction in chronic inflammation and a significant increase in peroxisome proliferator-activated receptor-γ activity in the fat-1 liver tissue, hepatic insulin signaling was sharply elevated. The activities of prolipogenic regulators, such as liver X receptor, stearoyl-CoA desaturase-1, and sterol regulatory element binding protein-1 were sharply decreased, whereas the activity of peroxisome proliferator-activated receptor-α, a nuclear receptor that facilitates lipid β-oxidation, was markedly increased. Thus, endogenous conversion of ω-6 to ω-3 PUFAs via fat-1 strongly protects against obesity, diabetes, inflammation, and dyslipidemia and may represent a novel therapeutic modality to treat these prevalent

  6. Transgenic potato plants expressing cry3A gene confer resistance to Colorado potato beetle.

    Science.gov (United States)

    Mi, Xiaoxiao; Ji, Xiangzhuo; Yang, Jiangwei; Liang, Lina; Si, Huaijun; Wu, Jiahe; Zhang, Ning; Wang, Di

    2015-07-01

    The Colorado potato beetle (Leptinotarsa decemlineata Say, CPB) is a fatal pest, which is a quarantine pest in China. The CPB has now invaded the Xinjiang Uygur Autonomous Region and is constantly spreading eastward in China. In this study, we developed transgenic potato plants expressing cry3A gene. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis indicated that the cry3A gene expressed in leaves, stems and roots of the transgenic plants under the control of CaMV 35S promoter, while they expressed only in leaves and stems under the control of potato leaf and stem-specific promoter ST-LS1. The mortality of the larvae was higher (28% and 36%) on the transgenic plant line 35S1 on the 3rd and 4th days, and on ST3 (48%) on the 5th day after inoculation with instar larvae. Insect biomass accumulation on the foliage of the transgenic plant lines 35S1, 35S2 and ST3 was significantly lower (0.42%, 0.43% and 0.42%). Foliage consumption was lowest on transgenic lines 35S8 and ST2 among all plant foliage (7.47 mg/larvae/day and 12.46 mg/larvae/day). The different transgenic plant foliages had varied inhibition to larval growth. The survivors on the transgenic lines obviously were smaller than their original size and extremely weak. The transgenic potato plants with CPB resistance could be used to develop germplasms or varieties for controlling CPB damage and halting its spread in China. Copyright © 2015 Académie des sciences. Published by Elsevier SAS. All rights reserved.

  7. Genetic characterisation of antimicrobial resistance and virulence genes in Staphylococcus aureus isolated from commercial broiler chickens in the Durban metropolitan area, South Africa

    Directory of Open Access Journals (Sweden)

    Nelisiwe Mkize

    2017-05-01

    Full Text Available Antimicrobial resistance of Staphylococcus aureus in human and veterinary medicine is a serious worldwide problem. The aim of this study was to investigate the prevalence of S. aureus in commercial broiler chickens as well as to establish antimicrobial susceptibility and the distribution of genetic determinants conferring resistance and virulence. One hundred and ninety-four samples were aseptically collected from broiler chicken slaughterhouses and retail outlets around the Durban metropolitan area in South Africa. Microbiological and molecular methods were used to detect the presence of S. aureus as well as its resistance- and virulence-associated genes. Polymerase chain reaction (PCR was used to confirm the presence of S. aureus by amplifying the nuc gene. Approximately 54% of 194 samples were positive for S. aureus. The disc diffusion technique was used to investigate antimicrobial susceptibility profiles of the S. aureus isolates to a battery of 10 antimicrobial agents, namely ampicillin, chloramphenicol, gentamicin, erythromycin, cefoxitin, kanamycin, streptomycin, tetracycline, vancomycin and trimethoprim. The results demonstrated that S. aureus isolates of abattoir origin had a high level (79.4% of resistance to tetracycline, followed by ampicillin, vancomycin, cefoxitin, trimethoprim, erythromycin and streptomycin with resistance rates of 65.1%, 61.9%, 60.3%, 58.7%, 57.1% and 46.0%, respectively. Staphylococcus aureus isolates of retail origin exhibited higher antimicrobial resistance prevalence rates than those of abattoir origin. Tetracycline had the highest resistance rate (100%, followed by cefoxitin (91.7%, erythromycin (83.3%, streptomycin (83.3% and kanamycin (66.7%. All isolates were resistant to two or more antimicrobial agents. Out of the four virulence genes that were screened, only two were detected (coagulase and protein A; however, their prevalence rates were very low. All antimicrobial resistance genes screened were

  8. High-resolution mapping reveals linkage between genes in common bean cultivar Ouro Negro conferring resistance to the rust, anthracnose, and angular leaf spot diseases.

    Science.gov (United States)

    Valentini, Giseli; Gonçalves-Vidigal, Maria Celeste; Hurtado-Gonzales, Oscar P; de Lima Castro, Sandra Aparecida; Cregan, Perry B; Song, Qijian; Pastor-Corrales, Marcial A

    2017-08-01

    Co-segregation analysis and high-throughput genotyping using SNP, SSR, and KASP markers demonstrated genetic linkage between Ur-14 and Co-3 4 /Phg-3 loci conferring resistance to the rust, anthracnose and angular leaf spot diseases of common bean. Rust, anthracnose, and angular leaf spot are major diseases of common bean in the Americas and Africa. The cultivar Ouro Negro has the Ur-14 gene that confers broad spectrum resistance to rust and the gene cluster Co-3 4 /Phg-3 containing two tightly linked genes conferring resistance to anthracnose and angular leaf spot, respectively. We used co-segregation analysis and high-throughput genotyping of 179 F 2:3 families from the Rudá (susceptible) × Ouro Negro (resistant) cross-phenotyped separately with races of the rust and anthracnose pathogens. The results confirmed that Ur-14 and Co-3 4 /Phg-3 cluster in Ouro Negro conferred resistance to rust and anthracnose, respectively, and that Ur-14 and the Co-3 4 /Phg-3 cluster were closely linked. Genotyping the F 2:3 families, first with 5398 SNPs on the Illumina BeadChip BARCBEAN6K_3 and with 15 SSR, and eight KASP markers, specifically designed for the candidate region containing Ur-14 and Co-3 4 /Phg-3, permitted the creation of a high-resolution genetic linkage map which revealed that Ur-14 was positioned at 2.2 cM from Co-3 4 /Phg-3 on the short arm of chromosome Pv04 of the common bean genome. Five flanking SSR markers were tightly linked at 0.1 and 0.2 cM from Ur-14, and two flanking KASP markers were tightly linked at 0.1 and 0.3 cM from Co-3 4 /Phg-3. Many other SSR, SNP, and KASP markers were also linked to these genes. These markers will be useful for the development of common bean cultivars combining the important Ur-14 and Co-3 4 /Phg-3 genes conferring resistance to three of the most destructive diseases of common bean.

  9. Cereal cyst nematode resistance conferred by the Cre7 gene from Aegilops triuncialis and its relationship with Cre genes from Australian wheat cultivars

    OpenAIRE

    Montes, Maria Jesus; Andrés, María Fe; Sin, E.; Lopez Braña, Isidoro; Martín-Sánchez, J.A.; Romero, M.D.; Delibes Castro, Angeles

    2008-01-01

    Cereal cyst nematode (CCN; Heterodera avenae Woll.) is a root pathogen of cereal crops that can cause severe yield losses in wheat (Triticum aestivum). Differential host–nematode interactions occur in wheat cultivars carrying different CCN resistance (Cre) genes. The objective of this study was to determine the CCN resistance conferred by the Cre7 gene from Aegilops triuncialis in a 42-chromosome introgression line and to assess the effects of the Cre1, Cre3, Cre4, and Cre8 genes present in A...

  10. Resistance to nitrofurantoin and UV-irradiation in recA; uvrA; and uvrA, lexA, Escherichia coli mutants conferred by an R-plasmid from an Escherichia coli clinical isolate

    International Nuclear Information System (INIS)

    Obaseiki-Ebor, E.E.

    1984-01-01

    There have been some reports of R-plasmids conferring nitrofuran resistance by decreasing the reduction of nitrofurantoin. The mechanism by which these R-plasmids mediate nitrofurantoin resistance is still not properly understood. Since DNA repair mutants are very sensitive to nitrofurantoin, it was therefore of interest to see whether R-plasmids conferring nitrofurantoin resistance affected the nitrofurantoin sensitivity of recA; uvrA and uvrA, lexA strains of E. coli K-12. Protection against UV-irradiation was also estimated. The experiments showed that the nitrofurantoin resistance conferred by R-plasmid pBN105 was not due to defective nitrofurantoin reduction or altered permeability of the cell. Because it is known that repair-deficient bacteria have increased susceptibility to nitrofurantoin, it may be suggested that the mechanisms of UV and nitrofurantoin protection conferred by pBN105 to the DNA repair mutant strains are related. (Auth.)

  11. From multidrug-resistant to extensively drug-resistant tuberculosis in Lisbon, Portugal: the stepwise mode of resistance acquisition.

    Science.gov (United States)

    Perdigão, João; Macedo, Rita; Silva, Carla; Machado, Diana; Couto, Isabel; Viveiros, Miguel; Jordao, Luisa; Portugal, Isabel

    2013-01-01

    The development and transmission of extensively drug-resistant (XDR) tuberculosis (TB) constitutes a serious threat to the effective control of TB in several countries. Here, in an attempt to further elucidate the dynamics of the acquisition of resistance to second-line drugs and investigate an eventual role for eis promoter mutations in aminoglycoside resistance, we have studied a set of multidrug-resistant (MDR)/XDR-TB isolates circulating in Lisbon, Portugal. Forty-four MDR-TB or XDR-TB isolates were genotyped and screened for mutations in genes associated with second-line drug resistance, namely tlyA, gyrA, rrs and eis. The most prevalent mutations found in each gene were Ins755GT in tlyA, A1401G in rrs, G-10A in eis and S91P in gyrA. Additionally, two genetic clusters were found in this study: Lisboa3 and Q1. The characteristic mutational profile found among recent XDR-TB circulating in Lisbon was also found in MDR-TB strains isolated in the 1990s. Also investigated was the resistance level conferred by eis G-10A mutations, revealing that eis G-10A mutations may result in amikacin resistance undetectable by widely used phenotypic assays. The analysis of the distribution of the mutations found by genetic clustering showed that in the Q1 cluster, two mutations, gyrA D94A and rrs A1401G, were enough to ensure development of XDR-TB from an MDR strain. Moreover, in the Lisboa3 cluster it was possible to elaborate a model in which the development of low-level kanamycin resistance was at the origin of the emergence of XDR-TB strains that can be discriminated by tlyA mutations.

  12. Identification of QTL conferring resistance to stripe rust (Puccinia striiformis f. sp. hordei) and leaf rust (Puccinia hordei) in barley using nested association mapping (NAM).

    Science.gov (United States)

    Vatter, Thomas; Maurer, Andreas; Perovic, Dragan; Kopahnke, Doris; Pillen, Klaus; Ordon, Frank

    2018-01-01

    The biotrophic rust fungi Puccinia hordei and Puccinia striiformis are important barley pathogens with the potential to cause high yield losses through an epidemic spread. The identification of QTL conferring resistance to these pathogens is the basis for targeted breeding approaches aiming to improve stripe rust and leaf rust resistance of modern cultivars. Exploiting the allelic richness of wild barley accessions proved to be a valuable tool to broaden the genetic base of resistance of barley cultivars. In this study, SNP-based nested association mapping (NAM) was performed to map stripe rust and leaf rust resistance QTL in the barley NAM population HEB-25, comprising 1,420 lines derived from BC1S3 generation. By scoring the percentage of infected leaf area, followed by calculation of the area under the disease progress curve and the average ordinate during a two-year field trial, a large variability of resistance across and within HEB-25 families was observed. NAM based on 5,715 informative SNPs resulted in the identification of twelve and eleven robust QTL for resistance against stripe rust and leaf rust, respectively. Out of these, eight QTL for stripe rust and two QTL for leaf rust are considered novel showing no overlap with previously reported resistance QTL. Overall, resistance to both pathogens in HEB-25 is most likely due to the accumulation of numerous small effect loci. In addition, the NAM results indicate that the 25 wild donor QTL alleles present in HEB-25 strongly differ in regard to their individual effect on rust resistance. In future, the NAM concept will allow to select and combine individual wild barley alleles from different HEB parents to increase rust resistance in barley. The HEB-25 results will support to unravel the genetic basis of rust resistance in barley, and to improve resistance against stripe rust and leaf rust of modern barley cultivars.

  13. Identification of QTL conferring resistance to stripe rust (Puccinia striiformis f. sp. hordei) and leaf rust (Puccinia hordei) in barley using nested association mapping (NAM)

    Science.gov (United States)

    Vatter, Thomas; Maurer, Andreas; Perovic, Dragan; Kopahnke, Doris; Pillen, Klaus

    2018-01-01

    The biotrophic rust fungi Puccinia hordei and Puccinia striiformis are important barley pathogens with the potential to cause high yield losses through an epidemic spread. The identification of QTL conferring resistance to these pathogens is the basis for targeted breeding approaches aiming to improve stripe rust and leaf rust resistance of modern cultivars. Exploiting the allelic richness of wild barley accessions proved to be a valuable tool to broaden the genetic base of resistance of barley cultivars. In this study, SNP-based nested association mapping (NAM) was performed to map stripe rust and leaf rust resistance QTL in the barley NAM population HEB-25, comprising 1,420 lines derived from BC1S3 generation. By scoring the percentage of infected leaf area, followed by calculation of the area under the disease progress curve and the average ordinate during a two-year field trial, a large variability of resistance across and within HEB-25 families was observed. NAM based on 5,715 informative SNPs resulted in the identification of twelve and eleven robust QTL for resistance against stripe rust and leaf rust, respectively. Out of these, eight QTL for stripe rust and two QTL for leaf rust are considered novel showing no overlap with previously reported resistance QTL. Overall, resistance to both pathogens in HEB-25 is most likely due to the accumulation of numerous small effect loci. In addition, the NAM results indicate that the 25 wild donor QTL alleles present in HEB-25 strongly differ in regard to their individual effect on rust resistance. In future, the NAM concept will allow to select and combine individual wild barley alleles from different HEB parents to increase rust resistance in barley. The HEB-25 results will support to unravel the genetic basis of rust resistance in barley, and to improve resistance against stripe rust and leaf rust of modern barley cultivars. PMID:29370232

  14. Identification of QTL conferring resistance to stripe rust (Puccinia striiformis f. sp. hordei and leaf rust (Puccinia hordei in barley using nested association mapping (NAM.

    Directory of Open Access Journals (Sweden)

    Thomas Vatter

    Full Text Available The biotrophic rust fungi Puccinia hordei and Puccinia striiformis are important barley pathogens with the potential to cause high yield losses through an epidemic spread. The identification of QTL conferring resistance to these pathogens is the basis for targeted breeding approaches aiming to improve stripe rust and leaf rust resistance of modern cultivars. Exploiting the allelic richness of wild barley accessions proved to be a valuable tool to broaden the genetic base of resistance of barley cultivars. In this study, SNP-based nested association mapping (NAM was performed to map stripe rust and leaf rust resistance QTL in the barley NAM population HEB-25, comprising 1,420 lines derived from BC1S3 generation. By scoring the percentage of infected leaf area, followed by calculation of the area under the disease progress curve and the average ordinate during a two-year field trial, a large variability of resistance across and within HEB-25 families was observed. NAM based on 5,715 informative SNPs resulted in the identification of twelve and eleven robust QTL for resistance against stripe rust and leaf rust, respectively. Out of these, eight QTL for stripe rust and two QTL for leaf rust are considered novel showing no overlap with previously reported resistance QTL. Overall, resistance to both pathogens in HEB-25 is most likely due to the accumulation of numerous small effect loci. In addition, the NAM results indicate that the 25 wild donor QTL alleles present in HEB-25 strongly differ in regard to their individual effect on rust resistance. In future, the NAM concept will allow to select and combine individual wild barley alleles from different HEB parents to increase rust resistance in barley. The HEB-25 results will support to unravel the genetic basis of rust resistance in barley, and to improve resistance against stripe rust and leaf rust of modern barley cultivars.

  15. The Effect of Kanamycin and Tetracycline on Growth and Photosynthetic Activity of Two Chlorophyte Algae

    Directory of Open Access Journals (Sweden)

    Khawaja Muhammad Imran Bashir

    2016-01-01

    Full Text Available Antibiotics are routinely used in microalgae culture screening, stock culture maintenance, and genetic transformation. By studying the effect of antibiotics on microalgae growth, we can estimate the least value to inhibit growth of undesired pathogens in algal culture. We studied the effect of kanamycin and tetracycline on the growth and photosynthetic activity of two chlorophyte microalgae, Dictyosphaerium pulchellum and Micractinium pusillum. We measured CFU mL−1 on agar plates, optical density, fluorescence yields, and photosynthetic inhibition. Our results showed a significant effect of kan and tet on the tested microalgae species except tet, which showed a minor effect on M. pusillum. Both antibiotics are believed to interact with the protein synthesis machinery; hence, the inhibitory effect of the tested antibiotics was further confirmed by isolation and quantification of the whole cell protein. A significant reduction in protein quantity was observed at concentrations more than 5 mg L−1, except M. pusillum, which showed only a slight reduction in protein quantity even at the maximum tested concentration of tet (30 mg L−1. This study can further aid in aquaculture industry, for the maintenance of the microalgae stock cultures and it can also help the microalgae genetic engineers in the construction of molecular markers.

  16. Preparation and Microbiological Evaluation of Amphiphilic Kanamycin-Lipoamino Acid Ion-Pairs

    Directory of Open Access Journals (Sweden)

    Rosario Pignatello

    2014-05-01

    Full Text Available Amphiphilic ion-pairs of kanamycin (KAN were prepared by evaporation of a water-ethanol co-solution of KAN base and a lipoamino acid bearing a 12-carbon atoms alkyl side chain (LAA12, at different molar ratios. Infrared spectroscopy confirmed the structure of ion-pairs, while differential scanning calorimetry (DSC and powder X-ray diffractometry (PXRD studies supported the formation of new saline species with a different crystalline structure than the starting components. The solubility pattern shown in a range of both aqueous and organic solvents confirmed that the ion-pairs possess an amphiphilic character. The LAA12 counter-ion showed not to improve the antibacterial activity of KAN, suggesting that such chemical strategy is not able to favor the penetration of this drug inside the bacteria cells. Nevertheless, a slight improving, i.e., a one-fold dilution, was observed in E. coli. The present study can also serve as the basis for a further evaluation of LAA ion-pairing of antibiotics, as a means to improve the loading of hydrophilic drugs into lipid-based nanocarriers.

  17. Alteration of TEAD1 expression levels confers apoptotic resistance through the transcriptional up-regulation of Livin.

    Directory of Open Access Journals (Sweden)

    André Landin Malt

    Full Text Available BACKGROUND: TEA domain (TEAD proteins are highly conserved transcription factors involved in embryonic development and differentiation of various tissues. More recently, emerging evidences for a contribution of these proteins towards apoptosis and cell proliferation regulation have also been proposed. These effects appear to be mediated by the interaction between TEAD and its co-activator Yes-Associated Protein (YAP, the downstream effector of the Hippo tumour suppressor pathway. METHODOLOGY/PRINCIPAL FINDINGS: We further investigated the mechanisms underlying TEAD-mediated apoptosis regulation and showed that overexpression or RNAi-mediated silencing of the TEAD1 protein is sufficient to protect mammalian cell lines from induced apoptosis, suggesting a proapoptotic function for TEAD1 and a non physiological cytoprotective effect for overexpressed TEAD1. Moreover we show that the apoptotic resistance conferred by altered TEAD1 expression is mediated by the transcriptional up-regulation of Livin, a member of the Inhibitor of Apoptosis Protein (IAP family. In addition, we show that overexpression of a repressive form of TEAD1 can induce Livin up-regulation, indicating that the effect of TEAD1 on Livin expression is indirect and favoring a model in which TEAD1 activates a repressor of Livin by interacting with a limiting cofactor that gets titrated upon TEAD1 up-regulation. Interestingly, we show that overexpression of a mutated form of TEAD1 (Y421H implicated in Sveinsson's chorioretinal atrophy that strongly reduces its interaction with YAP as well as its activation, can induce Livin expression and protect cells from induced apoptosis, suggesting that YAP is not the cofactor involved in this process. CONCLUSIONS/SIGNIFICANCE: Taken together our data reveal a new, Livin-dependent, apoptotic role for TEAD1 in mammals and provide mechanistic insight downstream of TEAD1 deregulation in cancers.

  18. Induction of Streptomycin Uptake in Resistant Strains of Escherichia coli

    Science.gov (United States)

    Höltje, Joachim-Volker

    1979-01-01

    Different streptomycin-resistant strains of Escherichia coli, including an R100 plasmid-carrying strain of E. coli W3110, the ribosomally resistant mutant SM10, and the spontaneous revertant from dependence to independence d1023, exhibited poor accumulation capacity for aminoglycoside antibiotics. This was due to a failure of these mutants to induce the general polyamine transport system that is utilized by streptomycin to enter the cell. It is shown that the aminoglycoside kanamycin, which is effective on these streptomycin-resistant strains, was capable of inducing the uptake of streptomycin, thus giving rise to streptomycin accumulation up to wild-type levels. Plasmid-determined resistance, which has been speculated to be the result of a blockage of the uptake system by modified antibiotic molecules, cannot be overcome by the induction of streptomycin transport. Increase in permeability of the antibiotic does not affect the susceptibility of the bacteria. It is shown that all of the antibiotic taken up was enzymatically modified. R-plasmid-conferred resistance to aminoglycosides is therefore explained by the inactivation of the antibiotic entering the bacterial cell. PMID:371542

  19. Invasive breast cancer induces laminin-332 upregulation and integrin β4 neoexpression in myofibroblasts to confer an anoikis-resistant phenotype during tissue remodeling.

    Science.gov (United States)

    Kim, Baek Gil; Gao, Ming-Qing; Choi, Yoon Pyo; Kang, Suki; Park, Haeng Ran; Kang, Kyu Sub; Cho, Nam Hoon

    2012-06-06

    Although development of anoikis-resistant myofibroblasts during tissue remodeling is known to be associated with tumor invasion, the mechanism by which myofibroblasts become resistant to anoikis is unknown. We previously demonstrated laminin-332 upregulation in the fibrosis around invasive ductal carcinoma (IDC). Because laminin-332 promotes cell survival through binding to integrins, we hypothesized that invasive breast cancer cells confer an anoikis-resistant phenotype on myofibroblasts by upregulating laminin-332 expression during tissue remodeling. Here, we demonstrate that invasive breast cancer cells induce laminin-332 upregulation and integrin β4 neoexpression in myofibroblasts to confer an anoikis-resistant phenotype. Three types of fibroblasts were isolated from the tumor burden, the fibrosis, and normal tissue of patients with early stage IDC (less than 10 mm diameter), designated cancer-associated fibroblasts (CAFs), interface fibroblasts (InFs), and normal breast fibroblasts (NBFs), respectively. To investigate direct and indirect crosstalk with tumor cells, fibroblasts were co-cultured with invasive MDA-MB-231 or noninvasive MCF7 cells or in conditioned medium. Anoikis resistance of fibroblasts was measured by cell viability and caspase-3 activity after incubation on poly-HEMA coated plates for 72 hours. Involvement of laminin-332/integrin α3β1 or α6β4 signaling in anoikis resistance was confirmed by treatment with purified laminin-332 or blocking antibodies against laminin-332, integrin β1, or integrin β4. MDA-MB-231 cells induced laminin-332 upregulation and integrin β4 neoexpression in fibroblasts, leading to anoikis resistance. InFs showed a higher endogenous level of laminin-332 than did CAFs and NBFs. After stimulation with MDA-MB-231-conditioned medium, laminin-332 expression of InFs was dramatically increased and maintained under anoikis conditions. Laminin-332 upregulation was also observed in CAFs and NBFs, but at a lower level than

  20. Autocrine production of TGF-β confers resistance to apoptosis after an epithelial-mesenchymal transition process in hepatocytes: Role of EGF receptor ligands

    International Nuclear Information System (INIS)

    Castillo, Gaelle del; Murillo, Miguel M.; Alvarez-Barrientos, Alberto; Bertran, Esther; Fernandez, Margarita; Sanchez, Aranzazu; Fabregat, Isabel

    2006-01-01

    Transforming growth factor-beta (TGF-β) induces apoptosis in fetal rat hepatocytes. However, a subpopulation of these cells survives, concomitant with changes in phenotype, reminiscent of an epithelial-mesenchymal transition (EMT). We have previously suggested that EMT might confer cell resistance to apoptosis (Valdes et al., Mol. Cancer Res., 1: 68-78, 2002). However, the molecular mechanisms responsible for this resistance are not explored yet. In this work, we have isolated and subcultured the population of hepatocytes that suffered the EMT process and are resistant to apoptosis (TGF-β-treated fetal hepatocytes: TβT-FH). We prove that they secrete mitogenic and survival factors, as analyzed by the proliferative and survival capacity of conditioned medium. Inhibition of the epidermal growth factor receptor (EGFR) sensitizes TβT-FH to die after serum withdrawal. TβT-FH expresses high levels of transforming growth factor-alpha (TGF-α) and heparin-binding EGF-like growth factor (HB-EGF) and shows constitutive activation of the EGFR pathway. A blocking anti-TGF-α antibody restores the capacity of cells to die. TGF-β, which is expressed by TβT-FH, mediates up-regulation of TGF-α and HB-EGF expression in those cells. In summary, results suggest that an autocrine loop of TGF-β confers resistance to apoptosis after an EMT process in hepatocytes, through the increase in the expression of EGFR ligands

  1. Pancreatic adenocarcinoma upregulated factor (PAUF) confers resistance to pancreatic cancer cells against oncolytic parvovirus H-1 infection through IFNA receptor-mediated signaling

    Energy Technology Data Exchange (ETDEWEB)

    Kaowinn, Sirichat; Cho, Il-Rae; Moon, Jeong; Jun, Seung Won; Kim, Chang Seok [BK21+, Department of Cogno-Mechatronics Engineering, Pusan National University, Busan 609-736 (Korea, Republic of); Kang, Ho Young [Department of Microbiology, Pusan National University, Busan 609-736 (Korea, Republic of); Kim, Manbok [Department of Medical Science, Dankook University College of Medicine, Cheonan 330-714 (Korea, Republic of); Koh, Sang Seok [Department of Biological Sciences, Dong-A University, Busan 604-714 (Korea, Republic of); Chung, Young-Hwa, E-mail: younghc@pusan.ac.kr [BK21+, Department of Cogno-Mechatronics Engineering, Pusan National University, Busan 609-736 (Korea, Republic of)

    2015-04-03

    Pancreatic adenocarcinoma upregulated factor (PAUF), a novel oncogene, plays a crucial role in the development of pancreatic cancer, including its metastasis and proliferation. Therefore, PAUF-expressing pancreatic cancer cells could be important targets for oncolytic virus-mediated treatment. Panc-1 cells expressing PAUF (Panc-PAUF) showed relative resistance to parvovirus H-1 infection compared with Panc-1 cells expressing an empty vector (Panc-Vec). Of interest, expression of type I IFN-α receptor (IFNAR) was higher in Panc-PAUF cells than in Panc-Vec cells. Increased expression of IFNAR in turn increased the activation of Stat1 and Tyk2 in Panc-PAUF cells compared with that in Panc-Vec cells. Suppression of Tyk2 and Stat1, which are important downstream molecules for IFN-α signaling, sensitized pancreatic cancer cells to parvovirus H-1-mediated apoptosis. Further, constitutive suppression of PAUF sensitized Bxpc3 pancreatic cancer cells to parvovirus H-1 infection. Taken together, these results suggested that PAUF conferred resistance to pancreatic cancer cells against oncolytic parvovirus H-1 infection through IFNAR-mediated signaling. - Highlights: • PAUF confers resistance against oncolytic parvovirus H-1 infection. • PAUF enhances the expression of IFNAR in Panc-1 cells. • Increased activation of Tyk2 or Stat1 by PAUF provides resistance to parvovirus H-1-mediated apoptosis. • Constitutive inhibition of PAUF enhances parvovirus H-1-mediated oncolysis of Bxpc3 pancreatic cancer cells.

  2. One shot-two pathogens blocked: exposure of Arabidopsis to hexadecane, a long chain volatile organic compound, confers induced resistance against both Pectobacterium carotovorum and Pseudomonas syringae.

    Science.gov (United States)

    Park, Hyo Bee; Lee, Boyoung; Kloepper, Joseph W; Ryu, Choong-Min

    2013-07-01

    Bacteria and plant derived volatile organic compounds have been reported as the chemical triggers that elicit induced resistance in plants. Previously, volatile organic compounds (VOCs), including acetoin and 2,3-butanediol, were found to be emitted from plant growth-promoting rhizobacteria (PGPR) Bacillus subtilis GB03, which had been shown to elicit ISR and plant growth promotion. More recently, we reported data that stronger induced resistance could be elicited against Pseudomonas syringae pv maculicola ES4326 in plants exposed to C13 VOC from another PGPR Paenibacillus polymyxa E681 compared with that of strain GB03. Here, we assessed whether another long hydrocarbon C16 hexadecane (HD) conferred protection to Arabidopsis from infection of a biotrophic pathogen, P. syringae pv maculicola and a necrotrophic pathogen, Pectobacterium carotovorum subsp carotovorum. Collectively, long-chain VOCs can be linked to a plant resistance activator for protecting plants against both biotrophic and necrotrophic pathogens at the same time.

  3. Structural Studies of Bacterial Enzymes and their Relation to Antibiotic Resistance Mechanisms - Final Paper

    Energy Technology Data Exchange (ETDEWEB)

    Maltz, Lauren [SLAC National Accelerator Lab., Menlo Park, CA (United States)

    2015-08-27

    By using protein crystallography and X-ray diffraction, structures of bacterial enzymes were solved to gain a better understanding of how enzymatic modification acts as an antibacterial resistance mechanism. Aminoglycoside phosphotransferases (APHs) are one of three aminoglycoside modifying enzymes that confer resistance to the aminoglycoside antibiotics via enzymatic modification, rendering many drugs obsolete. Specifically, the APH(2”) family vary in their substrate specificities and also in their preference for the phosphate donor (ADP versus GDP). By solving the structures of members of the APH(2”) family of enzymes, we can see how domain movements are important to their substrate specificity. Our structure of the ternary complex of APH(2”)-IIIa with GDP and kanamycin, when compared to the known structures of APH(2”)-IVa, reveals that there are real physical differences between these two enzymes, a structural finding that explains why the two enzymes differ in their preferences for certain aminoglycosides. Another important group of bacterial resistance enzymes are the Class D β- lactamases. Oxacillinase carbapenemases (OXAs) are part of this enzyme class and have begun to confer resistance to ‘last resort’ drugs, most notably carbapenems. Our structure of OXA-143 shows that the conformational flexibility of a conserved hydrophobic residue in the active site (Val130) serves to control the entry of a transient water molecule responsible for a key step in the enzyme’s mechanism. Our results provide insight into the structural mechanisms of these two different enzymes

  4. Novel Peptide Vaccine GV1001 Rescues Hearing in Kanamycin/Furosemide-Treated Mice

    Directory of Open Access Journals (Sweden)

    Shin Hye Kim

    2018-01-01

    Full Text Available The cell-penetrating peptide GV1001 has been investigated as an anticancer agent and recently demonstrated anti-oxidant and anti-inflammatory effects. It has shown a protective effect on a kanamycin (KM-induced ototoxicity mouse model. In the present study, we administered GV1001 at different time points after inducing hair cell damage, and examined if it rescues hair cell loss and restores hearing. A deaf mouse model was created by intraperitoneal injection of KM and furosemide. First, to test the early temporal change of hearing and extent of hair cell damage after KM and furosemide injection, hearing and outer hair cells (OHCs morphology were evaluated on day 1, day 2 and day 3 after injection. In the second experiment, following KM and furosemide injection, GV1001, dexamethasone, or saline were given for three consecutive days at different time points: D0 group (days 0, 1, and 2, D1 group (days 1, 2, and 3, D3 group (days 3, 4, and 5 and D7 group (days 7, 8, and 9. The hearing thresholds were measured at 8, 16, and 32 kHz before ototoxic insult, and 7 days and 14 days after KM and furosemide injection. After 14 days, each turn of the cochlea was imaged to evaluate OHCs damage. GV1001-treated mice showed significantly less hearing loss and OHCs damage than the saline control group in the D0, D1 and D3 groups (p < 0.0167. However, there was no hearing restoration or intact hair cell in the D7 group. GV1001 protected against cochlear hair cell damage, and furthermore, delayed administration of GV1001 up to 3 days rescued hair cell damage and hearing loss in KM/furosemide-induced deaf mouse model.

  5. Transgenic expression of the rice Xa21 pattern-recognition receptor in banana (Musa sp.) confers resistance to Xanthomonas campestris pv. musacearum.

    Science.gov (United States)

    Tripathi, Jaindra N; Lorenzen, Jim; Bahar, Ofir; Ronald, Pamela; Tripathi, Leena

    2014-08-01

    Banana Xanthomonas wilt (BXW), caused by the bacterium Xanthomonas campestris pv. musacearum (Xcm), is the most devastating disease of banana in east and central Africa. The spread of BXW threatens the livelihood of millions of African farmers who depend on banana for food security and income. There are no commercial chemicals, biocontrol agents or resistant cultivars available to control BXW. Here, we take advantage of the robust resistance conferred by the rice pattern-recognition receptor (PRR), XA21, to the rice pathogen Xanthomonas oryzae pv. oryzae (Xoo). We identified a set of genes required for activation of Xa21-mediated immunity (rax) that were conserved in both Xoo and Xcm. Based on the conservation, we hypothesized that intergeneric transfer of Xa21 would confer resistance to Xcm. We evaluated 25 transgenic lines of the banana cultivar 'Gonja manjaya' (AAB) using a rapid bioassay and 12 transgenic lines in the glasshouse for resistance against Xcm. About 50% of the transgenic lines showed complete resistance to Xcm in both assays. In contrast, all of the nontransgenic control plants showed severe symptoms that progressed to complete wilting. These results indicate that the constitutive expression of the rice Xa21 gene in banana results in enhanced resistance against Xcm. Furthermore, this work demonstrates the feasibility of PRR gene transfer between monocotyledonous species and provides a valuable new tool for controlling the BXW pandemic of banana, a staple food for 100 million people in east Africa. © 2014 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

  6. Experimental evolution, genetic analysis and genome re-sequencing reveal the mutation conferring artemisinin resistance in an isogenic lineage of malaria parasites

    KAUST Repository

    Hunt, Paul

    2010-09-16

    Background: Classical and quantitative linkage analyses of genetic crosses have traditionally been used to map genes of interest, such as those conferring chloroquine or quinine resistance in malaria parasites. Next-generation sequencing technologies now present the possibility of determining genome-wide genetic variation at single base-pair resolution. Here, we combine in vivo experimental evolution, a rapid genetic strategy and whole genome re-sequencing to identify the precise genetic basis of artemisinin resistance in a lineage of the rodent malaria parasite, Plasmodium chabaudi. Such genetic markers will further the investigation of resistance and its control in natural infections of the human malaria, P. falciparum.Results: A lineage of isogenic in vivo drug-selected mutant P. chabaudi parasites was investigated. By measuring the artemisinin responses of these clones, the appearance of an in vivo artemisinin resistance phenotype within the lineage was defined. The underlying genetic locus was mapped to a region of chromosome 2 by Linkage Group Selection in two different genetic crosses. Whole-genome deep coverage short-read re-sequencing (IlluminaSolexa) defined the point mutations, insertions, deletions and copy-number variations arising in the lineage. Eight point mutations arise within the mutant lineage, only one of which appears on chromosome 2. This missense mutation arises contemporaneously with artemisinin resistance and maps to a gene encoding a de-ubiquitinating enzyme.Conclusions: This integrated approach facilitates the rapid identification of mutations conferring selectable phenotypes, without prior knowledge of biological and molecular mechanisms. For malaria, this model can identify candidate genes before resistant parasites are commonly observed in natural human malaria populations. 2010 Hunt et al; licensee BioMed Central Ltd.

  7. Experimental evolution, genetic analysis and genome re-sequencing reveal the mutation conferring artemisinin resistance in an isogenic lineage of malaria parasites

    Directory of Open Access Journals (Sweden)

    Hunt Paul

    2010-09-01

    Full Text Available Abstract Background Classical and quantitative linkage analyses of genetic crosses have traditionally been used to map genes of interest, such as those conferring chloroquine or quinine resistance in malaria parasites. Next-generation sequencing technologies now present the possibility of determining genome-wide genetic variation at single base-pair resolution. Here, we combine in vivo experimental evolution, a rapid genetic strategy and whole genome re-sequencing to identify the precise genetic basis of artemisinin resistance in a lineage of the rodent malaria parasite, Plasmodium chabaudi. Such genetic markers will further the investigation of resistance and its control in natural infections of the human malaria, P. falciparum. Results A lineage of isogenic in vivo drug-selected mutant P. chabaudi parasites was investigated. By measuring the artemisinin responses of these clones, the appearance of an in vivo artemisinin resistance phenotype within the lineage was defined. The underlying genetic locus was mapped to a region of chromosome 2 by Linkage Group Selection in two different genetic crosses. Whole-genome deep coverage short-read re-sequencing (Illumina® Solexa defined the point mutations, insertions, deletions and copy-number variations arising in the lineage. Eight point mutations arise within the mutant lineage, only one of which appears on chromosome 2. This missense mutation arises contemporaneously with artemisinin resistance and maps to a gene encoding a de-ubiquitinating enzyme. Conclusions This integrated approach facilitates the rapid identification of mutations conferring selectable phenotypes, without prior knowledge of biological and molecular mechanisms. For malaria, this model can identify candidate genes before resistant parasites are commonly observed in natural human malaria populations.

  8. Role of a Novel I1781T Mutation and Other Mechanisms in Conferring Resistance to Acetyl-CoA Carboxylase Inhibiting Herbicides in a Black-Grass Population

    Science.gov (United States)

    Kaundun, Shiv Shankhar; Hutchings, Sarah-Jane; Dale, Richard P.; McIndoe, Eddie

    2013-01-01

    Background Knowledge of the mechanisms of herbicide resistance is important for designing long term sustainable weed management strategies. Here, we have used an integrated biology and molecular approach to investigate the mechanisms of resistance to acetyl-CoA carboxylase inhibiting herbicides in a UK black-grass population (BG2). Methodology/Principal Findings Comparison between BG2 phenotypes using single discriminant rates of herbicides and genotypes based on ACCase gene sequencing showed that the I1781L, a novel I1781T, but not the W2027C mutations, were associated with resistance to cycloxydim. All plants were killed with clethodim and a few individuals containing the I1781L mutation were partially resistant to tepraloxydim. Whole plant dose response assays demonstrated that a single copy of the mutant T1781 allele conferred fourfold resistance levels to cycloxydim and clodinafop-propargyl. In contrast, the impact of the I1781T mutation was low (Rf = 1.6) and non-significant on pinoxaden. BG2 was also characterised by high levels of resistance, very likely non-target site based, to the two cereal selective herbicides clodinafop-propargyl and pinoxaden and not to the poorly metabolisable cyclohexanedione herbicides. Analysis of 480 plants from 40 cycloxydim resistant black grass populations from the UK using two very effective and high throughput dCAPS assays established for detecting any amino acid changes at the 1781 ACCase codon and for positively identifying the threonine residue, showed that the occurrence of the T1781 is extremely rare compared to the L1781 allele. Conclusion/Significance This study revealed a novel mutation at ACCase codon position 1781 and adequately assessed target site and non-target site mechanisms in conferring resistance to several ACCase herbicides in a black-grass population. It highlights that over time the level of suspected non-target site resistance to some cereal selective ACCase herbicides have in some instances

  9. Conjugal transfer of aac(6')Ie-aph(2″)Ia gene from native species and mechanism of regulation and cross resistance in Enterococcus faecalis MCC3063 by real time-PCR.

    Science.gov (United States)

    Jaimee, G; Halami, P M

    2017-09-01

    High level aminoglycoside resistance (HLAR) in the lactic acid bacteria (LAB) derived from food animals is detrimental. The aim of this study was to investigate the localization and conjugal transfer of aminoglycoside resistance genes, aac(6')Ie-aph(2″)Ia and aph(3')IIIa in different Enterococcus species. The cross resistance patterns in Enterococcus faecalis MCC3063 to clinically important aminoglycosides by real time PCR were also studied. Southern hybridization experiments revealed the presence of aac(6')Ie-aph(2 ″ )Ia and aph(3')IIIa genes conferring HLAR in high molecular weight plasmids except in Lactobacillus plantarum. The plasmid encoded bifunctional aac(6')Ie-aph(2″)Ia gene was transferable from Enterococcus avium (n = 2), E. cecorum (n = 1), E. faecalis (n = 1) and Pediococcus lolii (n = 1) species into the recipient strain; E. faecalis JH2-2 by filter mating experiments thus indicating the possible risks of gene transfer into pathogenic strains. Molecular analysis of cross resistance patterns in native isolate of E. faecalis MCC3063 carrying aac(6')Ie-aph(2″)Ia and aph(3')IIIa gene was displayed by quantification of the mRNA levels in this study. For this, the culture was induced with increasing concentrations of gentamicin, kanamycin and streptomycin (2048, 4096, 8192, 16384 μg/mL) individually. The increasing concentrations of gentamicin and kanamycin induced the expression of the aac(6')Ie-aph(2″)Ia and aph(3')IIIa resistance genes, respectively. Interestingly, it was observed that induction with streptomycin triggered a significant fold increase in the expression of the aph(3')IIIa gene which otherwise was not known to modify the aminoglycoside. This is noteworthy as streptomycin was found to confer cross resistance to structurally unrelated kanamycin. Also, expression of the aph(3')IIIa gene when induced with streptomycin, revealed that bacteria harbouring this gene will be able to overcome streptomycin bactericidal action at

  10. The oncogenic phosphatase PPM1D confers cisplatin resistance in ovarian carcinoma cells by attenuating checkpoint kinase 1 and p53 activation.

    Science.gov (United States)

    Ali, A Y; Abedini, M R; Tsang, B K

    2012-04-26

    Cisplatin (CDDP: cis-diamminedichloroplatinum) resistance is a major hurdle in the treatment of human ovarian cancer (OVCA). A better understanding of the mechanisms of CDDP resistance can greatly improve therapeutic outcome for patients. A determinant of CDDP sensitivity in OVCA, p53, is activated by checkpoint kinase 1 (Chk1) in response to DNA damage. Although the oncogenic phosphatase protein phosphatase magnesium-dependent 1 (PPM1D) can deactivate both p53 and Chk1 through site-specific dephosphorylation, whether PPM1D has a role in CDDP resistance is unknown. Here, using pair-matched wild-type p53 CDDP-sensitive (OV2008) and -resistant (C13*) cells, and p53-compromised CDDP-resistant cells (A2780cp, OCC-1, OVCAR-3 and SKOV3), we have demonstrated (i) the existence of site-specific differences in phospho-Ser-Chk1 content between sensitive and resistant cells in response to CDDP; (ii) PPM1D, but not phosphoinositide-3-kinase-related kinase Ataxia Telangiectasia and Rad3 related protein (ATR), is important in the regulation of CDDP-induced Chk1 activation and OVCA cell chemosensitivity; (iii) PPM1D downregulation sensitizes resistant cells to CDDP primarily by activating Chk1 and p53. Our findings establish for the first time that PPM1D confers CDDP resistance in OVCA cells through attenuating CDDP-induced, Chk1-mediated, p53-dependent apoptosis. These findings extend the current knowledge on the molecular and cellular basis of cisplatin resistance and offer the rationale for PPMID as a potential target for treatment of chemoresistant OVCA.

  11. 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.

  12. Plasmid-free CRISPR/Cas9 genome editing in Plasmodium falciparum confirms mutations conferring resistance to the dihydroisoquinolone clinical candidate SJ733.

    Directory of Open Access Journals (Sweden)

    Emily D Crawford

    Full Text Available Genetic manipulation of the deadly malaria parasite Plasmodium falciparum remains challenging, but the rise of CRISPR/Cas9-based genome editing tools is increasing the feasibility of altering this parasite's genome in order to study its biology. Of particular interest is the investigation of drug targets and drug resistance mechanisms, which have major implications for fighting malaria. We present a new method for introducing drug resistance mutations in P. falciparum without the use of plasmids or the need for cloning homologous recombination templates. We demonstrate this method by introducing edits into the sodium efflux channel PfATP4 by transfection of a purified CRISPR/Cas9-guide RNA ribonucleoprotein complex and a 200-nucleotide single-stranded oligodeoxynucleotide (ssODN repair template. Analysis of whole genome sequencing data with the variant-finding program MinorityReport confirmed that only the intended edits were made, and growth inhibition assays confirmed that these mutations confer resistance to the antimalarial SJ733. The method described here is ideally suited for the introduction of mutations that confer a fitness advantage under selection conditions, and the novel finding that an ssODN can function as a repair template in P. falciparum could greatly simplify future editing attempts regardless of the nuclease used or the delivery method.

  13. High frequency of resistance, lack of clinical benefit, and poor outcomes in capreomycin treated South African patients with extensively drug-resistant tuberculosis.

    Directory of Open Access Journals (Sweden)

    Elize Pietersen

    Full Text Available There are limited data about the epidemiology and treatment-related outcomes associated with capreomycin resistance in patients with XDR-TB. Capreomycin achieves high serum concentrations relative to MIC but whether capreomycin has therapeutic benefit despite microbiological resistance remains unclear.We reviewed the susceptibility profiles and outcomes associated with capreomycin usage in patients diagnosed with XDR-TB between August 2002 and October 2012 in two provinces of South Africa. Patients whose isolates were genotypically tested for capreomycin resistance were included in the analysis.Of 178 XDR-TB patients 41% were HIV-infected. 87% (154/178 isolates contained a capreomycin resistance-conferring mutation [80% (143/178 rrs A1401G and 6% (11/178 were heteroresistant (containing both the rrs A1401G mutation and wild-type sequences]. Previous MDR-TB treatment, prior usage of kanamycin, or strain type was not associated with capreomycin resistance. 92% (163/178 of XDR-TB patients were empirically treated with capreomycin. Capreomycin resistance decreased the odds of sputum culture conversion. In capreomycin sensitive and resistant persons combined weight at diagnosis was the only independent predictor for survival (p=<0.001. By contrast, HIV status and use of co-amoxicillin/clavulanic acid were independent predictors of mortality (p=<0.05. Capreomycin usage was not associated with survival or culture conversion when the analysis was restricted to those whose isolates were resistant to capreomycin.In South Africa the frequency of capreomycin conferring mutations was extremely high in XDR-TB isolates. In those with capreomycin resistance there appeared to be no therapeutic benefit of using capreomycin. These data inform susceptibility testing and the design of treatment regimens for XDR-TB in TB endemic settings.

  14. Association mapping of North American spring wheat breeding germplasm reveals loci conferring resistance to Ug99 and other African stem rust races.

    Science.gov (United States)

    Bajgain, P; Rouse, M N; Bulli, P; Bhavani, S; Gordon, T; Wanyera, R; Njau, P N; Legesse, W; Anderson, J A; Pumphrey, M O

    2015-10-14

    The recently identified Puccinia graminis f. sp. tritici (Pgt) race TTKSK (Ug99) poses a severe threat to global wheat production because of its broad virulence on several widely deployed resistance genes. Additional virulences have been detected in the Ug99 group of races, and the spread of this race group has been documented across wheat growing regions in Africa, the Middle East (Yemen), and West Asia (Iran). Other broadly virulent Pgt races, such as TRTTF and TKTTF, present further difficulties in maintaining abundant genetic resistance for their effective use in wheat breeding against this destructive fungal disease of wheat. In an effort to identify loci conferring resistance to these races, a genome-wide association study was carried out on a panel of 250 spring wheat breeding lines from the International Maize and Wheat Improvement Center (CIMMYT), six wheat breeding programs in the United States and three wheat breeding programs in Canada. The lines included in this study were grouped into two major clusters, based on the results of principal component analysis using 23,976 SNP markers. Upon screening for adult plant resistance (APR) to Ug99 during 2013 and 2014 in artificial stem rust screening nurseries at Njoro, Kenya and at Debre Zeit, Ethiopia, several wheat lines were found to exhibit APR. The lines were also screened for resistance at the seedling stage against races TTKSK, TRTTF, and TKTTF at USDA-ARS Cereal Disease Laboratory in St. Paul, Minnesota; and only 9 of the 250 lines displayed seedling resistance to all the races. Using a mixed linear model, 27 SNP markers associated with APR against Ug99 were detected, including markers linked with the known APR gene Sr2. Using the same model, 23, 86, and 111 SNP markers associated with seedling resistance against races TTKSK, TRTTF, and TKTTF were identified, respectively. These included markers linked to the genes Sr8a and Sr11 providing seedling resistance to races TRTTF and TKTTF, respectively. We

  15. Alanine to valine substitutions in the pore helix IIIP1 and linker-helix IIIL45 confer cockroach sodium channel resistance to DDT and pyrethroids.

    Science.gov (United States)

    Chen, Mengli; Du, Yuzhe; Nomura, Yoshiko; Zhu, Guonian; Zhorov, Boris S; Dong, Ke

    2017-05-01

    Pyrethroid insecticides exert toxic effects by prolonging the opening of voltage-gated sodium channels. More than 20 sodium channel mutations from arthropod pests and disease vectors have been confirmed to confer pyrethroid resistance. These mutations have been valuable in elucidating the molecular interaction between pyrethroids and sodium channels, including identification of two pyrethroid receptor sites. Previously, two alanine to valine substitutions, one in the pore helix IIIP1 and the other in the linker-helix connecting S4 and S5 in domain III (IIIL45), were found in Drosophila melanogaster mutants that are resistant to DDT and deltamethrin (a type II pyrethroid with an α-cyano group at the phenylbenzyl alcohol position, which is lacking in type I pyrethroids), but their role in target-site-mediated insecticide resistance has not been functionally confirmed. In this study, we functionally examined the two mutations in cockroach sodium channels expressed in Xenopus laevis oocytes. Both mutations caused depolarizing shifts in the voltage dependence of activation, conferred DDT resistance and also resistance to two Type I pyrethroids by almost abolishing the tail currents induced by Type I pyrethroids. In contrast, neither mutation reduced the amplitude of tail currents induced by the Type II pyrethroids, deltamethrin or cypermethrin. However, both mutations accelerated the decay of Type II pyrethroid-induced tail currents, which normally decay extremely slowly. These results provided new insight into the molecular basis of different actions of Type I and Type II pyrethroids on sodium channels. Computer modeling predicts that both mutations may allosterically affect pyrethroid binding. Copyright © 2016 Elsevier B.V. All rights reserved.

  16. Stable gene transfer of CCR5 and CXCR4 siRNAs by sleeping beauty transposon system to confer HIV-1 resistance

    Directory of Open Access Journals (Sweden)

    Akkina Ramesh

    2008-07-01

    Full Text Available Abstract Background Thus far gene therapy strategies for HIV/AIDS have used either conventional retroviral vectors or lentiviral vectors for gene transfer. Although highly efficient, their use poses a certain degree of risk in terms of viral mediated oncogenesis. Sleeping Beauty (SB transposon system offers a non-viral method of gene transfer to avoid this possible risk. With respect to conferring HIV resistance, stable knock down of HIV-1 coreceptors CCR5 and CXCR4 by the use of lentiviral vector delivered siRNAs has proved to be a promising strategy to protect cells from HIV-1 infection. In the current studies our aim is to evaluate the utility of SB system for stable gene transfer of CCR5 and CXCR4 siRNA genes to derive HIV resistant cells as a first step towards using this system for gene therapy. Results Two well characterized siRNAs against the HIV-1 coreceptors CCR5 and CXCR4 were chosen based on their previous efficacy for the SB transposon gene delivery. The siRNA transgenes were incorporated individually into a modified SB transfer plasmid containing a FACS sortable red fluorescence protein (RFP reporter and a drug selectable neomycin resistance gene. Gene transfer was achieved by co-delivery with a construct expressing a hyperactive transposase (HSB5 into the GHOST-R3/X4/R5 cell line, which expresses the major HIV receptor CD4 and and the co-receptors CCR5 and CXCR4. SB constructs expressing CCR5 or CXCR4 siRNAs were also transfected into MAGI-CCR5 or MAGI-CXCR4 cell lines, respectively. Near complete downregulation of CCR5 and CXCR4 surface expression was observed in transfected cells. During viral challenge with X4-tropic (NL4.3 or R5-tropic (BaL HIV-1 strains, the respective transposed cells showed marked viral resistance. Conclusion SB transposon system can be used to deliver siRNA genes for stable gene transfer. The siRNA genes against HIV-1 coreceptors CCR5 and CXCR4 are able to downregulate the respective cell surface proteins

  17. Cell Death Is Not Sufficient for the Restriction of Potato Virus Y Spread in Hypersensitive Response-Conferred Resistance in Potato.

    Science.gov (United States)

    Lukan, Tjaša; Baebler, Špela; Pompe-Novak, Maruša; Guček, Katja; Zagorščak, Maja; Coll, Anna; Gruden, Kristina

    2018-01-01

    Hypersensitive response (HR)-conferred resistance to viral infection restricts the virus spread and is accompanied by the induction of cell death, manifested as the formation of necrotic lesions. While it is known that salicylic acid is the key component in the orchestration of the events restricting viral spread in HR, the exact function of the cell death in resistance is still unknown. We show that potato virus Y (PVY) can be detected outside the cell death zone in Ny-1 -mediated HR in potato plants (cv. Rywal), observed as individual infected cells or small clusters of infected cells outside the cell death zone. By exploiting the features of temperature dependent Ny-1 -mediated resistance, we confirmed that the cells at the border of the cell death zone are alive and harbor viable PVY that is able to reinitiate infection. To get additional insights into this phenomenon we further studied the dynamics of both cell death zone expansion and occurrence of viral infected cell islands outside it. We compared the response of Rywal plants to their transgenic counterparts, impaired in SA accumulation (NahG-Rywal), where the lesions occur but the spread of the virus is not restricted. We show that the virus is detected outside the cell death zone in all lesion developmental stages of HR lesions. We also measured the dynamics of lesions expansion in both genotypes. We show that while rapid lesion expansion is observed in SA-depleted plants, virus spread is even faster. On the other hand the majority of analyzed lesions slowly expand also in HR-conferred resistance opening the possibility that the infected cells are eventually engulfed by cell death zone. Taken altogether, we suggest that the HR cell death is separated from the resistance mechanisms which lead to PVY restriction in Ny-1 genetic background. We propose that HR should be regarded as a process where the dynamics of events is crucial for effectiveness of viral arrest albeit the exact mechanism conferring this

  18. Insights into the structure, function and evolution of the radical-SAM 23S rRNA methyltransferase Cfr that confers antibiotic resistance in bacteria

    DEFF Research Database (Denmark)

    Karminska, K. H.; Purta, E.; Hansen, L .H.

    2010-01-01

    The Cfr methyltransferase confers combined resistance to five classes of antibiotics that bind to the peptidyl tranferase center of bacterial ribosomes by catalyzing methylation of the C-8 position of 23S rRNA nucleotide A2503. The same nucleotide is targeted by the housekeeping methyltransferase...... of a 4Fe-4S cluster, a SAM molecule coordinated to the iron-sulfur cluster (SAM1) and a SAM molecule that is the putative methyl group donor (SAM2). All mutations at predicted functional sites affect Cfr activity significantly as assayed by antibiotic susceptibility testing and primer extension analysis...

  19. Identification and characterization of integron-mediated antibiotic resistance in the phytopathogen Xanthomonas oryzae pv. oryzae.

    Directory of Open Access Journals (Sweden)

    Ying Xu

    Full Text Available Four streptomycin-resistant isolates of Xanthomonas oryzae pv. oryzae (YNA7-1, YNA10-2, YNA11-2, and YNA12-2 were examined via PCR amplification for the presence of class 1, class 2, and class 3 integrons and aadA1 and aadA2 genes, which confer resistance to streptomycin and spectinomycin. The class 1 integrase gene intI1 and the aminoglycoside adenylyltransferase gene aadA1 were identified in all four resistant isolates but not in 25 sensitive isolates. PCR amplifications showed that 7790-bp, 7162-bp, 7790-bp, and 7240-bp resistance integrons with transposition gene modules (tni module in 3' conserved segments existed in YNA7-1, YNA10-2, YNA11-2, and YNA12-2, respectively. Subsequent analysis of sequences indicated that the integrons of YNA7-1 and YNA11-2 carried three gene cassettes in the order |aacA3|arr3|aadA1|. The integron of YNA10-2 carried only |arr3|aadA1| gene cassettes. The integron of YNA12-2 lacked a 550-bp sequence including part of intI1 but it still carried |aacA3|arr3|aadA1| gene cassettes. The analysis of inactive mutants and complementation tests confirmed that the aacA3 gene conferred resistance to tobramycin, kanamycin, gentamicin and netilmicin; the arr3 gene conferred resistance to rifampicin; and the aadA1 gene conferred resistance to streptomycin and spectinomycin. The resistance phenotypes of the four isolates corresponded with their resistance gene cassettes, except that YNA7-1 and YNA12-2 did not show rifampicin resistance. Sequence comparison revealed that no gene cassette array in GenBank was in the same order as in the integrons of the four resistant isolates in this study and the aadA1, which was identical in the four resistant isolates, showed 99% identity with aadA1 sequences in GenBank. The result of a stability test showed that the resistance phenotype, the aadA1 gene, and the intI1 gene were completely stable in YNA7-1 and YNA12-2 but unstable in YNA10-2 and YNA11-2. To our knowledge, this is the first

  20. Identification and Characterization of Integron-Mediated Antibiotic Resistance in the Phytopathogen Xanthomonas oryzae pv. oryzae

    Science.gov (United States)

    Zhou, Ming-guo

    2013-01-01

    Four streptomycin-resistant isolates of Xanthomonas oryzae pv. oryzae (YNA7-1, YNA10-2, YNA11-2, and YNA12-2) were examined via PCR amplification for the presence of class 1, class 2, and class 3 integrons and aadA1 and aadA2 genes, which confer resistance to streptomycin and spectinomycin. The class 1 integrase gene intI1 and the aminoglycoside adenylyltransferase gene aadA1 were identified in all four resistant isolates but not in 25 sensitive isolates. PCR amplifications showed that 7790-bp, 7162-bp, 7790-bp, and 7240-bp resistance integrons with transposition gene modules (tni module) in 3′ conserved segments existed in YNA7-1, YNA10-2, YNA11-2, and YNA12-2, respectively. Subsequent analysis of sequences indicated that the integrons of YNA7-1 and YNA11-2 carried three gene cassettes in the order |aacA3|arr3|aadA1|. The integron of YNA10-2 carried only |arr3|aadA1| gene cassettes. The integron of YNA12-2 lacked a 550-bp sequence including part of intI1 but it still carried |aacA3|arr3|aadA1| gene cassettes. The analysis of inactive mutants and complementation tests confirmed that the aacA3 gene conferred resistance to tobramycin, kanamycin, gentamicin and netilmicin; the arr3 gene conferred resistance to rifampicin; and the aadA1 gene conferred resistance to streptomycin and spectinomycin. The resistance phenotypes of the four isolates corresponded with their resistance gene cassettes, except that YNA7-1 and YNA12-2 did not show rifampicin resistance. Sequence comparison revealed that no gene cassette array in GenBank was in the same order as in the integrons of the four resistant isolates in this study and the aadA1, which was identical in the four resistant isolates, showed 99% identity with aadA1 sequences in GenBank. The result of a stability test showed that the resistance phenotype, the aadA1 gene, and the intI1 gene were completely stable in YNA7-1 and YNA12-2 but unstable in YNA10-2 and YNA11-2. To our knowledge, this is the first report of resistance

  1. Functional metagenomic characterization of antibiotic resistance genes in agricultural soils from China.

    Science.gov (United States)

    Su, Jian Qiang; Wei, Bei; Xu, Chun Yan; Qiao, Min; Zhu, Yong Guan

    2014-04-01

    Soil has been regarded as a rich source of antibiotic resistance genes (ARGs) due to the complex microbial community and diverse antibiotic-producing microbes in soil, however, little is known about the ARGs in unculturable bacteria. To investigate the diversity and distribution of ARGs in soil and assess the impact of agricultural practice on the ARGs, we screened soil metagenomic library constructed using DNA from four different agricultural soil for ARGs. We identified 45 clones conferring resistance to minocycline, tetracycline, streptomycin, gentamicin, kanamycin, amikacin, chloramphenicol and rifampicin. The similarity of identified ARGs with the closest protein in GenBank ranged from 26% to 92%, with more than 60% of identified ARGs had low similarity less than 60% at amino acid level. The identified ARGs include aminoglycoside acetyltransferase, aminoglycoside 6-adenyltransferase, ADP-ribosyl transferase, ribosome protection protein, transporters and other antibiotic resistant determinants. The identified ARGs from the soil with manure application account for approximately 70% of the total ARGs in this study, implying that manure amendment may increase the diversity of antibiotic resistance genes in soil bacteria. These results suggest that antibiotic resistance in soil remains unexplored and functional metagenomic approach is powerful in discovering novel ARGs and resistant mechanisms. Copyright © 2013 Elsevier Ltd. All rights reserved.

  2. Identification and distribution of a GABA receptor mutation conferring dieldrin resistance in the malaria vector Anopheles funestus in Africa

    OpenAIRE

    Wondji, Charles S.; Dabire, Roch K.; Tukur, Zainab; Irving, Helen; Djouaka, Rousseau; Morgan, John C.

    2011-01-01

    Growing problems of pyrethroid resistance in Anopheles funestus have intensified efforts to identify alternative insecticides. Many agrochemicals target the GABA receptors, but cross-resistance from dieldrin resistance may preclude their introduction. Dieldrin resistance was detected in An. funestus populations from West (Burkina Faso) and central (Cameroon) Africa, but populations from East (Uganda) and Southern Africa (Mozambique and Malawi) were fully susceptible to this insecticide. Parti...

  3. The Distribution of Fitness Costs of Resistance-Conferring Mutations Is a Key Determinant for the Future Burden of Drug-Resistant Tuberculosis: A Model-Based Analysis

    NARCIS (Netherlands)

    Knight, Gwenan M.; Colijn, Caroline; Shrestha, Sourya; Fofana, Mariam; Cobelens, Frank; White, Richard G.; Dowdy, David W.; Cohen, Ted

    2015-01-01

    Drug resistance poses a serious challenge for the control of tuberculosis in many settings. It is well established that the expected future trend in resistance depends on the reproductive fitness of drug-resistant Mycobacterium tuberculosis. However, the variability in fitness between strains with

  4. Systematic review of allelic exchange experiments aimed at identifying mutations that confer drug resistance in Mycobacterium tuberculosis.

    Science.gov (United States)

    Nebenzahl-Guimaraes, Hanna; Jacobson, Karen R; Farhat, Maha R; Murray, Megan B

    2014-02-01

    Improving our understanding of the relationship between the genotype and the drug resistance phenotype of Mycobacterium tuberculosis will aid the development of more accurate molecular diagnostics for drug-resistant tuberculosis. Studies that use direct genetic manipulation to identify the mutations that cause M. tuberculosis drug resistance are superior to associational studies in elucidating an individual mutation's contribution to the drug resistance phenotype. We systematically reviewed the literature for publications reporting allelic exchange experiments in any of the resistance-associated M. tuberculosis genes. We included studies that introduced single point mutations using specialized linkage transduction or site-directed/in vitro mutagenesis and documented a change in the resistance phenotype. We summarize evidence supporting the causal relationship of 54 different mutations in eight genes (katG, inhA, kasA, embB, embC, rpoB, gyrA and gyrB) and one intergenic region (furA-katG) with resistance to isoniazid, the rifamycins, ethambutol and fluoroquinolones. We observed a significant role for the strain genomic background in modulating the resistance phenotype of 21 of these mutations and found examples of where the same drug resistance mutations caused varying levels of resistance to different members of the same drug class. This systematic review highlights those mutations that have been shown to causally change phenotypic resistance in M. tuberculosis and brings attention to a notable lack of allelic exchange data for several of the genes known to be associated with drug resistance.

  5. Expression of self-complementary hairpin RNA under the control of the rolC promoter confers systemic disease resistance to plum pox virus without preventing local infection.

    Science.gov (United States)

    Pandolfini, Tiziana; Molesini, Barbara; Avesani, Linda; Spena, Angelo; Polverari, Annalisa

    2003-06-25

    Homology-dependent selective degradation of RNA, or post-transcriptional gene silencing (PTGS), is involved in several biological phenomena, including adaptative defense mechanisms against plant viruses. Small interfering RNAs mediate the selective degradation of target RNA by guiding a multicomponent RNAse. Expression of self-complementary hairpin RNAs within two complementary regions separated by an intron elicits PTGS with high efficiency. Plum pox virus (PPV) is the etiological agent of sharka disease in Drupaceae, although it can also be transmitted to herbaceous species (e.g. Nicotiana benthamiana). Once inside the plant, PPV is transmitted via plasmodesmata from cell to cell, and at longer distances, via phloem. The rolC promoter drives expression in phloem cells. RolC expression is absent in both epidermal and mesophyll cells. The aim of the present study was to confer systemic disease resistance without preventing local viral infection. In the ihprolC-PP197 gene (intron hair pin rolC PPV 197), a 197 bp sequence homologous to the PPV RNA genome (from base 134 to 330) was placed as two inverted repeats separated by the DNA sequence of the rolA intron. This hairpin construct is under the control of the rolC promoter.N. benthamiana plants transgenic for the ihprolC-PP197 gene contain siRNAs homologous to the 197 bp sequence. The transgenic progeny of ihprolC-PP197 plants are resistant to PPV systemic infection. Local infection is unaffected. Most (80%) transgenic plants are virus free and symptomless. Some plants (20%) contain virus in uninoculated apical leaves; however they show only mild symptoms of leaf mottling. PPV systemic resistance cosegregates with the ihprolC-PP197 transgene and was observed in progeny plants of all independent transgenic lines analyzed. SiRNAs of 23-25 nt homologous to the PPV sequence used in the ihprolC-PP197 construct were detected in transgenic plants before and after inoculation. Transitivity of siRNAs was observed in

  6. Expression of self-complementary hairpin RNA under the control of the rolC promoter confers systemic disease resistance to plum pox virus without preventing local infection

    Directory of Open Access Journals (Sweden)

    Spena Angelo

    2003-06-01

    Full Text Available Abstract Background Homology-dependent selective degradation of RNA, or post-transcriptional gene silencing (PTGS, is involved in several biological phenomena, including adaptative defense mechanisms against plant viruses. Small interfering RNAs mediate the selective degradation of target RNA by guiding a multicomponent RNAse. Expression of self-complementary hairpin RNAs within two complementary regions separated by an intron elicits PTGS with high efficiency. Plum pox virus (PPV is the etiological agent of sharka disease in Drupaceae, although it can also be transmitted to herbaceous species (e.g. Nicotiana benthamiana. Once inside the plant, PPV is transmitted via plasmodesmata from cell to cell, and at longer distances, via phloem. The rolC promoter drives expression in phloem cells. RolC expression is absent in both epidermal and mesophyll cells. The aim of the present study was to confer systemic disease resistance without preventing local viral infection. Results In the ihprolC-PP197 gene (intron hair pin rolC PPV 197, a 197 bp sequence homologous to the PPV RNA genome (from base 134 to 330 was placed as two inverted repeats separated by the DNA sequence of the rolA intron. This hairpin construct is under the control of the rolC promoter.N. benthamiana plants transgenic for the ihprolC-PP197 gene contain siRNAs homologous to the 197 bp sequence. The transgenic progeny of ihprolC-PP197 plants are resistant to PPV systemic infection. Local infection is unaffected. Most (80% transgenic plants are virus free and symptomless. Some plants (20% contain virus in uninoculated apical leaves; however they show only mild symptoms of leaf mottling. PPV systemic resistance cosegregates with the ihprolC-PP197 transgene and was observed in progeny plants of all independent transgenic lines analyzed. SiRNAs of 23–25 nt homologous to the PPV sequence used in the ihprolC-PP197 construct were detected in transgenic plants before and after inoculation

  7. The actin filament cross-linker L-plastin confers resistance to TNF-α in MCF-7 breast cancer cells in a phosphorylation-dependent manner

    Science.gov (United States)

    Janji, Bassam; Vallar, Laurent; Tanoury, Ziad Al; Bernardin, François; Vetter, Guillaume; Schaffner-Reckinger, Elisabeth; Berchem, Guy; Friederich, Evelyne; Chouaib, Salem

    2010-01-01

    Abstract We used a tumour necrosis factor (TNF)-α resistant breast adenocarcinoma MCF-7 cell line to investigate the involvement of the actin cytoskeleton in the mechanism of cell resistance to this cytokine. We found that TNF resistance correlates with the loss of cell epithelial properties and the gain of a mesenchymal phenotype, reminiscent of an epithelial-to-mesenchymal transition (EMT). Morphological changes were associated with a profound reorganization of the actin cytoskeleton and with a change in the repertoire of expressed actin cytoskeleton genes and EMT markers, as revealed by DNA microarray-based expression profiling. L-plastin, an F-actin cross-linking and stabilizing protein, was identified as one of the most significantly up-regulated genes in TNF-resistant cells. Knockdown of L-plastin in these cells revealed its crucial role in conferring TNF resistance. Importantly, overexpression of wild-type L-plastin in TNF-sensitive MCF-7 cells was sufficient to protect them against TNF-mediated cell death. Furthermore, we found that this effect is dependent on serine-5 phosphorylation of L-plastin and that non-conventional protein kinase C isoforms and the ceramide pathway may regulate its phosphorylation state. The protective role of L-plastin was not restricted to TNF-α resistant MCF-7 cells because a correlation between the expression of L-plastin and the resistance to TNF-α was observed in other breast cancer cell lines. Together, our study discloses a novel unexpected role of the actin bundling protein L-plastin as a cell protective protein against TNF-cytotoxicity. PMID:19799649

  8. Isolation and validation of a candidate Rsv3 gene from a soybean genotype that confers strain-specific resistance to soybean mosaic virus.

    Science.gov (United States)

    Tran, Phu-Tri; Widyasari, Kristin; Seo, Jang-Kyun; Kim, Kook-Hyung

    2018-01-01

    Soybean mosaic virus (SMV), a member of the genus Potyvirus, significantly reduces soybean production worldwide. Rsv3, which confers strain-specific resistance to SMV, was previously mapped between the markers A519F/R and M3Satt in chromosome 14 of the soybean [Glycine max (L.) Merr.] genotype L29. Analysis of the soybean genome database revealed that five different NBS-LRR sequences exist between the flanking markers. Among these candidate Rsv3 genes, the full-length cDNA of the Glyma.14g204700 was successfully cloned from L29. Over-expression of Glyma.14g204700 in leaves inoculated with SMV inhibited viral infection in a soybean genotype lacking Rsv3. In addition, the transient silencing of the candidate gene caused a high accumulation of an avirulent strain in L29 carrying Rsv3. Our results therefore provide additional line of evidence to support that Glyma.14g204700 is likely Rsv3 gene that confers strain-specific resistance to SMV. Copyright © 2017 Elsevier Inc. All rights reserved.

  9. CRISPR/Cas9-Mediated CCR5 Ablation in Human Hematopoietic Stem/Progenitor Cells Confers HIV-1 Resistance In Vivo.

    Science.gov (United States)

    Xu, Lei; Yang, Huan; Gao, Yang; Chen, Zeyu; Xie, Liangfu; Liu, Yulin; Liu, Ying; Wang, Xiaobao; Li, Hanwei; Lai, Weifeng; He, Yuan; Yao, Anzhi; Ma, Liying; Shao, Yiming; Zhang, Bin; Wang, Chengyan; Chen, Hu; Deng, Hongkui

    2017-08-02

    Transplantation of hematopoietic stem cells (HSCs) with a naturally occurring CCR5 mutation confers a loss of detectable HIV-1 in the patient, making ablation of the CCR5 gene in HSCs an ideal therapy for an HIV-1 cure. Although CCR5 disruption has been attempted in CD4 + T cells and hematopoietic stem/progenitor cells (HSPCs), efficient gene editing with high specificity and long-term therapeutic potential remains a major challenge for clinical translation. Here, we established a CRISPR/Cas9 gene editing system in human CD34 + HSPCs and achieved efficient CCR5 ablation evaluated in long-term reconstituted NOD/Prkdc scid /IL-2Rγ null mice. The CCR5 disruption efficiency in our system remained robust in secondary transplanted repopulating hematopoietic cells. More importantly, an HIV-1 resistance effect was observed as indicated by significant reduction of virus titration and enrichment of human CD4 + T cells. Hence, we successfully established a CRISPR/Cas9 mediated CCR5 ablating system in long-term HSCs, which confers HIV-1 resistance in vivo. Our study provides evidence for translating CCR5 gene-edited HSC transplantation for an HIV cure to the clinic. Copyright © 2017 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.

  10. Novel resistance to Cydia pomonella granulovirus (CpGV in codling moth shows autosomal and dominant inheritance and confers cross-resistance to different CpGV genome groups.

    Directory of Open Access Journals (Sweden)

    Annette J Sauer

    Full Text Available Commercial Cydia pomonella granulovirus (CpGV products have been successfully applied to control codling moth (CM in organic and integrated fruit production for more than 30 years. Since 2005, resistance against the widely used isolate CpGV-M has been reported from different countries in Europe. The inheritance of this so-called type I resistance is dominant and linked to the Z chromosome. Recently, a second form (type II of CpGV resistance in CM was reported from a field population (NRW-WE in Germany. Type II resistance confers reduced susceptibility not only to CpGV-M but to most known CpGV isolates and it does not follow the previously described Z-linked inheritance of type I resistance. To further analyze type II resistance, two CM strains, termed CpR5M and CpR5S, were generated from parental NRW-WE by repeated mass crosses and selection using the two isolates CpGV-M and CpGV-S, respectively. Both CpR5M and CpR5S were considered to be genetically homogeneous for the presence of the resistance allele(s. By crossing and backcrossing experiments with a susceptible CM strain, followed by resistance testing of the offspring, an autosomal dominant inheritance of resistance was elucidated. In addition, cross-resistance to CpGV-M and CpGV-S was detected in both strains, CpR5M and CpR5S. To test the hypothesis that the autosomal inheritance of type II resistance was caused by a large interchromosomal rearrangement involving the Z chromosome, making type I resistance appear to be autosomal in these strains; fluorescence in situ hybridization with bacterial artificial chromosome probes (BAC-FISH was used to physically map the Z chromosomes of different CM strains. Conserved synteny of the Z-linked genes in CpR5M and other CM strains rejects this hypothesis and argues for a novel genetic and functional mode of resistance in CM populations with type II resistance.

  11. Novel resistance to Cydia pomonella granulovirus (CpGV) in codling moth shows autosomal and dominant inheritance and confers cross-resistance to different CpGV genome groups.

    Science.gov (United States)

    Sauer, Annette J; Fritsch, Eva; Undorf-Spahn, Karin; Nguyen, Petr; Marec, Frantisek; Heckel, David G; Jehle, Johannes A

    2017-01-01

    Commercial Cydia pomonella granulovirus (CpGV) products have been successfully applied to control codling moth (CM) in organic and integrated fruit production for more than 30 years. Since 2005, resistance against the widely used isolate CpGV-M has been reported from different countries in Europe. The inheritance of this so-called type I resistance is dominant and linked to the Z chromosome. Recently, a second form (type II) of CpGV resistance in CM was reported from a field population (NRW-WE) in Germany. Type II resistance confers reduced susceptibility not only to CpGV-M but to most known CpGV isolates and it does not follow the previously described Z-linked inheritance of type I resistance. To further analyze type II resistance, two CM strains, termed CpR5M and CpR5S, were generated from parental NRW-WE by repeated mass crosses and selection using the two isolates CpGV-M and CpGV-S, respectively. Both CpR5M and CpR5S were considered to be genetically homogeneous for the presence of the resistance allele(s). By crossing and backcrossing experiments with a susceptible CM strain, followed by resistance testing of the offspring, an autosomal dominant inheritance of resistance was elucidated. In addition, cross-resistance to CpGV-M and CpGV-S was detected in both strains, CpR5M and CpR5S. To test the hypothesis that the autosomal inheritance of type II resistance was caused by a large interchromosomal rearrangement involving the Z chromosome, making type I resistance appear to be autosomal in these strains; fluorescence in situ hybridization with bacterial artificial chromosome probes (BAC-FISH) was used to physically map the Z chromosomes of different CM strains. Conserved synteny of the Z-linked genes in CpR5M and other CM strains rejects this hypothesis and argues for a novel genetic and functional mode of resistance in CM populations with type II resistance.

  12. Two non-target recessive genes confer resistance to the anti-oomycete microtubule inhibitor zoxamide in Phytophthora capsici.

    Directory of Open Access Journals (Sweden)

    Yang Bi

    Full Text Available This study characterized isolates of P. capsici that had developed a novel mechanism of resistance to zoxamide, which altered the minimum inhibition concentration (MIC but not the EC50. Molecular analysis revealed that the β-tubulin gene of the resistant isolates contained no mutations and was expressed at the same level as in zoxamide-sensitive isolates. This suggested that P. capsici had developed a novel non-target-site-based resistance to zoxamide. Analysis of the segregation ratio of zoxamide-resistance in the sexual progeny of the sensitive isolates PCAS1 and PCAS2 indicated that the resistance to zoxamide was controlled by one or more recessive nuclear genes. Furthermore, the segregation of resistance in the F1, F2, and BC1 progeny was in accordance with the theoretical ratios of the χ(2 test (P>0.05, which suggested that the resistance to zoxamide was controlled by two recessive genes, and that resistance to zoxamide occurred when at least one pair of these alleles was homozygous. This implies that the risk of zoxamide-resistance in P. capsici is low to moderate. Nevertheless this potential for resistance should be monitored closely, especially if two compatible mating types co-exist in the same field.

  13. Resistance to the macrolide antibiotic tylosin is conferred by single methylations at 23S rRNA nucleotides G748 and A2058 acting in synergy

    Science.gov (United States)

    Liu, Mingfu; Douthwaite, Stephen

    2002-01-01

    The macrolide antibiotic tylosin has been used extensively in veterinary medicine and exerts potent antimicrobial activity against Gram-positive bacteria. Tylosin-synthesizing strains of the Gram-positive bacterium Streptomyces fradiae protect themselves from their own product by differential expression of four resistance determinants, tlrA, tlrB, tlrC, and tlrD. The tlrB and tlrD genes encode methyltransferases that add single methyl groups at 23S rRNA nucleotides G748 and A2058, respectively. Here we show that methylation by neither TlrB nor TlrD is sufficient on its own to give tylosin resistance, and resistance is conferred by the G748 and A2058 methylations acting together in synergy. This synergistic mechanism of resistance is specific for the macrolides tylosin and mycinamycin that possess sugars extending from the 5- and 14-positions of the macrolactone ring and is not observed for macrolides, such as carbomycin, spiramycin, and erythromycin, that have different constellations of sugars. The manner in which the G748 and A2058 methylations coincide with the glycosylation patterns of tylosin and mycinamycin reflects unambiguously how these macrolides fit into their binding site within the bacterial 50S ribosomal subunit. PMID:12417742

  14. The Arabidopsis genes RPW8.1 and RPW8.2 confer induced resistance to powdery mildew diseases in tobacco.

    Science.gov (United States)

    Xiao, Shunyuan; Charoenwattana, Piyavadee; Holcombe, Lucy; Turner, John G

    2003-04-01

    Plant disease resistance (R) gene products recognize pathogen avirulence (Avr) gene products and induce defense responses. It is not known if an R gene can function in different plant families, however. The Arabidopsis thaliana R genes RPW8.1 and RPW8.2 confer resistance to the powdery mildew pathogens Erysiphe orontii, E. cichoracearum, and Oidium lycopersici, which also infect plants from other families. We produced transgenic Nicotiana tabacum, N. benthamiana, and Lycopersicon esculentum plants containing RPW8.1 and RPW8.2. Transgenic N. tabacum plants had increased resistance to E. orontii and O. lycopersici, transgenic N. benthamiana plants had increased resistance to E. cichoracearum, but transgenic L. esculentum plants remained susceptible to these pathogens. The defense responses induced in transgenic N. tabacum and N. benthamiana were similar to those mediated by RPW8.1 and RPW8.2 in Arabidopsis. Apparently, RPW8.1 and RPW8.2 could be used to control powdery mildew diseases of plants from other families.

  15. The MLA6 coiled-coil, NBS-LRR protein confers AvrMla6-dependent resistance specificity to Blumeria graminis f. sp. hordei in barley and wheat.

    Science.gov (United States)

    Halterman, D; Zhou, F; Wei, F; Wise, R P; Schulze-Lefert, P

    2001-02-01

    The barley Mla locus confers multiple resistance specificities to the obligate fungal biotroph, Blumeria (= Erysiphe) graminis f. sp. hordei. Interspersed within the 240 kb Mla complex are three families of resistance gene homologs (RGHs). Probes from the Mla-RGH1 family were used to identify three classes of cDNAs. The first class is predicted to encode a full-length CC-NBS-LRR protein and the other two classes contain alternatively spliced, truncated variants. Utilizing a cosmid that contains a gene corresponding to the full-length candidate cDNA, two single-cell expression assays were used to demonstrate complementation of AvrMla6-dependent, resistance specificity to B. graminis in barley and wheat. The first of these assays was also used to substantiate previous genetic data that the Mla6 allele requires the signaling pathway component, Rar1, for function. Computational analysis of MLA6 and the Rar1-independent, MLA1 protein reveals 91.2% identity and shows that the LRR domain is subject to diversifying selection. Our findings demonstrate that highly related CC-NBS-LRR proteins encoded by alleles of the Mla locus can dictate similar powdery mildew resistance phenotypes yet still require distinct downstream signaling components.

  16. The targeted inhibition of mitochondrial Hsp90 overcomes the apoptosis resistance conferred by Bcl-2 in Hep3B cells via necroptosis

    International Nuclear Information System (INIS)

    Yan, Chunlan; Oh, Joon Seok; Yoo, Seung Hee; Lee, Jee Suk; Yoon, Young Geol; Oh, Yoo Jin; Jang, Min Seok; Lee, Sang Yeob; Yang, Jun; Lee, Sang Hwa; Kim, Hye Young; Yoo, Young Hyun

    2013-01-01

    Previous studies have reported that a Gamitrinib variant containing triphenylphosphonium (G-TPP) binds to mitochondrial Hsp90 and rapidly inhibits its activity, thus inducing the apoptotic pathway in the cells. Accordingly, G-TPP shows a potential as a promising drug for the treatment of cancer. A cell can die from different types of cell death such as apoptosis, necrosis, necroptosis, and autophagic cell death. In this study, we further investigated the mechanisms and modes of cell death in the G-TPP-treated Hep3B and U937 cell lines. We discovered that G-TPP kills the U937 cells through the apoptotic pathway and the overexpression of Bcl-2 significantly inhibits U937 cell death to G-TPP. We further discovered that G-TPP kills the Hep3B cells by activating necroptosis in combination with the partial activation of caspase-dependent apoptosis. Importantly, G-TPP overcomes the apoptosis resistance conferred by Bcl-2 in Hep3B cells via necroptosis. We also observed that G-TPP induces compensatory autophagy in the Hep3B cell line. We further found that whereas there is a Bcl-2-Beclin 1 interaction in response to G-TPP, silencing the beclin 1 gene failed to block LC3-II accumulation in the Hep3B cells, indicating that G-TPP triggers Beclin 1-independent protective autophagy in Hep3B cells. Taken together, these data reveal that G-TPP induces cell death through a combination of death pathways, including necroptosis and apoptosis, and overcomes the apoptosis resistance conferred by Bcl-2 in Hep3B cells via necroptosis. These findings are important for the therapeutic exploitation of necroptosis as an alternative cell death program to bypass the resistance to apoptosis. Highlights: ► G-TPP binds to mitochondrial Hsp90. ► G-TPP induces apoptosis in U937 human leukemia cancer cells. ► G-TPP induces combination of death pathways in Hep3B cell. ► G-TPP overcomes the resistance conferred by Bcl-2 in Hep3B cells via necroptosis. ► G-TPP triggers Beclin 1-independent

  17. Multidrug-Resistant Salmonella enterica Serovar Muenchen from Pigs and Humans and Potential Interserovar Transfer of Antimicrobial Resistance

    OpenAIRE

    Gebreyes, Wondwossen A.; Thakur, Siddhartha

    2005-01-01

    Salmonella serovars are important reservoirs of antimicrobial resistance. Recently, we reported on multidrug-resistant (MDR) Salmonella enterica serovar Typhimurium strains among pigs with resistance to ampicillin, kanamycin, streptomycin, sulfamethoxazole, and tetracycline (resistance [R] type AKSSuT) and resistance to amoxicillin-clavulanic acid, ampicillin, chloramphenicol, streptomycin, sulfamethoxazole, and tetracycline (R type AxACSSuT). In the present study, 67 isolates (39 from humans...

  18. Precise gene editing of chicken Na+/H+ exchange type 1 (chNHE1) confers resistance to avian leukosis virus subgroup J (ALV-J).

    Science.gov (United States)

    Lee, Hong Jo; Lee, Kyung Youn; Jung, Kyung Min; Park, Kyung Je; Lee, Ko On; Suh, Jeong-Yong; Yao, Yongxiu; Nair, Venugopal; Han, Jae Yong

    2017-12-01

    Avian leukosis virus subgroup J (ALV-J), first isolated in the late 1980s, has caused economic losses to the poultry industry in many countries. As all chicken lines studied to date are susceptible to ALV infection, there is enormous interest in developing resistant chicken lines. The ALV-J receptor, chicken Na + /H + exchange 1 (chNHE1) and the critical amino acid sequences involved in viral attachment and entry have already been characterized. However, there are no reported attempts to induce resistance to the virus by targeted genome modification of the receptor sequences. In an attempt to induce resistance to ALV-J infection, we used clustered regularly interspaced short palindromic repeats (CRISPR)-associated (CRISPR/Cas9)-based genome editing approaches to modify critical residues of the chNHE1 receptor in chicken cells. The susceptibility of the modified cell lines to ALV-J infection was examined using enhanced green fluorescent protein (EGFP)-expressing marker viruses. We showed that modifying the chNHE1 receptor by artificially generating a premature stop codon induced absolute resistance to viral infection, with mutations of the tryptophan residue at position 38 (Trp38) being very critical. Single-stranded oligodeoxynucleotide (ssODN)-mediated targeted recombination of the Trp38 region revealed that deletions involving the Trp38 residue were most effective in conferring resistance to ALV-J. Moreover, protein structure analysis of the chNHE1 receptor sequence suggested that its intrinsically disordered region undergoes local conformational changes through genetic alteration. Collectively, these results demonstrate that targeted mutations on chNHE1 alter the susceptibility to ALV-J and the technique is expected to contribute to develop disease-resistant chicken lines. Copyright © 2017 Elsevier Ltd. All rights reserved.

  19. Technical note: Occurrence in fecal microbiota of genes conferring resistance to both macrolide-lincosamide-streptogramin B and tetracyclines concomitant with feeding of beef cattle with tylosin.

    Science.gov (United States)

    Chen, J; Fluharty, F L; St-Pierre, N; Morrison, M; Yu, Z

    2008-09-01

    Development of antimicrobial resistance in food animals receiving antimicrobials has been well documented among bacterial isolates, especially pathogens, but information on development of antimicrobial resistance at the microbial community level during long-term feeding of antimicrobials is lacking. The objective of this study was to examine the association between inclusion of tylosin in feed and occurrence of resistance to macrolide-lincosamide-streptogramin B (MLS(B)) in the entire fecal microbial communities of beef cattle over a feeding study of 168 d. A completely randomized design included 6 pens housed together in 1 barn, with each pen housing 10 to 11 steers. The control and tylosin groups each had 3 pens, with the former receiving no antimicrobial whereas the latter received both tylosin and monensin (11 and 29.9 mg/ kg of feed, respectively, DM) in feed. The abundance of genes conferring resistance to MLS(B) (erm genes) and tetracyclines (tet genes) were quantified using class-specific, real-time PCR assays. The abundances of erm and tet genes were analyzed with pens as experimental units using the MIXED procedure of SAS. Correlations between abundance of different resistance genes were calculated using the CORR procedure of SAS. We identified 4 classes (B, F, T, and X) of erm genes in fresh fecal samples collected at wk 2, 17, and 21 of feeding. From wk 2 to 17, the abundance of erm(T) and erm(X) increased (P tylosin feeding. Such co-selection of multiresistance at community level by one antimicrobial drug used in animals has the important implication that future studies should examine resistance to not only the antimicrobials used in animals, but also other antimicrobials, especially those used in human medicine, to fully assess the potential risk associated with antimicrobial use in animals. Both the erm and tet genes appeared to be disseminated among the microbial populations in all steers housed together.

  20. Ectopic expression of MgSM1, a Cerato-platanin family protein from Magnaporthe grisea, confers broad-spectrum disease resistance in Arabidopsis.

    Science.gov (United States)

    Yang, Yayun; Zhang, Huijuan; Li, Guojun; Li, Wei; Wang, Xiao'e; Song, Fengming

    2009-10-01

    Proteins belonging to the newly identified Cerato-platanin (CP) family have been shown to have elicitor activity in inducing disease resistance responses in various plants. In this study, we characterized a gene, MgSM1, from Magnaporthe grisea, encoding a putative small protein belonging to the CP family. MgSM1 was constitutively expressed not only in different fungal growth stages but also during its infection process in rice plants. Agrobacterium-mediated transient expression of MgSM1 in Arabidopsis resulted in hypersensitive response in the infiltrated local leaves and enhanced disease resistance against Botrytis cinerea and Pseudomonas syringae pv. tomato (Pst) DC3000 in upper leaves of plants, accompanyed by up-regulated expression of defense genes (PR-1, PR-5 and PDF1.2). Transgenic Arabidopsis plants expressing MgSM1 under control of a dexamethasone (DEX)-inducible promoter were generated. Expression of MgSM1 in transgenic plants was induced by exogenous application of DEX. MgSM1-expressing plants showed normal growth with application of resistance against B. cinerea, Alternaria brassicicola and Psto DC3000 as well as up-regulated expression of some of defense genes. Moreover, accumulation of reactive oxygen species was observed in MgSM1-expressing plants. These results collectively suggest that ectopic expression of MgSM1 in transgenic plants confers broad-spectrum resistance against different types of pathogens. Our study also provides a novel strategy to generate environment-friendly crops with enhanced broad-spectrum resistance through ectopic expression of microbe-derived disease resistance-inducing proteins.

  1. Identification and mapping of Sr46 from Aegilops tauschii accession CIae 25 conferring resistance to race TTKSK (Ug99) of wheat stem rust pathogen.

    Science.gov (United States)

    Yu, Guotai; Zhang, Qijun; Friesen, Timothy L; Rouse, Matthew N; Jin, Yue; Zhong, Shaobin; Rasmussen, Jack B; Lagudah, Evans S; Xu, Steven S

    2015-03-01

    Mapping studies confirm that resistance to Ug99 race of stem rust pathogen in Aegilops tauschii accession Clae 25 is conditioned by Sr46 and markers linked to the gene were developed for marker-assisted selection. The race TTKSK (Ug99) of Puccinia graminis f. sp. tritici, the causal pathogen for wheat stem rust, is considered as a major threat to global wheat production. To address this threat, researchers across the world have been devoted to identifying TTKSK-resistant genes. Here, we report the identification and mapping of a stem rust resistance gene in Aegilops tauschii accession CIae 25 that confers resistance to TTKSK and the development of molecular markers for the gene. An F2 population of 710 plants from an Ae. tauschii cross CIae 25 × AL8/78 were first evaluated against race TPMKC. A set of 14 resistant and 116 susceptible F2:3 families from the F2 plants were then evaluated for their reactions to TTKSK. Based on the tests, 179 homozygous susceptible F2 plants were selected as the mapping population to identify the simple sequence repeat (SSR) and sequence tagged site (STS) markers linked to the gene by bulk segregant analysis. A dominant stem rust resistance gene was identified and mapped with 16 SSR and five new STS markers to the deletion bin 2DS5-0.47-1.00 of chromosome arm 2DS in which Sr46 was located. Molecular marker and stem rust tests on CIae 25 and two Ae. tauschii accessions carrying Sr46 confirmed that the gene in CIae 25 is Sr46. This study also demonstrated that Sr46 is temperature-sensitive being less effective at low temperatures. The marker validation indicated that two closely linked markers Xgwm210 and Xwmc111 can be used for marker-assisted selection of Sr46 in wheat breeding programs.

  2. Emergence of a new mutation and its accumulation in the topoisomerase IV gene confers high levels of resistance to fluoroquinolones in Escherichia coli isolates.

    Science.gov (United States)

    Moon, Dong Chan; Seol, Sung Yong; Gurung, Mamata; Jin, Jong Sook; Choi, Chul Hee; Kim, Jungmin; Lee, Yoo Chul; Cho, Dong Taek; Lee, Je Chul

    2010-01-01

    Mutations in DNA gyrase and topoisomerase IV genes are the main mechanisms of resistance to quinolones. In this study, we determined mutations in gyrA, gyrB, parC and parE among 57 ciprofloxacin-resistant Escherichia coli isolates from a South Korean hospital and analysed the relationship between the minimal inhibitory concentrations (MICs) of fluoroquinolones and mutations in the topoisomerase IV gene. All ciprofloxacin-resistant E. coli isolates carried double mutations in gyrA and at least a single mutation in parC; some isolates also carried a single mutation in parE. The most common mutations were S83L and D87N in gyrA, S80I in parC and S458A in parE, which accounted for 25% of isolates. Single mutations in parE at L445I, S458P and S458W were identified for the first time. Double mutations in parC and a combination of single mutations in parC and parE significantly increased the MIC values of fluoroquinolones. In vitro induction of resistance to ciprofloxacin showed that double mutations in gyrA were a prerequisite to conferring a resistant phenotype to fluoroquinolones, and an additional mutation in the topoisomerase IV gene increased the MIC values of ciprofloxacin. In conclusion, emergence of a new mutation in parC and parE and its accumulation induces high levels of resistance to fluoroquinolones in E. coli.

  3. Inducible Expression of both ermB and ermT Conferred High Macrolide Resistance in Streptococcus gallolyticus subsp. pasteurianus Isolates in China

    Directory of Open Access Journals (Sweden)

    Meixia Li

    2016-09-01

    Full Text Available Streptococcus gallolyticus subsp. pasteurianus is an under-recognized pathogen and zoonotic agent causing opportunistic infections in humans. Despite increasing recognition of this subspecies as a cause for human infectious diseases, limited information is known about its antibiotic resistance mechanism. In this study, we aim to identify the molecular mechanism underlying the high macrolide resistance of six S. gallolyticus subsp. pasteurianus isolates from dead ducklings collected in several natural outbreaks in China during 2010–2013. All isolates exhibited multi-drug resistance including high macrolide resistance (MIC ≥ 1024 mg/L for erythromycin, and 512 mg/L for clarithromycin. Efflux-encoding mefA and mefE genes were not detectable in these isolates. The presence of 23S rRNA mutations in specific isolates did not significantly change macrolide MICs. No nucleotide substitutions were found in genes encoding ribosomal proteins L4 or L22. The ermB and ermT genes were found in the genomes of all isolates. These two genes were acquired independently in one highly virulent isolate AL101002, and clustered with Tn916 and IS1216, respectively. The expression of both ermB and ermT in all isolates was erythromycin inducible and yielded comparable macrolide MICs in all six isolates. Taken together, inducible expression of both ermB and ermT conferred high macrolide resistance in these S. gallolyticus subsp. pasterianus isolates. Our findings reveal new macrolide resistance features in S. gallolyticus subsp. pasteurianus by both ermB and ermT.

  4. Transposon Tn5393e carrying the aphA1-containing transposon Tn6023 upstream of strAB does not confer resistance to streptomycin.

    Science.gov (United States)

    Cain, Amy K; Hall, Ruth M

    2011-09-01

    The simplest form of transposon Tn5393 carries the strA and strB genes that confer resistance to streptomycin, in addition to its transposition determinants. Tn5393e, made up of Tn5393 and a second transposon, Tn6023, was found in an IncHI2 plasmid, pSRC125, recovered from a multiply antibiotic-resistant Salmonella enterica serovar Typhimurium isolate of bovine origin. Tn6023 is made up of the aphA1b gene flanked by two inversely oriented copies of insertion sequence (IS) IS26 and is flanked by an 8 bp duplication. It is related to several other transposons that carry aphA1b in fragments of differing length, also flanked by copies of IS26. Tn6023 is located in the tnpR gene of Tn5393, which lies upstream of the strA and strB genes, and the combined structure was designated Tn5393e. Although neither strA nor strB contain any mutations that would inactivate them, pSRC125 does not confer resistance to streptomycin, indicating that the strA and strB genes are not expressed. In Tn5393, strA and strB are transcribed from the tnpR promoter, and in Tn5393e neither this transcript nor the transcript from the aphA1b promoter in Tn6023 must reach strAB. Tn5393e was previously found in different locations in Corynebacteria, indicating that it can move and suggesting a wide distribution. The structures of several further variants of Tn5393 found in GenBank were analyzed and assigned variant designations Tn5393f-Tn5393i.

  5. Overexpression of BSR1 confers broad-spectrum resistance against two bacterial diseases and two major fungal diseases in rice.

    Science.gov (United States)

    Maeda, Satoru; Hayashi, Nagao; Sasaya, Takahide; Mori, Masaki

    2016-06-01

    Broad-spectrum disease resistance against two or more types of pathogen species is desirable for crop improvement. In rice, Xanthomonas oryzae pv. oryzae (Xoo), the causal bacteria of rice leaf blight, and Magnaporthe oryzae, the fungal pathogen causing rice blast, are two of the most devastating pathogens. We identified the rice BROAD-SPECTRUM RESISTANCE 1 (BSR1) gene for a BIK1-like receptor-like cytoplasmic kinase using the FOX hunting system, and demonstrated that BSR1-overexpressing (OX) rice showed strong resistance to the bacterial pathogen, Xoo and the fungal pathogen, M. oryzae. Here, we report that BSR1-OX rice showed extended resistance against two other different races of Xoo, and to at least one other race of M. oryzae. In addition, the rice showed resistance to another bacterial species, Burkholderia glumae, which causes bacterial seedling rot and bacterial grain rot, and to Cochliobolus miyabeanus, another fungal species causing brown spot. Furthermore, BSR1-OX rice showed slight resistance to rice stripe disease, a major viral disease caused by rice stripe virus. Thus, we demonstrated that BSR1-OX rice shows remarkable broad-spectrum resistance to at least two major bacterial species and two major fungal species, and slight resistance to one viral pathogen.

  6. Menoctone Resistance in Malaria Parasites Is Conferred by M133I Mutations in Cytochrome b That Are Transmissible through Mosquitoes.

    Science.gov (United States)

    Blake, Lynn D; Johnson, Myles E; Siegel, Sasha V; McQueen, Adonis; Iyamu, Iredia D; Shaikh, Abdul Kadar; Shultis, Michael W; Manetsch, Roman; Kyle, Dennis E

    2017-08-01

    Malaria-related mortality has slowly decreased over the past decade; however, eradication of malaria requires the development of new antimalarial chemotherapies that target liver stages of the parasite and combat the emergence of drug resistance. The diminishing arsenal of anti-liver-stage compounds sparked our interest in reviving the old and previously abandoned compound menoctone. In support of these studies, we developed a new convergent synthesis method that was facile, required fewer steps, produced better yields, and utilized less expensive reagents than the previously published method. Menoctone proved to be highly potent against liver stages of Plasmodium berghei (50 percent inhibitory concentration [IC 50 ] = 0.41 nM) and erythrocytic stages of Plasmodium falciparum (113 nM). We selected for resistance to menoctone and found M133I mutations in cytochrome b of both P. falciparum and P. berghei The same mutation has been observed previously in atovaquone resistance, and we confirmed cross-resistance between menoctone and atovaquone in vitro (for P. falciparum ) and in vivo (for P. berghei ). Finally, we assessed the transmission potential of menoctone-resistant P. berghei and found that the M133I mutant parasites were readily transmitted from mouse to mosquitoes and back to mice. In each step, the M133I mutation in cytochrome b , inducing menoctone resistance, was confirmed. In summary, this study is the first to show the mechanism of resistance to menoctone and that menoctone and atovaquone resistance is transmissible through mosquitoes. Copyright © 2017 American Society for Microbiology.

  7. Phosphoprotein enriched in diabetes (PED/PEA15) promotes migration in hepatocellular carcinoma and confers resistance to sorafenib

    DEFF Research Database (Denmark)

    Quintavalle, Cristina; Hindupur, Sravanth Kumar; Quagliata, Luca

    2017-01-01

    Hepatocellular carcinoma (HCC) is the third-leading cause of cancer-related death with limited treatment options and frequent resistance to sorafenib, the only drug currently approved for first-line therapy. Therefore, better understanding of HCC tumor biology and its resistance to treatment is u...

  8. Mapping quantitative trait loci conferring resistance to a widely virulent isolate of Cochliobolus sativus in wild barley accession PI 466423.

    Science.gov (United States)

    Haas, Matthew; Menke, Jon; Chao, Shiaoman; Steffenson, Brian J

    2016-10-01

    This research characterized the genetics of resistance of wild barley accession PI 466423 to a widely virulent pathotype of Cochliobolus sativus . Breeding lines were identified that combine the Midwest Six-rowed Durable Resistance Haplotype and resistance to the virulent isolate ND4008. Spot blotch, caused by Cochliobolus sativus, is a historically important foliar disease of barley (Hordeum vulgare L.) in the Upper Midwest region of the USA. However, for the last 50 years this disease has been of little consequence due to the deployment of resistant six-rowed malting cultivars. These durably resistant cultivars carry the Midwest Six-rowed Durable Resistant Haplotype (MSDRH) comprised of three Quantitative Trait Loci (QTL) on chromosomes 1H, 3H and 7H, originally contributed by breeding line NDB112. Recent reports of C. sativus isolates (e.g. ND4008) with virulence on NDB112 indicate that widely grown cultivars of the region are vulnerable to spot blotch epidemics. Wild barley (H. vulgare ssp. spontaneum), the progenitor of cultivated barley, is a rich source of novel alleles, especially for disease resistance. Wild barley accession PI 466423 is highly resistant to C. sativus isolate ND4008. To determine the genetic architecture of resistance to isolate ND4008 in PI 466423, we phenotyped and genotyped an advanced backcross population (N = 244) derived from the wild accession and the recurrent parent 'Rasmusson', a Minnesota cultivar with the MSDRH. Disease phenotyping was done on BC2F4 seedlings in the greenhouse using isolate ND4008. The Rasmusson/PI 466423 population was genotyped with 7842 single nucleotide polymorphic markers. QTL analysis using composite interval mapping revealed four resistance loci on chromosomes 1H, 2H, 4H and 5H explaining 10.3, 7.4, 6.4 and 8.4 % of the variance, respectively. Resistance alleles on chromosomes 1H, 4H and 5H were contributed by PI 466423, whereas the one on chromosome 2H was contributed by Rasmusson. All four

  9. Cuticle genes CpCPR63 and CpCPR47 may confer resistance to deltamethrin in Culex pipiens pallens.

    Science.gov (United States)

    Sun, Xueli; Guo, Juxin; Ye, Wenyun; Guo, Qin; Huang, Yun; Ma, Lei; Zhou, Dan; Shen, Bo; Sun, Yan; Zhu, Changliang

    2017-08-01

    Cuticular proteins (CPs) are implicated in insecticide resistance in mosquito populations. Here, we investigated the role of cuticular genes in regulation of insecticide resistance in Culex pipiens pallens. We identified two CpCPRs (CpCPR63 and CpCPR47) that exhibited higher transcript levels in pyrethroid-resistant strains than in susceptible strains. Mosquito mortality was increased after knockdown of CpCPR genes by dsRNA injection. The RNA interference experiment suggested an interaction between CpCPR63 and CpCPR47, as silencing of one gene resulted in decreased expression of the other. These findings revealed that CpCPRs may regulate pyrethroid resistance and could be used as a potential genetic marker to monitor pyrethroid resistance in mosquitoes.

  10. Arabidopsis nonhost resistance gene PSS1 confers immunity against an oomycete and a fungal pathogen but not a bacterial pathogen that cause diseases in soybean

    Directory of Open Access Journals (Sweden)

    Sumit Rishi

    2012-06-01

    Full Text Available Abstract Background Nonhost resistance (NHR provides immunity to all members of a plant species against all isolates of a microorganism that is pathogenic to other plant species. Three Arabidopsis thaliana PEN (penetration deficient genes, PEN1, 2 and 3 have been shown to provide NHR against the barley pathogen Blumeria graminis f. sp. hordei at the prehaustorial level. Arabidopsis pen1-1 mutant lacking the PEN1 gene is penetrated by the hemibiotrophic oomycete pathogen Phytophthora sojae, the causal organism of the root and stem rot disease in soybean. We investigated if there is any novel nonhost resistance mechanism in Arabidopsis against the soybean pathogen, P. sojae. Results The P.sojaesusceptible (pss 1 mutant was identified by screening a mutant population created in the Arabidopsis pen1-1 mutant that lacks penetration resistance against the non adapted barley biotrophic fungal pathogen, Blumeria graminis f. sp. hordei. Segregation data suggested that PEN1 is not epistatic to PSS1. Responses of pss1 and pen1-1 to P. sojae invasion were distinct and suggest that PSS1 may act at both pre- and post-haustorial levels, while PEN1 acts at the pre-haustorial level against this soybean pathogen. Therefore, PSS1 encodes a new form of nonhost resistance. The pss1 mutant is also infected by the necrotrophic fungal pathogen, Fusarium virguliforme, which causes sudden death syndrome in soybean. Thus, a common NHR mechanism is operative in Arabidopsis against both hemibiotrophic oomycetes and necrotrophic fungal pathogens that are pathogenic to soybean. However, PSS1 does not play any role in immunity against the bacterial pathogen, Pseudomonas syringae pv. glycinea, that causes bacterial blight in soybean. We mapped PSS1 to a region very close to the southern telomere of chromosome 3 that carries no known disease resistance genes. Conclusions The study revealed that Arabidopsis PSS1 is a novel nonhost resistance gene that confers a new form of

  11. Arabidopsis nonhost resistance gene PSS1 confers immunity against an oomycete and a fungal pathogen but not a bacterial pathogen that cause diseases in soybean.

    Science.gov (United States)

    Sumit, Rishi; Sahu, Binod B; Xu, Min; Sandhu, Devinder; Bhattacharyya, Madan K

    2012-06-13

    Nonhost resistance (NHR) provides immunity to all members of a plant species against all isolates of a microorganism that is pathogenic to other plant species. Three Arabidopsis thaliana PEN (penetration deficient) genes, PEN1, 2 and 3 have been shown to provide NHR against the barley pathogen Blumeria graminis f. sp. hordei at the prehaustorial level. Arabidopsis pen1-1 mutant lacking the PEN1 gene is penetrated by the hemibiotrophic oomycete pathogen Phytophthora sojae, the causal organism of the root and stem rot disease in soybean. We investigated if there is any novel nonhost resistance mechanism in Arabidopsis against the soybean pathogen, P. sojae. The P.sojaesusceptible (pss) 1 mutant was identified by screening a mutant population created in the Arabidopsis pen1-1 mutant that lacks penetration resistance against the non adapted barley biotrophic fungal pathogen, Blumeria graminis f. sp. hordei. Segregation data suggested that PEN1 is not epistatic to PSS1. Responses of pss1 and pen1-1 to P. sojae invasion were distinct and suggest that PSS1 may act at both pre- and post-haustorial levels, while PEN1 acts at the pre-haustorial level against this soybean pathogen. Therefore, PSS1 encodes a new form of nonhost resistance. The pss1 mutant is also infected by the necrotrophic fungal pathogen, Fusarium virguliforme, which causes sudden death syndrome in soybean. Thus, a common NHR mechanism is operative in Arabidopsis against both hemibiotrophic oomycetes and necrotrophic fungal pathogens that are pathogenic to soybean. However, PSS1 does not play any role in immunity against the bacterial pathogen, Pseudomonas syringae pv. glycinea, that causes bacterial blight in soybean. We mapped PSS1 to a region very close to the southern telomere of chromosome 3 that carries no known disease resistance genes. The study revealed that Arabidopsis PSS1 is a novel nonhost resistance gene that confers a new form of nonhost resistance against both a hemibiotrophic oomycete

  12. Discovery of a Novel er1 Allele Conferring Powdery Mildew Resistance in Chinese Pea (Pisum sativum L.) Landraces.

    Science.gov (United States)

    Sun, Suli; Fu, Haining; Wang, Zhongyi; Duan, Canxing; Zong, Xuxiao; Zhu, Zhendong

    2016-01-01

    Pea powdery mildew, caused by Erysiphe pisi D.C., is an important disease worldwide. Deployment of resistant varieties is the main way to control this disease. This study aimed to screen Chinese pea (Pisum sativum L.) landraces resistant to E. pisi, and to characterize the resistance gene(s) at the er1 locus in the resistant landraces, and to develop functional marker(s) specific to the novel er1 allele. The 322 landraces showed different resistance levels. Among them, 12 (3.73%), 4 (1.24%) and 17 (5.28%) landraces showed immunity, high resistance and resistance to E. pisi, respectively. The other landraces appeared susceptible or highly susceptible to E. pisi. Most of the immune and highly resistant landraces were collected from Yunnan province. To characterize the resistance gene at the er1 locus, cDNA sequences of PsMLO1 gene were determined in 12 immune and four highly resistant accessions. The cDNAs of PsMLO1 from the immune landrace G0005576 produced three distinct transcripts, characterized by a 129-bp deletion, and 155-bp and 220-bp insertions, which were consistent with those of er1-2 allele. The PsMLO1 cDNAs in the other 15 resistant landraces produced identical transcripts, which had a new point mutation (T→C) at position 1121 of PsMLO1, indicating a novel er1 allele, designated as er1-6. This mutation caused a leucine to proline change in the amino acid sequence. Subsequently, the resistance allele er1-6 in landrace G0001778 was confirmed by resistance inheritance analysis and genetic mapping on the region of the er1 locus using populations derived from G0001778 × Bawan 6. Finally, a functional marker specific to er1-6, SNP1121, was developed using the high-resolution melting technique, which could be used in pea breeding via marker-assisted selection. The results described here provide valuable genetic information for Chinese pea landraces and a powerful tool for pea breeders.

  13. Discovery of a Novel er1 Allele Conferring Powdery Mildew Resistance in Chinese Pea (Pisum sativum L. Landraces.

    Directory of Open Access Journals (Sweden)

    Suli Sun

    Full Text Available Pea powdery mildew, caused by Erysiphe pisi D.C., is an important disease worldwide. Deployment of resistant varieties is the main way to control this disease. This study aimed to screen Chinese pea (Pisum sativum L. landraces resistant to E. pisi, and to characterize the resistance gene(s at the er1 locus in the resistant landraces, and to develop functional marker(s specific to the novel er1 allele. The 322 landraces showed different resistance levels. Among them, 12 (3.73%, 4 (1.24% and 17 (5.28% landraces showed immunity, high resistance and resistance to E. pisi, respectively. The other landraces appeared susceptible or highly susceptible to E. pisi. Most of the immune and highly resistant landraces were collected from Yunnan province. To characterize the resistance gene at the er1 locus, cDNA sequences of PsMLO1 gene were determined in 12 immune and four highly resistant accessions. The cDNAs of PsMLO1 from the immune landrace G0005576 produced three distinct transcripts, characterized by a 129-bp deletion, and 155-bp and 220-bp insertions, which were consistent with those of er1-2 allele. The PsMLO1 cDNAs in the other 15 resistant landraces produced identical transcripts, which had a new point mutation (T→C at position 1121 of PsMLO1, indicating a novel er1 allele, designated as er1-6. This mutation caused a leucine to proline change in the amino acid sequence. Subsequently, the resistance allele er1-6 in landrace G0001778 was confirmed by resistance inheritance analysis and genetic mapping on the region of the er1 locus using populations derived from G0001778 × Bawan 6. Finally, a functional marker specific to er1-6, SNP1121, was developed using the high-resolution melting technique, which could be used in pea breeding via marker-assisted selection. The results described here provide valuable genetic information for Chinese pea landraces and a powerful tool for pea breeders.

  14. Two Cassava Basic Leucine Zipper (bZIP Transcription Factors (MebZIP3 and MebZIP5 Confer Disease Resistance against Cassava Bacterial Blight

    Directory of Open Access Journals (Sweden)

    Xiaolin Li

    2017-12-01

    Full Text Available Basic domain-leucine zipper (bZIP transcription factor, one type of conserved gene family, plays an important role in plant development and stress responses. Although 77 MebZIPs have been genome-wide identified in cassava, their in vivo roles remain unknown. In this study, we analyzed the expression pattern and the function of two MebZIPs (MebZIP3 and MebZIP5 in response to pathogen infection. Gene expression analysis indicated that MebZIP3 and MebZIP5 were commonly regulated by flg22, Xanthomonas axonopodis pv. manihotis (Xam, salicylic acid (SA, and hydrogen peroxide (H2O2. Subcellular localization analysis showed that MebZIP3 and MebZIP5 are specifically located in cell nucleus. Through overexpression in tobacco, we found that MebZIP3 and MebZIP5 conferred improved disease resistance against cassava bacterial blight, with more callose depositions. On the contrary, MebZIP3- and MebZIP5-silenced plants by virus-induced gene silencing (VIGS showed disease sensitive phenotype, lower transcript levels of defense-related genes and less callose depositions. Taken together, this study highlights the positive role of MebZIP3 and MebZIP5 in disease resistance against cassava bacterial blight for further utilization in genetic improvement of cassava disease resistance.

  15. Conjugative IncF and IncI1 plasmids with tet(A) and class 1 integron conferring multidrug resistance in F18(+) porcine enterotoxigenic E. coli.

    Science.gov (United States)

    Szmolka, Ama; Lestár, Barbara; Pászti, Judit; Fekete, Péter; Nagy, Béla

    2015-12-01

    Enterotoxigenic E. coli (ETEC) bacteria frequently cause watery diarrhoea in newborn and weaned pigs. Plasmids carrying genes of different enterotoxins and fimbrial adhesins, as well as plasmids conferring antimicrobial resistance are of prime importance in the epidemiology and pathogenesis of ETEC. Recent studies have revealed the significance of the porcine ETEC plasmid pTC, carrying tetracycline resistance gene tet(B) with enterotoxin genes. In contrast, the role of tet(A) plasmids in transferring resistance of porcine ETEC is less understood. The objective of the present study was to provide a comparative analysis of antimicrobial resistance and virulence gene profiles of porcine post-weaning ETEC strains representing pork-producing areas in Central Europe and in the USA, with special attention to plasmids carrying the tet(A) gene. Antimicrobial resistance phenotypes and genotypes of 87 porcine ETEC strains isolated from cases of post-weaning diarrhoea in Austria, the Czech Republic, Hungary and the Midwest USA was determined by disk diffusion and by PCR. Central European strains carrying tet(A) or tet(B) were further subjected to molecular characterisation of their tet plasmids. Results indicated that > 90% of the ETEC strains shared a common multidrug resistant (MDR) pattern of sulphamethoxazole (91%), tetracycline (84%) and streptomycin (80%) resistance. Tetracycline resistance was most frequently determined by the tet(B) gene (38%), while tet(A) was identified in 26% of all isolates with wide ranges for both tet gene types between some countries and with class 1 integrons and resistance genes co-transferred by conjugation. The virulence gene profiles included enterotoxin genes (lt, sta and/or stb), as well as adhesin genes (k88/f4, f18). Characterisation of two representative tet(A) plasmids of porcine F18(+) ETEC from Central Europe revealed that the IncF plasmid (pES11732) of the Czech strain (~120 kb) carried tet(A) in association with catA1 for

  16. Alterations in penicillin-binding protein 1A confer resistance to beta-lactam antibiotics in Helicobacter pylori

    NARCIS (Netherlands)

    M.M. Gerrits (Monique); D. Schuijffel; A.A. van Zwet (Anton); E.J. Kuipers (Ernst); C.M.J.E. Vandenbroucke-Grauls (Christina); J.G. Kusters (Johannes)

    2002-01-01

    textabstractMost Helicobacter pylori strains are susceptible to amoxicillin, an important component of combination therapies for H. pylori eradication. The isolation and initial characterization of the first reported stable amoxicillin-resistant clinical H. pylori isolate (the

  17. Alterations in penicillin-binding protein 1A confer resistance to beta-lactam antibiotics in Helicobacter pylori

    NARCIS (Netherlands)

    Gerrits, M. M.; Schuijffel, D.; van Zwet, A. A.; Kuipers, E. J.; Vandenbroucke-Grauls, C. M. J. E.; Kusters, J. G.

    2002-01-01

    Most Helicobacter pylori strains are susceptible to amoxicillin, an important component of combination therapies for H. pylori eradication. The isolation and initial characterization of the first reported stable amoxicillin-resistant clinical H. pylori isolate (the Hardenberg strain) have been

  18. Insights into the Enhanced in vivo Fitness of Neisseria gonorrhoeae Driven by a Fluoroquinolone Resistance-Conferring Mutant DNA Gyrase

    Science.gov (United States)

    2015-02-05

    93 xi CHAPTER 1: Introduction Neisseria gonorrhoeae The majority of the 19 species within the genus Neisseria are...families: the resistance- nodulation-cell division (RND) family, the ATP-binding cassette (ABC) family, the small multi- drug resistance family, the...urine from men are the specimens of choice for NAAT-based detection of N. gonorrhoeae (23). Although not cleared by the Food and Drug Administration

  19. Mutation in elongation factor G confers resistance to the antibiotic argyrin in the opportunistic pathogen Pseudomonas aeruginosa.

    Science.gov (United States)

    Bielecki, Piotr; Lukat, Peer; Hüsecken, Kristina; Dötsch, Andreas; Steinmetz, Heinrich; Hartmann, Rolf W; Müller, Rolf; Häussler, Susanne

    2012-11-05

    The natural myxobacterial product argyrin is a cyclic peptide exhibiting immunosuppressive activity as well as antibacterial activity directed against the highly intrinsically resistant opportunistic pathogen Pseudomonas aeruginosa. In this study, we used whole-genome sequencing technology as a powerful tool to determine the mode of action of argyrin. Sequencing of argyrin-resistant P. aeruginosa isolates selected in vitro uncovered six point mutations that distinguished the resistant mutants from their susceptible parental strain. All six mutations were localized within one gene: fusA1, which encodes for the elongation factor EF-G. After the reintroduction of selected mutations into the susceptible wild type, the strain became resistant to argyrin. Surface plasmon resonance experiments confirmed the interaction of argyrin A with FusA1. Interestingly, EF-G has been previously shown to be the target of the anti-Staphylococcus antibiotic fusidic acid. Mapping of the mutations onto a structural model of EF-G revealed that the mutations conveying resistance against argyrin were clustered within domain III on the side opposite to that involved in fusidic acid binding, thus indicating that argyrin exhibits a new mode of protein synthesis inhibition. Although no mutations causing argyrin resistance have been found in other genes of P. aeruginosa, analysis of the sequence identity in EF-G and its correlation with argyrin resistance in different bacteria imply that additional factors such as uptake of argyrin play a role in the argyrin resistance of other organisms. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  20. Label-free image-based detection of drug resistance with optofluidic time-stretch microscopy (Conference Presentation)

    Science.gov (United States)

    Kobayashi, Hirofumi; Lei, Cheng; Mao, Ailin; Jiang, Yiyue; Guo, Baoshan; Ozeki, Yasuyuki; Goda, Keisuke

    2017-02-01

    Acquired drug resistance is a fundamental predicament in cancer therapy. Early detection of drug-resistant cancer cells during or after treatment is expected to benefit patients from unnecessary drug administration and thus play a significant role in the development of a therapeutic strategy. However, the development of an effective method of detecting drug-resistant cancer cells is still in its infancy due to their complex mechanism in drug resistance. To address this problem, we propose and experimentally demonstrate label-free image-based drug resistance detection with optofluidic time-stretch microscopy using leukemia cells (K562 and K562/ADM). By adding adriamycin (ADM) to both K562 and K562/ADM (ADM-resistant K562 cells) cells, both types of cells express unique morphological changes, which are subsequently captured by an optofluidic time-stretch microscope. These unique morphological changes are extracted as image features and are subjected to supervised machine learning for cell classification. We hereby have successfully differentiated K562 and K562/ADM solely with label-free images, which suggests that our technique is capable of detecting drug-resistant cancer cells. Our optofluidic time-stretch microscope consists of a time-stretch microscope with a high spatial resolution of 780 nm at a 1D frame rate of 75 MHz and a microfluidic device that focuses and orders cells. We compare various machine learning algorithms as well as various concentrations of ADM for cell classification. Owing to its unprecedented versatility of using label-free image and its independency from specific molecules, our technique holds great promise for detecting drug resistance of cancer cells for which its underlying mechanism is still unknown or chemical probes are still unavailable.

  1. High Prevalence and Predominance of the aph(2″)-If Gene Conferring Aminoglycoside Resistance in Campylobacter

    OpenAIRE

    Yao, Hong; Liu, Dejun; Wang, Yang; Zhang, Qijing; Shen, Zhangqi

    2017-01-01

    Campylobacter is a major foodborne pathogen, and previous studies revealed that Campylobacter isolates from food-producing animals are increasingly resistant to gentamicin in China. The molecular epidemiology and genetic mechanisms responsible for gentamicin resistance in China have not been well understood. In this study, 607 Campylobacter isolates of chicken and swine origins collected in 2014 were analyzed, revealing that 15.6% (25/160) of the Campylobacter jejuni isolates and 79.9% (357/4...

  2. Combining the FtsZ-Targeting Prodrug TXA709 and the Cephalosporin Cefdinir Confers Synergy and Reduces the Frequency of Resistance in Methicillin-Resistant Staphylococcus aureus.

    Science.gov (United States)

    Kaul, Malvika; Mark, Lilly; Parhi, Ajit K; LaVoie, Edmond J; Pilch, Daniel S

    2016-07-01

    Combination therapy of bacterial infections with synergistic drug partners offers distinct advantages over monotherapy. Among these advantages are (i) a reduction of the drug dose required for efficacy, (ii) a reduced potential for drug-induced toxicity, and (iii) a reduced potential for the emergence of resistance. Here, we describe the synergistic actions of the third-generation oral cephalosporin cefdinir and TXA709, a new, FtsZ-targeting prodrug that we have developed with improved pharmacokinetics and enhanced in vivo efficacy against methicillin-resistant Staphylococcus aureus (MRSA) relative to earlier agents. We show that the active product of TXA709 (TXA707) acts synergistically with cefdinir in vitro against clinical isolates of MRSA, vancomycin-intermediate S. aureus (VISA), vancomycin-resistant S. aureus (VRSA), and linezolid-resistant S. aureus (LRSA). In addition, relative to TXA707 alone, the combination of TXA707 and cefdinir significantly reduces or eliminates the detectable emergence of resistance. We also demonstrate synergy in vivo with oral administration of the prodrug TXA709 and cefdinir in mouse models of both systemic and tissue (thigh) infections with MRSA. This synergy reduces the dose of TXA709 required for efficacy 3-fold. Viewed as a whole, our results highlight the potential of TXA709 and cefdinir as a promising combination for the treatment of drug-resistant staphylococcal infections. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  3. Disease resistance conferred by expression of a gene encoding H2O2-generating glucose oxidase in transgenic potato plants.

    Science.gov (United States)

    Wu, G; Shortt, B J; Lawrence, E B; Levine, E B; Fitzsimmons, K C; Shah, D M

    1995-09-01

    Plant defense responses to pathogen infection involve the production of active oxygen species, including hydrogen peroxide (H2O2). We obtained transgenic potato plants expressing a fungal gene encoding glucose oxidase, which generates H2O2 when glucose is oxidized. H2O2 levels were elevated in both leaf and tuber tissues of these plants. Transgenic potato tubers exhibited strong resistance to a bacterial soft rot disease caused by Erwinia carotovora subsp carotovora, and disease resistance was sustained under both aerobic and anaerobic conditions of bacterial infection. This resistance to soft rot was apparently mediated by elevated levels of H2O2, because the resistance could be counteracted by exogenously added H2O2-degrading catalase. The transgenic plants with increased levels of H2O2 also exhibited enhanced resistance to potato late blight caused by Phytophthora infestans. The development of lesions resulting from infection by P. infestans was significantly delayed in leaves of these plants. Thus, the expression of an active oxygen species-generating enzyme in transgenic plants represents a novel approach for engineering broad-spectrum disease resistance in plants.

  4. Shp2 confers cisplatin resistance in small cell lung cancer via an AKT-mediated increase in CA916798.

    Science.gov (United States)

    Yang, Xuemei; Tang, Chunlan; Luo, Hu; Wang, Haijing; Zhou, Xiangdong

    2017-04-04

    The tyrosine phosphatase Shp2 is associated with tumorigenesis in small cell lung cancer (SCLC). However, the relationship between Shp2 and resistance to chemotherapy remains unclear. Here, we show that Shp2 plays an important role in inducing resistance to cisplatin-based chemotherapy via the SHP2-AKT-CA916798 pathway. In an SCLC cell line, overexpression of Shp2 induced cisplatin resistance and the increased expression of AKT, pAKT, pmTOR, and CA916798. Conversely, depletion of Shp2 in a cisplatin-resistant cell line via RNA interference increased cisplatin sensitivity and decreased AKT, pAKT, pmTOR, and CA916798 expression levels. Activation of AKT stimulated CA916798 expression and altered the level of Shp2. A mouse xenograft model verified the results obtained from the in vitro experiments. In addition, we collected and analyzed clinical SCLC specimens and found that Shp2 levels correlated with CA916798 expression in tumor tissues. Importantly, higher levels of Shp2 or CA916798 were associated with a poorer prognosis in SCLC patients who received chemotherapy. Together, our findings indicate that Shp2 induces cisplatin resistance in SCLC patients via the SHP2-AKT-CA916798 pathway. Therefore, Shp2 and CA916798 may be promising biomarkers for predicting resistance to chemotherapy and may function as targets for enhancing treatments.

  5. Previously undescribed plasmids recovered from activated sludge confer tetracycline resistance and phenotypic changes to Acinetobacter oleivorans DR1.

    Science.gov (United States)

    Hong, Hyerim; Ko, Hyeok-Jin; Choi, In-Geol; Park, Woojun

    2014-02-01

    We used culture-dependent and culture-independent methods to extract previously undescribed plasmids harboring tetracycline (TC) resistance genes from activated sludge. The extracted plasmids were transformed into naturally competent Acinetobacter oleivorans DR1 to recover a non-Escherichia coli-based plasmid. The transformed cells showed 80-100-fold higher TC resistance than the wild-type strain. Restriction length polymorphism performed using 30 transformed cells showed four different types of plasmids. Illumina-based whole sequencing of the four plasmids identified three previously unreported plasmids and one previously reported plasmid. All plasmids carried TC resistance-related genes (tetL, tetH), tetracycline transcriptional regulators (tetR), and mobilization-related genes. As per expression analysis, TC resistance genes were functional in the presence of TC. The recovered plasmids showed mosaic gene acquisition through horizontal gene transfer. Membrane fluidity, hydrophobicity, biofilm formation, motility, growth rate, sensitivity to stresses, and quorum sensing signals of the transformed cells were different from those of the wild-type cells. Plasmid-bearing cells seemed to have an energy burden for maintaining and expressing plasmid genes. Our data showed that acquisition of TC resistance through plasmid uptake is related to loss of biological fitness. Thus, cells acquiring antibiotic resistance plasmids can survive in the presence of antibiotics, but must pay ecological costs.

  6. Insights into the structure, function and evolution of the radical-SAM 23S rRNA methyltransferase Cfr that confers antibiotic resistance in bacteria

    DEFF Research Database (Denmark)

    Karminska, K. H.; Purta, E.; Hansen, L .H.

    2010-01-01

    The Cfr methyltransferase confers combined resistance to five classes of antibiotics that bind to the peptidyl tranferase center of bacterial ribosomes by catalyzing methylation of the C-8 position of 23S rRNA nucleotide A2503. The same nucleotide is targeted by the housekeeping methyltransferase....... The investigation has identified essential amino acids and Cfr variants with altered reaction mechanisms and represents a first step towards understanding the structural basis of Cfr activity....... of a 4Fe-4S cluster, a SAM molecule coordinated to the iron-sulfur cluster (SAM1) and a SAM molecule that is the putative methyl group donor (SAM2). All mutations at predicted functional sites affect Cfr activity significantly as assayed by antibiotic susceptibility testing and primer extension analysis...

  7. Expression, purification, crystallization, and preliminary X-ray crystallographic analysis of OXA-17, an extended-spectrum β-lactamase conferring severe antibiotic resistance

    International Nuclear Information System (INIS)

    Lee, J. H.; Sohn, S. G.; Jung, H. I.; An, Y. J.; Lee, S. H.

    2013-01-01

    OXA-17, an extended-spectrum β-lactamase (ESBL) conferring severe antibiotic resistance, hydrolytically inactivates β-lactam antibiotics, inducing a lack of eradication of pathogenic bacteria by oxyimino β-lactams and not helping hospital infection control. Thus, the enzyme is a potential target for developing antimicrobial agents against pathogens producing ESBLs. OXA-17 was purified and crystallized at 298 K. X-ray diffraction data from OXA-17 crystal have been collected to 1.85 Å resolution using synchrotron radiation. The crystal of OXA-17 belongs to space group P2 1 2 1 2 1 , with unit-cell parameters a = 48.37, b = 101.12, and c = 126.07 Å. Analysis of the packing density shows that the asymmetric unit probably contains two molecules with a solvent content of 54.6%

  8. Expression, purification, crystallization, and preliminary X-ray crystallographic analysis of OXA-17, an extended-spectrum {beta}-lactamase conferring severe antibiotic resistance

    Energy Technology Data Exchange (ETDEWEB)

    Lee, J. H., E-mail: msgjhlee@mju.ac.kr; Sohn, S. G., E-mail: sgsohn@mju.ac.kr; Jung, H. I., E-mail: jhinumber1@hanmail.net; An, Y. J., E-mail: anyj0120@hanmail.net; Lee, S. H., E-mail: sangheelee@mju.ac.kr [Myongji University, Drug Resistance Proteomics Laboratory, Department of Biological Sciences (Korea, Republic of)

    2013-07-15

    OXA-17, an extended-spectrum {beta}-lactamase (ESBL) conferring severe antibiotic resistance, hydrolytically inactivates {beta}-lactam antibiotics, inducing a lack of eradication of pathogenic bacteria by oxyimino {beta}-lactams and not helping hospital infection control. Thus, the enzyme is a potential target for developing antimicrobial agents against pathogens producing ESBLs. OXA-17 was purified and crystallized at 298 K. X-ray diffraction data from OXA-17 crystal have been collected to 1.85 A resolution using synchrotron radiation. The crystal of OXA-17 belongs to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 48.37, b = 101.12, and c = 126.07 A. Analysis of the packing density shows that the asymmetric unit probably contains two molecules with a solvent content of 54.6%.

  9. Transgenic expression of gallerimycin, a novel antifungal insect defensin from the greater wax moth Galleria mellonella, confers resistance to pathogenic fungi in tobacco.

    Science.gov (United States)

    Langen, Gregor; Imani, Jafargholi; Altincicek, Boran; Kieseritzky, Gernot; Kogel, Karl-Heinz; Vilcinskas, Andreas

    2006-05-01

    A cDNA encoding gallerimycin, a novel antifungal peptide from the greater wax moth Galleria mellonella, was isolated from a cDNA library of genes expressed during innate immune response in the caterpillars. Upon ectopic expression of gallerimycin in tobacco, using Agrobacterium tumefaciens as a vector, gallerimycin conferred resistance to the fungal pathogens Erysiphe cichoracearum and Sclerotinia minor. Quantification of gallerimycin mRNA in transgenic tobacco by real-time PCR confirmed transgenic expression under control of the inducible mannopine synthase promoter. Leaf sap and intercellular washing fluid from transgenic tobacco inhibited in vitro germination and growth of the fungal pathogens, demonstrating that gallerimycin is secreted into intercellular spaces. The feasibility of the use of gallerimycin to counteract fungal diseases in crop plants is discussed.

  10. 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

  11. Identification and distribution of a GABA receptor mutation conferring dieldrin resistance in the malaria vector Anopheles funestus in Africa.

    Science.gov (United States)

    Wondji, Charles S; Dabire, Roch K; Tukur, Zainab; Irving, Helen; Djouaka, Rousseau; Morgan, John C

    2011-07-01

    Growing problems of pyrethroid resistance in Anopheles funestus have intensified efforts to identify alternative insecticides. Many agrochemicals target the GABA receptors, but cross-resistance from dieldrin resistance may preclude their introduction. Dieldrin resistance was detected in An. funestus populations from West (Burkina Faso) and central (Cameroon) Africa, but populations from East (Uganda) and Southern Africa (Mozambique and Malawi) were fully susceptible to this insecticide. Partial sequencing of the dieldrin target site, the γ-aminobutyric acid (GABA) receptor, identified two amino acid substitutions, A296S and V327I. The A296S mutation has been associated with dieldrin resistance in other species. The V327I mutations was detected in the resistant sample from Burkina Faso and Cameroon and consistently associated with the A296S substitution. The full-length of the An. funestus GABA-receptor gene, amplified by RT-PCR, generated a sequence of 1674 bp encoding 557 amino acid of the protein in An. funestus with 98% similarity to that of Anopheles gambiae. Two diagnostic assays were developed to genotype the A296S mutation (pyrosequencing and PCR-RFLP), and use of these assays revealed high frequency of the resistant allele in Burkina Faso (60%) and Cameroon (82%), moderate level in Benin (16%) while low frequency or absence of the mutation was observed respectively in Uganda (7.5%) or 0% in Malawi and Mozambique. The distribution of the Rdl(R) mutation in An. funestus populations in Africa suggests extensive barriers to gene flow between populations from different regions. Copyright © 2011 Elsevier Ltd. All rights reserved.

  12. Molecular Mapping of PMR1, a Novel Locus Conferring Resistance to Powdery Mildew in Pepper (Capsicum annuum

    Directory of Open Access Journals (Sweden)

    Jinkwan Jo

    2017-12-01

    Full Text Available Powdery mildew, caused by Leveillula taurica, is a major fungal disease affecting greenhouse-grown pepper (Capsicum annuum. Powdery mildew resistance has a complex mode of inheritance. In the present study, we investigated a novel powdery mildew resistance locus, PMR1, using two mapping populations: 102 ‘VK515' F2:3 families (derived from a cross between resistant parental line ‘VK515R' and susceptible parental line ‘VK515S' and 80 ‘PM Singang' F2 plants (derived from the F1 ‘PM Singang' commercial hybrid. Genetic analysis of the F2:3 ‘VK515' and F2 ‘PM Singang' populations revealed a single dominant locus for inheritance of the powdery mildew resistance trait. Genetic mapping showed that the PMR1 locus is located on syntenic regions of pepper chromosome 4 in a 4-Mb region between markers CZ2_11628 and HRM4.1.6 in ‘VK515R'. Six molecular markers including one SCAR marker and five SNP markers were localized to a region 0 cM from the PMR1 locus. Two putative nucleotide-binding site leucine-rich repeat (NBS-LRR-type disease resistance genes were identified in this PMR1 region. Genotyping-by-sequencing (GBS and genetic mapping analysis revealed suppressed recombination in the PMR1 region, perhaps due to alien introgression. In addition, a comparison of species-specific InDel markers as well as GBS-derived SNP markers indicated that C. baccatum represents a possible source of such alien introgression of powdery mildew resistance into ‘VK515R'. The molecular markers developed in this study will be especially helpful for marker-assisted selection in pepper breeding programs for powdery mildew resistance.

  13. Shotgun label-free proteomic analysis of clubroot (Plasmodiophora brassicae resistance conferred by the gene Rcr1 in Brassica rapa

    Directory of Open Access Journals (Sweden)

    Tao Song

    2016-07-01

    Full Text Available Clubroot, caused by the plasmodiophorid pathogen Plasmodiophora brassicae, is one of the most serious diseases on Brassica crops worldwide and a major threat to canola production in western Canada. Host resistance is the key strategy for clubroot management on canola. Several clubroot resistance (CR genes have been identified, but the mechanisms associated with these CR genes are poorly understood. In the current study, a label-free shotgun proteomic approach was used to profile and compare the proteomes of B. rapa carrying and not carrying the CR gene Rcr1 upon P. brassicae infection. A total of 527 differentially accumulated proteins (DAPs were identified between the resistant and susceptible samples, and functional annotation of these DAPs indicates that the perception of P. brassicae and activation of defense responses is triggered via an unique signaling pathway distinct from common modes of recognition receptors reported with many other plant-pathogen interactions; this pathway appears to act in a calcium-independent manner through a not-well defined cascade of mitogen-activated protein kinases and may require the ubiquitin-26S proteasome related to abiotic stresses, especially the cold-stress tolerance. Both up-regulation of defense-related and down-regulation of pathogenicity-related metabolism were observed in plants carrying Rcr1, and these functions may all contribute to the clubroot resistance mediated by this CR gene. These results, combined with those of transcriptomic analysis reported earlier, improved our understanding of molecular mechanisms associated with Rcr1 and clubroot resistance at large, and identified candidate metabolites or pathways for further confirmation of specific resistance mechanisms. Deploying CR genes with different modes of action may help improve the durability of clubroot resistance.

  14. Host-dependent transposon Tn5-mediated streptomycin resistance.

    OpenAIRE

    De Vos, G F; Finan, T M; Signer, E R; Walker, G C

    1984-01-01

    Transposon Tn5 encodes streptomycin resistance in addition to kanamycin-neomycin resistance. This resistance was not detectable in Escherichia coli but was efficiently expressed in Rhizobium meliloti and certain other strains. By analysis of cloned Tn5 restriction endonuclease fragments, the streptomycin resistance (str) gene was located in the right-hand side of the central region as the transposon is conventionally drawn. Transcription of str appeared to originate at pL, the promoter for th...

  15. Genetic mapping, marker assisted selection and allelic relationships for the Pu 6 gene conferring rust resistance in sunflower.

    Science.gov (United States)

    Bulos, Mariano; Vergani, Pablo Nicolas; Altieri, Emiliano

    2014-09-01

    Rust resistance in the sunflower line P386 is controlled by Pu 6 , a gene which was reported to segregate independently from other rust resistant genes, such as R 4 . The objectives of this work were to map Pu 6 , to provide and validate molecular tools for its identification, and to determine the linkage relationship of Pu 6 and R 4 . Genetic mapping of Pu 6 with six markers covered 24.8 cM of genetic distance on the lower end of linkage Group 13 of the sunflower consensus map. The marker most closely linked to Pu 6 was ORS316 at 2.5 cM in the distal position. ORS316 presented five alleles when was assayed with a representative set of resistant and susceptible lines. Allelism test between Pu 6 and R 4 indicated that both genes are linked at a genetic distance of 6.25 cM. This is the first confirmation based on an allelism test that at least two members of the R adv /R 4 /R 11 / R 13a /R 13b /Pu 6 cluster of genes are at different loci. A fine elucidation of the architecture of this complex locus will allow designing and constructing completely new genomic regions combining genes from different resistant sources and the elimination of the linkage drag around each resistant gene.

  16. Transgenic expression of antimicrobial peptide D2A21 confers resistance to diseases incited by Pseudomonas syringae pv. tabaci and Xanthomonas citri, but not Candidatus Liberibacter asiaticus.

    Directory of Open Access Journals (Sweden)

    Guixia Hao

    Full Text Available Citrus Huanglongbing (HLB associated with 'Candidatus Liberibacter asiaticus' (Las and citrus canker disease incited by Xanthomonas citri are the most devastating citrus diseases worldwide. To control citrus HLB and canker disease, we previously screened over forty antimicrobial peptides (AMPs in vitro for their potential application in genetic engineering. D2A21 was one of the most active AMPs against X. citri, Agrobacterium tumefaciens and Sinorhizobium meliloti with low hemolysis activity. Therefore, we conducted this work to assess transgenic expression of D2A21 peptide to achieve citrus resistant to canker and HLB. We generated a construct expressing D2A21 and initially transformed tobacco as a model plant. Transgenic tobacco expressing D2A21 was obtained by Agrobacterium-mediated transformation. Successful transformation and D2A21 expression was confirmed by molecular analysis. We evaluated disease development incited by Pseudomonas syringae pv. tabaci in transgenic tobacco. Transgenic tobacco plants expressing D2A21 showed remarkable disease resistance compared to control plants. Therefore, we performed citrus transformations with the same construct and obtained transgenic Carrizo citrange. Gene integration and gene expression in transgenic plants were determined by PCR and RT-qPCR. Transgenic Carrizo expressing D2A21 showed significant canker resistance while the control plants showed clear canker symptoms following both leaf infiltration and spray inoculation with X. citri 3213. Transgenic Carrizo plants were challenged for HLB evaluation by grafting with Las infected rough lemon buds. Las titer was determined by qPCR in the leaves and roots of transgenic and control plants. However, our results showed that transgenic plants expressing D2A21 did not significantly reduce Las titer compared to control plants. We demonstrated that transgenic expression of D2A21 conferred resistance to diseases incited by P. syringae pv. tabaci and X. citri

  17. A stilbene synthase allele from a Chinese wild grapevine confers resistance to powdery mildew by recruiting salicylic acid signalling for efficient defence.

    Science.gov (United States)

    Jiao, Yuntong; Xu, Weirong; Duan, Dong; Wang, Yuejin; Nick, Peter

    2016-10-01

    Stilbenes are central phytoalexins in Vitis, and induction of the key enzyme stilbene synthase (STS) is pivotal for disease resistance. Here, we address the potential for breeding resistance using an STS allele isolated from Chinese wild grapevine Vitis pseudoreticulata (VpSTS) by comparison with its homologue from Vitis vinifera cv. 'Carigane' (VvSTS). Although the coding regions of both alleles are very similar (>99% identity on the amino acid level), the promoter regions are significantly different. By expression in Arabidopsis as a heterologous system, we show that the allele from the wild Chinese grapevine can confer accumulation of stilbenes and resistance against the powdery mildew Golovinomyces cichoracearum, whereas the allele from the vinifera cultivar cannot. To dissect the upstream signalling driving the activation of this promoter, we used a dual-luciferase reporter system in a grapevine cell culture. We show elevated responsiveness of the promoter from the wild grape to salicylic acid (SA) and to the pathogen-associated molecular pattern (PAMP) flg22, equal induction of both alleles by jasmonic acid (JA), and a lack of response to the cell death-inducing elicitor Harpin. This elevated SA response of the VpSTS promoter depends on calcium influx, oxidative burst by RboH, mitogen-activated protein kinase (MAPK) signalling, and JA synthesis. We integrate the data in the context of a model where the resistance of V. pseudoreticulata is linked to a more efficient recruitment of SA signalling for phytoalexin synthesis. © The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology.

  18. Multiple Patterns of Regulation and Overexpression of a Ribonuclease-Like Pathogenesis-Related Protein Gene, OsPR10a, Conferring Disease Resistance in Rice and Arabidopsis.

    Directory of Open Access Journals (Sweden)

    Li-Fen Huang

    Full Text Available An abundant 17 kDa RNase, encoded by OsPR10a (also known as PBZ1, was purified from Pi-starved rice suspension-cultured cells. Biochemical analysis showed that the range of optimal temperature for its RNase activity was 40-70°C and the optimum pH was 5.0. Disulfide bond formation and divalent metal ion Mg2+ were required for the RNase activity. The expression of OsPR10a::GUS in transgenic rice was induced upon phosphate (Pi starvation, wounding, infection by the pathogen Xanthomonas oryzae pv. oryzae (Xoo, leaf senescence, anther, style, the style-ovary junction, germinating embryo and shoot. We also provide first evidence in whole-plant system, demonstrated that OsPR10a-overexpressing in rice and Arabidopsis conferred significant level of enhanced resistance to infection by the pathogen Xoo and Xanthomona campestris pv. campestris (Xcc, respectively. Transgenic rice and Arabidopsis overexpressing OsPR10a significantly increased the length of primary root under phosphate deficiency (-Pi condition. These results showed that OsPR10a might play multiple roles in phosphate recycling in phosphate-starved cells and senescing leaves, and could improve resistance to pathogen infection and/or against chewing insect pests. It is possible that Pi acquisition or homeostasis is associated with plant disease resistance. Our findings suggest that gene regulation of OsPR10a could act as a good model system to unravel the mechanisms behind the correlation between Pi starvation and plant-pathogen interactions, and also provides a potential application in crops disease resistance.

  19. Identification of Nine Pathotype-Specific Genes Conferring Resistance to Fusiform Rust in Loblolly Pine (Pinus taeda L.

    Directory of Open Access Journals (Sweden)

    Henry V. Amerson

    2015-08-01

    Full Text Available Nearly two decades of research on the host-pathogen interaction in fusiform rust of loblolly pine is detailed. Results clearly indicate that pathotype-specific genes in the host interacting with pathogen avirulence cause resistance as defined by the non-gall phenotype under favorable environmental conditions for disease development. In particular, nine fusiform rust resistance genes (Fr genes are described here including the specific methods to determine each and their localization on the reference genetic map of loblolly pine. Understanding how these and other apparent Fr genes in loblolly pine and other rust-susceptible pines impact resistance screening, parental and progeny selection, and family and clonal deployment is an important area in forest genetics research and operational tree breeding. The documentation of these Fr genes is a key piece of information towards gaining that understanding and ultimately improving breeding and deployment strategies.

  20. The Cfr rRNA methyltransferase confers resistance to Phenicols, Lincosamides, Oxazolidinones, Pleuromutilins, and Streptogramin A antibiotics

    DEFF Research Database (Denmark)

    Long, K. S.; Poehlsgaard, Jacob; Kehrenberg, C.

    2006-01-01

    and recently shown to encode a methyltransferase that modifies 23S rRNA at A2503. Antimicrobial susceptibility testing shows that S. aureus and E. coli strains expressing the cfr gene exhibit elevated MICs to a number of chemically unrelated drugs. The phenotype is named PhLOPSA for resistance to the following......A novel multidrug resistance phenotype mediated by the Cfr rRNA methyltransferase is observed in Staphylococcus aureus and Escherichia coli. The cfr gene has previously been identified as a phenicol and lincosamide resistance gene on plasmids isolated from Staphylococcus spp. of animal origin...... drug classes: Phenicols, Lincosamides, Oxazolidinones, Pleuromutilins, and Streptogramin A antibiotics. Each of these five drug classes contains important antimicrobial agents that are currently used in human and/or veterinary medicine. We find that binding of the PhLOPSA drugs, which bind...

  1. Altered viral fitness and drug susceptibility in HIV-1 carrying mutations that confer resistance to nonnucleoside reverse transcriptase and integrase strand transfer inhibitors.

    Science.gov (United States)

    Hu, Zixin; Kuritzkes, Daniel R

    2014-08-01

    . Nonnucleoside reverse transcriptase inhibitors and integrase inhibitors are used to treat infection with HIV-1. Mutations that confer resistance to these drugs reduce the ability of HIV-1 to reproduce (that is, they decrease viral fitness). It is known that reverse transcriptase and integrase interact and that some mutations can disrupt their interaction, which is necessary for proper functioning of these two enzymes. To determine whether resistance mutations in these enzymes interact, we investigated their effects on drug sensitivity and viral fitness. Although individual drug resistance mutations usually reduced viral fitness, certain combinations of mutations increased fitness. When present in certain combinations, some integrase inhibitor resistance mutations increased resistance to nonnucleoside reverse transcriptase inhibitors and vice versa. Because these drugs are sometimes used together in the treatment of HIV-1 infection, these interactions could make viruses more resistant to both drugs, further limiting their clinical benefit. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  2. Stepwise Development of a Homozygous S80P Substitution in Fks1p, Conferring Echinocandin Resistance in Candida tropicalis

    DEFF Research Database (Denmark)

    Jensen, Rasmus Hare; Johansen, Helle Krogh; Arendrup, Maiken Cavling

    2013-01-01

    Three Candida tropicalis isolates were obtained from a patient with acute lymphoblastic leukemia. The first isolate was susceptible to all drug classes, while isolates 2 and 3, obtained after 8 and 8.5 weeks of caspofungin treatment, respectively, were resistant to the three echinocandins...

  3. Mutations Conferring Resistance to Viral DNA Polymerase Inhibitors in Camelpox Virus Give Different Drug-Susceptibility Profiles in Vaccinia Virus

    Czech Academy of Sciences Publication Activity Database

    Duraffour, S.; Andrei, G.; Topalis, D.; Krečmerová, Marcela; Crance, J. M.; Garin, D.; Snoeck, R.

    2012-01-01

    Roč. 86, č. 13 (2012), s. 7310-7325 ISSN 0022-538X Institutional support: RVO:61388963 Keywords : camelpox virus * CMLV * vaccinia virus VACV * acyclic nucleoside phosphonates * HPMPDAP * cidofovir * drug resistance Subject RIV: CC - Organic Chemistry Impact factor: 5.076, year: 2012

  4. Identification of nine pathotype-specific genes conferring resistance to fusiform rust in loblolly pine (Pinus taeda L.)

    Science.gov (United States)

    Henry Amerson; C. Dana Nelson; Thomas L. Kubisiak; E.George Kuhlman; Saul Garcia

    2015-01-01

    Nearly two decades of research on the host-pathogen interaction in fusiform rust of loblolly pine is detailed. Results clearly indicate that pathotype-specific genes in the host interacting with pathogen avirulence cause resistance as defined by the non-gall phenotype under favorable environmental conditions for disease development. In particular, nine fusiform rust...

  5. Molecular and cytogenetic characterization of a durum wheat Aegilops speltoides chromosome translocation conferring resistance to stem rust

    Science.gov (United States)

    Stem rust is a serious disease of wheat that has caused historical epidemics, but it has not been a threat in recent decades in North America due to the eradication of the alternate host and deployment of resistant cultivars. However, the recent emergence of Ug99 (or race TTKS) poses a threat to glo...

  6. AFLP markers for the R-gene in the flea beetle, Phyllotreta nemorum, conferring resistance to defenses in Barbarea vulgaris

    NARCIS (Netherlands)

    Breuker, C.J.; Victoir, K.; Jong, de P.W.; Meijden, van der E.; Brakefield, P.M.; Vrieling, K.

    2005-01-01

    A so-called R-gene renders the yellow-striped flea beetle Phyllotreta nemorum L. (Coleoptera: Chrysomelidae: Alticinae) resistant to the defenses of the yellow rocket Barbarea vulgaris R.Br. (Brassicacea) and enables it to use it as a host plant in Denmark. In this study, genetic markers for an

  7. Mapping of a Leishmania major gene/locus that confers pentamidine resistance by deletion and insertion of transposable element

    Directory of Open Access Journals (Sweden)

    Coelho Adriano C.

    2004-01-01

    Full Text Available Pentamidine (PEN is an alternative compound to treat antimony-resistant leishmaniasis patients, which cellular target remains unclear. One approach to the identification of prospective targets is to identify genes able to mediate PEN resistance following overexpression. Starting from a genomic library of transfected parasites bearing a multicopy episomal cosmid vector containing wild-type Leishmania major DNA, we isolated one locus capable to render PEN resistance to wild type cells after DNA transfection. In order to map this Leishmania locus, cosmid insert was deleted by two successive sets of partial digestion with restriction enzymes, followed by transfection into wild type cells, overexpression, induction and functional tests in the presence of PEN. To determine the Leishmania gene related to PEN resistance, nucleotide sequencing experiments were done through insertion of the transposon Mariner element of Drosophila melanogaster (mosK into the deleted insert to work as primer island. Using general molecular techniques, we described here this method that permits a quickly identification of a functional gene facilitating nucleotide sequence experiments from large DNA fragments. Followed experiments revealed the presence of a P-Glycoprotein gene in this locus which role in Leishmania metabolism has now been analyzed.

  8. Strigolactone deficiency confers resistance in tomato line SL-ORT1 to the parasitic weeds Phelipanche and Orobanche spp.

    Science.gov (United States)

    Dor, Evgenia; Yoneyama, Koichi; Wininger, Smadar; Kapulnik, Yoram; Yoneyama, Kaori; Koltai, Hinanit; Xie, Xiaonan; Hershenhorn, Joseph

    2011-02-01

    The parasitic flowering plants of the genera Orobanche and Phelipanche (broomrape species) are obligatory chlorophyll-lacking root-parasitic weeds that infect dicotyledonous plants and cause heavy economic losses in a wide variety of plant species in warm-temperate and subtropical regions. One of the most effective strategies for broomrape control is crop breeding for broomrape resistance. Previous efforts to find natural broomrape-resistant tomato (Solanum lycopersicon) genotypes were unsuccessful, and no broomrape resistance was found in any wild tomato species. Recently, however, the fast-neutron-mutagenized tomato mutant SL-ORT1 was found to be highly resistant to various Phelipanche and Orobanche spp. Nevertheless, SL-ORT1 plants were parasitized by Phelipanche aegyptiaca if grown in pots together with the susceptible tomato cv. M-82. In the present study, no toxic activity or inhibition of Phelipanche seed germination could be detected in the SL-ORT1 root extracts. SL-ORT1 roots did not induce Phelipanche seed germination in pots but they were parasitized, at the same level as M-82, after application of the synthetic germination stimulant GR24 to the rhizosphere. Whereas liquid chromatography coupled to tandem mass spectrometry analysis of root exudates of M-82 revealed the presence of the strigolactones orobanchol, solanacol, and didehydro-orobanchol isomer, these compounds were not found in the exudates of SL-ORT1. It can be concluded that SL-ORT1 resistance results from its inability to produce and secrete natural germination stimulants to the rhizosphere.

  9. Pedigree-Based Analysis in a Multiparental Population of Octoploid Strawberry Reveals QTL Alleles Conferring Resistance to Phytophthora cactorum

    Science.gov (United States)

    Mangandi, Jozer; Verma, Sujeet; Osorio, Luis; Peres, Natalia A.; van de Weg, Eric; Whitaker, Vance M.

    2017-01-01

    Understanding the genetic architecture of traits in breeding programs can be critical for making genetic progress. Important factors include the number of loci controlling a trait, allele frequencies at those loci, and allele effects in breeding germplasm. To this end, multiparental populations offer many advantages for quantitative trait locus (QTL) analyses compared to biparental populations. These include increased power for QTL detection, the ability to sample a larger number of segregating loci and alleles, and estimation of allele effects across diverse genetic backgrounds. Here, we investigate the genetic architecture of resistance to crown rot disease caused by Phytophthora cactorum in strawberry (Fragaria × ananassa), using connected full-sib families from a breeding population. Clonal replicates of > 1100 seedlings from 139 full-sib families arising from 61 parents were control-inoculated during two consecutive seasons. Subgenome-specific single nucleotide polymorphism (SNP) loci were mapped in allo-octoploid strawberry (2n = 8 × = 56), and FlexQTL software was utilized to perform a Bayesian, pedigree-based QTL analysis. A major locus on linkage group (LG) 7D, which we name FaRPc2, accounts for most of the genetic variation for resistance. Four predominant SNP haplotypes were detected in the FaRPc2 region, two of which are strongly associated with two different levels of resistance, suggesting the presence of multiple resistance alleles. The phenotypic effects of FaRPc2 alleles across trials and across numerous genetic backgrounds make this locus a highly desirable target for genetic improvement of resistance in cultivated strawberry. PMID:28592652

  10. Characterization of a new Pm2 allele conferring powdery mildew resistance in the wheat germplasm line FG-1

    Directory of Open Access Journals (Sweden)

    Pengtao eMa

    2016-04-01

    Full Text Available Powdery mildew has a negative impact on wheat production. Novel host resistance increases the diversity of resistance genes and helps to control the disease. In this study, wheat line FG-1 imported from France showed a high level of powdery mildew resistance at both the seedling and adult stages. An F2 population and F2:3 families from the cross FG-1 × Mingxian 169 both fit Mendelian ratios for a single dominant resistance gene when tested against multiple avirulent Blumeria tritici f. sp. tritici (Bgt races. This gene was temporarily designated PmFG. PmFG was mapped on the multi-allelic Pm2 locus of chromosome 5DS using seven SSR, ten SNP-derived and two SCAR markers with the flanking markers Xbwm21/Xcfd81/Xscar112 (distal and Xbwm25 (proximal at 0.3 and 0.5 cM being the closest. Marker SCAR203 co-segregated with PmFG. Allelism tests between PmFG and documented Pm2 alleles confirmed that PmFG was allelic with Pm2. Line FG-1 produced a significantly different reaction pattern compared to other lines with genes at or near Pm2 when tested against 49 Bgt isolates. The PmFG-linked marker alleles detected by the SNP-derived markers revealed significant variation between FG-1 and other lines with genes at or near Pm2. It was concluded that PmFG is a new allele at the Pm2 locus. Data from seven closely linked markers tested on 31 wheat cultivars indicated opportunities for marker-assisted pyramiding of this gene with other genes for powdery mildew resistance and additional traits.

  11. Multi-agent chemotherapy overcomes glucocorticoid resistance conferred by a BIM deletion polymorphism in pediatric acute lymphoblastic leukemia.

    Science.gov (United States)

    Soh, Sheila Xinxuan; Lim, Joshua Yew Suang; Huang, John W J; Jiang, Nan; Yeoh, Allen Eng Juh; Ong, S Tiong

    2014-01-01

    A broad range of anti-cancer agents, including glucocorticoids (GCs) and tyrosine kinase inhibitors (TKIs), kill cells by upregulating the pro-apoptotic BCL2 family member, BIM. A common germline deletion in the BIM gene was recently shown to favor the production of non-apoptotic BIM isoforms, and to predict inferior responses in TKI-treated chronic myeloid leukemia (CML) and EGFR-driven lung cancer patients. Given that both in vitro and in vivo GC resistance are predictive of adverse outcomes in acute lymphoblastic leukemia (ALL), we hypothesized that this polymorphism would mediate GC resistance, and serve as a biomarker of poor response in ALL. Accordingly, we used zinc finger nucleases to generate ALL cell lines with the BIM deletion, and confirmed the ability of the deletion to mediate GC resistance in vitro. In contrast to CML and lung cancer, the BIM deletion did not predict for poorer clinical outcome in a retrospective analysis of 411 pediatric ALL patients who were uniformly treated with GCs and chemotherapy. Underlying the lack of prognostic significance, we found that the chemotherapy agents used in our cohort (vincristine, L-asparaginase, and methotrexate) were each able to induce ALL cell death in a BIM-independent fashion, and resensitize BIM deletion-containing cells to GCs. Together, our work demonstrates how effective therapy can overcome intrinsic resistance in ALL patients, and suggests the potential of using combinations of drugs that work via divergent mechanisms of cell killing to surmount BIM deletion-mediated drug resistance in other cancers.

  12. Identification and characterization of a novel aac(6')-Iag associated with the blaIMP-1-integron in a multidrug-resistant Pseudomonas aeruginosa.

    Science.gov (United States)

    Kobayashi, Kanao; Hayashi, Ikue; Kouda, Syuntaro; Kato, Fuminori; Fujiwara, Tamaki; Kayama, Shizuo; Hirakawa, Hideki; Itaha, Hideyuki; Ohge, Hiroki; Gotoh, Naomasa; Usui, Tsuguru; Matsubara, Akio; Sugai, Motoyuki

    2013-01-01

    In a continuing study from Dec 2006 to Apr 2008, we characterized nine multi-drug resistant Pseudomonas aeruginosa strains isolated from four patients in a ward at the Hiroshima University Hospital, Japan. Pulsed-field gel electrophoresis of SpeI-digested genomic DNAs from the isolates suggested the clonal expansion of a single strain; however, only one strain, NK0009, was found to produce metallo-β-lactamase. PCR and subsequent sequencing analysis indicated NK0009 possessed a novel class 1 integron, designated as In124, that carries an array of four gene cassettes: a novel aminoglycoside (AG) resistance gene, aac(6')-Iag, blaIMP-1, a truncated form of blaIMP-1, and a truncated form of aac(6')-Iag. The aac(6')-Iag encoded a 167-amino-acid protein that shows 40% identity with AAC(6')-Iz. Recombinant AAC(6')-Iag protein showed aminoglycoside 6'-N-acetyltransferase activity using thin-layer chromatography (TLC) and MS spectrometric analysis. Escherichia coli carrying aac(6')-Iag showed resistance to amikacin, arbekacin, dibekacin, isepamicin, kanamycin, sisomicin, and tobramycin; but not to gentamicin. A conjugation experiment and subsequent Southern hybridization with the gene probes for blaIMP-1 and aac(6')-Ig strongly suggested In124 is on a conjugal plasmid. Transconjugants acquired resistance to gentamicin and were resistant to virtually all AGs, suggesting that the In124 conjugal plasmid also possesses a gene conferring resistance to gentamicin.

  13. Erythromycin Resistance-Conferring Plasmid pRSB105, Isolated from a Sewage Treatment Plant, Harbors a New Macrolide Resistance Determinant, an Integron-Containing Tn402-Like Element, and a Large Region of Unknown Function▿

    Science.gov (United States)

    Schlüter, A.; Szczepanowski, R.; Kurz, N.; Schneiker, S.; Krahn, I.; Pühler, A.

    2007-01-01

    The erythromycin resistance plasmid pRSB105 was previously isolated from an activated sludge bacterial community of a municipal wastewater treatment plant. Compilation of the complete pRSB105 nucleotide sequence revealed that the plasmid is 57,137 bp in size and has a mean G+C content of 56.66 mol%. The pRSB105 backbone is composed of two different replication and/or partitioning modules and a functional mobilization region encoding the mobilization genes mobCDE and mobBA. The first replicon (Rep1) is nearly identical to the corresponding replication module of the multiresistance plasmid pRSB101 isolated from an unknown activated sludge bacterium. Accordingly, pRSB101 and pRSB105 are sister plasmids belonging to a new plasmid family. The second replicon (Rep2) of pRSB105 was classified as a member of the IncP-6 group. While Rep1 confers replication ability only in γ-proteobacteria, Rep2 extents the host range of the plasmid since it is also functional in the β-proteobacterium Ralstonia eutropha. Plasmid pRSB105 harbors the macrolide resistance genes mel and mph, encoding, respectively, a predicted ABC-type efflux permease and a macrolide-2′-phosphotransferase. Erythromycin resistance is mainly attributed to mel, whereas mph contributes to erythromycin resistance to a lesser extent. The second resistance region, represented by an integron-containing Tn402-like element, includes a β-lactam (oxa10) and a trimethoprim (dfrB2) resistance gene cassette. In addition to antibiotic resistance modules, pRSB105 encodes a functional restriction/modification system and two nonresistance regions of unknown function. The presence of different mobile genetic elements that flank resistance and nonresistance modules on pRSB105 indicates that these elements were involved in acquisition of accessory plasmid modules. Comparative genomics of pRSB105 and related plasmids elucidated that pRSB105 evolved by integration of distinct modules from different plasmid sources, including

  14. Study of the resistance mechanisms to ultraviolet light in Escherichia Coli. III. Genetic mapping of a mutation that confers resistance to radiation

    International Nuclear Information System (INIS)

    Alcantara D, D.

    1996-08-01

    Inside this third work with respect to the investigation of the process of adaptation of different subcultures of Escherichia coli, to the UV light, its are present the obtained results in the identification of the gene or genes implied in the resistance to UV of one of the 5 populations derived for repeated exposure to growing dose of UV light of E. coli PQ30. Those obtained data indicate that the causing mutation of the phenotype is located in a single gene, since the insert of the fragment of DNA miniTn5 in this gene, causes the lost one from the resistance and the return to the original phenotype. The gene in question is denominated radA, located in the minute 99.6 of the chromosome of E. coli and required for the efficient repair of double ruptures in the DNA. (Author)

  15. Beta4 integrin-dependent formation of polarized three-dimensionalarchitecture confers resistance to apoptosis in normal and malignantmammary epithelium

    Energy Technology Data Exchange (ETDEWEB)

    Weaver, Valerie M.; Lelievre, Sophie; Lakins, Johnathon N.; Chrenek, Micah A.; Jones, Jonathan C.R.; Giancotti, Filippo; Werb, Zena; Bissell, Mina J.

    2002-08-27

    Tumor cells can evade chemotherapy by acquiring resistanceto apoptosis. We investigated the molecular mechanism whereby malignantand nonmalignant mammary epithelial cells become insensitive toapoptosis. We show that regardless of growth status formation ofpolarized, three-dimensional structures driven by basement membraneconfers protection to apoptosis in both nonmalignant and malignantmammary epithelial cells. By contrast, irrespective of their malignantstatus, nonpolarized structures are sensitive to induction of apoptosis.Resistance to apoptosis requires ligation of beta4 integrins, whichregulates tissue polarity, hemidesmosome formation and NFkB activation.Expression of beta4 integrin that lacks the hemidesmosome targetingdomain interferes with tissue polarity and NFkB activation and permitsapoptosis. These results indicate that integrin-induced polarity maydrive tumor cell resistance to apoptosis-inducing agents via effects onNFkB.

  16. A cfr-Like Gene from Clostridium difficile Confers Multiple Antibiotic Resistance by the Same Mechanism as the cfr Gene

    DEFF Research Database (Denmark)

    Hansen, Lykke H; Vester, Birte

    2015-01-01

    The Cfr RNA methyltransferase causes multiple resistances to peptidyl transferase inhibitors by methylation of A2503 23S rRNA. Many cfr-like gene sequences in the databases code for unknown functions. This study confirms that a Cfr-like protein from a Peptoclostridium difficile (formerly Clostrid......The Cfr RNA methyltransferase causes multiple resistances to peptidyl transferase inhibitors by methylation of A2503 23S rRNA. Many cfr-like gene sequences in the databases code for unknown functions. This study confirms that a Cfr-like protein from a Peptoclostridium difficile (formerly...... Clostridium difficile) strain does function as a Cfr protein. The enzyme is expressed in Escherichia coli and shows elevated MICs for five classes of antibiotics. A primer extension stop indicates a modification at A2503 in 23S rRNA....

  17. Constitutive Activity of the Arabidopsis MAP Kinase 3 Confers Resistance to Pseudomonas syringae and Drives Robust Immune Responses

    KAUST Repository

    Lang, Julien

    2017-08-02

    Mitogen Activated Protein Kinases (MAPKs) are known to be important mediators of plant responses to biotic and abiotic stresses. In a recent report, we enlarged the understanding of the Arabidopsis thaliana MPK3 functions showing that the expression of a constitutively active (CA) form of the protein led to auto-immune phenotypes. CA-MPK3 plants are dwarf and display defense responses that are characterized by the accumulation of salicylic acid and phytoalexins as well as by the upregulation of several defense genes. Consistently with these data, we present here results demonstrating that, compared to wild type controls, CA-MPK3 plants are more resistant to the hemibiotrophic pathogen Pseudomonas syringae DC3000. Based on our previous work, we also discuss the mechanisms of robust plant immunity controlled by sustained MPK3 activity, focusing especially on the roles of disease resistance proteins.

  18. Target site insensitivity mutations in the AChE enzyme confer resistance to organophosphorous insecticides in Leptinotarsa decemlineata (Say).

    Science.gov (United States)

    Malekmohammadi, M; Galehdari, H

    2016-01-01

    In the present study, we demonstrated the use and optimization of the tetra-primer ARMS-PCR procedure to detect and analyze the frequency of the R30K and I392T mutations in resistant field populations of CPB. The R30K mutation was detected in 72%, 84%, 52% and 64% of Bahar, Dehpiaz, Aliabad and Yengijeh populations, respectively. Overall frequencies of the I392T mutation were 12%, 8% and 16% of Bahar, Aliabad and Yengijeh populations, respectively. No I392T point mutation was found among samples from Dehpiaz field population. Moreover, only 31% and 2% of samples from the resistant field populations were homozygous for R30K and I392T mutations, respectively. No individual simultaneously had both I392T and S291G/R30K point mutations. The incidence of individuals with both S291G and R30K point mutations in the samples from Bahar, Dehpiaz, Aliabad, and Yengijeh populations were 31.5%, 44.7%, 41.6%, and 27.3% respectively. Genotypes determined by the tetra-primer ARMS-PCR method were consistent with those determined by PCR sequencing. There was no significant correlation between the mutation frequencies and resistance levels in the resistant populations, indicating that other mutations may contribute to this variation. Polymorphism in the partial L. decemlineata cDNA AChE gene Ldace2 of four field populations was identified by direct sequencing of PCR-amplified fragments. Among 45 novel mutations detected in this study, T29P mutation was found across all four field populations that likely contribute to the AChE insensitivity. Site-directed mutagenesis and protein expression experiments are needed for a more complete evaluation. Copyright © 2015 Elsevier Inc. All rights reserved.

  19. Overcoming imatinib resistance conferred by the BIM deletion polymorphism in chronic myeloid leukemia with splice-switching antisense oligonucleotides

    Science.gov (United States)

    Liu, Jun; Bhadra, Malini; Sinnakannu, Joanna Rajeswary; Yue, Wan Lin; Tan, Cheryl Weiqi; Rigo, Frank; Ong, S.Tiong; Roca, Xavier

    2017-01-01

    Many tyrosine kinase-driven cancers, including chronic myeloid leukemia (CML), are characterized by high response rates to specific tyrosine kinase inhibitors (TKIs) like imatinib. In East Asians, primary imatinib resistance is caused by a deletion polymorphism in Intron 2 of the BIM gene, whose product is required for TKI-induced apoptosis. The deletion biases BIM splicing from exon 4 to exon 3, generating splice isoforms lacking the exon 4-encoded pro-apoptotic BH3 domain, which impairs the ability of TKIs to induce apoptosis. We sought to identify splice-switching antisense oligonucleotides (ASOs) that block exon 3 but enhance exon 4 splicing, and thereby resensitize BIM deletion-containing cancers to imatinib. First, we mapped multiple cis-acting splicing elements around BIM exon 3 by minigene mutations, and found an exonic splicing enhancer acting via SRSF1. Second, by a systematic ASO walk, we isolated ASOs that corrected the aberrant BIM splicing. Eight of 67 ASOs increased exon 4 levels in BIM deletion-containing cells, and restored imatinib-induced apoptosis and TKI sensitivity. This proof-of-principle study proves that resistant CML cells by BIM deletion polymorphism can be resensitized to imatinib via splice-switching BIM ASOs. Future optimizations might yield a therapeutic ASO as precision-medicine adjuvant treatment for BIM-polymorphism-associated TKI-resistant CML and other cancers. PMID:29100409

  20. Marker-Assisted Selection of Xa21 Conferring Resistance to Bacterial Leaf Blight in indica Rice Cultivar LT2

    Directory of Open Access Journals (Sweden)

    Hue Thi Nguyen

    2018-01-01

    Full Text Available Bacterial leaf blight of rice (BLB, caused by Xanthomonas oryzae pv. oryzae, is one of the most destructive diseases in Asian rice fields. A high-quality rice variety, LT2, was used as the recipient parent. IRBB21, which carries the Xa21 gene, was used as the donor parent. The resistance gene Xa21 was introduced into LT2 by marker-assisted backcrossing. Three Xoo races were used to inoculate the improved lines following the clipping method. Eleven BC3F3 lines carrying Xa21 were obtained based on molecular markers and agronomic performance. The 11 lines were then inoculated with the three Xoo races. All the 11 improved lines showed better resistance to BLB than the recipient parent LT2. Based on the level of resistance to BLB and their agronomic performance, five lines (BC3F3 5.1.5.1, BC3F3 5.1.5.12, BC3F3 8.5.6.44, BC3F3 9.5.4.1 and BC3F3 9.5.4.23 were selected as the most promising for commercial release. These improved lines could contribute to rice production in terms of food security.

  1. Overcoming imatinib resistance conferred by the BIM deletion polymorphism in chronic myeloid leukemia with splice-switching antisense oligonucleotides.

    Science.gov (United States)

    Liu, Jun; Bhadra, Malini; Sinnakannu, Joanna Rajeswary; Yue, Wan Lin; Tan, Cheryl Weiqi; Rigo, Frank; Ong, S Tiong; Roca, Xavier

    2017-09-29

    Many tyrosine kinase-driven cancers, including chronic myeloid leukemia (CML), are characterized by high response rates to specific tyrosine kinase inhibitors (TKIs) like imatinib. In East Asians, primary imatinib resistance is caused by a deletion polymorphism in Intron 2 of the BIM gene, whose product is required for TKI-induced apoptosis. The deletion biases BIM splicing from exon 4 to exon 3, generating splice isoforms lacking the exon 4-encoded pro-apoptotic BH3 domain, which impairs the ability of TKIs to induce apoptosis. We sought to identify splice-switching antisense oligonucleotides (ASOs) that block exon 3 but enhance exon 4 splicing, and thereby resensitize BIM deletion-containing cancers to imatinib. First, we mapped multiple cis -acting splicing elements around BIM exon 3 by minigene mutations, and found an exonic splicing enhancer acting via SRSF1. Second, by a systematic ASO walk, we isolated ASOs that corrected the aberrant BIM splicing. Eight of 67 ASOs increased exon 4 levels in BIM deletion-containing cells, and restored imatinib-induced apoptosis and TKI sensitivity. This proof-of-principle study proves that resistant CML cells by BIM deletion polymorphism can be resensitized to imatinib via splice-switching BIM ASOs. Future optimizations might yield a therapeutic ASO as precision-medicine adjuvant treatment for BIM -polymorphism-associated TKI-resistant CML and other cancers.

  2. Linc-ROR confers gemcitabine resistance to pancreatic cancer cells via inducing autophagy and modulating the miR-124/PTBP1/PKM2 axis.

    Science.gov (United States)

    Li, Chenggang; Zhao, Zhiming; Zhou, Zhipeng; Liu, Rong

    2016-12-01

    In this study, we investigated the regulation of linc-ROR on autophagy and gemcitabine resistance of pancreatic cancer cells and further studied the underlying involvement of the miR-124/PTBP1/PKM2 axis in this regulation. Pancreatic cancer cell lines PANC-1 and MIAPaCa-2 cells were used as in vitro model. Autophagy was assessed by western blot of LC3 I/II and observation GFP-LC3 puncta. Cell viability was examined using CCK-8 assay. Cell apoptosis was examined by flow cytometric analysis of Annexin V/PI staining. QRT-PCR, RNA fluorescence in situ hybridization and dual luciferase assay were used to study the expression and the binding between linc-ROR and miR-124. Linc-ROR siRNA significantly sensitized PANC-1 and MIAPaCa-2 cells to gemcitabine, while linc-ROR overexpression significantly reduced the sensitivity. Linc-ROR knockdown reduced basal autophagy, while linc-ROR overexpression markedly increased basal autophagy in the cells. Linc-ROR siRNA showed similar effect as 3-MA on enhancing gemcitabine-induced cell apoptosis and also reduced PKM2 expression. MiR-124 overexpression restored PKM1 and reduced PKM2 levels in the cells. In addition, miR-124 mimics also alleviated autophagy in pancreatic cancer cells. Both miR-124 mimics and PKM2 siRNA enhanced gemcitabine-induced cell apoptosis. In both pancreatic cell lines and PADC tissues, linc-ROR is negatively correlated with miR-124 expression. In addition, dual luciferase assay verified two 8mer binding sites between miR-124 and linc-ROR. Linc-ROR confers gemcitabine resistance to pancreatic cancer cells at least partly via inducing autophagy. There is a linc-ROR/miR-124/PTBP1/PKM2 axis involved in regulation of gemcitabine resistance in pancreatic cancer cells.

  3. Expression of the Grape VqSTS21 Gene in Arabidopsis Confers Resistance to Osmotic Stress and Biotrophic Pathogens but Not Botrytis cinerea.

    Science.gov (United States)

    Huang, Li; Zhang, Songlin; Singer, Stacy D; Yin, Xiangjing; Yang, Jinhua; Wang, Yuejin; Wang, Xiping

    2016-01-01

    Stilbene synthase ( STS ) is a key gene in the biosynthesis of various stilbenoids, including resveratrol and its derivative glucosides (such as piceid), that has been shown to contribute to disease resistance in plants. However, the mechanism behind such a role has yet to be elucidated. Furthermore, the function of STS genes in osmotic stress tolerance remains unclear. As such, we sought to elucidate the role of STS genes in the defense against biotic and abiotic stress in the model plant Arabidopsis thaliana . Expression profiling of 31 VqSTS genes from Vitis quinquangularis revealed that VqSTS21 was up-regulated in response to powdery mildew (PM) infection. To provide a deeper understanding of the function of this gene, we cloned the full-length coding sequence of VqSTS21 and overexpressed it in Arabidopsis thaliana via Agrobacterium -mediated transformation. The resulting VqSTS21 Arabidopsis lines produced trans -piceid rather than resveratrol as their main stilbenoid product and exhibited improved disease resistance to PM and Pseudomonas syringae pv. tomato DC3000, but displayed increased susceptibility to Botrytis cinerea . In addition, transgenic Arabidopsis lines were found to confer tolerance to salt and drought stress from seed germination through plant maturity. Intriguingly, qPCR assays of defense-related genes involved in salicylic acid, jasmonic acid, and abscisic acid-induced signaling pathways in these transgenic lines suggested that VqSTS21 plays a role in various phytohormone-related pathways, providing insight into the mechanism behind VqSTS21 -mediated resistance to biotic and abiotic stress.

  4. Expression of the grape VqSTS21 gene in Arabidopsis confers resistance to osmotic stress and biotrophic pathogens but not Botrytis cinerea

    Directory of Open Access Journals (Sweden)

    Li Huang

    2016-09-01

    Full Text Available Stilbene synthase (STS is a key gene in the biosynthesis of various stilbenoids, including resveratrol and its derivative glucosides (such as piceid, that has been shown to contribute to disease resistance in plants. However, the mechanism behind such a role has yet to be elucidated. Furthermore, the function of STS genes in osmotic stress tolerance remains unclear. As such, we sought to elucidate the role of STS genes in the defense against biotic and abiotic stress in the model plant Arabidopsis thaliana. Expression profiling of 31 VqSTS genes from Vitis quinquangularis revealed that VqSTS21 was up-regulated in response to powdery mildew (PM infection. To provide a deeper understanding of the function of this gene, we cloned the full-length coding sequence of VqSTS21 and overexpressed it in Arabidopsis thaliana via Agrobacterium-mediated transformation. The resulting VqSTS21 Arabidopsis lines produced trans-piceid rather than resveratrol as their main stilbenoid product and exhibited improved disease resistance to PM and Pseudomonas syringae pv. tomato DC3000, but displayed increased susceptibility to Botrytis cinerea. In addition, transgenic Arabidopsis lines were found to confer tolerance to salt and drought stress from seed germination through plant maturity. Intriguingly, qPCR assays of defense-related genes involved in salicylic acid, jasmonic acid, and abscisic acid-induced signaling pathways in these transgenic lines suggested that VqSTS21 plays a role in various phytohormone-related pathways, providing insight into the mechanism behind VqSTS21-mediated resistance to biotic and abiotic stress.

  5. Inducible expression of Bs2 R gene from Capsicum chacoense in sweet orange (Citrus sinensis L. Osbeck) confers enhanced resistance to citrus canker disease.

    Science.gov (United States)

    Sendín, Lorena Noelia; Orce, Ingrid Georgina; Gómez, Rocío Liliana; Enrique, Ramón; Grellet Bournonville, Carlos Froilán; Noguera, Aldo Sergio; Vojnov, Adrián Alberto; Marano, María Rosa; Castagnaro, Atilio Pedro; Filippone, María Paula

    2017-04-01

    Transgenic expression of the pepper Bs2 gene confers resistance to Xanthomonas campestris pv. vesicatoria (Xcv) pathogenic strains which contain the avrBs2 avirulence gene in susceptible pepper and tomato varieties. The avrBs2 gene is highly conserved among members of the Xanthomonas genus, and the avrBs2 of Xcv shares 96% homology with the avrBs2 of Xanthomonas citri subsp. citri (Xcc), the causal agent of citrus canker disease. A previous study showed that the transient expression of pepper Bs2 in lemon leaves reduced canker formation and induced plant defence mechanisms. In this work, the effect of the stable expression of Bs2 gene on citrus canker resistance was evaluated in transgenic plants of Citrus sinensis cv. Pineapple. Interestingly, Agrobacterium-mediated transformation of epicotyls was unsuccessful when a constitutive promoter (2× CaMV 35S) was used in the plasmid construction, but seven transgenic lines were obtained with a genetic construction harbouring Bs2 under the control of a pathogen-inducible promoter, from glutathione S-transferase gene from potato. A reduction of disease symptoms of up to 70% was observed in transgenic lines expressing Bs2 with respect to non-transformed control plants. This reduction was directly dependent on the Xcc avrBs2 gene since no effect was observed when a mutant strain of Xcc with a disruption in avrBs2 gene was used for inoculations. Additionally, a canker symptom reduction was correlated with levels of the Bs2 expression in transgenic plants, as assessed by real-time qPCR, and accompanied by the production of reactive oxygen species. These results indicate that the pepper Bs2 resistance gene is also functional in a family other than the Solanaceae, and could be considered for canker control.

  6. Integration of auxin/indole-3-acetic acid 17 and RGA-LIKE3 confers salt stress resistance through stabilization by nitric oxide in Arabidopsis.

    Science.gov (United States)

    Shi, Haitao; Liu, Wen; Wei, Yunxie; Ye, Tiantian

    2017-02-01

    Plants have developed complex mechanisms to respond to salt stress, depending on secondary messenger-mediated stress perception and signal transduction. Nitric oxide (NO) is widely known as a 'jack-of-all-trades' in stress responses. However, NO-mediated crosstalk between plant hormones remains unclear. In this study, we found that salt stabilized both AUXIN/INDOLE-3-ACETIC ACID 17 (Aux/IAA17) and RGA-LIKE3 (RGL3) proteins due to salt-induced NO production. Salt-induced NO overaccumulation and IAA17 overexpression decreased the transcripts of GA3ox genes, resulting in lower bioactive GA4. Further investigation showed that IAA17 directly interacted with RGL3 and increased its protein stability. Consistently, RGL3 stabilized IAA17 protein through inhibiting the interaction of TIR1 and IAA17 by competitively binding to IAA17. Moreover, both IAA17 and RGL3 conferred salt stress resistance. Overexpression of IAA17 and RGL3 partially alleviated the inhibitory effect of NO deficiency on salt resistance, whereas the iaa17 and rgl3 mutants displayed reduced responsiveness to NO-promoted salt resistance. Thus, the associations between IAA17 and gibberellin (GA) synthesis and signal transduction, and between the IAA17-interacting complex and the NO-mediated salt stress response were revealed based on physiological and genetic approaches. We conclude that integration of IAA17 and RGL3 is an essential component of NO-mediated salt stress response. © The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  7. HIV-1 resistance conferred by siRNA cosuppression of CXCR4 and CCR5 coreceptors by a bispecific lentiviral vector

    Directory of Open Access Journals (Sweden)

    Akkina Ramesh

    2005-01-01

    Full Text Available Abstract Background RNA interference (RNAi mediated by small interfering RNAs (siRNAs has proved to be a highly effective gene silencing mechanism with great potential for HIV/AIDS gene therapy. Previous work with siRNAs against cellular coreceptors CXCR4 and CCR5 had shown that down regulation of these surface molecules could prevent HIV-1 entry and confer viral resistance. Since monospecific siRNAs targeting individual coreceptors are inadequate in protecting against both T cell tropic (X4 and monocyte tropic (R5 viral strains simultaneously, bispecific constructs with dual specificity are required. For effective long range therapy, the bispecific constructs need to be stably transduced into HIV-1 target cells via integrating viral vectors. Results To achieve this goal, lentiviral vectors incorporating both CXCR4 and CCR5 siRNAs of short hairpin design were constructed. The CXCR4 siRNA was driven by a U6 promoter whereas the CCR5 siRNA was driven by an H1 promoter. A CMV promoter driven EGFP reporter gene is also incorporated in the bispecific construct. High efficiency transduction into coreceptor expressing Magi and Ghost cell lines with a concomitant down regulation of respective coreceptors was achieved with lentiviral vectors. When the siRNA expressing transduced cells were challenged with X4 and R5 tropic HIV-1, they demonstrated marked viral resistance. HIV-1 resistance was also observed in bispecific lentiviral vector transduced primary PBMCs. Conclusions Both CXCR4 and CCR5 coreceptors could be simultaneously targeted for down regulation by a single combinatorial lentiviral vector incorporating respective anti-coreceptor siRNAs. Stable down regulation of both the coreceptors protects cells against infection by both X4 and R5 tropic HIV-1. Stable down regulation of cellular molecules that aid in HIV-1 infection will be an effective strategy for long range HIV gene therapy.

  8. RAGE Deletion Confers Renoprotection by Reducing Responsiveness to Transforming Growth Factor-β and Increasing Resistance to Apoptosis.

    Science.gov (United States)

    Hagiwara, Shinji; Sourris, Karly; Ziemann, Mark; Tieqiao, Wu; Mohan, Muthukumar; McClelland, Aaron D; Brennan, Eoin; Forbes, Josephine; Coughlan, Melinda; Harcourt, Brooke; Penfold, Sally; Wang, Bo; Higgins, Gavin; Pickering, Raelene; El-Osta, Assam; Thomas, Merlin C; Cooper, Mark E; Kantharidis, Phillip

    2018-02-15

    Signalling via the receptor of advanced glycation end-products (RAGE) although complex and not fully elucidated in the setting of diabetes, is considered a key injurious pathway in the development of diabetic nephropathy (DN). We report here that RAGE deletion resulted in increased expression of fibrotic (collagen I and IV, fibronectin) and the inflammatory marker, MCP-1 in primary mouse mesangial cells (MC) and in kidney cortex. RNA-seq analysis in MCs from RAGE -/- and wild type mice confirmed these observations. Nevertheless, despite these gene expression changes a decreased responsiveness to transforming growth factor-β was identified in RAGE -/- mice. Furthermore, RAGE deletion conferred a more proliferative phenotype in MCs and reduced susceptibility to staurosporine-induced apoptosis. RAGE restoration experiments in RAGE -/- MCs largely reversed these gene expression changes resulting in reduced expression of fibrotic and inflammatory markers. This study highlights that protection against DN in RAGE KO mice is likely in part to be due the result of decreased responsiveness to growth factor stimulation and an anti-apoptotic phenotype in mesangial cells. Furthermore, it extends our understanding of the role of RAGE in the progression of DN since RAGE appears to play a key role in modulating the sensitivity of the kidney to injurious stimuli such as prosclerotic cytokines. © 2018 by the American Diabetes Association.

  9. Frequency of alleles conferring resistance to the Bacillus thuringiensis toxins Cry1Ac and Cry2Ab in Australian populations of Helicoverpa punctigera (Lepidoptera: Noctuidae) from 2002 to 2006.

    Science.gov (United States)

    Downes, S; Parker, T L; Mahon, R J

    2009-04-01

    Helicoverpa punctigera and Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) are important pests of field and horticultural crops in Australia. The former is endemic to the continent, whereas the latter is also distributed in Africa and Asia. Although H. armigera rapidly developed resistance to virtually every group of insecticide used against it, there is only one report of resistance to an insecticide in H. punctigera. In 1996 the Australian cotton industry adopted Ingard, which expresses the Bacillus thuringiensis (Bt) toxin gene cry1Ac. In 2004/2005, Bollgard II (which expresses Cry1Ac and Cry2Ab) replaced Ingard and has subsequently been grown on 80% of the area planted to cotton, Gossypium hirsutum L. From 2002/2003 to 2006/2007, F2 screens were used to detect resistance to Cry1Ac or Cry2Ab. We detected no alleles conferring resistance to Cry1Ac; the frequency was < 0.0005 (n = 2,180 alleles), with a 95% credibility interval between 0 and 0.0014. However, during the same period, we detected alleles that confer resistance to Cry2Ab at a frequency of 0.0018 (n = 2,192 alleles), with a 95% credibility interval between 0.0005 and 0.0040. For both toxins, the experiment-wise detection probability was 94%, i.e., if there actually was a resistance allele in any tested lines, we would have detected it 94% of the time. The first isolation of Cry2Ab resistance in H. punctigera was before the widespread deployment of Bollgard II. This finding supports our published notion for H. armigera that alleles conferring resistance to Cry2Ab may be present at detectable frequencies in populations before selection by transgenic crops.

  10. Over-Expression of Rice CBS Domain Containing Protein, OsCBSX3, Confers Rice Resistance to Magnaporthe oryzae Inoculation

    Directory of Open Access Journals (Sweden)

    Shaoling Mou

    2015-07-01

    Full Text Available Cystathionine β-synthase (CBS domain containing proteins (CDCPs constitute a big family in plants and some members in this family have been implicated in a variety of biological processes, but the precise functions and the underlying mechanism of the majority of this family in plant immunity remain to be elucidated. In the present study, a CBS domain containing protein gene, OsCBSX3, is functionally characterized in rice resistance against Magnaporthe oryzae (M. oryzae. By quantitative real-time PCR, transcripts of OsCBSX3 are up-regulated significantly by inoculation of M. oryzae and the exogenously applied salicylic acid (SA and methyl jasmonate (MeJA. OsCBSX3 is exclusively localized to the plasma membrane by transient expression of OsCBSX3 fused to green fluorescent protein (GFP through approach of Agrobacterium infiltration in Nicotiana benthamiana leaves. The plants of homozygous T3 transgenic rice lines of over-expressing OsCBSX3 exhibit significant enhanced resistance to M. oryzae inoculation, manifested by decreased disease symptoms, and inhibition of pathogen growth detected in DNA. Consistently, the over-expression of OsCBSX3 enhances the transcript levels of immunity associated marker genes including PR1a, PR1b, PR5, AOS2, PAL, NH1, and OsWRKY13 in plants inoculated with M. oryzae. These results suggest that OsCBSX3 acts as a positive regulator in resistance of rice to M. oryzae regulated by SA and JA-mediated signaling pathways synergistically.

  11. Expression of antimicrobial peptides under control of a camalexin-biosynthetic promoter confers enhanced resistance against Pseudomonas syringae.

    Science.gov (United States)

    Chapman, Alexandra; Lindermayr, Christian; Glawischnig, Erich

    2016-02-01

    In Arabidopsis thaliana phytoalexin biosynthesis is tightly regulated. The camalexin biosynthetic gene CYP71B15/PAD3 is highly expressed in response to pathogens and specific abiotic triggers, while constitutive expression is very low. Based on this property we expressed artificial antimicrobial peptides under control of the CYP71B15 promoter avoiding potential toxic effects to the plant related to constitutive expression. Significant and substantial growth inhibition of Pseudomonas syringae was observed, demonstrating that expression of these peptides under control of a phytoalexin promoter is an effective approach for enhancement of resistance against bacterial pathogens. Copyright © 2016 Elsevier Ltd. All rights reserved.

  12. Molecular organization of small plasmids bearing blaTEM-1 and conferring resistance to β-lactams in Haemophilus influenzae.

    Science.gov (United States)

    Søndergaard, Annette; San Millan, Alvaro; Santos-Lopez, Alfonso; Nielsen, Signe M; Gonzalez-Zorn, Bruno; Nørskov-Lauritsen, Niels

    2012-09-01

    TEM-1 is the dominant β-lactamase of Haemophilus influenzae and can be located on small plasmids. Three distinct plasmids with sizes from 4,304 to 5,646 nucleotides (nt) were characterized: pA1606, pA1209, and pPN223. In addition to TEM-1 and a replication enzyme of the Rep 3 superfamily, pA1606 carries a Tn3 resolvase gene and pA1606 and pA1209 carry an open reading frame (ORF) similar to a plasmid recombination enzyme gene described in Gram-positive bacteria. The plasmids transformed strain Rd to the ampicillin-resistant phenotype.

  13. Conference Interpreters

    DEFF Research Database (Denmark)

    Leal Lobato, Ana Isabel

    2017-01-01

    Conference Interpreters: How to serve the cause of minorized communities in the new postmonolingual / ‘postmonodiscoursive’ order,......Conference Interpreters: How to serve the cause of minorized communities in the new postmonolingual / ‘postmonodiscoursive’ order,...

  14. Interaction of reverse transcriptase (RT) mutations conferring resistance to lamivudine and etravirine: effects on fitness and RT activity of human immunodeficiency virus type 1.

    Science.gov (United States)

    Hu, Zixin; Kuritzkes, Daniel R

    2011-11-01

    Resistance to the nonnucleoside reverse transcriptase inhibitors etravirine and rilpivirine (RPV) is conferred by the E138K mutation in human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT). Clinical trials of RPV administered with lamivudine or emtricitabine showed the emergence of E138K together with M184I, which confers lamivudine and emtricitabine resistance in most patients with virologic failure. To understand why M184I was favored over M184V, we determined the drug susceptibility, infectivity, relative fitness, and reverse transcriptase activity of HIV-1 carrying E138K/M184I or E138K/M184V mutations. Whereas the replication capacity (RC) of the single mutants was reduced compared to that of the wild type (WT), the RC of the two double mutants was comparable to that of the WT in the absence of drug. The RC of the E138K/M184I mutant in the presence of etravirine was significantly greater than that of the E138K and E138K/M184V mutants; the RC of the double mutants was greater than that of the M184I or M184V mutant. Fitness profiles and growth competition experiments showed that the E138K/M184I mutant had a significant replicative advantage over the E138K/M184V mutant in the presence of etravirine and lamivudine. The virion-associated RT activity of the E138K, M184I, or M184V virus was significantly reduced compared to that of the WT, whereas the RT activity of the E138K/M184I virus was significantly greater than that of the WT or E138K/M184V virus. These results suggest that the E138K and M184I/V mutations are mutually compensatory and may explain the frequent occurrence of E138K/M184I after the virologic failure of rilpivirine-, lamivudine-, and emtricitabine-containing regimens.

  15. Interaction of Reverse Transcriptase (RT) Mutations Conferring Resistance to Lamivudine and Etravirine: Effects on Fitness and RT Activity of Human Immunodeficiency Virus Type 1 ▿

    Science.gov (United States)

    Hu, Zixin; Kuritzkes, Daniel R.

    2011-01-01

    Resistance to the nonnucleoside reverse transcriptase inhibitors etravirine and rilpivirine (RPV) is conferred by the E138K mutation in human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT). Clinical trials of RPV administered with lamivudine or emtricitabine showed the emergence of E138K together with M184I, which confers lamivudine and emtricitabine resistance in most patients with virologic failure. To understand why M184I was favored over M184V, we determined the drug susceptibility, infectivity, relative fitness, and reverse transcriptase activity of HIV-1 carrying E138K/M184I or E138K/M184V mutations. Whereas the replication capacity (RC) of the single mutants was reduced compared to that of the wild type (WT), the RC of the two double mutants was comparable to that of the WT in the absence of drug. The RC of the E138K/M184I mutant in the presence of etravirine was significantly greater than that of the E138K and E138K/M184V mutants; the RC of the double mutants was greater than that of the M184I or M184V mutant. Fitness profiles and growth competition experiments showed that the E138K/M184I mutant had a significant replicative advantage over the E138K/M184V mutant in the presence of etravirine and lamivudine. The virion-associated RT activity of the E138K, M184I, or M184V virus was significantly reduced compared to that of the WT, whereas the RT activity of the E138K/M184I virus was significantly greater than that of the WT or E138K/M184V virus. These results suggest that the E138K and M184I/V mutations are mutually compensatory and may explain the frequent occurrence of E138K/M184I after the virologic failure of rilpivirine-, lamivudine-, and emtricitabine-containing regimens. PMID:21849432

  16. The overexpression of an Amaranthus hypochondriacus NF-YC gene modifies growth and confers water deficit stress resistance in Arabidopsis.

    Science.gov (United States)

    Palmeros-Suárez, Paola A; Massange-Sánchez, Julio A; Martínez-Gallardo, Norma A; Montero-Vargas, Josaphat M; Gómez-Leyva, Juan F; Délano-Frier, John P

    2015-11-01

    Nuclear factor-Y (NF-Y), is a plant heterotrimeric transcription factor constituted by NF-YA, NF-YB and NF-YC subunits. The function of many NF-Y subunits, mostly of the A and B type, has been studied in plants, but knowledge regarding the C subunit remains fragmentary. Here, a water stress-induced NF-YC gene from Amaranthus hypochondriacus (AhNF-YC) was further characterized by its overexpression in transgenic Arabidospis thaliana plants. A role in development was inferred from modified growth rates in root, rosettes and inflorescences recorded in AhNF-YC overexpressing Arabidopsis plants, in addition to a delayed onset of flowering. Also, the overexpression of AhNF-YC caused increased seedling sensitivity to abscisic acid (ABA), and influenced the expression of several genes involved in secondary metabolism, development and ABA-related responses. An altered expression of the latter in water stressed and recovered transgenic plants, together with the observed increase in ABA sensitivity, suggested that their increased water stress resistance was partly ABA-dependent. An untargeted metabolomic analysis also revealed an altered metabolite pattern, both in normal and water stress/recovery conditions. These results suggest that AhNF-YC may play an important regulatory role in both development and stress, and represents a candidate gene for the engineering of abiotic stress resistance in commercial crops. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  17. Expression of tomato prosystemin gene in Arabidopsis reveals systemic translocation of its mRNA and confers necrotrophic fungal resistance.

    Science.gov (United States)

    Zhang, Haiyan; Yu, Pengli; Zhao, Jiuhai; Jiang, Hongling; Wang, Haiyang; Zhu, Yingfang; Botella, Miguel A; Šamaj, Jozef; Li, Chuanyou; Lin, Jinxing

    2018-01-01

    Systemin (SYS), an octadecapeptide hormone processed from a 200-amino-acid precursor (prosystemin, PS), plays a central role in the systemic activation of defense genes in tomato in response to herbivore and pathogen attacks. However, whether PS mRNA is transferable and its role in systemic defense responses remain unknown. We created the transgenic tomato PS gene tagged with the green fluorescent protein (PS-GFP) using a shoot- or root-specific promoter, and the constitutive 35S promoter in Arabidopsis. Subcellular localization of PS-/SYS-GFP was observed using confocal laser scanning microscopy and gene transcripts were determined using quantitative real-time PCR. In Arabidopsis, PS protein can be processed and SYS is secreted. Shoot-/root-specific expression of PS-GFP in Arabidopsis, and grafting experiments, revealed that the PS mRNA moves in a bi-directional manner. We also found that ectopic expression of PS improves Arabidopsis resistance to the necrotrophic fungus Botrytis cinerea, consistent with substantial upregulation of the transcript levels of specific pathogen-responsive genes. Our results provide novel insights into the multifaceted mechanism of SYS signaling transport and its potential application in genetic engineering for increasing pathogen resistance across diverse plant families. © 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

  18. Amplification of the Gp41 gene for detection of mutations conferring resistance to HIV-1 fusion inhibitors on genotypic assay

    Science.gov (United States)

    Tanumihardja, J.; Bela, B.

    2017-08-01

    Fusion inhibitors have potential for future use in HIV control programs in Indonesia, so the capacity to test resistance to such drugs needs to be developed. Resistance-detection with a genotypic assay began with amplification of the target gene, gp41. Based on the sequence of the two most common HIV subtypes in Indonesia, AE and B, a primer pair was designed. Plasma samples containing both subtypes were extracted to obtain HIV RNA. Using PCR, the primer pair was used to produce the amplification product, the identity of which was checked based on length under electrophoresis. Eleven plasma samples were included in this study. One-step PCR using the primer pair was able to amplify gp41 from 54.5% of the samples, and an unspecific amplification product was seen in 1.1% of the samples. Amplification failed in 36.4% of the samples, which may be due to an inappropriate primer sequence. It was also found that the optimal annealing temperature for producing the single expected band was 57.2 °C. With one-step PCR, the designed primer pair amplified the HIV-1 gp41 gene from subtypes AE and B. However, further research should be done to determine the conditions that will increase the sensitivity and specificity of the amplification process.

  19. Elevated BCRP/ABCG2 expression confers acquired resistance to gefitinib in wild-type EGFR-expressing cells.

    Directory of Open Access Journals (Sweden)

    Yun-Ju Chen

    Full Text Available The sensitivity of non-small cell lung cancer (NSCLC patients to EGFR tyrosine kinase inhibitors (TKIs is strongly associated with activating EGFR mutations. Although not as sensitive as patients harboring these mutations, some patients with wild-type EGFR (wtEGFR remain responsive to EGFR TKIs, suggesting that the existence of unexplored mechanisms renders most of wtEGFR-expressing cancer cells insensitive.Here, we show that acquired resistance of wtEGFR-expressing cancer cells to an EGFR TKI, gefitinib, is associated with elevated expression of breast cancer resistance protein (BCRP/ABCG2, which in turn leads to gefitinib efflux from cells. In addition, BCRP/ABCG2 expression correlates with poor response to gefitinib in both cancer cell lines and lung cancer patients with wtEGFR. Co-treatment with BCRP/ABCG2 inhibitors enhanced the anti-tumor activity of gefitinib.Thus, BCRP/ABCG2 expression may be a predictor for poor efficacy of gefitinib treatment, and targeting BCRP/ABCG2 may broaden the use of gefitinib in patients with wtEGFR.

  20. Transgenic expression in citrus of single-chain antibody fragments specific to Citrus tristeza virus confers virus resistance.

    Science.gov (United States)

    Cervera, Magdalena; Esteban, Olga; Gil, Maite; Gorris, M Teresa; Martínez, M Carmen; Peña, Leandro; Cambra, Mariano

    2010-12-01

    Citrus tristeza virus (CTV) causes one of the most destructive viral diseases of citrus worldwide. Generation of resistant citrus genotypes through genetic engineering could be a good alternative to control CTV. To study whether production of single-chain variable fragment (scFv) antibodies in citrus could interfere and immunomodulate CTV infection, transgenic Mexican lime plants expressing two different scFv constructs, separately and simultaneously, were generated. These constructs derived from the well-referenced monoclonal antibodies 3DF1 and 3CA5, specific against CTV p25 major coat protein, whose mixture is able to detect all CTV isolates characterized so far. ScFv accumulation levels were low and could be readily detected just in four transgenic lines. Twelve homogeneous and vigorous lines were propagated and CTV-challenged by graft inoculation with an aggressive CTV strain. A clear protective effect was observed in most transgenic lines, which showed resistance in up to 40-60% of propagations. Besides, both a delay in symptom appearance and attenuation of symptom intensity were observed in infected transgenic plants compared with control plants. This effect was more evident in lines carrying the 3DF1scFv transgene, being probably related to the biological functions of the epitope recognized by this antibody. This is the first report describing successful protection against a pathogen in woody transgenic plants by ectopic expression of scFv recombinant antibodies.

  1. Antibiotic resistance marker genes as environmental pollutants in GMO-pristine agricultural soils in Austria.

    Science.gov (United States)

    Woegerbauer, Markus; Zeinzinger, Josef; Gottsberger, Richard Alexander; Pascher, Kathrin; Hufnagl, Peter; Indra, Alexander; Fuchs, Reinhard; Hofrichter, Johannes; Kopacka, Ian; Korschineck, Irina; Schleicher, Corina; Schwarz, Michael; Steinwider, Johann; Springer, Burkhard; Allerberger, Franz; Nielsen, Kaare M; Fuchs, Klemens

    2015-11-01

    Antibiotic resistance genes may be considered as environmental pollutants if anthropogenic emission and manipulations increase their prevalence above usually occurring background levels. The prevalence of aph(3')-IIa/nptII and aph(3')-IIIa/nptIII - frequent marker genes in plant biotechnology conferring resistance to certain aminoglycosides - was determined in Austrian soils from 100 maize and potato fields not yet exposed to but eligible for GMO crop cultivation. Total soil DNA extracts were analysed by nptII/nptIII-specific TaqMan real time PCR. Of all fields 6% were positive for nptII (median: 150 copies/g soil; range: 31-856) and 85% for nptIII (1190 copies/g soil; 13-61600). The copy-number deduced prevalence of nptIII carriers was 14-fold higher compared to nptII. Of the cultivable kanamycin-resistant soil bacteria 1.8% (95% confidence interval: 0-3.3%) were positive for nptIII, none for nptII (0-0.8%). The nptII-load of the studied soils was low rendering nptII a typical candidate as environmental pollutant upon anthropogenic release into these ecosystems. Copyright © 2015 Elsevier Ltd. All rights reserved.

  2. Conference Planning.

    Science.gov (United States)

    Burke, W. Warner, Ed.; Beckhard, Richard, Ed.

    This book, written to instruct in the use of a conference as a medium of social intercourse, is divided into four sections. Section I, which contains five articles, deals with factors to be considered in planning a conference. Specific techniques one can employ to improve a conference and several different techniques for evaluating the…

  3. Irradiation-resistance conferred by superoxide dismutase: possible adaptive role of a natural polymorphism in Drosophila melanogaster

    International Nuclear Information System (INIS)

    Peng, T.X.; Moya, A.; Ayala, F.J.

    1986-01-01

    The toxic effects of ionizing radiation to DNA are thought to be due to the generation of the superoxide radical, 02-. Superoxide dismutase (SOD), which scavenges 02-., has been invoked as a protecting enzyme against ionizing radiation in viruses, bacteria, mammalian cells in culture, and live mice. We now demonstrate that SOD is involved in the resistance of Drosophila melanogaster against irradiation. The protection is greatest when flies carry the S form of the enzyme (which exhibits highest in vitro specific activity), intermediate when they carry the F form of the enzyme, and lowest when they are homozygous for N, an allele that reduces the amount of the enzyme to 3.5% of the normal level. Natural selection experiments show that the fitness of the high-activity S allele is increased in an irradiated population relative to the nonirradiated control. These results point towards a possible adaptive function of the S/F polymorphism found in natural populations of D. melanogaster

  4. Get Tough, Get Toxic, or Get a Bodyguard: Identifying Candidate Traits Conferring Belowground Resistance to Herbivores in Grasses.

    Science.gov (United States)

    Moore, Ben D; Johnson, Scott N

    2016-01-01

    Grasses (Poaceae) are the fifth-largest plant family by species and their uses for crops, forage, fiber, and fuel make them the most economically important. In grasslands, which broadly-defined cover 40% of the Earth's terrestrial surface outside of Greenland and Antarctica, 40-60% of net primary productivity and 70-98% of invertebrate biomass occurs belowground, providing extensive scope for interactions between roots and rhizosphere invertebrates. Grasses invest 50-70% of fixed carbon into root construction, which suggests roots are high value tissues that should be defended from herbivores, but we know relatively little about such defenses. In this article, we identify candidate grass root defenses, including physical (tough) and chemical (toxic) resistance traits, together with indirect defenses involving recruitment of root herbivores' natural enemies. We draw on relevant literature to establish whether these defenses are present in grasses, and specifically in grass roots, and which herbivores of grasses are affected by these defenses. Physical defenses could include structural macro-molecules such as lignin, cellulose, suberin, and callose in addition to silica and calcium oxalate. Root hairs and rhizosheaths, a structural adaptation unique to grasses, might also play defensive roles. To date, only lignin and silica have been shown to negatively affect root herbivores. In terms of chemical resistance traits, nitrate, oxalic acid, terpenoids, alkaloids, amino acids, cyanogenic glycosides, benzoxazinoids, phenolics, and proteinase inhibitors have the potential to negatively affect grass root herbivores. Several good examples demonstrate the existence of indirect defenses in grass roots, including maize, which can recruit entomopathogenic nematodes (EPNs) via emission of (E)-β-caryophyllene, and similar defenses are likely to be common. In producing this review, we aimed to equip researchers with candidate root defenses for further research.

  5. Overexpression of miR529a confers enhanced resistance to oxidative stress in rice (Oryza sativa L.).

    Science.gov (United States)

    Yue, Erkui; Liu, Zhen; Li, Chao; Li, Yu; Liu, Qiuxiang; Xu, Jian-Hong

    2017-07-01

    Overexpressing miR529a can enhance oxidative stress resistance by targeting OsSPL2 and OsSPL14 genes that can regulate the expression of their downstream SOD and POD related genes. MicroRNAs are involved in the regulation of plant developmental and physiological processes, and their expression can be altered when plants suffered environment stresses, including salt, oxidative, drought and Cadmium. The expression of microRNA529 (miR529) can be induced under oxidative stress. However, its biological function under abiotic stress responses is still unclear. In this study, miR529a was overexpressed to investigate the function of miR529a under oxidative stress in rice. Our results demonstrated that the expression of miR529a can be induced by exogenous H 2 O 2 , and overexpressing miR529a can increase plant tolerance to high level of H 2 O 2 , resulting in increased seed germination rate, root tip cell viability, reduced leaf rolling rate and chlorophyll retention. The expression of oxidative stress responsive genes and the activities of superoxide dismutase (SOD) and peroxidase (POD) were increased in miR529a overexpression plant, which could help to reduce redundant reactive oxygen species (ROS). Furthermore, only OsSPL2 and OsSPL14 were targeted by miR529a in rice seedlings, repressing their expression in miR529aOE plants could lead to strengthen plant tolerance to oxidation stress. Our study provided the evidence that overexpression of miR529a could strengthen oxidation resistance, and its target genes OsSPL2 and OsSPL14 were responsible for oxidative tolerance, implied the manipulation of miR529a and its target genes regulation on H 2 O 2 related response genes could improve oxidative stress tolerance in rice.

  6. A cost-effective smartphone-based antimicrobial susceptibility test reader for drug resistance testing (Conference Presentation)

    Science.gov (United States)

    Feng, Steve W.; Tseng, Derek; Di Carlo, Dino; Garner, Omai B.; Ozcan, Aydogan

    2017-03-01

    Antimicrobial susceptibility testing (AST) is commonly used for determining microbial drug resistance, but routine testing, which can significantly reduce the spread of multi-drug resistant organisms, is not regularly performed in resource-limited and field-settings due to technological challenges and lack of trained diagnosticians. We developed a portable cost-effective smartphone-based colorimetric 96-well microtiter plate (MTP) reader capable of automated AST without the need for a trained diagnostician. This system is composed of a smartphone used in conjunction with a 3D-printed opto-mechanical attachment, which holds a set of inexpensive light-emitting-diodes and fiber-optic cables coupled to the 96-well MTP for enabling the capture of the transmitted light through each well by the smartphone camera. Images of the MTP plate are captured at multiple exposures and uploaded to a local or remote server (e.g., a laptop) for automated processing/analysis of the results using a custom-designed smartphone application. Each set of images are combined to generate a high dynamic-range image and analyzed for well turbidity (indicative of bacterial growth), followed by interpretative analysis per plate to determine minimum inhibitory concentration (MIC) and drug susceptibility for the specific bacterium. Results are returned to the originating device within 1 minute and shown to the user in tabular form. We demonstrated the capability of this platform using MTPs prepared with 17 antibiotic drugs targeting Gram-negative bacteria and tested 82 patient isolate MTPs of Klebsiella pneumoniae, achieving well turbidity accuracy of 98.19%, MIC accuracy of 95.15%, and drug susceptibility interpretation accuracy of 99.06%, meeting the FDA defined criteria for AST.

  7. Ectopic expression of ubiquitin-conjugating enzyme gene from wild rice, OgUBC1, confers resistance against UV-B radiation and Botrytis infection in Arabidopsis thaliana

    International Nuclear Information System (INIS)

    Jeon, En Hee; Pak, Jung Hun; Kim, Mi Jin; Kim, Hye Jeong; Shin, Sang Hyun; Lee, Jai Heon; Kim, Doh Hoon; Oh, Ju Sung; Oh, Boung-Jun; Jung, Ho Won; Chung, Young Soo

    2012-01-01

    Highlights: ► We isolated a novel E2 ubiquitin-conjugating enzyme from leaves of wild rice plants. ► The OgUBC1 was highly expressed in leaves treated with SA and UV-B radiation. ► The recombinant OgUBC1 has an enzymatic activity of E2 in vitro. ► The OgUBC1 could protect disruption of plant cells by UV-B radiation. ► OgUBC1 confers disease resistance and UV-B tolerance in transgenic Arabidopsis plants. -- Abstract: A previously unidentified gene encoding ubiquitin-conjugating enzyme was isolated from leaves of wild rice plant treated with wounding and microbe-associated molecular patterns. The OgUBC1 gene was composed of 148 amino acids and contained a typical active site and 21 ubiquitin thioester intermediate interaction residues and 4 E3 interaction residues. Both exogenous application of salicylic acid and UV-B irradiation triggered expression of OgUBC1 in leaves of wild rice. Recombinant OgUBC1 proteins bound to ubiquitins in vitro, proposing that the protein might act as E2 enzyme in planta. Heterologous expression of the OgUBC1 in Arabidopsis thaliana protected plants from cellular damage caused by an excess of UV-B radiation. A stable expression of chalcone synthase gene was detected in leaves of OgUBC1-expressing Arabidopsis, resulting in producing higher amounts of anthocyanin than those in wild-type Col-0 plants. Additionally, both pathogenesis-related gene1 and 5 were transcribed in the transgenic Arabidopsis in the absence of pathogen infection. The OgUBC1-expressing plants were resistant to the infection of Botrytis cinerea. Taken together, we suggested that the OgUBC1 is involved in ubiquitination process important for cellular response against biotic and abiotic stresses in plants.

  8. Obesogenic memory can confer long-term increases in adipose tissue but not liver inflammation and insulin resistance after weight loss.

    Science.gov (United States)

    Schmitz, J; Evers, N; Awazawa, M; Nicholls, H T; Brönneke, H S; Dietrich, A; Mauer, J; Blüher, M; Brüning, J C

    2016-05-01

    Obesity represents a major risk factor for the development of type 2 diabetes mellitus, atherosclerosis and certain cancer entities. Treatment of obesity is hindered by the long-term maintenance of initially reduced body weight, and it remains unclear whether all pathologies associated with obesity are fully reversible even upon successfully maintained weight loss. We compared high fat diet-fed, weight reduced and lean mice in terms of body weight development, adipose tissue and liver insulin sensitivity as well as inflammatory gene expression. Moreover, we assessed similar parameters in a human cohort before and after bariatric surgery. Compared to lean animals, mice that demonstrated successful weight reduction showed increased weight gain following exposure to ad libitum control diet. However, pair-feeding weight-reduced mice with lean controls efficiently stabilized body weight, indicating that hyperphagia was the predominant cause for the observed weight regain. Additionally, whereas glucose tolerance improved rapidly after weight loss, systemic insulin resistance was retained and ameliorated only upon prolonged pair-feeding. Weight loss enhanced insulin action and resolved pro-inflammatory gene expression exclusively in the liver, whereas visceral adipose tissue displayed no significant improvement of metabolic and inflammatory parameters compared to obese mice. Similarly, bariatric surgery in humans (n = 55) resulted in massive weight reduction, improved hepatic inflammation and systemic glucose homeostasis, while adipose tissue inflammation remained unaffected and adipocyte-autonomous insulin action only exhibit minor improvements in a subgroup of patients (42%). These results demonstrate that although sustained weight loss improves systemic glucose homeostasis, primarily through improved inflammation and insulin action in liver, a remarkable obesogenic memory can confer long-term increases in adipose tissue inflammation and insulin resistance in mice as

  9. Ectopic expression of ubiquitin-conjugating enzyme gene from wild rice, OgUBC1, confers resistance against UV-B radiation and Botrytis infection in Arabidopsis thaliana

    Energy Technology Data Exchange (ETDEWEB)

    Jeon, En Hee; Pak, Jung Hun; Kim, Mi Jin; Kim, Hye Jeong [Department of Genetic Engineering, Dong-A University, Busan 604-714 (Korea, Republic of); Shin, Sang Hyun [National Crop Experiment Station, Rural Development Administration, Suwon 441-100 (Korea, Republic of); Lee, Jai Heon; Kim, Doh Hoon; Oh, Ju Sung [Department of Genetic Engineering, Dong-A University, Busan 604-714 (Korea, Republic of); Oh, Boung-Jun [BioControl Center, Jeonnam 516-942 (Korea, Republic of); Jung, Ho Won, E-mail: hwjung@dau.ac.kr [Department of Genetic Engineering, Dong-A University, Busan 604-714 (Korea, Republic of); Chung, Young Soo, E-mail: chungys@dau.ac.kr [Department of Genetic Engineering, Dong-A University, Busan 604-714 (Korea, Republic of)

    2012-10-19

    Highlights: Black-Right-Pointing-Pointer We isolated a novel E2 ubiquitin-conjugating enzyme from leaves of wild rice plants. Black-Right-Pointing-Pointer The OgUBC1 was highly expressed in leaves treated with SA and UV-B radiation. Black-Right-Pointing-Pointer The recombinant OgUBC1 has an enzymatic activity of E2 in vitro. Black-Right-Pointing-Pointer The OgUBC1 could protect disruption of plant cells by UV-B radiation. Black-Right-Pointing-Pointer OgUBC1 confers disease resistance and UV-B tolerance in transgenic Arabidopsis plants. -- Abstract: A previously unidentified gene encoding ubiquitin-conjugating enzyme was isolated from leaves of wild rice plant treated with wounding and microbe-associated molecular patterns. The OgUBC1 gene was composed of 148 amino acids and contained a typical active site and 21 ubiquitin thioester intermediate interaction residues and 4 E3 interaction residues. Both exogenous application of salicylic acid and UV-B irradiation triggered expression of OgUBC1 in leaves of wild rice. Recombinant OgUBC1 proteins bound to ubiquitins in vitro, proposing that the protein might act as E2 enzyme in planta. Heterologous expression of the OgUBC1 in Arabidopsis thaliana protected plants from cellular damage caused by an excess of UV-B radiation. A stable expression of chalcone synthase gene was detected in leaves of OgUBC1-expressing Arabidopsis, resulting in producing higher amounts of anthocyanin than those in wild-type Col-0 plants. Additionally, both pathogenesis-related gene1 and 5 were transcribed in the transgenic Arabidopsis in the absence of pathogen infection. The OgUBC1-expressing plants were resistant to the infection of Botrytis cinerea. Taken together, we suggested that the OgUBC1 is involved in ubiquitination process important for cellular response against biotic and abiotic stresses in plants.

  10. Exogenous application of double-stranded RNA molecules from TMV p126 and CP genes confers resistance against TMV in tobacco.

    Science.gov (United States)

    Konakalla, Naga Charan; Kaldis, Athanasios; Berbati, Margarita; Masarapu, Hema; Voloudakis, Andreas E

    2016-10-01

    External application of dsRNA molecules from Tobacco mosaic virus (TMV) p126 and CP genes confers significant resistance against TMV infection. Exogenously applied dsRNA exhibits a rapid systemic trafficking in planta , and it is processed successfully by DICER-like proteins producing small interfering RNAs. RNA interference (RNAi) is a sequence-specific, post-transcriptional gene silencing mechanism, induced by double-stranded RNA (dsRNA), which protects eukaryotic cells against invasive nucleic acids like viruses and transposons. In the present study, we used a non-transgenic strategy to induce RNAi in Nicotiana tabacum cv. Xanthi plants against TMV. DsRNA molecules for the p126 (TMV silencing suppressor) and coat protein (CP) genes were produced by a two-step PCR approach followed by in vitro transcription. The application of TMV p126 dsRNA onto tobacco plants induced greater resistance against TMV infection as compared to CP dsRNA (65 vs. 50 %). This study also reported the fast systemic spread of TMV p126 dsRNA from the treated (local) to non-treated (systemic) leaves beginning from 1 h post-application, confirmed by both conventional and real-time RT-PCR. Furthermore, we employed a stem-loop RT-PCR and confirmed the presence of a putative viral siRNA for up to 9 days in local leaves and up to 6 days in systemic leaves post-application. The approach employed could represent a simple and environmentally safe way for the control of plant viruses in future agriculture.

  11. The mthA mutation conferring low-level resistance to streptomycin enhances antibiotic production in Bacillus subtilis by increasing the S-adenosylmethionine pool size.

    Science.gov (United States)

    Tojo, Shigeo; Kim, Ji-Yun; Tanaka, Yukinori; Inaoka, Takashi; Hiraga, Yoshikazu; Ochi, Kozo

    2014-04-01

    Certain Str(r) mutations that confer low-level streptomycin resistance result in the overproduction of antibiotics by Bacillus subtilis. Using comparative genome-sequencing analysis, we successfully identified this novel mutation in B. subtilis as being located in the mthA gene, which encodes S-adenosylhomocysteine/methylthioadenosine nucleosidase, an enzyme involved in the S-adenosylmethionine (SAM)-recycling pathways. Transformation experiments showed that this mthA mutation was responsible for the acquisition of low-level streptomycin resistance and overproduction of bacilysin. The mthA mutant had an elevated level of intracellular SAM, apparently acquired by arresting SAM-recycling pathways. This increase in the SAM level was directly responsible for bacilysin overproduction, as confirmed by forced expression of the metK gene encoding SAM synthetase. The mthA mutation fully exerted its effect on antibiotic overproduction in the genetic background of rel(+) but not the rel mutant, as demonstrated using an mthA relA double mutant. Strikingly, the mthA mutation activated, at the transcription level, even the dormant ability to produce another antibiotic, neotrehalosadiamine, at concentrations of 150 to 200 μg/ml, an antibiotic not produced (<1 μg/ml) by the wild-type strain. These findings establish the significance of SAM in initiating bacterial secondary metabolism. They also suggest a feasible methodology to enhance or activate antibiotic production, by introducing either the rsmG mutation to Streptomyces or the mthA mutation to eubacteria, since many eubacteria have mthA homologues.

  12. An Enterobacter Plasmid as a New Genetic Background for the Transposon Tn1331

    Science.gov (United States)

    2011-11-25

    presence of ampicillin - and kanamycin- resistant bacteria by antibiotic-selective agar method. One of the resistant colonies was subjected to 16S... bacteria from complex wounds, an Enterobacter species was isolated that harbors antibiotic- resistant plasmids conferring resistance to Escherichia...TOP10 cells (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. The transformed cells were selected on kanamycin- and ampicillin

  13. A cysteine-rich antimicrobial peptide from Pinus monticola (PmAMP1) confers resistance to multiple fungal pathogens in canola (Brassica napus).

    Science.gov (United States)

    Verma, Shiv S; Yajima, William R; Rahman, Muhammad H; Shah, Saleh; Liu, Jun-Jun; Ekramoddoullah, Abul K M; Kav, Nat N V

    2012-05-01

    Canola (Brassica napus), an agriculturally important oilseed crop, can be significantly affected by diseases such as sclerotinia stem rot, blackleg, and alternaria black spot resulting in significant loss of crop productivity and quality. Cysteine-rich antimicrobial peptides isolated from plants have emerged as a potential resource for protection of plants against phytopathogens. Here we report the significance of an antimicrobial peptide, PmAMP1, isolated from western white pine (Pinus monticola), in providing canola with resistance against multiple phytopathogenic fungi. The cDNA encoding PmAMP1 was successfully incorporated into the genome of B. napus, and it's in planta expression conferred greater protection against Alternaria brassicae, Leptosphaeria maculans and Sclerotinia sclerotiorum. In vitro experiments with proteins extracted from transgenic canola expressing Pm-AMP1 demonstrated its inhibitory activity by reducing growth of fungal hyphae. In addition, the in vitro synthesized peptide also inhibited the growth of the fungi. These results demonstrate that generating transgenic crops expressing PmAMP1 may be an effective and versatile method to protect susceptible crops against multiple phytopathogens.

  14. qnrD, a Novel Gene Conferring Transferable Quinolone Resistance in Salmonella enterica Serovar Kentucky and Bovismorbificans Strains of Human Origin

    DEFF Research Database (Denmark)

    Cavaco, Lina; Hasman, Henrik; Xia, S.

    2009-01-01

    In a previous study, four Salmonella isolates from humans in the Henan province of China showed reduced susceptibility to ciprofloxacin (MIC, 0.125 to 0.25 mu g/ml) but were susceptible to nalidixic acid ( MIC, 4 to 8 mu g/ml). All isolates were negative for known qnr genes ( A, B, and S), aac(6......')Ib-cr, and mutations in gyrA and parC. Plasmid DNA was extracted from all four isolates and transformed into Escherichia coli TG1 and DH10B cells by electroporation, and transformants were selected on 0.06 mu g/ml ciprofloxacin containing brain heart infusion agar plates. Resistance to ciprofloxacin...... qnrD, showed 48% similarity to qnrA1, 61% similarity to qnrB1, and 41% similarity to qnrS1. Further subcloning of the qnrD coding region into the constitutively expressed tetA gene of vector pBR322 showed that the gene conferred an increase in the MIC of ciprofloxacin by a factor of 32 ( from an MIC...

  15. Overexpression of rice serotonin N-acetyltransferase 1 in transgenic rice plants confers resistance to cadmium and senescence and increases grain yield.

    Science.gov (United States)

    Lee, Kyungjin; Back, Kyoungwhan

    2017-04-01

    While ectopic overexpression of serotonin N-acetyltransferase (SNAT) in plants has been accomplished using animal SNAT genes, ectopic overexpression of plant SNAT genes in plants has not been investigated. Because the plant SNAT protein differs from that of animals in its subcellular localization and enzyme kinetics, its ectopic overexpression in plants would be expected to give outcomes distinct from those observed from overexpression of animal SNAT genes in transgenic plants. Consistent with our expectations, we found that transgenic rice plants overexpressing rice (Oryza sativa) SNAT1 (OsSNAT1) did not show enhanced seedling growth like that observed in ovine SNAT-overexpressing transgenic rice plants, although both types of plants exhibited increased melatonin levels. OsSNAT1-overexpressing rice plants did show significant resistance to cadmium and senescence stresses relative to wild-type controls. In contrast to tomato, melatonin synthesis in rice seedlings was not induced by selenium and OsSNAT1 transgenic rice plants did not show tolerance to selenium. T 2 homozygous OsSNAT1 transgenic rice plants exhibited increased grain yield due to increased panicle number per plant under paddy field conditions. These benefits conferred by ectopic overexpression of OsSNAT1 had not been observed in transgenic rice plants overexpressing ovine SNAT, suggesting that plant SNAT functions differently from animal SNAT in plants. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  16. Combined phylogeny and neighborhood analysis of the evolution of the ABC transporters conferring multiple drug resistance in hemiascomycete yeasts

    Directory of Open Access Journals (Sweden)

    Goffeau André

    2009-10-01

    Full Text Available Abstract Background Pleiotropic Drug Resistant transporters (PDR are members of the ATP-Binding Cassette (ABC subfamily which export antifungals and other xenobiotics in fungi and plants. This subfamily of transmembrane transporters has nine known members in Saccharomyces cerevisiae. We have analyzed the complex evolution of the pleiotropic drug resistance proteins (Pdrp subfamily where gene duplications and deletions occur independently in individual genomes. This study was carried out on 62 Pdrp from nine hemiascomycetous species, seven of which span 6 of the 14 clades of the Saccharomyces complex while the two others species, Debaryomyces hansenii and Yarrowia lipolytica, are further apart from an evolutive point of view. Results Combined phylogenetic and neighborhood analyses enabled us to identify five Pdrp clusters in the Saccharomyces complex. Three of them comprise orthologs of the Pdrp sensu stricto, Pdr5p, Pdr10p, Pdr12p, Pdr15p, Snq2p and YNR070wp. The evolutive pathway of the orthologs of Snq2 and YNR070w is particularly complex due to a tandem gene array in Eremothecium gossypii, Kluyveromyces lactis and Saccharomyces (Lachancea kluyveri. This pathway and different cases of duplications and deletions were clarified by using a neighborhood analysis based on synteny. For the two distant species, Yarrowia lipolytica and Debaryomyces hansenii, no neighborhood evidence is available for these clusters and many homologs of Pdr5 and Pdr15 are phylogenetically assigned to species-based clusters. Two other clusters comprise the orthologs of the sensu lato Pdrp, Aus1p/Pdr11p and YOL075cp respectively. The evolutionary pathway of these clusters is simpler. Nevertheless, orthologs of these genes are missing in some species. Conclusion Numerous duplications were traced among the Hemiascomycetous Pdrp studied. The role of the Whole Genome Duplication (WGD is sorted out and our analyses confirm the common ancestrality of Pdr5p and Pdr15p. A tandem

  17. Combined phylogeny and neighborhood analysis of the evolution of the ABC transporters conferring multiple drug resistance in hemiascomycete yeasts.

    Science.gov (United States)

    Seret, Marie-Line; Diffels, Julie F; Goffeau, André; Baret, Philippe V

    2009-10-01

    Pleiotropic Drug Resistant transporters (PDR) are members of the ATP-Binding Cassette (ABC) subfamily which export antifungals and other xenobiotics in fungi and plants. This subfamily of transmembrane transporters has nine known members in Saccharomyces cerevisiae. We have analyzed the complex evolution of the pleiotropic drug resistance proteins (Pdrp) subfamily where gene duplications and deletions occur independently in individual genomes. This study was carried out on 62 Pdrp from nine hemiascomycetous species, seven of which span 6 of the 14 clades of the Saccharomyces complex while the two others species, Debaryomyces hansenii and Yarrowia lipolytica, are further apart from an evolutive point of view. Combined phylogenetic and neighborhood analyses enabled us to identify five Pdrp clusters in the Saccharomyces complex. Three of them comprise orthologs of the Pdrp sensu stricto, Pdr5p, Pdr10p, Pdr12p, Pdr15p, Snq2p and YNR070wp. The evolutive pathway of the orthologs of Snq2 and YNR070w is particularly complex due to a tandem gene array in Eremothecium gossypii, Kluyveromyces lactis and Saccharomyces (Lachancea) kluyveri. This pathway and different cases of duplications and deletions were clarified by using a neighborhood analysis based on synteny. For the two distant species, Yarrowia lipolytica and Debaryomyces hansenii, no neighborhood evidence is available for these clusters and many homologs of Pdr5 and Pdr15 are phylogenetically assigned to species-based clusters. Two other clusters comprise the orthologs of the sensu lato Pdrp, Aus1p/Pdr11p and YOL075cp respectively. The evolutionary pathway of these clusters is simpler. Nevertheless, orthologs of these genes are missing in some species. Numerous duplications were traced among the Hemiascomycetous Pdrp studied. The role of the Whole Genome Duplication (WGD) is sorted out and our analyses confirm the common ancestrality of Pdr5p and Pdr15p. A tandem gene array is observed in Eremothecium gossypii. One

  18. vanG element insertions within a conserved chromosomal site conferring vancomycin resistance to Streptococcus agalactiae and Streptococcus anginosus.

    Science.gov (United States)

    Srinivasan, Velusamy; Metcalf, Benjamin J; Knipe, Kristen M; Ouattara, Mahamoudou; McGee, Lesley; Shewmaker, Patricia L; Glennen, Anita; Nichols, Megin; Harris, Carol; Brimmage, Mary; Ostrowsky, Belinda; Park, Connie J; Schrag, Stephanie J; Frace, Michael A; Sammons, Scott A; Beall, Bernard

    2014-07-22

    Three vancomycin-resistant streptococcal strains carrying vanG elements (two invasive Streptococcus agalactiae isolates [GBS-NY and GBS-NM, both serotype II and multilocus sequence type 22] and one Streptococcus anginosus [Sa]) were examined. The 45,585-bp elements found within Sa and GBS-NY were nearly identical (together designated vanG-1) and shared near-identity over an ~15-kb overlap with a previously described vanG element from Enterococcus faecalis. Unexpectedly, vanG-1 shared much less homology with the 49,321-bp vanG-2 element from GBS-NM, with widely different levels (50% to 99%) of sequence identity shared among 44 related open reading frames. Immediately adjacent to both vanG-1 and vanG-2 were 44,670-bp and 44,680-bp integrative conjugative element (ICE)-like sequences, designated ICE-r, that were nearly identical in the two group B streptococcal (GBS) strains. The dual vanG and ICE-r elements from both GBS strains were inserted at the same position, between bases 1328 and 1329, within the identical RNA methyltransferase (rumA) genes. A GenBank search revealed that although most GBS strains contained insertions within this specific site, only sequence type 22 (ST22) GBS strains contained highly related ICE-r derivatives. The vanG-1 element in Sa was also inserted within this position corresponding to its rumA homolog adjacent to an ICE-r derivative. vanG-1 insertions were previously reported within the same relative position in the E. faecalis rumA homolog. An ICE-r sequence perfectly conserved with respect to its counterpart in GBS-NY was apparent within the same site of the rumA homolog of a Streptococcus dysgalactiae subsp. equisimilis strain. Additionally, homologous vanG-like elements within the conserved rumA target site were evident in Roseburia intestinalis. Importance: These three streptococcal strains represent the first known vancomycin-resistant strains of their species. The collective observations made from these strains reveal a specific

  19. Prevalent HLA Class II Alleles in Mexico City Appear to Confer Resistance to the Development of Amebic Liver Abscess.

    Science.gov (United States)

    Hernández, Eric G; Granados, Julio; Partida-Rodríguez, Oswaldo; Valenzuela, Olivia; Rascón, Edgar; Magaña, Ulises; Escamilla-Tilch, Mónica; López-Reyes, Alberto; Nieves-Ramírez, Miriam; González, Enrique; Morán, Patricia; Rojas, Liliana; Valadez, Alicia; Luna, Alexandra; Estrada, Francisco J; Maldonado, Carmen; Ximénez, Cecilia

    2015-01-01

    Amebiasis is an endemic disease and a public health problem throughout Mexico, although the incidence rates of amebic liver abscess (ALA) vary among the geographic regions of the country. Notably, incidence rates are high in the northwestern states (especially Sonora with a rate of 12.57/100,000 inhabitants) compared with the central region (Mexico City with a rate of 0.69/100,000 inhabitants). These data may be related to host genetic factors that are partially responsible for resistance or susceptibility. Therefore, we studied the association of the HLA-DRB1 and HLA-DQB1 alleles with resistance or susceptibility to ALA in two Mexican populations, one each from Mexico City and Sonora. Ninety ALA patients were clinically diagnosed by serology and sonography. Genomic DNA was extracted from peripheral blood mononuclear cells. To establish the genetic identity of both populations, 15 short tandem repeats (STRs) were analyzed with multiplexed PCR, and the allelic frequencies of HLA were studied by PCR-SSO using LUMINEX technology. The allele frequencies obtained were compared to an ethnically matched healthy control group (146 individuals). We observed that both affected populations differed genetically from the control group. We also found interesting trends in the population from Mexico City. HLA-DQB1*02 allele frequencies were higher in ALA patients compared to the control group (0.127 vs 0.047; p= 0.01; pc= NS; OR= 2.9, 95% CI= 1.09-8.3). The less frequent alleles in ALA patients were HLA-DRB1*08 (0.118 vs 0.238 in controls; p= 0.01; pc= NS; OR= 0.42, 95% CI= 0.19-0.87) and HLA-DQB1*04 (0.109 vs 0.214; p= 0.02; pc= NS; OR= 0.40, 95% CI= 0.20-0.94). The haplotype HLA-DRB1*08/-DQB1*04 also demonstrated a protective trend against the development of this disease (0.081 vs. 0.178; p=0.02; pc=NS; OR= 0.40, 95% CI= 0.16-0.93). These trends suggest that the prevalent alleles in the population of Mexico City may be associated with protection against the development of ALA.

  20. Prevalence and molecular characterization of pyrazinamide resistance among multidrug-resistant Mycobacterium tuberculosis isolates from Southern China.

    Science.gov (United States)

    Pang, Yu; Zhu, Damian; Zheng, Huiwen; Shen, Jing; Hu, Yan; Liu, Jie; Zhao, Yanlin

    2017-11-06

    Pyrazinamide (PZA) plays a unique role in the treatment for multidrug-resistant tuberculosis (MDR-TB) in both first- and second-line regimens. The aim of this study was to investigate the prevalence and molecular characterization of PZA resistance among MDR-TB isolates collected in Chongqing municipality. A total of 133 MDR-TB isolates were collected from the smear-positive tuberculosis patients who were registered at local TB dispensaries of Chongqing. PZA susceptibility testing was determined with a Bactec MGIT 960 system. In addition, the genes conferring for PZA resistance were screened by DNA sequencing. Of these 133 MDR-TB isolates, 83 (62.4%) were determined as PZA-resistant by MGIT 960. In addition, streptomycin- (83.1% vs. 56.0%, P < 0.01), ofloxacin- (51.8% vs. 18.0%, P < 0.01), kanamycin- (22.9% vs. 2.0%, P < 0.01), amikacin- (18.1% vs. 2.0%, P = 0.01), capromycin-resistance (12.0% vs. 2.0%, P = 0.05), were more frequently observed among PZA-resistant isolates compared with PZA-susceptible isolates. Sequence analysis revealed that 73 out of 83 (88.0%) MDR strains harbored a mutation located in the pncA gene, including 55 (75.3%, 55/73) of single nucleotide substitutions and 18 (24.7%, 18/73) of frameshift mutation, while no genetic mutation associated with PZA resistance was found in the rpsA gene. The pncA expression of strains harboring substitution from A to G at position -11 in the promoter region of pncA was significantly lower than that of H37Rv (P < 0.01). In conclusion, our data have demonstrated that the analysis of the pncA gene rather than rpsA gene provides rapid and accurate information regarding PZA susceptibility for MDR-TB isolates in Chongqing. In addition, loss of pncA expression caused by promoter mutation confers PZA resistance in MDR-TB isolates.

  1. Prevalence and molecular characterization of pyrazinamide resistance among multidrug-resistant Mycobacterium tuberculosis isolates from Southern China

    Directory of Open Access Journals (Sweden)

    Yu Pang

    2017-11-01

    Full Text Available Abstract Background Pyrazinamide (PZA plays a unique role in the treatment for multidrug-resistant tuberculosis (MDR-TB in both first- and second-line regimens. The aim of this study was to investigate the prevalence and molecular characterization of PZA resistance among MDR-TB isolates collected in Chongqing municipality. Methods A total of 133 MDR-TB isolates were collected from the smear-positive tuberculosis patients who were registered at local TB dispensaries of Chongqing. PZA susceptibility testing was determined with a Bactec MGIT 960 system. In addition, the genes conferring for PZA resistance were screened by DNA sequencing. Results Of these 133 MDR-TB isolates, 83 (62.4% were determined as PZA-resistant by MGIT 960. In addition, streptomycin- (83.1% vs. 56.0%, P < 0.01, ofloxacin- (51.8% vs. 18.0%, P < 0.01, kanamycin- (22.9% vs. 2.0%, P < 0.01, amikacin- (18.1% vs. 2.0%, P = 0.01, capromycin-resistance (12.0% vs. 2.0%, P = 0.05, were more frequently observed among PZA-resistant isolates compared with PZA-susceptible isolates. Sequence analysis revealed that 73 out of 83 (88.0% MDR strains harbored a mutation located in the pncA gene, including 55 (75.3%, 55/73 of single nucleotide substitutions and 18 (24.7%, 18/73 of frameshift mutation, while no genetic mutation associated with PZA resistance was found in the rpsA gene. The pncA expression of strains harboring substitution from A to G at position −11 in the promoter region of pncA was significantly lower than that of H37Rv (P < 0.01. Conclusions In conclusion, our data have demonstrated that the analysis of the pncA gene rather than rpsA gene provides rapid and accurate information regarding PZA susceptibility for MDR-TB isolates in Chongqing. In addition, loss of pncA expression caused by promoter mutation confers PZA resistance in MDR-TB isolates.

  2. Loss of Hepatic Mitochondrial Long-Chain Fatty Acid Oxidation Confers Resistance to Diet-Induced Obesity and Glucose Intolerance.

    Science.gov (United States)

    Lee, Jieun; Choi, Joseph; Selen Alpergin, Ebru S; Zhao, Liang; Hartung, Thomas; Scafidi, Susanna; Riddle, Ryan C; Wolfgang, Michael J

    2017-07-18

    The liver has a large capacity for mitochondrial fatty acid β-oxidation, which is critical for systemic metabolic adaptations such as gluconeogenesis and ketogenesis. To understand the role of hepatic fatty acid oxidation in response to a chronic high-fat diet (HFD), we generated mice with a liver-specific deficiency of mitochondrial long-chain fatty acid β-oxidation (Cpt2 L-/- mice). Paradoxically, Cpt2 L-/- mice were resistant to HFD-induced obesity and glucose intolerance with an absence of liver damage, although they exhibited serum dyslipidemia, hepatic oxidative stress, and systemic carnitine deficiency. Feeding an HFD induced hepatokines in mice, with a loss of hepatic fatty acid oxidation that enhanced systemic energy expenditure and suppressed adiposity. Additionally, the suppression in hepatic gluconeogenesis was sufficient to improve HFD-induced glucose intolerance. These data show that inhibiting hepatic fatty acid oxidation results in a systemic hormetic response that protects mice from HFD-induced obesity and glucose intolerance. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  3. Expression of a cystatin transgene can confer resistance to root lesion nematodes in Lilium longiflorum cv. 'Nellie White'.

    Science.gov (United States)

    Vieira, Paulo; Wantoch, Sarah; Lilley, Catherine J; Chitwood, David J; Atkinson, Howard J; Kamo, Kathryn

    2015-06-01

    Lilium longiflorum cv. 'Nellie White' assumes a great economic importance as cut flowers, being one of the most valuable species (annual pot plants value above $20,000,000) in terms of wholesales in the US. The root lesion nematode Pratylenchus penetrans (RLN) constitutes one of the main pests for lily producers due to the significant root damage it causes. Our efforts have focused on the generation of soybean hairy roots (as a transient test model) and stable transgenic lilies overexpressing a modified rice cystatin (Oc-IΔD86) transgene and challenged with root lesion nematodes. Lily transformation was achieved by gene gun co-bombardment using both a pBluescript-based vector containing the cystatin gene and pDM307 that contains a bar gene for phosphinothricin selection. Both soybean hairy roots and lilies overexpressing the OcIΔD86 transgene exhibited enhanced resistance to RLN infection by means of nematode reduction up to 75 ± 5% on the total number of nematodes. In addition, lily plants overexpressing OcIΔD86 displayed an increase of plant mass and better growth performance in comparison to wild-type plants, thereby demonstrating an alternative strategy for increasing the yield and reducing nematode damage to this important floral crop.

  4. Loss of Hepatic Mitochondrial Long-Chain Fatty Acid Oxidation Confers Resistance to Diet-Induced Obesity and Glucose Intolerance

    Directory of Open Access Journals (Sweden)

    Jieun Lee

    2017-07-01

    Full Text Available The liver has a large capacity for mitochondrial fatty acid β-oxidation, which is critical for systemic metabolic adaptations such as gluconeogenesis and ketogenesis. To understand the role of hepatic fatty acid oxidation in response to a chronic high-fat diet (HFD, we generated mice with a liver-specific deficiency of mitochondrial long-chain fatty acid β-oxidation (Cpt2L−/− mice. Paradoxically, Cpt2L−/− mice were resistant to HFD-induced obesity and glucose intolerance with an absence of liver damage, although they exhibited serum dyslipidemia, hepatic oxidative stress, and systemic carnitine deficiency. Feeding an HFD induced hepatokines in mice, with a loss of hepatic fatty acid oxidation that enhanced systemic energy expenditure and suppressed adiposity. Additionally, the suppression in hepatic gluconeogenesis was sufficient to improve HFD-induced glucose intolerance. These data show that inhibiting hepatic fatty acid oxidation results in a systemic hormetic response that protects mice from HFD-induced obesity and glucose intolerance.

  5. Targeting chitinase gene of Helicoverpa armigera by host-induced RNA interference confers insect resistance in tobacco and tomato.

    Science.gov (United States)

    Mamta; Reddy, K R K; Rajam, M V

    2016-02-01

    Helicoverpa armigera Hübner (Lepidoptera: Noctuidae) is a devastating agricultural insect pest with broad spectrum of host range, causing million dollars crop loss annually. Limitations in the present conventional and transgenic approaches have made it crucial to develop sustainable and environmental friendly methods for crop improvement. In the present study, host-induced RNA interference (HI-RNAi) approach was used to develop H. armigera resistant tobacco and tomato plants. Chitinase (HaCHI) gene, critically required for insect molting and metamorphosis was selected as a potential target. Hair-pin RNAi construct was prepared from the conserved off-target free partial HaCHI gene sequence and was used to generate several HaCHI-RNAi tobacco and tomato plants. Northern hybridization confirmed the production of HaCHI gene-specific siRNAs in HaCHI-RNAi tobacco and tomato lines. Continuous feeding on leaves of RNAi lines drastically reduced the target gene transcripts and consequently, affected the overall growth and survival of H. armigera. Various developmental deformities were also manifested in H. armigera larvae after feeding on the leaves of RNAi lines. These results demonstrated the role of chitinase in insect development and potential of HI-RNAi for effective management of H. armigera.

  6. Disease resistance conferred by the expression of a gene encoding a synthetic peptide in transgenic cotton (Gossypium hirsutum L.) plants.

    Science.gov (United States)

    Rajasekaran, Kanniah; Cary, Jeffrey W; Jaynes, Jesse M; Cleveland, Thomas E

    2005-11-01

    Fertile, transgenic cotton plants expressing the synthetic antimicrobial peptide, D4E1, were produced through Agrobacterium-mediated transformation. PCR products and Southern blots confirmed integration of the D4E1 gene, while RT-PCR of cotton RNA confirmed the presence of D4E1 transcripts. In vitro assays with crude leaf protein extracts from T0 and T1 plants confirmed that D4E1 was expressed at sufficient levels to inhibit the growth of Fusarium verticillioides and Verticillium dahliae compared to extracts from negative control plants transformed with pBI-d35S(Omega)-uidA-nos (CGUS). Although in vitro assays did not show control of pre-germinated spores of Aspergillus flavus, bioassays with cotton seeds in situ or in planta, inoculated with a GFP-expressing A. flavus, indicated that the transgenic cotton seeds inhibited extensive colonization and spread by the fungus in cotyledons and seed coats. In planta assays with the fungal pathogen, Thielaviopsis basicola, which causes black root rot in cotton, showed typical symptoms such as black discoloration and constriction on hypocotyls, reduced branching of roots in CGUS negative control T1 seedlings, while transgenic T1 seedlings showed a significant reduction in disease symptoms and increased seedling fresh weight, demonstrating tolerance to the fungal pathogen. Significant advantages of synthetic peptides in developing transgenic crop plants that are resistant to diseases and mycotoxin-causing fungal pathogens are highlighted in this report.

  7. Transgenic Cotton Plants Expressing the HaHR3 Gene Conferred Enhanced Resistance to Helicoverpa armigera and Improved Cotton Yield.

    Science.gov (United States)

    Han, Qiang; Wang, Zhenzhen; He, Yunxin; Xiong, Yehui; Lv, Shun; Li, Shupeng; Zhang, Zhigang; Qiu, Dewen; Zeng, Hongmei

    2017-08-30

    RNA interference (RNAi) has been developed as an efficient technology. RNAi insect-resistant transgenic plants expressing double-stranded RNA (dsRNA) that is ingested into insects to silence target genes can affect the viability of these pests or even lead to their death. HaHR3 , a molt-regulating transcription factor gene, was previously selected as a target expressed in bacteria and tobacco plants to control Helicoverpa armigera by RNAi technology. In this work, we selected the dsRNA- HaHR3 fragment to silence HaHR3 in cotton bollworm for plant mediated-RNAi research. A total of 19 transgenic cotton lines expressing HaHR3 were successfully cultivated, and seven generated lines were used to perform feeding bioassays. Transgenic cotton plants expressing ds HaHR3 were shown to induce high larval mortality and deformities of pupation and adult eclosion when used to feed the newly hatched larvae, and 3rd and 5th instar larvae of H. armigera . Moreover, HaHR3 transgenic cotton also demonstrated an improved cotton yield when compared with controls.

  8. Caprolactam-silica network, a strong potentiator of the antimicrobial activity of kanamycin against gram-positive and gram-negative bacterial strains.

    Science.gov (United States)

    Voicu, Georgeta; Grumezescu, Valentina; Andronescu, Ecaterina; Grumezescu, Alexandru Mihai; Ficai, Anton; Ficai, Denisa; Ghitulica, Cristina Daniela; Gheorghe, Irina; Chifiriuc, Mariana Carmen

    2013-03-25

    Here, we report the fabrication of a novel ε-caprolactam-silica (ε-SiO2) network and assessed its biocompatibility and ability to improve the antimicrobial activity of kanamycin. The results of the quantitative antimicrobial assay demonstrate that the obtained ε-SiO2 network has efficiently improved the kanamycin activity on Staphylococcus aureus ATCC 25923 and Escherichia coli ATCC 25922 strains, with a significant decrease of the minimum inhibitory concentration. The ε-SiO2 network could be feasibly obtained and represents an alternative for the design of new antibiotic drug carriers or delivery systems to control bacterial infections. Copyright © 2013 Elsevier B.V. All rights reserved.

  9. Role of the two component signal transduction system CpxAR in conferring cefepime and chloramphenicol resistance in Klebsiella pneumoniae NTUH-K2044.

    Directory of Open Access Journals (Sweden)

    Vijaya Bharathi Srinivasan

    Full Text Available BACKGROUND: Klebsiella pneumoniae is a gram-negative, non-motile, facultative anaerobe belonging to the Enterobacteriaceae family of the γ-Proteobacteria class in the phylum Proteobacteria. Multidrug resistant K. pneumoniae have caused major therapeutic problems worldwide due to emergence of extended-spectrum β-lactamase producing strains. Two-component systems serve as a basic stimulus-response coupling mechanism to allow organisms to sense and respond to changes in many different environmental conditions including antibiotic stress. PRINCIPAL FINDINGS: In the present study, we investigated the role of an uncharacterized cpxAR operon in bacterial physiology and antimicrobial resistance by generating isogenic mutant (ΔcpxAR deficient in the CpxA/CpxR component derived from the hyper mucoidal K1 strain K. pneumoniae NTUH-K2044. The behaviour of ΔcpxAR was determined under hostile conditions, reproducing stresses encountered in the gastrointestinal environment and deletion resulted in higher sensitivity to bile, osmotic and acid stresses. The ΔcpxAR was more susceptible to β-lactams and chloramphenicol than the wild-type strain, and complementation restored the altered phenotypes. The relative change in expression of acrB, acrD, eefB efflux genes were decreased in cpxAR mutant as evidenced by qRT-PCR. Comparison of outer membrane protein profiles indicated a conspicuous difference in the knock out background. Gel shift assays demonstrated direct binding of CpxR(KP to promoter region of ompC(KP in a concentration dependent manner. CONCLUSIONS AND SIGNIFICANCE: The Cpx envelope stress response system is known to be activated by alterations in pH, membrane composition and misfolded proteins, and this systematic investigation reveals its direct involvement in conferring antimicrobial resistance against clinically significant antibiotics for the very first time. Overall results displayed in this report reflect the pleiotropic role of the Cpx

  10. Single mutation confers vanadate resistance to the plasma membrane H+-ATPase from the yeast Schizosaccharomyces pombe

    International Nuclear Information System (INIS)

    Ulaszewski, S.; Van Herck, J.C.; Dufour, J.P.; Kulpa, J.; Nieuwenhuis, B.; Goffeau, A.

    1987-01-01

    A single-gene nuclear mutant has been selected from the yeast Schizosaccharomyces pombe for growth resistance to Dio-9, a plasma membrane H+-ATPase inhibitor. From this mutant, called pma1, an ATPase activity has been purified. It contains a Mr = 100,000 major polypeptide which is phosphorylated by [gamma- 32 P] ATP. Proton pumping is not impaired since the isolated mutant ATPase is able, in reconstituted proteoliposomes, to quench the fluorescence of the delta pH probe 9-amino-6-chloro-2-methoxy acridine. The isolated mutant ATPase is sensitive to Dio-9 as well as to seven other plasma membrane H+-ATPase inhibitors. The mutant H+-ATPase activity tested in vitro is, however, insensitive to vanadate. Its Km for MgATP is modified and its ATPase specific activity is decreased. The pma1 mutation decreases the rate of extracellular acidification induced by glucose when cells are incubated at pH 4.5 under nongrowing conditions. During growth, the intracellular mutant pH is more acid than the wild type one. The derepression by ammonia starvation of methionine transport is decreased in the mutant. The growth rate of pma1 mutants is reduced in minimal medium compared to rich medium, especially when combined to an auxotrophic mutation. It is concluded that the H+-ATPase activity from yeast plasma membranes controls the intracellular pH as well as the derepression of amino acid, purine, and pyrimidine uptakes. The pma1 mutation modifies several transport properties of the cells including those responsible for the uptake of Dio-9 and other inhibitors

  11. Rhodococcus equi's extreme resistance to hydrogen peroxide is mainly conferred by one of its four catalase genes.

    Science.gov (United States)

    Bidaud, Pauline; Hébert, Laurent; Barbey, Corinne; Appourchaux, Anne-Cécile; Torelli, Riccardo; Sanguinetti, Maurizio; Laugier, Claire; Petry, Sandrine

    2012-01-01

    Rhodococcus equi is one of the most widespread causes of disease in foals aged from 1 to 6 months. R. equi possesses antioxidant defense mechanisms to protect it from reactive oxygen metabolites such as hydrogen peroxide (H(2)O(2)) generated during the respiratory burst of phagocytic cells. These defense mechanisms include enzymes such as catalase, which detoxify hydrogen peroxide. Recently, an analysis of the R. equi 103 genome sequence revealed the presence of four potential catalase genes. We first constructed ΔkatA-, ΔkatB-, ΔkatC-and ΔkatD-deficient mutants to study the ability of R. equi to survive exposure to H(2)O(2)in vitro and within mouse peritoneal macrophages. Results showed that ΔkatA and, to a lesser extent ΔkatC, were affected by 80 mM H(2)O(2). Moreover, katA deletion seems to significantly affect the ability of R. equi to survive within murine macrophages. We finally investigated the expression of the four catalases in response to H(2)O(2) assays with a real time PCR technique. Results showed that katA is overexpressed 367.9 times (± 122.6) in response to exposure to 50 mM of H(2)O(2) added in the stationary phase, and 3.11 times (± 0.59) when treatment was administered in the exponential phase. In untreated bacteria, katB, katC and katD were overexpressed from 4.3 to 17.5 times in the stationary compared to the exponential phase. Taken together, our results show that KatA is the major catalase involved in the extreme H(2)O(2) resistance capability of R. equi.

  12. Elimination of Gut Microbes with Antibiotics Confers Resistance to Bacillus thuringiensis Toxin Proteins in Helicoverpa armigera (Hubner).

    Science.gov (United States)

    Visweshwar, R; Sharma, H C; Akbar, S M D; Sreeramulu, K

    2015-12-01

    Helicoverpa armigera is one of the most important pests worldwide. Transgenic crops with toxin genes from Bacillus thuringiensis (Bt) have been deployed on a large scale to control this pest. The insecticidal activity of Bt is probably influenced by the insect midgut microbes, which vary across crop hosts and locations. Therefore, we examined the role of gut microbes in pathogenicity of Bt toxins in the H. armigera. Antibiotic cocktail was used for the complete elimination of the H. armigera gut microbes. Activated Cry1Ac, Bt formulation, and transgenic cotton resulted in larval weight loss and increase in mortality, but pretreatment of larvae with antibiotic cocktail significantly decreased larval mortality and increased the larval weight gain. Activated Cry1Ac and Bt formulation inhibited the activity of proteases in midgut of H. armigera larvae but showed no such effect in the larvae pretreated with antibiotic cocktail. Five protease bands in activated Cry1Ac and two in Bt formulation-treated larvae were inhibited but no such effect in the larvae pretreated with antibiotic cocktail. Cry1Ac protein was detected in Bt/Cry1Ac protoxin-fed larval gut extract in the absence of antibiotic cocktail, but fewer in larvae pretreated with antibiotic cocktail. The activity of antioxidant enzymes and aminopeptidases increased in larvae fed on Bt toxin, but there was no significant increase in antioxidant enzymes in larvae reared on toxin protein in combination with antibiotic cocktail. The results suggest that gut microbes exercise a significant influence on the toxicity of Cry1Ac and Bt formulation in H. armigera larvae. The implications of these results have been discussed in relation to development of insect resistance to Bt transgenic crops deployed for pest management.

  13. Lambda-Cyhalothrin Resistance in the Lady Beetle Eriopis connexa (Coleoptera: Coccinellidae) Confers Tolerance to Other Pyrethroids.

    Science.gov (United States)

    Torres, J B; Rodrigues, A R S; Barros, E M; Santos, D S

    2015-02-01

    Pyrethroid insecticides are widely recommended to control insect defoliators but lack efficacy against most aphid species. Thus, conserving aphid predators such as the lady beetle Eriopis connexa (Germar) is important to pest management in crop ecosystems that require pyrethroid sprays. In a greenhouse, early fourth-instar larvae and 5-day-old adults from susceptible (S) and resistant (R) E. connexa populations were caged on lambda-cyhalothrin-treated cotton plants, after which survival and egg production (for those caged at adult stage) were assessed. In the laboratory, similar groups were subjected to dried residues and topical treatment with one of eight pyrethroids (alpha-cypermethrin, bifenthrin, deltamethrin, esfenvalerate, fenpropathrin, permethrin, zeta-cypermethrin, and lambda-cyhalothrin), the organophosphate methidathion, or water and wetting agent. After caging on treated cotton terminals, 66% of the R-population larvae survived to adulthood, compared with 2% of those from the S-population. At 12 d after caging at adult stage under the same conditions, 64% of the females from the R-population survived and laid eggs, compared with 100% mortality and no oviposition for the S-females. In trials involving dried insecticide residues, gain in survival based on the survival difference (percentage for R-population minus percentage for S-population) across all tested pyrethroids varied from 3 to 63% for larvae and from 3 to 70% for adults. In trials involving topical sprays of the tested pyrethroids, survival differences ranged from 36 to 96% for larvae and from 21 to 82% for adults. Fenpropathrin and bifenthrin were the least and most toxic, respectively. © The Authors 2015. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  14. Antibiotic Resistance in Staphylococcus aureus and Coagulase Negative Staphylococci Isolated from Goats with Subclinical Mastitis

    Directory of Open Access Journals (Sweden)

    Salvatore Virdis

    2010-01-01

    Full Text Available Antimicrobial resistance patterns and gene coding for methicillin resistance (mecA were determined in 25 S. aureus and 75 Coagulase Negative Staphylococci (CNS strains isolates from half-udder milk samples collected from goats with subclinical mastitis. Fourteen (56.0% S. aureus and thirty-one (41.3% CNS isolates were resistant to one or more antimicrobial agents. S. aureus showed the highest resistance rate against kanamycin (28.0%, oxytetracycline (16.0%, and ampicillin (12.0%. The CNS tested were more frequently resistant to ampicillin (36.0% and kanamycin (6.7%. Multiple antimicrobial resistance was observed in eight isolates, and one Staphylococcus epidermidis was found to be resistant to six antibiotics. The mecA gene was not found in any of the tested isolates. Single resistance against β-lactamics or aminoglicosides is the most common trait observed while multiresistance is less frequent.

  15. Conference summaries

    International Nuclear Information System (INIS)

    1991-01-01

    This volume contains conference summaries for the 31. annual conference of the Canadian Nuclear Association and the 12. annual conference of the Canadian Nuclear Society. Topics of discussion include: reactor physics; thermalhydraulics; industrial irradiation; computer applications; fuel channel analysis; small reactors; severe accidents; fuel behaviour under accident conditions; reactor components, safety related computer software; nuclear fuel management; fuel behaviour and performance; reactor safety; reactor engineering; nuclear waste management; and, uranium mining and processing

  16. Personalized Cancer Care Conference

    OpenAIRE

    Zänker, Kurt S.; Mihich, Enrico; Huber, Hans-Peter; Borresen-Dale, Anne-Lise

    2013-01-01

    The Oslo University Hospital (Norway), the K.G. Jebsen Centre for Breast Cancer Research (Norway), The Radiumhospital Foundation (Norway) and the Fritz-Bender-Foundation (Germany) designed under the conference chairmen (E. Mihich, K.S. Zänker, A.L. Borresen-Dale) and advisory committee (A. Borg, Z. Szallasi, O. Kallioniemi, H.P. Huber) a program at the cutting edge of “PERSONALIZED CANCER CARE: Risk prediction, early diagnosis, progression and therapy resistance.” The conference was held in O...

  17. MCF7/LCC9: an antiestrogen-resistant MCF-7 variant in which acquired resistance to the steroidal antiestrogen ICI 182,780 confers an early cross-resistance to the nonsteroidal antiestrogen tamoxifen

    DEFF Research Database (Denmark)

    Brünner, N; Boysen, B; Jirus, S

    1997-01-01

    Acquired resistance to antiestrogens is a major problem in the clinical management of initially endocrine responsive metastatic breast cancer. We have shown previously that estrogen-independent and -responsive MCF7/LCC1 human breast cancer cells selected for resistance to the triphenylethylene...... tamoxifen produce a variant (MCF7/LCC2) that retains sensitivity to the steroidal antiestrogen ICI 182,780 (N. Brunner et al., Cancer Res., 53: 3229-3232, 1993). We have now applied stepwise selections in vitro from 10 pM to 1 microM ICI 182,780 against MCF7/LCC1 and obtained a stable ICI 182,780-resistant...... variant designated MCF7/LCC9. In contrast to 4-hydroxytamoxifen-selected MCF7/LCC2 cells, MCF7/LCC9 cells exhibit full cross-resistance to tamoxifen, despite never having been exposed to this drug. Significantly, tamoxifen cross-resistance arose early in the selection, appearing following selection...

  18. Loss of expression of a new member of the DNAJ protein family confers resistance to chemotherapeutic agents used in the treatment of ovarian cancer.

    Science.gov (United States)

    Shridhar, V; Bible, K C; Staub, J; Avula, R; Lee, Y K; Kalli, K; Huang, H; Hartmann, L C; Kaufmann, S H; Smith, D I

    2001-05-15

    Differential display-PCR between ovarian tumor cell lines and short-term cultures of normal ovarian epithelial cell brushings was used to isolate a differentially expressed transcript and its corresponding gene. The gene, which mapped to 13q14.1, has partial homology in the DNAJ domain to a number of proteins with a similar domain and was designated as methylation-controlled J protein (MCJ). MCJ has the highest similarity to a functionally undefined protein from Caenorhabditis elegans. MCJ is expressed as a 1.2-kb transcript in several adult tissues, with testis showing the highest level of expression. Expression of MCJ was absent in three of seven ovarian cancer cell lines. Similarly, expression analysis using semiquantitative reverse transcription-PCR indicated that 12 of 18 primary ovarian tumors examined had either a complete absence or lower levels of expression of this gene. 5-Aza-2'-deoxycytidine treatment of the OV202 cell line induced MCJ expression in a dose-dependent manner, implicating methylation in this induction. Loss of heterozygosity and methylation-specific PCR analysis revealed that the loss of MCJ expression in primary tumors and cell lines was attributable to deletion of one allele and methylation of the other. To assess the potential functional significance of MCJ down-regulation, the sensitivity of parental (MCJ-nonexpressing) and MCJ-transfected OV167 cells to antineoplastic agents was evaluated. MCJ expression was associated with enhanced sensitivity to paclitaxel, topotecan, and cisplatin, suggesting that MCJ loss may play a role in de novo chemoresistance in ovarian carcinoma. These observations raise the possibility that MCJ loss may: (a) have potential prognostic significance in ovarian cancer; and (b) contribute to the malignant phenotype by conferring resistance to the most commonly used chemotherapeutic agents for ovarian cancer.

  19. Incidence and characterisation of methicillin-resistant Staphylococcus aureus (MRSA from nasal colonisation in participants attending a cattle veterinary conference in the UK.

    Directory of Open Access Journals (Sweden)

    Gavin K Paterson

    Full Text Available We sought to determine the prevalence of nasal colonisation with methicillin-resistant Staphylococcus aureus among cattle veterinarians in the UK. There was particular interest in examining the frequency of colonisation with MRSA harbouring mecC, as strains with this mecA homologue were originally identified in bovine milk and may represent a zoonotic risk to those in contact with dairy livestock. Three hundred and seven delegates at the British Cattle Veterinarian Association (BCVA Congress 2011 in Southport, UK were screening for nasal colonisation with MRSA. Isolates were characterised by whole genome sequencing and antimicrobial susceptibility testing. Eight out of three hundred and seven delegates (2.6% were positive for nasal colonisation with MRSA. All strains were positive for mecA and none possessed mecC. The time since a delegate's last visit to a farm was significantly shorter in the MRSA-positive group than in MRSA-negative counterparts. BCVA delegates have an increased risk of MRSA colonisation compared to the general population but their frequency of colonisation is lower than that reported from other types of veterinarian conference, and from that seen in human healthcare workers. The results indicate that recent visitation to a farm is a risk factor for MRSA colonisation and that mecC-MRSA are rare among BCVA delegates (<1% based on sample size. Contact with livestock, including dairy cattle, may still be a risk factor for human colonisation with mecC-MRSA but occurs at a rate below the lower limit of detection available in this study.

  20. Functional Screening of Antibiotic Resistance Genes from a Representative Metagenomic Library of Food Fermenting Microbiota

    Directory of Open Access Journals (Sweden)

    Chiara Devirgiliis

    2014-01-01

    Full Text Available Lactic acid bacteria (LAB represent the predominant microbiota in fermented foods. Foodborne LAB have received increasing attention as potential reservoir of antibiotic resistance (AR determinants, which may be horizontally transferred to opportunistic pathogens. We have previously reported isolation of AR LAB from the raw ingredients of a fermented cheese, while AR genes could be detected in the final, marketed product only by PCR amplification, thus pointing at the need for more sensitive microbial isolation techniques. We turned therefore to construction of a metagenomic library containing microbial DNA extracted directly from the food matrix. To maximize yield and purity and to ensure that genomic complexity of the library was representative of the original bacterial population, we defined a suitable protocol for total DNA extraction from cheese which can also be applied to other lipid-rich foods. Functional library screening on different antibiotics allowed recovery of ampicillin and kanamycin resistant clones originating from Streptococcus salivarius subsp. thermophilus and Lactobacillus helveticus genomes. We report molecular characterization of the cloned inserts, which were fully sequenced and shown to confer AR phenotype to recipient bacteria. We also show that metagenomics can be applied to food microbiota to identify underrepresented species carrying specific genes of interest.

  1. MCF7/LCC9: an antiestrogen-resistant MCF-7 variant in which acquired resistance to the steroidal antiestrogen ICI 182,780 confers an early cross-resistance to the nonsteroidal antiestrogen tamoxifen

    DEFF Research Database (Denmark)

    Brünner, N; Boysen, B; Jirus, S

    1997-01-01

    variant designated MCF7/LCC9. In contrast to 4-hydroxytamoxifen-selected MCF7/LCC2 cells, MCF7/LCC9 cells exhibit full cross-resistance to tamoxifen, despite never having been exposed to this drug. Significantly, tamoxifen cross-resistance arose early in the selection, appearing following selection...... growth of MCF7/LCC9 tumors is not affected by treatment with ICI 182,780. Although there is some evidence of tamoxifen stimulation of tumor growth, this did not reach statistical significance. If this pattern of cross-resistance occurs in some breast cancer patients, administering triphenylethylene...

  2. Conference summaries

    International Nuclear Information System (INIS)

    1986-01-01

    This volume contains conference summaries of the international conference on radioactive waste management of the Canadian Nuclear Society. Topics of discussion include: storage and disposal; hydrogeology and geochemistry; transportation; buffers and backfill; public attitudes; tailings; site investigations and geomechanics; concrete; economics; licensing; matrix materials and container design; durability of fuel; biosphere modelling; radioactive waste processing; and, future options

  3. Antimicrobial resistances and virulence markers in Methicillin-resistant Staphylococcus aureus from broiler and turkey: A molecular view from farm to fork.

    Science.gov (United States)

    Kraushaar, Britta; Ballhausen, Britta; Leeser, Daniel; Tenhagen, Bernd-Alois; Käsbohrer, Annemarie; Fetsch, Alexandra

    2017-02-01

    Little is known about the characteristics of MRSA occurring along the broiler and turkey production chains. The aim of this study was to characterise and compare MRSA of turkey and broiler origin sampled on different production levels using a DNA microarray and antimicrobial susceptibility testing. Main differences could be observed in the prevalence of the resistance genes erm(C), aacA-aphD and tet(K) and the number of non-wild type strains with minimum inhibitory concentration values (MICs) above the epidemiological cut-off values (ECOFFs) for gentamicin and kanamycin. Overall, MRSA with non-wild type phenotype for the macrolide-lincosamide-streptogramin group, tetracycline, and trimethoprim were found in more than 70% of poultry isolates. Non-wild type isolates carrying the qacC gene conferring resistance to quaternary ammonium compound disinfectants were found at all production stages in similar frequencies. Regarding the presence of enterotoxin genes in poultry-derived MRSA the enterotoxin gene cluster (egc) was only found in Non-CC (clonal complex) 398 strains. Three CC398 strains harboured the genes sed (from turkey at slaughter and broiler meat) and sea-N315 (from a chicken carcass). One Non-CC398 strain isolated from turkey meat was found positive for the seb gene and also enterotoxin production. Similarity analysis based on selected resistance and virulence markers revealed a high clonality among Non-CC398 isolates. Isolates of the same clonal complex clustered together according to their common virulence and resistance profiles. Strains of CC9 were grouped in two subclusters due to different resistance genes. Our results underline, that there are other poultry associated clones of MRSA (mainly CC9 and CC5) besides the predominant CC398 which are similar in both poultry species. Copyright © 2016 Elsevier B.V. All rights reserved.

  4. Chronological Change of Resistance to β-Lactams in Salmonella enterica serovar Infantis Isolated from Broilers in Japan.

    Science.gov (United States)

    Chuma, Takehisa; Miyasako, Daisuke; Dahshan, Hesham; Takayama, Tomoko; Nakamoto, Yuko; Shahada, Francis; Akiba, Masato; Okamoto, Karoku

    2013-01-01

    Epidemiologic surveillance study was conducted in southern Japan to determine the antimicrobial resistance phenotypes and characterize the β-lactamase genes and the plasmids harboring these genes in Salmonella enterica serovar Infantis (S. Infantis) isolates from broilers. Between January, 2007 and December, 2008, a total of 1,472 fecal samples were collected and examined at the Laboratory of Veterinary Public Health, Kagoshima University, Japan. In 93 (6.3%) isolates recovered, 33 (35.5%) isolates showed resistance to cefotaxime, an extended-spectrum cephalosporin (ESC), conferred by TEM-20, TEM-52 and CTX-M-25 extended-spectrum β-lactamases (ESBLs). In addition to ESC-resistance, eight (8.6%) isolates exhibited resistance to cefoxitin mediated by CMY-2 AmpC β-lactamase. Plasmid analysis and polymerase chain reaction replicon typing revealed the bla TEM-20 and bla CMY-2 genes were associated with IncP plasmids, bla TEM-52 was linked with a non-typable plasmid and bla CTX-M-25 was carried by an IncA/C plasmid. Non-β-lactam resistance to streptomycin, sulfamethoxazole, and oxytetracycline encoded by the aadA1, sul1, and tet(A) genes, respectively, was found in 86 (92.5%) isolates. Resistance to kanamycin and ofloxacin was exhibited in 12 (12.9%) and 11 (11.8%) isolates, respectively, the former was mediated by aphA1-Iab. These data indicate that S. Infantis isolates producing ESBLs and AmpC β-lactamase have spread among broiler farms in Japan. These data demonstrated that the incidence of ESC-resistant S. Infantis carrying bla TEM-52 remarkably increased and S. Infantis strains harboring bla CMY-2, bla TEM-20, or bla CTX-M-25 genes emerged from broilers in Japan for the first time in 2007 and 2008.

  5. Chronological Change of Resistance to β-Lactams in Salmonella enterica serovar Infantis Isolated from Broilers in Japan

    Directory of Open Access Journals (Sweden)

    Takehisa eChuma

    2013-05-01

    Full Text Available Epidemiologic surveillance study was conducted in southern Japan to determine the antimicrobial resistance phenotypes and characterize the β-lactamase genes and the plasmids harboring these genes in Salmonella enterica serovar Infantis (S. Infantis isolates from broilers. Between January, 2007 and December, 2008, a total of 1,472 fecal samples were collected and examined at the Laboratory of Veterinary Public Health, Kagoshima University, Japan. In 93 (6.3% isolates recovered, 33 (35.5% isolates showed resistance to cefotaxime, an extended-spectrum cephalosporin (ESC, conferred by TEM-20, TEM-52 and CTX-M-25 extended-spectrum β-lactamases (ESBLs. In addition to ESC-resistance, eight (8.6% isolates exhibited resistance to cefoxitin mediated by CMY-2 AmpC β-lactamase. Plasmid analysis and polymerase chain reaction replicon typing revealed the blaTEM-20 and blaCMY-2 genes were associated with IncP plasmids, blaTEM-52 was linked with a non-typable plasmid and blaCTX-M-25 was carried by an IncA/C plasmid. Non- β-lactam resistance to streptomycin, sulfamethoxazole and oxytetracycline encoded by the aadA1, sul1 and tet(A genes, respectively, was found in 86 (92.5% isolates. Resistance to kanamycin and ofloxacin was exhibited in 12 (12.9% and 11 (11.8% isolates, respectively, the former was mediated by aphA1-Iab. These data indicate that S. Infantis isolates producing ESBLs and AmpC β-lactamase have spread among broiler farms in Japan. These data demonstrated that the incidence of ESC-resistant S. Infantis carrying blaTEM-52 remarkably increased and S. Infantis strains harboring blaCMY-2, blaTEM-20 or blaCTX-M-25 genes emerged from broilers in Japan for the first time in 2007 and 2008.

  6. Conference summaries

    International Nuclear Information System (INIS)

    1988-01-01

    This volume contains conference summaries of the 28. annual conference of the Canadian Nuclear Association, and the 9. annual conference of the Canadian Nuclear Society. Topics of discussion include: power reactors; fuel cycles; nuclear power and public understanding; future trends; applications of nuclear technology; CANDU reactors; operational enhancements; design of small reactors; accident behaviour in fuel channels; fuel storage and waste management; reactor commissioning/decommissioning; nuclear safety experiments and modelling; the next generation reactors; advances in nuclear engineering education in Canada; safety of small reactors; current position and improvements of fuel channels; current issues in nuclear safety; and radiation applications - medical and industrial

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

    Indian Academy of Sciences (India)

    Madhu urs

    kanamycin) and hormones (1 mg/l zeatin and 0.1 mg/l indole acetic acid [IAA]) in a growth chamber. Shoots regenerating from the explants were subsequently transferred to MS ..... vegetables; Ann. Appl. Biol. 110 661–681. Varma A, Jain R K and Bhat A I 2002 Virus resistant transgenic plants for environmentally safe ...

  8. Characterization of Staphylococcus aureus Strains Isolated from Czech Cystic Fibrosis Patients: High Rate of Ribosomal Mutation Conferring Resistance to MLS(B) Antibiotics as a Result of Long-Term and Low-Dose Azithromycin Treatment.

    Science.gov (United States)

    Tkadlec, Jan; Vařeková, Eva; Pantůček, Roman; Doškař, Jiří; Růžičková, Vladislava; Botka, Tibor; Fila, Libor; Melter, Oto

    2015-08-01

    Staphylococcus aureus is one of the most frequent pathogens infecting the respiratory tract of patients with cystic fibrosis (CF). This study was the first to examine S. aureus isolates from CF patients in the Czech Republic. Among 100 S. aureus isolates from 92 of 107 observed patients, we found a high prevalence of resistance to macrolide-lincosamide-streptogramin B (MLS(B)) antibiotics (56%). More than half of the resistant strains (29 of 56) carried a mutation in the MLS(B) target site. The emergence of MLS(B) resistance and mutations conferring resistance to MLS(B) antibiotics was associated with azithromycin treatment (p=0.000000184 and p=0.000681, respectively). Methicillin resistance was only detected in 3% of isolates and the rate of resistance to other antibiotics did not exceed 12%. The prevalence of small-colony variant (SCV) strains was relatively low (9%) and eight of nine isolates with the SCV phenotype were thymidine dependent. The study population of S. aureus was heterogeneous in structure and both the most prevalent community-associated and hospital-acquired clonal lineages were represented. Of the virulence genes, enterotoxin genes seg (n=52), sei (n=49), and sec (n=16) were the most frequently detected among the isolates. The PVL genes (lukS-PV and lukF-PV) have not been revealed in any of the isolates.

  9. Nupe conference.

    Science.gov (United States)

    1991-04-24

    A charterfor nursing advocacy to strengthen the nurse's role in exercising accountability and professional responsibility has been called for in a motion to be debated at NUPE's national conference at Scarborough in May.

  10. R plasmid with carbadox resistance from Escherichia coli of porcine origin.

    OpenAIRE

    Ohmae, K; Yonezawa, S; Terakado, N

    1981-01-01

    Escherichia coli isolates of porcine fecal origin from a farm where the antibacterial agent carbadox was used were examined for resistance to carbadox (Cdxr). Of 72 strains examined, 24 showed resistance to this drug. All 24 Cdxr strains, except one, were also resistant to tetracycline (Tcr), streptomycin (Smr), spectinomycin (Spcr), sulfadimethoxine (Sur), kanamycin (Kmr), ampicillin (Apcr), or a combination of tetracycline, streptomycin, spectinomycin, sulfadimethoxine, and ampicillin. The ...

  11. A 2-4-Amino Acid Deletion in the V5 Region of HIV-1 Env gp120 Confers Viral Resistance to the Broadly Neutralizing Human Monoclonal Antibody, VRC01.

    Science.gov (United States)

    Tachibana, Shingo; Sasaki, Maho; Tanaka, Takako; Inoue, Mari; Ophinni, Youdiil; Kotaki, Tomohiro; Kameoka, Masanori

    2017-12-01

    The envelope glycoprotein (Env) gp120 of human immunodeficiency virus type 1 (HIV-1) plays a critical role in viral entry into host cells. The broadly neutralizing human monoclonal antibody VRC01, which recognizes the CD4 binding site on gp120, neutralizes more than 90% of HIV-1 isolates. However, some of the CRF01_AE viruses prevalent in Southeast Asia are resistant to VRC01-mediated neutralization. We previously reported that 3 amino acid residues at positions 185, 186, and 197 of gp120 played an important role in the VRC01 resistance of CRF01_AE Env (AE-Env) clones isolated from HIV-infected Thai individuals. However, the VRC01 susceptibility of AE-Env clones was not fully explained by mutations at these 3 residues. In the present study, we examined other factors involved in the acquisition of viral VRC01 resistance. Neutralization tests using lentiviral vectors expressing a series of mutant AE-Env clones revealed that the deletion of 2-4 amino acid residues on the loop structure in the V5 region of gp120 conferred VRC01 resistance to several AE-Env clones. Our results provide novel insights into the mechanisms underlying viral VRC01 resistance.

  12. Mendel conference

    CERN Document Server

    2015-01-01

    This book is a collection of selected accepted papers of Mendel conference that has been held in Brno, Czech Republic in June 2015. The book contents three chapters which represent recent advances in soft computing including intelligent image processing and bio-inspired robotics.: Chapter 1: Evolutionary Computing, and Swarm intelligence, Chapter 2: Neural Networks, Self-organization, and Machine Learning, and Chapter3: Intelligent Image Processing, and Bio-inspired Robotics. The Mendel conference was established in 1995, and it carries the name of the scientist and Augustinian priest Gregor J. Mendel who discovered the famous Laws of Heredity. In 2015 we are commemorating 150 years since Mendel's lectures, which he presented in Brno on February and March 1865. The main aim of the conference was to create a periodical possibility for students, academics and researchers to exchange their ideas and novel research methods.  .

  13. Mutations in an auxin receptor homolog AFB5 and in SGT1b confer resistance to synthetic picolinate auxins and not to 2,4-dichlorophenoxyacetic acid or indole-3-acetic acid in Arabidopsis.

    Science.gov (United States)

    Walsh, Terence A; Neal, Roben; Merlo, Ann Owens; Honma, Mary; Hicks, Glenn R; Wolff, Karen; Matsumura, Wendy; Davies, John P

    2006-10-01

    Although a wide range of structurally diverse small molecules can act as auxins, it is unclear whether all of these compounds act via the same mechanisms that have been characterized for 2,4-dichlorophenoxyacetic acid (2,4-D) and indole-3-acetic acid (IAA). To address this question, we used a novel member of the picolinate class of synthetic auxins that is structurally distinct from 2,4-D to screen for Arabidopsis (Arabidopsis thaliana) mutants that show chemically selective auxin resistance. We identified seven alleles at two distinct genetic loci that conferred significant resistance to picolinate auxins such as picloram, yet had minimal cross-resistance to 2,4-D or IAA. Double mutants had the same level and selectivity of resistance as single mutants. The sites of the mutations were identified by positional mapping as At4g11260 and At5g49980. At5g49980 is previously uncharacterized and encodes auxin signaling F-box protein 5, one of five homologs of TIR1 in the Arabidopsis genome. TIR1 is the recognition component of the Skp1-cullin-F-box complex associated with the ubiquitin-proteasome pathway involved in auxin signaling and has recently been shown to be a receptor for IAA and 2,4-D. At4g11260 encodes the tetratricopeptide protein SGT1b that has also been associated with Skp1-cullin-F-box-mediated ubiquitination in auxin signaling and other pathways. Complementation of mutant lines with their corresponding wild-type genes restored picolinate auxin sensitivity. These results show that chemical specificity in auxin signaling can be conferred by upstream components of the auxin response pathway. They also demonstrate the utility of genetic screens using structurally diverse chemistries to uncover novel pathway components.

  14. Mutations in an Auxin Receptor Homolog AFB5 and in SGT1b Confer Resistance to Synthetic Picolinate Auxins and Not to 2,4-Dichlorophenoxyacetic Acid or Indole-3-Acetic Acid in Arabidopsis[W

    Science.gov (United States)

    Walsh, Terence A.; Neal, Roben; Merlo, Ann Owens; Honma, Mary; Hicks, Glenn R.; Wolff, Karen; Matsumura, Wendy; Davies, John P.

    2006-01-01

    Although a wide range of structurally diverse small molecules can act as auxins, it is unclear whether all of these compounds act via the same mechanisms that have been characterized for 2,4-dichlorophenoxyacetic acid (2,4-D) and indole-3-acetic acid (IAA). To address this question, we used a novel member of the picolinate class of synthetic auxins that is structurally distinct from 2,4-D to screen for Arabidopsis (Arabidopsis thaliana) mutants that show chemically selective auxin resistance. We identified seven alleles at two distinct genetic loci that conferred significant resistance to picolinate auxins such as picloram, yet had minimal cross-resistance to 2,4-D or IAA. Double mutants had the same level and selectivity of resistance as single mutants. The sites of the mutations were identified by positional mapping as At4g11260 and At5g49980. At5g49980 is previously uncharacterized and encodes auxin signaling F-box protein 5, one of five homologs of TIR1 in the Arabidopsis genome. TIR1 is the recognition component of the Skp1-cullin-F-box complex associated with the ubiquitin-proteasome pathway involved in auxin signaling and has recently been shown to be a receptor for IAA and 2,4-D. At4g11260 encodes the tetratricopeptide protein SGT1b that has also been associated with Skp1-cullin-F-box-mediated ubiquitination in auxin signaling and other pathways. Complementation of mutant lines with their corresponding wild-type genes restored picolinate auxin sensitivity. These results show that chemical specificity in auxin signaling can be conferred by upstream components of the auxin response pathway. They also demonstrate the utility of genetic screens using structurally diverse chemistries to uncover novel pathway components. PMID:16920877

  15. Glycans flanking the hypervariable connecting peptide between the A and B strands of the V1/V2 domain of HIV-1 gp120 confer resistance to antibodies that neutralize CRF01_AE viruses.

    Science.gov (United States)

    O'Rourke, Sara M; Sutthent, Ruengpung; Phung, Pham; Mesa, Kathryn A; Frigon, Normand L; To, Briana; Horthongkham, Navin; Limoli, Kay; Wrin, Terri; Berman, Phillip W

    2015-01-01

    Understanding the molecular determinants of sensitivity and resistance to neutralizing antibodies is critical for the development of vaccines designed to prevent HIV infection. In this study, we used a genetic approach to characterize naturally occurring polymorphisms in the HIV envelope protein that conferred neutralization sensitivity or resistance. Libraries of closely related envelope genes, derived from virus quasi-species, were constructed from individuals infected with CRF01_AE viruses. The libraries were screened with plasma containing broadly neutralizing antibodies, and neutralization sensitive and resistant variants were selected for sequence analysis. In vitro mutagenesis allowed us to identify single amino acid changes in three individuals that conferred resistance to neutralization by these antibodies. All three mutations created N-linked glycosylation sites (two at N136 and one at N149) proximal to the hypervariable connecting peptide between the C-terminus of the A strand and the N-terminus of the B strand in the four-stranded V1/V2 domain β-sheet structure. Although N136 has previously been implicated