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Sample records for aminoglycoside resistance methyltransferase

  1. 30S Subunit-Dependent Activation of the Sorangium cellulosum So ce56 Aminoglycoside Resistance-Conferring 16S rRNA Methyltransferase Kmr

    Science.gov (United States)

    Savic, Miloje; Sunita, S.; Zelinskaya, Natalia; Desai, Pooja M.; Macmaster, Rachel; Vinal, Kellie

    2015-01-01

    Methylation of bacterial 16S rRNA within the ribosomal decoding center confers exceptionally high resistance to aminoglycoside antibiotics. This resistance mechanism is exploited by aminoglycoside producers for self-protection while functionally equivalent methyltransferases have been acquired by human and animal pathogenic bacteria. Here, we report structural and functional analyses of the Sorangium cellulosum So ce56 aminoglycoside resistance-conferring methyltransferase Kmr. Our results demonstrate that Kmr is a 16S rRNA methyltransferase acting at residue A1408 to confer a canonical aminoglycoside resistance spectrum in Escherichia coli. Kmr possesses a class I methyltransferase core fold but with dramatic differences in the regions which augment this structure to confer substrate specificity in functionally related enzymes. Most strikingly, the region linking core β-strands 6 and 7, which forms part of the S-adenosyl-l-methionine (SAM) binding pocket and contributes to base flipping by the m1A1408 methyltransferase NpmA, is disordered in Kmr, correlating with an exceptionally weak affinity for SAM. Kmr is unexpectedly insensitive to substitutions of residues critical for activity of other 16S rRNA (A1408) methyltransferases and also to the effects of by-product inhibition by S-adenosylhomocysteine (SAH). Collectively, our results indicate that adoption of a catalytically competent Kmr conformation and binding of the obligatory cosubstrate SAM must be induced by interaction with the 30S subunit substrate. PMID:25733511

  2. 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; Desmolaize, Benoît; Douthwaite, Stephen Roger; Mechulam, Yves; Courvalin, Patrice

    2011-01-01

    Aminoglycosides are ribosome-targeting antibiotics and a major drug group of choice in the treatment of serious enterococcal infections. Here we show that aminoglycoside resistance in Enterococcus faecium strain CIP 54-32 is conferred by the chromosomal gene efmM, encoding the E. faecium methyltr......Aminoglycosides are ribosome-targeting antibiotics and a major drug group of choice in the treatment of serious enterococcal infections. Here we show that aminoglycoside resistance in Enterococcus faecium strain CIP 54-32 is conferred by the chromosomal gene efmM, encoding the E. faecium...... locations are required for catalysis. The tertiary structure of EfmM is highly similar to that of RsmF, consistent with m(5)C formation at adjacent sites on the 30S subunit, while distinctive structural features account for the enzymes' respective specificities for nucleotides C1404 and C1407....

  3. The aminoglycoside resistance methyltransferase Sgm impedes RsmF methylation at an adjacent rRNA nucleotide in the ribosomal A site

    DEFF Research Database (Denmark)

    Cubrilo, Sonja; Babić, Fedora; Douthwaite, Stephen;

    2009-01-01

    methylated nucleotides including m(4)Cm1402 and m(5)C1407. Modification at m(5)C1407 by the methyltransferase RsmF is impeded as Sgm gains access to its adjacent G1405 target on the 30S ribosomal subunit. An Sgm mutant (G135A), which is impaired in S-adenosylmethionine binding and confers lower resistance......Ribosome-targeting antibiotics block protein synthesis by binding at functionally important regions of the bacterial rRNA. Resistance is often conferred by addition of a methyl group at the antibiotic binding site within an rRNA region that is already highly modified with several nucleotide...... methylations. In bacterial rRNA, each methylation requires its own specific methyltransferase enzyme, and this raises the question as to how an extra methyltransferase conferring antibiotic resistance can be accommodated and how it can gain access to its nucleotide target within a short and functionally...

  4. An aminoglycoside sensing riboswitch controls the expression of aminoglycoside resistance acetyltransferase and adenyltransferases.

    Science.gov (United States)

    Chen, Dongrong; Murchie, Alastair I H

    2014-10-01

    The emergence of antibiotic resistance in human pathogens is an increasing threat to public health. The fundamental mechanisms that control the high levels of expression of antibiotic resistance genes are not yet completely understood. The aminoglycosides are one of the earliest classes of antibiotics that were introduced in the 1940s. In the clinic aminoglycoside resistance is conferred most commonly through enzymatic modification of the drug although resistance through enzymatic modification of the target rRNA through methylation or the overexpression of efflux pumps is also appearing. An aminoglycoside sensing riboswitch has been identified that controls expression of the aminoglycoside resistance genes that encode the aminoglycoside acetyltransferase (AAC) and aminoglycoside nucleotidyltransferase (ANT) (adenyltransferase (AAD)) enzymes. AAC and ANT cause resistance to aminoglycoside antibiotics through modification of the drugs. Expression of the AAC and ANT resistance genes is regulated by aminoglycoside binding to the 5' leader RNA of the aac/aad genes. The aminoglycoside sensing RNA is also associated with the integron cassette system that captures antibiotic resistance genes. Specific aminoglycoside binding to the leader RNA induces a structural transition in the leader RNA, and consequently induction of resistance protein expression. Reporter gene expression, direct measurements of drug RNA binding, chemical probing and UV cross-linking combined with mutational analysis demonstrated that the leader RNA functioned as an aminoglycoside sensing riboswitch in which drug binding to the leader RNA leads to the induction of aminoglycoside antibiotic resistance. This article is part of a Special Issue entitled: Riboswitches. PMID:24631585

  5. Aminoglycoside resistance in clinical Gram-negative isolates from Norway

    OpenAIRE

    Haldorsen, Bjørg Christina

    2011-01-01

    Aminoglycosides represent an important class of antimicrobial agents. The prevalence of aminoglycoside resistance among Gram-negative bacteria in Norway is low, but an increased prevalence among clinical isolates of Escherichia coli has been observed during the last years. The most prevalent resistance mechanism is aminoglycoside modifying enzymes. In addition, resistance may occur when bacteria produces 16S rRNA methylases, which causes high level and broad-spectrum aminoglycoside resistance...

  6. Aminoglycoside resistance among isolates of nosocomial Enterobacteriaceae

    International Nuclear Information System (INIS)

    Fifty-seven gentamicin-resistant isolates of Enterobacteriaceae, obtained from patients attending hospital, were examined for the production of aminoglycoside-modifying enzymes. Of the 51 strains producing such enzymes, 34 were presumptively plasmid-mediated as indicated by conjugation experiments

  7. Association of the novel aminoglycoside resistance determinant RmtF with NDM carbapenemase in Enterobacteriaceae isolated in India and the UK

    DEFF Research Database (Denmark)

    Hidalgo, Laura; Hopkins, Katie L; Gutierrez, Belen; Ovejero, Cristina M; Shukla, Suruchi; Douthwaite, Stephen Roger; Prasad, Kashi N; Woodford, Neil; Gonzalez-Zorn, Bruno

    2013-01-01

    16S rRNA methyltransferases are an emerging mechanism conferring high-level resistance to clinically relevant aminoglycosides and have been associated with important mechanisms such as NDM-1. We sought genes encoding these enzymes in isolates highly resistant (MIC >200 mg/L) to gentamicin and ami...

  8. 16S ribosomal RNA methylation: emerging resistance mechanism against aminoglycosides.

    Science.gov (United States)

    Doi, Yohei; Arakawa, Yoshichika

    2007-07-01

    Methylation of 16S ribosomal RNA (rRNA) has recently emerged as a new mechanism of resistance against aminoglycosides among gram-negative pathogens belonging to the family Enterobacteriaceae and glucose-nonfermentative microbes, including Pseudomonas aeruginosa and Acinetobacter species. This event is mediated by a newly recognized group of 16S rRNA methylases, which share modest similarity to those produced by aminoglycoside-producing actinomycetes. Their presence confers a high level of resistance to all parenterally administered aminoglycosides that are currently in clinical use. The responsible genes are mostly located on transposons within transferable plasmids, which provides them with the potential to spread horizontally and may in part explain the already worldwide distribution of this novel resistance mechanism. Some of these organisms have been found to coproduce extended-spectrum beta-lactamases or metallo-beta-lactamases, contributing to their multidrug-resistant phenotypes. A 2-tiered approach, consisting of disk diffusion tests followed by confirmation with polymerase chain reaction, is recommended for detection of 16S rRNA methylase-mediated resistance. PMID:17554708

  9. Determination of aminoglycoside resistance in Staphylococcus aureus by DNA hybridization.

    OpenAIRE

    Dickgiesser, N; Kreiswirth, B N

    1986-01-01

    A method is described for identification of the genes conferring aminoglycoside resistance in Staphylococcus aureus by dot-blot and Southern blot techniques. As radioactive probes, fragments of plasmids pAT48, pUBH2, and pH13, carrying the genes for an aminocyclitol-3'-phosphotransferase, an aminocyclitol-4'-adenylyltransferase, and an aminocyclitol-2''-phosphotransferase-aminocyclitol-6'-acetyltransferase, respectively, were used.

  10. Genetic basis of aminoglycoside resistance following changes in aminoglycoside prescription patterns.

    Science.gov (United States)

    Kosmidis, Chris; Giannopoulou, Maria; Flountzi, Anastasia; Markogiannakis, Antonis; Goukos, Dimitris; Petrikkos, George; Daikos, George L; Tzanetou, Konstantina

    2013-08-01

    Aminoglycosides (AG) offer an important therapeutic option for the treatment of infections caused by multiresistant Enterobacteriaceae. We observed a change in AG usage patterns in our institution between 1997 and 2006, namely a reduction in use of all AG except amikacin. We studied the changes in AG susceptibility rates in these time periods and correlated with prevalence of different molecular resistance mechanisms. Enterobacteriaceae isolated from blood cultures from 1997 and 2006 were studied. Susceptibilities to AG were determined with the disk diffusion method. PCR was used to detect genes encoding AG-modifying enzymes and methylases. Gentamicin resistance rates dropped from 14·5 to 8·8%, whereas resistance rates to other AG remained unchanged. The AAC(6')-I+AAC(3)-I combination was more common in 1997, whereas AAC(6')-I was the most common mechanism in 2006. Reduction in gentamicin use may preserve the usefulness of this agent against severe infections by multiresistant bacteria such as carbapenemase-producing Enterobacteriaceae. PMID:23906075

  11. Synergistic effect of [10]-gingerol and aminoglycosides against vancomycin-resistant enterococci (VRE).

    Science.gov (United States)

    Nagoshi, Chihiro; Shiota, Sumiko; Kuroda, Teruo; Hatano, Tsutomu; Yoshida, Takashi; Kariyama, Reiko; Tsuchiya, Tomofusa

    2006-03-01

    An extract from ginger (root of Zingiber officinale) reduced the minimum inhibitory concentrations (MICs) of aminoglycosides in vancomycin-resistant enterococci (VRE). The effective compound was isolated and identified as [10]-gingerol. In the presence of [10]-gingerol at 1/10 concentration of its own MIC, the MIC of arbekacin was lowered by 1/32 to 1/16. [10]-Gingerol also reduced the MICs of other aminoglycosides, and of bacitracin and polymixin B, but not of other antimicrobial agents tested. Because [10]-gingerol reduced the MICs of several aminoglycosides both in strains possessing or lacking aminoglycoside-modification enzymes, it seems that the effect of [10]-gingerol is not related to these enzymes, which mainly confer bacterial resistance against aminoglycosides. It seemed that a detergent-like effect of [10]-gingerol potentiated the antimicrobial activity of the aminoglycosides. In fact, some detergents such as sodium dodecyl sulfate (SDS) and Triton X-100 reduced the MICs of aminoglycosides, bacitracin and polymixin B in VRE. Since the intrinsic resistance to aminoglycosides in enterococci is due to low level of entry of the drugs into the cells, increase in the membrane permeability caused by [10]-gingerol will enhance the influx of aminoglycosides into enterococcal cells. PMID:16508142

  12. Study of Pseudomonas Aeroginosa resistance to Penicillines, Cephalosporins and Aminoglycosides

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    Maleknezhad P

    1998-07-01

    Full Text Available Drug therapy and prophylaxy in infectious diseases, from hygienic and economical point of view, are very important. Infections caused by pseudomonas aeroginosa were particularly severe, with high mortality rates. In the recent years pseudomonas aeroginosa continued to cause the most severe, life-thereating infections in burned patients, in spite of the introduction of a wide variety of antibiotics advised specifically for their anti pseudomonal activity. The aim of this study, in which many cases of ps.aeroginosa infections are assessed is to identify the drug resistance of this bacteria to penicillines, cephalosporins and aminoglycosides by antibiotic sensitivity test (disk ager diffusion. Results as percent of resistance to each antibiotic were 89% to carbenicillin, 55% to piperacillin, 89% to mezlocillin, 89.5% to ticarcillin+clavulonic acid, 85% to ceftriaxone, 95% to tobramycin, 5% of all isolates were not sensitive to any antibiotics.

  13. The aac(6'Ib gene in Proteus mirabilis strains resistant to aminoglycosides.

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    Jerzy Ratajczak

    2009-01-01

    Full Text Available The aim of this study was to evaluate the presence of aac(6'-Ib gene conferring resistance to aminoglycosides in Proteus mirabilis strains. Five isolates had aac(6'-Ib gene. In one case the gene was no-expressed. Three isolates were resistant to all aminoglycosides and minimum inhibitory concentrations were > or = 256 microg/ml. Additionally, all positive strains were resistant to tetracycline and ciprofloxacin.

  14. Mechanism of Enhanced Activity of Liposome-Entrapped Aminoglycosides against Resistant Strains of Pseudomonas aeruginosa

    OpenAIRE

    Mugabe, Clement; Halwani, Majed; Azghani, Ali O.; Lafrenie, Robert M.; Omri, Abdelwahab

    2006-01-01

    Pseudomonas aeruginosa is inherently resistant to most conventional antibiotics. The mechanism of resistance of this bacterium is mainly associated with the low permeability of its outer membrane to these agents. We sought to assess the bactericidal efficacy of liposome-entrapped aminoglycosides against resistant clinical strains of P. aeruginosa and to define the mechanism of liposome-bacterium interactions. Aminoglycosides were incorporated into liposomes, and the bactericidal efficacies of...

  15. Strategies to overcome the action of aminoglycoside-modifying enzymes for treating resistant bacterial infections

    OpenAIRE

    Labby, Kristin J.; Garneau-Tsodikova, Sylvie

    2013-01-01

    Shortly after the discovery of the first antibiotics, bacterial resistance began to emerge. Many mechanisms give rise to resistance; the most prevalent mechanism of resistance to the aminoglycoside (AG) family of antibiotics is the action of aminoglycoside-modifying enzymes (AMEs). Since the identification of these modifying enzymes, many efforts have been put forth to prevent their damaging alterations of AGs. These diverse strategies are discussed within this review, including: creating new...

  16. Molecular Epidemiology of Aminoglycosides Resistance in Acinetobacter Spp. with Emergence of Multidrug-Resistant Strains

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    MH Nazem Shirazi

    2010-06-01

    Full Text Available Background: Acinetobacter spp. is characterized as an important nosocomial pathogen and increasing antimicrobial resistance. Our aim was to evaluate antimicrobial susceptibility and aminoglycosides resistance genes of Acinetobacter spp. isolated from hospitalized patients.Methods: Sixty isolates were identified as Acinetobacter species. The isolates were tested for antibiotic resistance by disc diffusion method for 12 antimicrobials. The presence of aphA6, aacC1 aadA1, and aadB genes were detected using PCR.Results: From the isolated Acinetobacter spp. the highest resistance rate showed against amikacin, tobramycin, and ceftazidim, respectively; while isolated bacteria were more sensitive to ampicillic/subactam. More than 66% of the isolates were resistant to at least three classes of antibiotics, and 27.5% of MDR strains were resistant to all seven tested classes of antimicrobials. The higher MDR rate presented in bacteria isolated from the ICU and blood samples. More than 60% of the MDR bacteria were resistance to amikacin, ceftazidim, ciprofloxacin, piperacillin/tazobactam, doxycycline, tobramycin and levofloxacin. Also, more than 60% of the isolates contained phosphotransferase aphA6, and acetyltransferase genes aacC1, but adenylyltransferase genes aadA1 (41.7%, and aadB (3.3% were less prominent. 21.7% of the strains contain three aminoglycoside resistance genes (aphA6, aacC1 and aadA1.Conclusion: The rising trend of resistance to aminoglycosides poses an alarming threat to treatment of such infections. The findings showed that clinical isolates of Acinetobacter spp. in our hospital carrying various kinds of aminoglycoside resistance genes.

  17. Emerging resistance to aminoglycosides in lactic acid bacteria of food origin-an impending menace.

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    Jaimee, G; Halami, P M

    2016-02-01

    Aminoglycosides are the most preferred choice of therapy against serious infections in humans. Therefore, its use in animal husbandry has been strictly regulated in the EU, UK, and USA to avoid the hazards of aminoglycoside resistance in gut microflora. Nevertheless, aminoglycosides are recommended for prophylaxis and therapeutics in food animals and agriculture owing to its bactericidal nature. In the recent past, the global surge in aminoglycoside-resistant lactic acid bacteria (LAB) from food sources has been noticed that might question its continued use in animal husbandry. Upon antibiotic administration, a selective pressure is created in the gut environment; in such instances, LAB could act as reservoirs of antibiotic resistance which may facilitate their transfer to pathogenic organisms contradicting its probiotic and industrial significance. This may be a risk to human health as the presence of one aminoglycoside resistance gene renders the bacteria tolerant to almost all antibiotics of the same class, thereby challenging its therapeutic efficacy. Low doses of aminoglycosides are recommended in farm animals due to its toxic nature and insolubility in blood. However, recent investigations indicate that use of aminoglycosides in sub-lethal concentrations can trigger the selection and conjugal transfer of aminoglycoside resistance in probiotic LAB. Resistance to erythromycin, tetracyclines, and fluoroquinolones in LAB were reported earlier to which immediate regulatory measures were adopted by some countries. Paradoxically, lack of regulations on antibiotic use in farms in most developing countries makes them a potential source of antibiotic resistance and its uncontrolled spread around the globe. The prevalence of aminoglycoside resistance was observed in enterococci from food origin earlier; however, its emergence in lactobacilli and pediococci suggests its spread in probiotic cultures which prompts immediate precautionary methods. This review highlights the

  18. Emergence of aminoglycoside resistance genes aadA and aadE in the genus Campylobacter.

    OpenAIRE

    Pinto-Alphandary, H; Mabilat, C; Courvalin, P

    1990-01-01

    Resistance to streptomycin or spectinomycin or both in five Campylobacter coli strains, two Campylobacter jejuni strains, and a Campylobacter-like strain was studied by enzymatic assays and dot blot hybridization. Resistance was due to 6- or 3",9-aminoglycoside adenylyltransferases and to new types of phospho- and adenylyltransferases.

  19. 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...

  20. Structural and Molecular Basis for Resistance to Aminoglycoside Antibiotics by the Adenylyltransferase ANT(2″)-Ia

    OpenAIRE

    Cox, Georgina; Peter J. Stogios; Savchenko, Alexei; Wright, Gerard D.

    2015-01-01

    ABSTRACT   The aminoglycosides are highly effective broad-spectrum antimicrobial agents. However, their efficacy is diminished due to enzyme-mediated covalent modification, which reduces affinity of the drug for the target ribosome. One of the most prevalent aminoglycoside resistance enzymes in Gram-negative pathogens is the adenylyltransferase ANT(2″)-Ia, which confers resistance to gentamicin, tobramycin, and kanamycin. Despite the importance of this enzyme in drug resistance, its structure...

  1. In vitro activities of quinolones against enterococci resistant to penicillin-aminoglycoside synergy.

    OpenAIRE

    Sahm, D F; Koburov, G T

    1989-01-01

    The MICs and MBCs of CI-934, ciprofloxacin, difloxacin (A-56619), A-56620, norfloxacin, enoxacin, amifloxacin, and coumermycin were determined for 43 clinical isolates of Enterococcus faecalis known to be resistant to penicillin-aminoglycoside synergy. Results were compared with those obtained for 37 synergy-susceptible E. faecalis and 22 Enterococcus faecium strains. Although no substantial differences in quinolone activities were observed between synergy-resistant and -susceptible E. faecal...

  2. Relationship between antimicrobial resistance and aminoglycoside-modifying enzyme gene expressions in Acinetobacter baumannii

    Institute of Scientific and Technical Information of China (English)

    SHI Wei-feng; JIANG Jian-ping; MI Zu-huang

    2005-01-01

    Background Acinetobacter baumannii is one of the main gram-negative bacilli in clinical practice. Nosocomial infections caused by multi-drug resistance Acinetobacter baumannii is very difficult to treat. This study was designed to investigate the antimicrobial resistance characteristics and four resistant gene expressions of aminoglycoside-modifying enzymes including N-acetyltransferases and O-phosphotransferases in Acinetobacter baumannii. Methods Bacterial identification and antimicrobial susceptibility test were performed by PhoenixTM system in 247 strains of Acinetobacter baumannii. Minimal inhibitory concentrations (MICs) of seven aminoglycosides including gentamicin, amikacin, kanamycin, tobramycin, netilmicin, neomycin and streptomycin in 15 strains of multi-drug resistant Acinetobacter baumannii were detected by agar dilution. Four aminoglycoside-modifying enzyme genes were amplified by polymerase chain reaction (PCR) and verified by DNA sequencer.Results The resistance rates of 247 strains of Acinetobacter baumannii against cefotaxime, levofloxacin, piperacillin, aztreonam, tetracycline, ciprofloxacin and chloramphenicol were more than 50%. Imipenem and meropenem showed high antibacterial activities with resistance rates of 3.2% and 4.1%. MIC50 and MIC90 of gentamicin, amikacin, streptomycin and kanamycin in 15 strains of multi-drug resistant Acinetobacter baumanii were all more than 1024 mg/L, and the resistance rates were 100%, 100%, 100% and 93.3%, respectively. But their resistance rates to tobramycin, netilmicin and neomycin were 86.7%, 93.3% and 46.7%, respectively. Three modifying enzyme genes, including aacC1, aacC2 and aacA4 genes, were found in 15 strains, but aphA6 had not been detected. Their positive rates were 93.3%, 20.0% and 20.0%, respectively. These three genes existed simultaneously in No.19 strain. Nucleotide sequences of aacC1, aacC2 and aacA4 genes shared 100%, 97.9% and 99.7% identities with GenBank genes (AY307113, S68058 and AY

  3. Resistance-Nodulation-Cell Division-Type Efflux Pump Involved in Aminoglycoside Resistance in Acinetobacter baumannii Strain BM4454

    OpenAIRE

    Magnet, Sophie; Courvalin, Patrice; Lambert, Thierry

    2001-01-01

    Multidrug-resistant strain Acinetobacter baumannii BM4454 was isolated from a patient with a urinary tract infection. The adeB gene, which encodes a resistance-nodulation-cell division (RND) protein, was detected in this strain by PCR with two degenerate oligodeoxynucleotides. Insertional inactivation of adeB in BM4454, which generated BM4454-1, showed that the corresponding protein was responsible for aminoglycoside resistance and was involved in the level of susceptibility to other drugs in...

  4. Worldwide Disseminated armA Aminoglycoside Resistance Methylase Gene Is Borne by Composite Transposon Tn1548

    OpenAIRE

    Galimand, M.; Sabtcheva, S.; Courvalin, P; Lambert, T.

    2005-01-01

    The armA (aminoglycoside resistance methylase) gene, which confers resistance to 4,6-disubstituted deoxystreptamines and fortimicin, was initially found in Klebsiella pneumoniae BM4536 on IncL/M plasmid pIP1204 of ca. 90 kb which also encodes the extended-spectrum β-lactamase CTX-M-3. Thirty-four enterobacteria from various countries that were likely to produce a CTX-M enzyme since they were more resistant to cefotaxime than to ceftazidime were studied. The armA gene was detected in 12 clinic...

  5. Dissecting the cosubstrate structure requirements of the Staphylococcus aureus aminoglycoside resistance enzyme ANT(4').

    Science.gov (United States)

    Porter, Vanessa R; Green, Keith D; Zolova, Olga E; Houghton, Jacob L; Garneau-Tsodikova, Sylvie

    2010-12-01

    Aminoglycosides are important antibiotics used against a wide range of pathogens. As a mechanism of defense, bacteria have evolved enzymes able to inactivate these drugs by regio-selectively adding a variety of functionalities (acetyl, phospho, and nucelotidyl groups) to their scaffolds. The aminoglycoside nucleotidyltransferase ANT(4') is one of the most prevalent and unique modifying-enzymes. Here, by TLC, HRMS, and colorimetric assays, we demonstrate that the resistance enzyme ANT(4') from Staphylococcus aureus is highly substrate and cosubstrate promiscuous. We show that deoxy-ribonucleotide triphosphates (dNTPs) are better cosubstrates than NTPs. We demonstrate that the position of the triphosphate group (5' and not 3') on the ribose/deoxyribose ring is important for recognition by ANT(4'), and that NTPs with larger substituents at the 3'-position of the ribose ring are not cosubstrates for ANT(4'). We confirm that for all aminoglycosides tested, the respective nucleotidylated products are completely inactive. These results provide valuable insights into the development of strategies to combat the ever-growing bacterial resistance problem. PMID:21040710

  6. Mechanism of enhanced activity of liposome-entrapped aminoglycosides against resistant strains of Pseudomonas aeruginosa.

    Science.gov (United States)

    Mugabe, Clement; Halwani, Majed; Azghani, Ali O; Lafrenie, Robert M; Omri, Abdelwahab

    2006-06-01

    Pseudomonas aeruginosa is inherently resistant to most conventional antibiotics. The mechanism of resistance of this bacterium is mainly associated with the low permeability of its outer membrane to these agents. We sought to assess the bactericidal efficacy of liposome-entrapped aminoglycosides against resistant clinical strains of P. aeruginosa and to define the mechanism of liposome-bacterium interactions. Aminoglycosides were incorporated into liposomes, and the bactericidal efficacies of both free and liposomal drugs were evaluated. To define the mechanism of liposome-bacterium interactions, transmission electron microscopy (TEM), flow cytometry, lipid mixing assay, and immunocytochemistry were employed. Encapsulation of aminoglycosides into liposomes significantly increased their antibacterial activity against the resistant strains used in this study (MICs of > or =32 versus < or =8 microg/ml). TEM observations showed that liposomes interact intimately with the outer membrane of P. aeruginosa, leading to the membrane deformation. The flow cytometry and lipid mixing assays confirmed liposome-bacterial membrane fusion, which increased as a function of incubation time. The maximum fusion rate was 54.3% +/- 1.5% for an antibiotic-sensitive strain of P. aeruginosa and 57.8% +/- 1.9% for a drug-resistant strain. The fusion between liposomes and P. aeruginosa significantly enhanced the antibiotics' penetration into the bacterial cells (3.2 +/- 2.3 versus 24.2 +/- 6.2 gold particles/bacterium, P < or = 0.001). Our data suggest that liposome-entrapped antibiotics could successfully resolve infections caused by antibiotic-resistant P. aeruginosa through an enhanced mechanism of drug entry into the bacterial cells. PMID:16723560

  7. Clonal origin of aminoglycoside-resistant Citrobacter freundii isolates in a Danish county

    DEFF Research Database (Denmark)

    Norskov-Lauritsen, N.; Sandvang, Dorthe; Hedegaard, J.; Fussing, V.; Mortensen, K.K.; Sperling-Petersen, H.U.; Schonheyder, H.C.

    2001-01-01

    During 1997, attention was drawn to an increased frequency of aminoglycoside-resistant Citrobacter freundii in a Danish county, when a total of 24 resistant C. freundii isolates was detected. In this study, 15 such isolates were typed by pulsed-field gel electrophoresis, riboprinting and partial ...... dihydrofolate reductase gene in a class I integron. The source of the strain remains unresolved. Representative isolates were obtained from various specimens from hospitals and general practice throughout the county, with no evidence of patient-to-patient transmission....

  8. Structural and molecular basis for resistance to aminoglycoside antibiotics by the adenylyltransferase ANT(2″)-Ia.

    Science.gov (United States)

    Cox, Georgina; Stogios, Peter J; Savchenko, Alexei; Wright, Gerard D

    2015-01-01

    The aminoglycosides are highly effective broad-spectrum antimicrobial agents. However, their efficacy is diminished due to enzyme-mediated covalent modification, which reduces affinity of the drug for the target ribosome. One of the most prevalent aminoglycoside resistance enzymes in Gram-negative pathogens is the adenylyltransferase ANT(2″)-Ia, which confers resistance to gentamicin, tobramycin, and kanamycin. Despite the importance of this enzyme in drug resistance, its structure and molecular mechanism have been elusive. This study describes the structural and mechanistic basis for adenylylation of aminoglycosides by the ANT(2″)-Ia enzyme. ANT(2″)-Ia confers resistance by magnesium-dependent transfer of a nucleoside monophosphate (AMP) to the 2″-hydroxyl of aminoglycoside substrates containing a 2-deoxystreptamine core. The catalyzed reaction follows a direct AMP transfer mechanism from ATP to the substrate antibiotic. Central to catalysis is the coordination of two Mg(2+) ions, positioning of the modifiable substrate ring, and the presence of a catalytic base (Asp86). Comparative structural analysis revealed that ANT(2″)-Ia has a two-domain structure with an N-terminal active-site architecture that is conserved among other antibiotic nucleotidyltransferases, including Lnu(A), LinB, ANT(4')-Ia, ANT(4″)-Ib, and ANT(6)-Ia. There is also similarity between the nucleotidyltransferase fold of ANT(2″)-Ia and DNA polymerase β. This similarity is consistent with evolution from a common ancestor, with the nucleotidyltransferase fold having adapted for activity against chemically distinct molecules. IMPORTANCE  : To successfully manage the threat associated with multidrug-resistant infectious diseases, innovative therapeutic strategies need to be developed. One such approach involves the enhancement or potentiation of existing antibiotics against resistant strains of bacteria. The reduction in clinical usefulness of the aminoglycosides is a particular

  9. Substrate recognition and modification by the nosiheptide resistance methyltransferase.

    Directory of Open Access Journals (Sweden)

    Sitao Yin

    Full Text Available The proliferation of antibiotic resistant pathogens is an increasing threat to the general public. Resistance may be conferred by a number of mechanisms including covalent or mutational modification of the antibiotic binding site, covalent modification of the drug, or the over-expression of efflux pumps. The nosiheptide resistance methyltransferase (NHR confers resistance to the thiazole antibiotic nosiheptide in the nosiheptide producer organism Streptomyces actuosus through 2'O-methylation of 23S rRNA at the nucleotide A1067. Although the crystal structures of NHR and the closely related thiostrepton-resistance methyltransferase (TSR in complex with the cofactor S-Adenosyl-L-methionine (SAM are available, the principles behind NHR substrate recognition and catalysis remain unclear.We have analyzed the binding interactions between NHR and model 58 and 29 nucleotide substrate RNAs by gel electrophoresis mobility shift assays (EMSA and fluorescence anisotropy. We show that the enzyme binds to RNA as a dimer. By constructing a hetero-dimer complex composed of one wild-type subunit and one inactive mutant NHR-R135A subunit, we show that only one functional subunit of the NHR homodimer is required for its enzymatic activity. Mutational analysis suggests that the interactions between neighbouring bases (G1068 and U1066 and A1067 have an important role in methyltransfer activity, such that the substitution of a deoxy sugar spacer (5' to the target nucleotide achieved near wild-type levels of methylation. A series of atomic substitutions at specific positions on the substrate adenine show that local base-base interactions between neighbouring bases are important for methylation.Taken together these data suggest that local base-base interactions play an important role in aligning the substrate 2' hydroxyl group of A1067 for methyl group transfer. Methylation of nucleic acids is playing an increasingly important role in fundamental biological processes

  10. Plasmid-Mediated High-Level Resistance to Aminoglycosides in Enterobacteriaceae Due to 16S rRNA Methylation

    OpenAIRE

    Galimand, Marc; Courvalin, Patrice; Lambert, Thierry

    2003-01-01

    A self-transferable plasmid of ca. 80 kb, pIP1204, conferred multiple-antibiotic resistance to Klebsiella pneumoniae BM4536, which was isolated from a urinary tract infection. Resistance to β-lactams was due to the blaTEM1 and blaCTX-M genes, resistance to trimethroprim was due to the dhfrXII gene, resistance to sulfonamides was due to the sul1 gene, resistance to streptomycin-spectinomycin was due to the ant3"9 gene, and resistance to nearly all remaining aminoglycosides was due to the aac3-...

  11. Appearance of amikacin and tobramycin resistance due to 4'-aminoglycoside nucleotidyltransferase [ANT(4')-II] in gram-negative pathogens.

    OpenAIRE

    Jacoby, G A; Blaser, M J; Santanam, P; Hächler, H; Kayser, F H; Hare, R S; Miller, G. H.

    1990-01-01

    Following the use of amikacin as the principal aminoglycoside at a Denver hospital, amikacin resistance appeared first in Pseudomonas aeruginosa and then in Escherichia coli, Klebsiella pneumoniae, and other enteric organisms from debilitated and compromised patients who had spent time in intensive care units and who had been treated with multiple antibiotics, usually including amikacin. In a P. aeruginosa isolate, resistance to amikacin and tobramycin was transferable by the IncP-2 plasmid p...

  12. Audiologic monitoring of multi-drug resistant tuberculosis patients on aminoglycoside treatment with long term follow-up

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    Sarkar Malay

    2007-11-01

    Full Text Available Abstract Background Multi-drug resistant tuberculosis has emerged as a significant problem with the resurfacing of tuberculosis and thus the need to use the second line drugs with the resultant increased incidence of adverse effects. We discuss the effect of second line aminoglycoside anti-tubercular drugs on the hearing status of MDR-TB patients. Methods Sixty four patients were put on second line aminoglycoside anti-TB drugs. These were divided into three groups: group I, 34 patients using amikacin, group II, 26 patients using kanamycin and group III, 4 patients using capreomycin. Results Of these, 18.75% of the patients developed sensorineural hearing loss involving higher frequencies while 6.25% had involvement of speech frequencies also. All patients were seen again approximately one year after aminoglycoside discontinuation and all hearing losses were permanent with no threshold improvement. Conclusion Aminoglycosides used in MDR-TB patients may result in irreversible hearing loss involving higher frequencies and can become a hearing handicap as speech frequencies are also involved in some of the patients thus underlining the need for regular audiologic evaluation in patients of MDR-TB during the treatment.

  13. Prevalence of plasmid-mediated quinolone resistance and aminoglycoside resistance determinants among carbapeneme non-susceptible Enterobacter cloacae.

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    Shifeng Huang

    Full Text Available BACKGROUND: Simultaneous resistance to aminoglycosides and fluoroquinolones in carbapeneme non-susceptible (CNS isolates will inevitably create problems. The present study was performed to characterize the prevalence of the plasmid-mediated quinolone resistance determinants (QRDs and aminoglycoside resistance determinants (ARDs among the CNS Enterobacter cloacae (E. cloacae isolates in a Chinese teaching hospital, and to acquire their molecular epidemiological characteristics. METHODS: The β-lactamases genes (including class A carbapenemase genes bla(KPC and bla(SME, metallo-β-lactamase genes (MBLs bla(IMP, bla(VIM and bla(NDM, and extended spectrum β-lactamases (ESBLs,bla(CTX-M, bla(TEM and bla(SHV, QRDs (including qnrA, qnrB, qnrS and aac(6'-Ib-cr and ARDs (including aac(6'-Ib, armA and rmtB of these 35 isolates were determined by PCR and sequenced bidirectionally. The clonal relatedness was investigated by pulsed-field gel electrophoresis (PFGE. RESULTS: Of the 35 isolates, 9 (25.7% harbored a carbapenemase gene; 23 (65.7% carried ESBLs; 24 (68.6% were QRD positive; and 27 (77.1% were ARD positive. Among the 5 bla(IMP-8 positive strains, 4 (80% contained both ESBL and QRD genes, and all the 5 (100% harbored ARD genes. Of the 23 ESBLs positive isolates, 6 (26.1% were carbapenemase positive, 14 (60.9% were QRD positive, and 18 (78.3% were ARD positive. PFGE revealed genetic diversity among the 35 isolates, indicating that the high prevalence of CNS E. cloacae isolates was not caused by clonal dissemination. CONCLUSION: QRD and ARD genes were highly prevalent among the CNS E. cloacae isolates. Multiple resistant genes were co-expressed in the same isolates. The CNS E. cloacae isolate co-expressing bla(NDM-1, bla(IMP-26, qnrA1 and qnrS1 was first reported.

  14. Isolation and speciation of Enterococci from various clinical samples and their antimicrobial susceptibility pattern with special reference to high level Aminoglycoside resistance

    Directory of Open Access Journals (Sweden)

    :Saroj Golia, Nirmala AR, Asha S Kamath B

    2014-07-01

    Full Text Available Background and Objectives: Enterococci are important nosocomial agents and strains resistant to penicillin and other antibiotics occur frequently. Enterococci are intrinsically resistant to cephalosporins and offer low level resistance to aminoglycosides. In penicillin sensitive strains, synergism occurs with combination treatment with penicillin and aminoglycoside. Serious infections caused by them are treated with penicillin and aminoglycoside combination. But the synergistic effect is lost, when the strain develops high level aminoglycoside resistance. The choice of drug for infections due to such strains is vancomycin. The present study was carried out to isolate and speciate Enterococci from various clinical samples, to know the susceptibility pattern of the isolates, to determine the High Level Aminoglycoside Resistance (HLAR among Enterococcal isolates. Methods: A total of One hundred Enterococcal species isolated from various clinical samples were identified by various biochemical reactions. Antimicrobial susceptibility testing and HLAR were determined by Kirby- Bauer disc diffusion method. Results: Out of 100 Enterococcal isolates, 59 were E. faecalis, 38 were E. faecium, 3 were other Enterococcal species. Among these 53 isolates showed High Level Aminoglycoside Resistance. Conclusion: Present study shows the presence of drug resistance to most of commonly used antibiotics and HLAR is also more in E.faecium compared to E.fecalis.

  15. Prevalence of resistance to aminoglycosides and fluoroquinolones among Pseudomonas aeruginosa strains in a University Hospital in Northeastern Poland

    OpenAIRE

    Anna Diana Michalska; Pawel Tomasz Sacha; Dominika Ojdana; * Anna Wieczorek; Elzbieta Tryniszewska

    2014-01-01

    The present study was conducted to investigate the prevalence of genes encoding resistance to aminoglycosides and fluoroquinolones among twenty-five Pseudomonas aeruginosa isolated between 2002 and 2009. In PCR, following genes were detected: ant(2″)-Ia in 9 (36.0%), aac(6′)-Ib in 7 (28.0%), qnrB in 5 (20.0%), aph(3″)-Ib in 2 (8.0%) of isolates.

  16. Development of aminoglycoside and β-lactamase resistance in intestinal microbiota of swine treated with lincomycin, chlorotetracycline and amoxicillin

    Directory of Open Access Journals (Sweden)

    Jian eSun

    2014-11-01

    Full Text Available Lincomycin, chlortetracycline, and amoxicillin are commonly used antimicrobials for growth promotion and infectious disease prophylaxis in swine production. In this study, we investigated the shifts and resistance development among intestinal microbiota in pregnant sows before and after lincomycin, chlortetracycline, and amoxicillin treatment by using phylogenetic analysis, bacterial enumeration, and PCR. After the antimicrobial treatment, shifts in microbial community, an increased proportion of resistant bacteria, and genes related to antimicrobial resistance as compared to the day before antimicrobial administration (day 0 were observed. Importantly, a positive correlation between antimicrobial resistance gene expression in different categories, especially those encoding aminoglycoside and β-lactamase and antimicrobial resistance, was observed. These findings demonstrate an important role of antimicrobial usage in animals in the development of antimicrobial resistance, and support the notion that prudent use of antimicrobials in swine is needed to reduce the risk of the emergence of multi-drug resistant (MDR zoonotic pathogens.

  17. Resistance-nodulation-cell division-type efflux pump involved in aminoglycoside resistance in Acinetobacter baumannii strain BM4454.

    Science.gov (United States)

    Magnet, S; Courvalin, P; Lambert, T

    2001-12-01

    Multidrug-resistant strain Acinetobacter baumannii BM4454 was isolated from a patient with a urinary tract infection. The adeB gene, which encodes a resistance-nodulation-cell division (RND) protein, was detected in this strain by PCR with two degenerate oligodeoxynucleotides. Insertional inactivation of adeB in BM4454, which generated BM4454-1, showed that the corresponding protein was responsible for aminoglycoside resistance and was involved in the level of susceptibility to other drugs including fluoroquinolones, tetracyclines, chloramphenicol, erythromycin, trimethoprim, and ethidium bromide. Study of ethidium bromide accumulation in BM4454 and BM4454-1, in the presence or in the absence of carbonyl cyanide m-chlorophenylhydrazone, demonstrated that AdeB was responsible for the decrease in intracellular ethidium bromide levels in a proton motive force-dependent manner. The adeB gene was part of a cluster that included adeA and adeC which encodes proteins homologous to membrane fusion and outer membrane proteins of RND-type three-component efflux systems, respectively. The products of two upstream open reading frames encoding a putative two-component regulatory system might be involved in the regulation of expression of the adeABC gene cluster. PMID:11709311

  18. The MisR Response Regulator Is Necessary for Intrinsic Cationic Antimicrobial Peptide and Aminoglycoside Resistance in Neisseria gonorrhoeae.

    Science.gov (United States)

    Kandler, Justin L; Holley, Concerta L; Reimche, Jennifer L; Dhulipala, Vijaya; Balthazar, Jacqueline T; Muszyński, Artur; Carlson, Russell W; Shafer, William M

    2016-08-01

    During infection, the sexually transmitted pathogen Neisseria gonorrhoeae (the gonococcus) encounters numerous host-derived antimicrobials, including cationic antimicrobial peptides (CAMPs) produced by epithelial and phagocytic cells. CAMPs have both direct and indirect killing mechanisms and help link the innate and adaptive immune responses during infection. Gonococcal CAMP resistance is likely important for avoidance of host nonoxidative killing systems expressed by polymorphonuclear granulocytes (e.g., neutrophils) and intracellular survival. Previously studied gonococcal CAMP resistance mechanisms include modification of lipid A with phosphoethanolamine by LptA and export of CAMPs by the MtrCDE efflux pump. In the related pathogen Neisseria meningitidis, a two-component regulatory system (2CRS) termed MisR-MisS has been shown to contribute to the capacity of the meningococcus to resist CAMP killing. We report that the gonococcal MisR response regulator but not the MisS sensor kinase is involved in constitutive and inducible CAMP resistance and is also required for intrinsic low-level resistance to aminoglycosides. The 4- to 8-fold increased susceptibility of misR-deficient gonococci to CAMPs and aminoglycosides was independent of phosphoethanolamine decoration of lipid A and the levels of the MtrCDE efflux pump and seemed to correlate with a general increase in membrane permeability. Transcriptional profiling and biochemical studies confirmed that expression of lptA and mtrCDE was not impacted by the loss of MisR. However, several genes encoding proteins involved in membrane integrity and redox control gave evidence of being MisR regulated. We propose that MisR modulates the levels of gonococcal susceptibility to antimicrobials by influencing the expression of genes involved in determining membrane integrity. PMID:27216061

  19. Involvement of aph(3‘-IIa in the formation of mosaic aminoglycoside resistance genes in natural environments

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    Markus eWoegerbauer

    2015-05-01

    Full Text Available Intragenic recombination leading to mosaic gene formation is known to alter resistance profiles for particular genes and bacterial species. Few studies have examined to what extent aminoglycoside resistance genes undergo intragenic recombination.We screened the GenBank database for mosaic gene formation in homologs of the aph(3’-IIa (nptII gene. APH(3’-IIa inactivates important aminoglycoside antibiotics. The gene is widely used as a selectable marker in biotechnology and enters the environment via laboratory discharges and the release of transgenic organisms. Such releases may provide opportunities for recombination in competent environmental bacteria.The retrieved GenBank sequences were grouped in 3 datasets comprising river water samples, duck pathogens and full-length variants from various bacterial genomes and plasmids. Analysis for recombination in these datasets was performed with the Recombination Detection Program, RDP4, and the Genetic Algorithm for Recombination Detection, GARD.From a total of 89 homologous sequences, 83% showed 99% - 100% sequence identity with aph(3’-IIa originally described as part of transposon Tn5. Fifty one were unique sequence variants eligible for recombination analysis. Only a single recombination event was identified with high confidence and indicated the involvement of aph(3’-IIa in the formation of a mosaic gene located on a plasmid of environmental origin in the multi-resistant isolate Pseudomonas aeruginosa PA96. The available data suggest that aph(3’-IIa is not an archetypical mosaic gene as the divergence between the described sequence variants and the number of detectable recombination events is low. This is in contrast to the numerous mosaic alleles reported for certain penicillin or tetracycline resistance determinants.

  20. Involvement of aph(3′)-IIa in the formation of mosaic aminoglycoside resistance genes in natural environments

    Science.gov (United States)

    Woegerbauer, Markus; Kuffner, Melanie; Domingues, Sara; Nielsen, Kaare M.

    2015-01-01

    Intragenic recombination leading to mosaic gene formation is known to alter resistance profiles for particular genes and bacterial species. Few studies have examined to what extent aminoglycoside resistance genes undergo intragenic recombination. We screened the GenBank database for mosaic gene formation in homologs of the aph(3′)-IIa (nptII) gene. APH(3′)-IIa inactivates important aminoglycoside antibiotics. The gene is widely used as a selectable marker in biotechnology and enters the environment via laboratory discharges and the release of transgenic organisms. Such releases may provide opportunities for recombination in competent environmental bacteria. The retrieved GenBank sequences were grouped in three datasets comprising river water samples, duck pathogens and full-length variants from various bacterial genomes and plasmids. Analysis for recombination in these datasets was performed with the Recombination Detection Program (RDP4), and the Genetic Algorithm for Recombination Detection (GARD). From a total of 89 homologous sequences, 83% showed 99–100% sequence identity with aph(3′)-IIa originally described as part of transposon Tn5. Fifty one were unique sequence variants eligible for recombination analysis. Only a single recombination event was identified with high confidence and indicated the involvement of aph(3′)-IIa in the formation of a mosaic gene located on a plasmid of environmental origin in the multi-resistant isolate Pseudomonas aeruginosa PA96. The available data suggest that aph(3′)-IIa is not an archetypical mosaic gene as the divergence between the described sequence variants and the number of detectable recombination events is low. This is in contrast to the numerous mosaic alleles reported for certain penicillin or tetracycline resistance determinants. PMID:26042098

  1. Partial characterization of an endemic strain of a methicillin- and aminoglycoside-resistant Staphylococcus aureus (MARSA) homogeneously resistant to beta-lactam antibiotics.

    Science.gov (United States)

    Jacob, J; Meers, P D

    1992-06-01

    Selected strains of methicillin- and aminoglycoside-resistant Staphylococcus aureus (MARSA) were subjected to a preliminary examination. They were representative of a larger group collected in a routine clinical microbiology laboratory over a period of 2 years. MARSA was endemic in the associated hospital. The characteristics investigated were antimicrobial resistance, the production of beta-lactamase, free and bound coagulase, protein A, DNA-ase, urease, lipase and pigment. The MARSA strains were generally indistinguishable, other than in their antimicrobial resistances. The resistance to methicillin was completely homogeneous. Except with imipenem, growth extended to the edge of discs containing methicillin and the other beta-lactam antibiotics tested when the strains were cultured at 37 degrees C on media without added salt. Homogeneous resistance may confer an epidemiological advantage on strains of this phenotype. PMID:1353087

  2. 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...

  3. Chemoprophylactic efficacy against experimental endocarditis caused by beta-lactamase-producing, aminoglycoside-resistant enterococci is associated with prolonged serum inhibitory activity.

    OpenAIRE

    Bayer, A S; Tu, J

    1990-01-01

    We studied the prevention of experimental aortic endocarditis caused by a beta-lactamase-producing, aminoglycoside-resistant strain of Enterococcus faecalis (HH22) in 146 catheterized rabbits. Both vancomycin and ampicillin-sulbactam readily killed this resistant enterococcus strain in vitro. At a challenge inoculum of approximately 10(9) CFU, vancomycin (40 mg/kg intravenously [i.v.]), ampicillin (40 mg/kg i.v.), or a combination of ampicillin plus a beta-lactamase inhibitor, sulbactam (20 m...

  4. Increasing Prevalence of Aminoglycoside-Resistant Enterococcus faecalis Isolates Due to the aac(6’)-aph(2”) Gene: A Therapeutic Problem in Kermanshah, Iran

    Science.gov (United States)

    Khani, Mitra; Fatollahzade, Mahdie; Pajavand, Hamid; Bakhtiari, Somaye; Abiri, Ramin

    2016-01-01

    Background: Enterococci are important pathogens in nosocomial infections. Various types of antibiotics, such as aminoglycosides, are used for treatment of these infections. Enterococci can acquire resistant traits, which can lead to therapeutic problems with aminoglycosides. Objectives: This study was designed to identify the prevalence of, and to compare, the aac(6’)-aph(2”) and aph(3)-IIIa genes and their antimicrobial resistance patterns among Enterococcus faecalis and E. faecium isolates from patients at Imam Reza hospital in Kermanshah in 2011 - 2012. Patients and Methods: One hundred thirty-eight clinical specimens collected from different wards of Imam Reza hospital were identified to the species level by biochemical tests. Antimicrobial susceptibility tests against kanamycin, teicoplanin, streptomycin, imipenem, ciprofloxacin, and ampicillin were performed by the disk diffusion method. The minimum inhibitory concentrations of gentamicin, streptomycin, kanamycin, and amikacin were evaluated with the microbroth dilution method. The aminoglycoside resistance genes aac(6’)-aph(2”) and aph(3”)-IIIa were analyzed with multiplex PCR. Results: The prevalence of isolates was 33 (24.1%) for E. faecium and 63 (46%) for E. faecalis. Eighty-nine percent of the isolates were high-level gentamicin resistant (HLGR), and 32.8% of E. faecium isolates and 67.2% of E. faecalis isolates carried aac(6’)-aph(2”). The prevalence of aph(3”)-IIIa among the E. faecalis and E. faecium isolates was 22.7% and 77.3%, respectively. Conclusions: Remarkably increased incidence of aac(6’)-aph(2”) among HLGR isolates explains the relationship between this gene and the high level of resistance to aminoglycosides. As the resistant gene among enterococci can be transferred, the use of new-generation antibiotics is necessary.

  5. Comparative Proteomic Analysis of Aminoglycosides Resistant and Susceptible Mycobacterium tuberculosis Clinical Isolates for Exploring Potential Drug Targets.

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    Divakar Sharma

    Full Text Available Aminoglycosides, amikacin (AK and kanamycin (KM are second line anti-tuberculosis drugs used to treat tuberculosis (TB and resistance to them affects the treatment. Membrane and membrane associated proteins have an anticipated role in biological processes and pathogenesis and are potential targets for the development of new diagnostics/vaccine/therapeutics. In this study we compared membrane and membrane associated proteins of AK and KM resistant and susceptible Mycobacterium tuberculosis isolates by 2DE coupled with MALDI-TOF/TOF-MS and bioinformatic tools. Twelve proteins were found to have increased intensities (PDQuest Advanced Software in resistant isolates and were identified as ATP synthase subunit alpha (Rv1308, Trigger factor (Rv2462c, Dihydrolipoyl dehydrogenase (Rv0462, Elongation factor Tu (Rv0685, Transcriptional regulator MoxR1(Rv1479, Universal stress protein (Rv2005c, 35kDa hypothetical protein (Rv2744c, Proteasome subunit alpha (Rv2109c, Putative short-chain type dehydrogenase/reductase (Rv0148, Bacterioferritin (Rv1876, Ferritin (Rv3841 and Alpha-crystallin/HspX (Rv2031c. Among these Rv2005c, Rv2744c and Rv0148 are proteins with unknown functions. Docking showed that both drugs bind to the conserved domain (Usp, PspA and SDR domain of these hypothetical proteins and GPS-PUP predicted potential pupylation sites within them. Increased intensities of these proteins and proteasome subunit alpha might not only be neutralized/modulated the drug molecules but also involved in protein turnover to overcome the AK and KM resistance. Besides that Rv1876, Rv3841 and Rv0685 were found to be associated with iron regulation signifying the role of iron in resistance. Further research is needed to explore how these potential protein targets contribute to resistance of AK and KM.

  6. Comparative Proteomic Analysis of Aminoglycosides Resistant and Susceptible Mycobacterium tuberculosis Clinical Isolates for Exploring Potential Drug Targets

    Science.gov (United States)

    Sharma, Divakar; Kumar, Bhavnesh; Lata, Manju; Joshi, Beenu; Venkatesan, Krishnamurthy; Shukla, Sangeeta; Bisht, Deepa

    2015-01-01

    Aminoglycosides, amikacin (AK) and kanamycin (KM) are second line anti-tuberculosis drugs used to treat tuberculosis (TB) and resistance to them affects the treatment. Membrane and membrane associated proteins have an anticipated role in biological processes and pathogenesis and are potential targets for the development of new diagnostics/vaccine/therapeutics. In this study we compared membrane and membrane associated proteins of AK and KM resistant and susceptible Mycobacterium tuberculosis isolates by 2DE coupled with MALDI-TOF/TOF-MS and bioinformatic tools. Twelve proteins were found to have increased intensities (PDQuest Advanced Software) in resistant isolates and were identified as ATP synthase subunit alpha (Rv1308), Trigger factor (Rv2462c), Dihydrolipoyl dehydrogenase (Rv0462), Elongation factor Tu (Rv0685), Transcriptional regulator MoxR1(Rv1479), Universal stress protein (Rv2005c), 35kDa hypothetical protein (Rv2744c), Proteasome subunit alpha (Rv2109c), Putative short-chain type dehydrogenase/reductase (Rv0148), Bacterioferritin (Rv1876), Ferritin (Rv3841) and Alpha-crystallin/HspX (Rv2031c). Among these Rv2005c, Rv2744c and Rv0148 are proteins with unknown functions. Docking showed that both drugs bind to the conserved domain (Usp, PspA and SDR domain) of these hypothetical proteins and GPS-PUP predicted potential pupylation sites within them. Increased intensities of these proteins and proteasome subunit alpha might not only be neutralized/modulated the drug molecules but also involved in protein turnover to overcome the AK and KM resistance. Besides that Rv1876, Rv3841 and Rv0685 were found to be associated with iron regulation signifying the role of iron in resistance. Further research is needed to explore how these potential protein targets contribute to resistance of AK and KM. PMID:26436944

  7. Enhancement of the antibiotic activity of aminoglycosides by extracts from Anadenanthera colubrine (Vell.) Brenan var. cebil against multi-drug resistant bacteria.

    Science.gov (United States)

    Barreto, Humberto M; Coelho, Kivia M R N; Ferreira, Josie H L; Dos Santos, Bernadete H C; de Abreu, Aislan P L; Coutinho, Henrique D M; da Silva, Romezio A C; de Sousa, Taciana O; Citó, Antonia M das G L; Lopes, José A D

    2016-06-01

    The aim of this work was to evaluate the antimicrobial activity of ethanol (EEAC) and hexane (HFAC) extracts from the stem bark of Anadenanthera colubrina (Vell.) Brenan var. cebil alone or in combination with aminoglycosides against multi-drug resistant (MDR) bacteria. Minimal inhibitory concentrations (MICs) of the extracts were determined by using microdilution assay. For the evaluation of extracts as modulators of antibiotic resistance, MICs of neomycin and amikacin were determined in presence or absence of each compound at sub-inhibitory concentrations. Both EEAC and HFAC did not show antimicrobial activity against MDR strains tested. However, the addition of EEAC and HFAC enhanced the activity of neomycin and amikacin against Staphylococcus aureus SA10 strain. When the natural products were replaced by chlorpromazine, the same effect was observed. Anadenanthera colubrine var. cebil may be a source of phytochemicals able to potentiate the aminoglycoside activity against MDR S. aureus by the inhibition of efflux pump. PMID:26158209

  8. Aminoglycosides: An Overview.

    Science.gov (United States)

    Krause, Kevin M; Serio, Alisa W; Kane, Timothy R; Connolly, Lynn E

    2016-01-01

    Aminoglycosides are natural or semisynthetic antibiotics derived from actinomycetes. They were among the first antibiotics to be introduced for routine clinical use and several examples have been approved for use in humans. They found widespread use as first-line agents in the early days of antimicrobial chemotherapy, but were eventually replaced in the 1980s with cephalosporins, carbapenems, and fluoroquinolones. Aminoglycosides synergize with a variety of other antibacterial classes, which, in combination with the continued increase in the rise of multidrug-resistant bacteria and the potential to improve the safety and efficacy of the class through optimized dosing regimens, has led to a renewed interest in these broad-spectrum and rapidly bactericidal antibacterials. PMID:27252397

  9. Development and evaluation of immunochromatography to detect Gram-negative bacteria producing ArmA 16S rRNA methylase responsible for aminoglycoside resistance.

    Science.gov (United States)

    Oshiro, Satoshi; Tada, Tatsuya; Kameoka, Yousuke; Suzuki, Kazuo; Ohmagari, Norio; Miyoshi-Akiyama, Tohru; Kirikae, Teruo

    2015-11-01

    Rapid and reliable detection of aminoglycoside-resistant bacteria is an important infection-control measure and a crucial aspect of antimicrobial chemotherapy. The enzyme 16S rRNA methylase has been shown to mediate aminoglycoside resistance in bacteria. This study describes a newly developed immunochromatographic assay using novel monoclonal antibodies (mAbs) that recognize ArmA 16S rRNA methylase. Epitope mapping showed that these mAbs recognized amino acids 1-93 of ArmA, which consists of 257 amino acids. Evaluation of the assay using ArmA producing and non-producing bacterial species, as well as bacteria producing other types of 16S rRNA methylases, indicated that immunochromatographic detection of the ArmA-type 16S rRNA methylase was fully consistent with PCR analysis for armA genes, with all immunochromatographically positive strains being resistant to aminoglycosides (MIC≥128μg/mL). The detection limit of the assay was 12ng ArmA. These findings indicate that this assay can be used for the rapid and reliable detection of the production of ArmA 16S rRNA methylase by Gram-negative bacteria, including Acinetobacter baumannii and Escherichia coli. PMID:26381663

  10. 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.; Schwarz, S.; Vester, B.

    2006-01-01

    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 and...

  11. A nanoplex PCR assay for the rapid detection of vancomycin and bifunctional aminoglycoside resistance genes in Enterococcus species

    Directory of Open Access Journals (Sweden)

    Ravichandran Manickam

    2007-12-01

    Full Text Available Abstract Background Enterococci have emerged as a significant cause of nosocomial infections in many parts of the world over the last decade. The most common enterococci strains present in clinical isolates are E. faecalis and E. faecium which have acquired resistant to either gentamicin or vancomycin. The conventional culture test takes 2–5 days to yield complete information of the organism and its antibiotic sensitivity pattern. Hence our present study was focused on developing a nanoplex PCR assay for the rapid detection of vancomycin and bifunctional aminoglycoside resistant enterococci (V-BiA-RE. This assay simultaneously detects 8 genes namely 16S rRNA of Enterococcus genus, ddl of E. faecalis and E. faecium, aacA-aphD that encodes high level gentamicin resistance (HLGR, multilevel vancomycin resistant genotypes such as vanA, vanB, vanC and vanD and one internal control gene. Results Unique and specific primer pairs were designed to amplify the 8 genes. The specificity of the primers was confirmed by DNA sequencing of the nanoplex PCR products and BLAST analysis. The sensitivity and specificity of V-BiA-RE nanoplex PCR assay was evaluated against the conventional culture method. The analytical sensitivity of the assay was found to be 1 ng at the DNA level while the analytical specificity was evaluated with 43 reference enterococci and non-enterococcal strains and was found to be 100%. The diagnostic accuracy was determined using 159 clinical specimens, which showed that 97% of the clinical isolates belonged to E. faecalis, of which 26% showed the HLGR genotype, but none were vancomycin resistant. The presence of an internal control in the V-BiA-RE nanoplex PCR assay helped us to rule out false negative cases. Conclusion The nanoplex PCR assay is robust and can give results within 4 hours about the 8 genes that are essential for the identification of the most common Enterococcus spp. and their antibiotic sensitivity pattern. The PCR assay

  12. 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

    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 a...

  13. Combinations of β-lactam or aminoglycoside antibiotics with plectasin are synergistic against methicillin-sensitive and methicillin-resistant Staphylococcus aureus.

    Directory of Open Access Journals (Sweden)

    Yanmin Hu

    Full Text Available Bacterial infections remain the leading killer worldwide which is worsened by the continuous emergence of antibiotic resistance. In particular, methicillin-sensitive (MSSA and methicillin-resistant Staphylococcus aureus (MRSA are prevalent and the latter can be difficult to treat. The traditional strategy of novel therapeutic drug development inevitably leads to emergence of resistant strains, rendering the new drugs ineffective. Therefore, rejuvenating the therapeutic potentials of existing antibiotics offers an attractive novel strategy. Plectasin, a defensin antimicrobial peptide, potentiates the activities of other antibiotics such as β-lactams, aminoglycosides and glycopeptides against MSSA and MRSA. We performed in vitro and in vivo investigations to test against genetically diverse clinical isolates of MSSA (n = 101 and MRSA (n = 115. Minimum inhibitory concentrations (MIC were determined by the broth microdilution method. The effects of combining plectasin with β-lactams, aminoglycosides and glycopeptides were examined using the chequerboard method and time kill curves. A murine neutropenic thigh model and a murine peritoneal infection model were used to test the effect of combination in vivo. Determined by factional inhibitory concentration index (FICI, plectasin in combination with aminoglycosides (gentamicin, neomycin or amikacin displayed synergistic effects in 76-78% of MSSA and MRSA. A similar synergistic response was observed when plectasin was combined with β-lactams (penicillin, amoxicillin or flucloxacillin in 87-89% of MSSA and MRSA. Interestingly, no such interaction was observed when plectasin was paired with vancomycin. Time kill analysis also demonstrated significant synergistic activities when plectasin was combined with amoxicillin, gentamicin or neomycin. In the murine models, plectasin at doses as low as 8 mg/kg augmented the activities of amoxicillin and gentamicin in successful treatment of MSSA and MRSA

  14. Assessment of hearing loss in multi-drug resistant tuberculosis (MDR-TB patients undergoing Aminoglycoside treatment

    Directory of Open Access Journals (Sweden)

    Sheikh Nizamuddin

    2015-07-01

    Conclusion: Aminoglycosides in MDR-TB patients may cause irreversible hearing loss involving higher frequencies and can become a hearing handicap as speech frequencies are too implied in more or less of the patients, thus underlining the need for regular audiologic evaluation in patients of MDR-TB during the treatment. [Int J Res Med Sci 2015; 3(7.000: 1734-1740

  15. 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.; Bujnicki, J. M.; Vester, B.; Long, Katherine

    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...

  16. Binding induced RNA conformational changes control substrate recognition and catalysis by the thiostrepton resistance methyltransferase (Tsr).

    Science.gov (United States)

    Kuiper, Emily G; Conn, Graeme L

    2014-09-19

    Ribosomal RNA (rRNA) post-transcriptional modifications are essential for ribosome maturation, translational fidelity, and are one mechanism used by both antibiotic-producing and pathogenic bacteria to resist the effects of antibiotics that target the ribosome. The thiostrepton producer Streptomyces azureus prevents self-intoxication by expressing the thiostrepton-resistance methyltransferase (Tsr), which methylates the 2'-hydroxyl of 23 S rRNA nucleotide adenosine 1067 within the thiostrepton binding site. Tsr is a homodimer with each protomer containing an L30e-like amino-terminal domain (NTD) and a SPOUT methyltransferase family catalytic carboxyl-terminal domain (CTD). We show that both enzyme domains are required for high affinity RNA substrate binding. The Tsr-CTD has intrinsic, weak RNA affinity that is necessary to direct the specific high-affinity Tsr-RNA interaction via NTDs, which have no detectable RNA affinity in isolation. RNA structure probing experiments identify the Tsr footprint on the RNA and structural changes in the substrate, induced specifically upon NTD binding, which are necessary for catalysis by the CTD. Additionally, we identify a key amino acid in each domain responsible for CTD-RNA binding and the observed NTD-dependent RNA structural changes. These studies allow us to develop a model for Tsr-RNA interaction in which the coordinated substrate recognition of each Tsr structural domain is an obligatory pre-catalytic recognition event. Our findings underscore the complexity of substrate recognition by RNA modification enzymes and the potential for direct involvement of the RNA substrate in controlling the process of its modification. PMID:25086036

  17. O6-methylguanine DNA methyltransferase as a promising target for the treatment of temozolomide-resistant gliomas

    OpenAIRE

    Fan, C-H; Liu, W-L; Cao, H.; Wen, C; Chen, L.; G. Jiang

    2013-01-01

    Temozolomide (TMZ) is an alkylating agent currently used as first-line therapy for gliomas treatment due to its DNA-damaging effect. However, drug resistance occurs, preventing multi-cycle use of this chemotherapeutic agent. One of the major mechanisms of cancer drug resistance is enhanced activity of a DNA repair enzyme, O6-methylguanine-DNA-methyltransferase (MGMT), which counteracts chemotherapy-induced DNA alkylation and is a key component of chemoresistance. MGMT repairs TMZ-induced DNA ...

  18. Distribution of 16S rRNA methylases among different species of Gram-negative bacilli with high-level resistance to aminoglycosides.

    Science.gov (United States)

    Zhou, Y; Yu, H; Guo, Q; Xu, X; Ye, X; Wu, S; Guo, Y; Wang, M

    2010-11-01

    16S rRNA methylases confer high-level resistance to most aminoglycosides in Gram-negative bacteria. Seven 16S rRNA methylase genes, armA, rmtA, rmtB, rmtC, rmtD, rmtE and npmA, have been identified since 2003. We studied the distribution of methylase genes in more than 200 aminoglycoside-resistant Gram-negative clinical isolates collected in 2007 at our hospital in Shanghai, China. 16S rRNA methylase genes were amplified by polymerase chain reaction (PCR) among 217 consecutive clinical isolates of Gram-negative bacilli resistant to gentamicin and amikacin by a disk diffusion method. 16S rRNA methylase genes were present in 97.5% (193/198) of clinical isolates highly resistant to amikacin (≥512 μg/ml), with armA and rmtB detected in 67.2 and 30.3% of strains, respectively, while no 16S rRNA methylase genes were detected in 19 strains with amikacin minimum inhibitory concentration (MIC) ≤256 μg/ml. armA or rmtB genes were detected in 100% of 104 strains of Enterobacteriaceae, and these two genes were equally represented (49 vs. 55 strains). Genes for armA or rmtB were detected in 94.7% (89/94) of Acinetobacter baumannii and Pseudomonas aeruginosa strains, and armA was predominant (84 vs. 5 strains with rmtB). No rmtA, rmtC, rmtD or npmA genes were found. Enterobacterial repetitive intergenic consensus sequence (ERIC-PCR) indicated that armA and rmtB genes were spread by both horizontal transfer and clonal dissemination. PMID:20614151

  19. Reprogramming metabolism by histone methyltransferase NSD2 drives endocrine resistance via coordinated activation of pentose phosphate pathway enzymes.

    Science.gov (United States)

    Wang, Junjian; Duan, Zhijian; Nugent, Zoann; Zou, June X; Borowsky, Alexander D; Zhang, Yanhong; Tepper, Clifford G; Li, Jian Jian; Fiehn, Oliver; Xu, Jianzhen; Kung, Hsing-Jien; Murphy, Leigh C; Chen, Hong-Wu

    2016-08-10

    Metabolic reprogramming such as the aerobic glycolysis or Warburg effect is well recognized as a common feature of tumorigenesis. However, molecular mechanisms underlying metabolic alterations for tumor therapeutic resistance are poorly understood. Through gene expression profiling analysis we found that histone H3K36 methyltransferase NSD2/MMSET/WHSC1 expression was highly elevated in tamoxifen-resistant breast cancer cell lines and clinical tumors. IHC analysis indicated that NSD2 protein overexpression was associated with the disease recurrence and poor survival. Ectopic expression of NSD2 wild type, but not the methylase-defective mutant, drove endocrine resistance in multiple cell models and xenograft tumors. Mechanistically, NSD2 was recruited to and methylated H3K36me2 at the promoters of key glucose metabolic enzyme genes. Its overexpression coordinately up-regulated hexokinase 2 (HK2) and glucose-6-phosphate dehydrogenase (G6PD), two key enzymes of glycolysis and the pentose phosphate pathway (PPP), as well as TP53-induced glycolysis regulatory phosphatase TIGAR. Consequently, NSD2-driven tamoxifen-resistant cells and tumors displayed heightened PPP activity, elevated NADPH production, and reduced ROS level, without significantly altered glycolysis. These results illustrate a coordinated, epigenetic activation of key glucose metabolic enzymes in therapeutic resistance and nominate methyltransferase NSD2 as a potential therapeutic target for endocrine resistant breast cancer. PMID:27164560

  20. The impact of methicillin- and aminoglycoside-resistant Staphylococcus aureus on the pattern of hospital-acquired infection in an acute hospital.

    Science.gov (United States)

    Meers, P D; Leong, K Y

    1990-10-01

    Infections due to methicillin- and aminoglycoside-resistant Staphylococcus aureus (MARSA) appeared in a new teaching hospital shortly after it opened. The effect this had on the pattern of hospital-acquired infections in the four years that followed is described. No control measures were applied and MARSA became endemic. New infections appeared at a rate of about four for each 1000 patients discharged. It established itself at different levels of incidence in various specialist units, patients under intensive care being most severely affected. MARSA was implicated in half of all hospital-acquired infections due to S. aureus but it was not more pathogenic than its more sensitive counterpart. It had little impact on the life of the hospital. PMID:1979573

  1. Pharmacokinetics of Aminoglycosides

    Institute of Scientific and Technical Information of China (English)

    Lokangu Lombo(Congo); HE Hua

    2004-01-01

    The Pharmacokinetics informations of aminoglycosides, their monograph and clinical Pharmacokinetics parameters are reported in this review. The Aminoglycosides are highly polarity and in reserve for serious infections caused by aerobic gram-negative bacteria and some gram-positive bacteria but their toxicity are major limitations in clinical use.

  2. A SAM-dependent methyltransferase cotranscribed with arsenate reductase alters resistance to peptidyl transferase center-binding antibiotics in Azospirillum brasilense Sp7.

    Science.gov (United States)

    Singh, Sudhir; Singh, Chhaya; Tripathi, Anil Kumar

    2014-05-01

    The genome of Azospirillum brasilense harbors a gene encoding S-adenosylmethionine-dependent methyltransferase, which is located downstream of an arsenate reductase gene. Both genes are cotranscribed and translationally coupled. When they were cloned and expressed individually in an arsenate-sensitive strain of Escherichia coli, arsenate reductase conferred tolerance to arsenate; however, methyltransferase failed to do so. Sequence analysis revealed that methyltransferase was more closely related to a PrmB-type N5-glutamine methyltransferase than to the arsenate detoxifying methyltransferase ArsM. Insertional inactivation of prmB gene in A. brasilense resulted in an increased sensitivity to chloramphenicol and resistance to tiamulin and clindamycin, which are known to bind at the peptidyl transferase center (PTC) in the ribosome. These observations suggested that the inability of prmB:km mutant to methylate L3 protein might alter hydrophobicity in the antibiotic-binding pocket of the PTC, which might affect the binding of chloramphenicol, clindamycin, and tiamulin differentially. This is the first report showing the role of PrmB-type N5-glutamine methyltransferases in conferring resistance to tiamulin and clindamycin in any bacterium. PMID:24573606

  3. A Site-Specific Integrative Plasmid Found in Pseudomonas aeruginosa Clinical Isolate HS87 along with A Plasmid Carrying an Aminoglycoside-Resistant Gene.

    Directory of Open Access Journals (Sweden)

    Dexi Bi

    Full Text Available Plasmids play critical roles in bacterial fitness and evolution of Pseudomonas aeruginosa. Here two plasmids found in a drug-resistant P. aeruginosa clinical isolate HS87 were completely sequenced. The pHS87b plasmid (11.2 kb carries phage-related genes and function-unknown genes. Notably, pHS87b encodes an integrase and has an adjacent tRNAThr-associated attachment site. A corresponding integrated form of pHS87b at the tRNAThr locus was identified on the chromosome of P. aeruginosa, showing that pHS87b is able to site-specifically integrate into the 3'-end of the tRNAThr gene. The pHS87a plasmid (26.8 kb displays a plastic structure containing a putative replication module, stability factors and a variable region. The RepA of pHS87a shows significant similarity to the replication proteins of pPT23A-family plasmids. pHS87a carries a transposon Tn6049, a truncated insertion sequence ΔIS1071 and a Tn402-like class 1 integron which contains an aacA4 cassette that may confer aminoglycoside resistance. Thus, pHS87b is a site-specific integrative plasmid whereas pHS87a is a plastic antibiotic resistance plasmid. The two native plasmids may promote the fitness and evolution of P. aeruginosa.

  4. A Site-Specific Integrative Plasmid Found in Pseudomonas aeruginosa Clinical Isolate HS87 along with A Plasmid Carrying an Aminoglycoside-Resistant Gene.

    Science.gov (United States)

    Bi, Dexi; Xie, Yingzhou; Tai, Cui; Jiang, Xiaofei; Zhang, Jie; Harrison, Ewan M; Jia, Shiru; Deng, Zixin; Rajakumar, Kumar; Ou, Hong-Yu

    2016-01-01

    Plasmids play critical roles in bacterial fitness and evolution of Pseudomonas aeruginosa. Here two plasmids found in a drug-resistant P. aeruginosa clinical isolate HS87 were completely sequenced. The pHS87b plasmid (11.2 kb) carries phage-related genes and function-unknown genes. Notably, pHS87b encodes an integrase and has an adjacent tRNAThr-associated attachment site. A corresponding integrated form of pHS87b at the tRNAThr locus was identified on the chromosome of P. aeruginosa, showing that pHS87b is able to site-specifically integrate into the 3'-end of the tRNAThr gene. The pHS87a plasmid (26.8 kb) displays a plastic structure containing a putative replication module, stability factors and a variable region. The RepA of pHS87a shows significant similarity to the replication proteins of pPT23A-family plasmids. pHS87a carries a transposon Tn6049, a truncated insertion sequence ΔIS1071 and a Tn402-like class 1 integron which contains an aacA4 cassette that may confer aminoglycoside resistance. Thus, pHS87b is a site-specific integrative plasmid whereas pHS87a is a plastic antibiotic resistance plasmid. The two native plasmids may promote the fitness and evolution of P. aeruginosa. PMID:26841043

  5. Outbreak of Serratia marcescens Coproducing ArmA and CTX-M-15 Mediated High Levels of Resistance to Aminoglycoside and Extended-Spectrum Beta-Lactamases, Algeria.

    Science.gov (United States)

    Batah, Rima; Loucif, Lotfi; Olaitan, Abiola Olumuyiwa; Boutefnouchet, Nafissa; Allag, Hamoudi; Rolain, Jean-Marc

    2015-08-01

    Serratia marcescens is one of the most important pathogens responsible for nosocomial infections worldwide. Here, we have investigated the molecular support of antibiotic resistance and genetic relationships in a series of 54 S. marcescens clinical isolates collected from Eastern Algeria between December 2011 and July 2013. The 54 isolates were identified by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS). Antibiotic susceptibility testing was performed by disc diffusion and E-test methods. Antibiotic resistance genes were detected by polymerase chain reaction (PCR). The genetic transfer of antibiotic resistance was performed by conjugation using azide-resistant Escherichia coli J53 as the recipient strain, and plasmid analysis was done by PCR-based replicon typing. The relatedness of our isolates was determined by phylogenetic analysis based on partial sequences of four protein-encoding genes (gyrB, rpoB, infB, and atpD) and then compared to MALDI-TOF MS clustering. Thirty-five out of 54 isolates yielded an extended-spectrum β-lactamase (ESBL) phenotype and carried bla(CTX-M-15) (n=32), bla(TEM-1) (n=26), bla(TEM-71) (n=1), bla(SHV-1a) (n=1), and bla(PER-2) (n=12). Among these isolates, we identified a cluster of 15 isolates from a urology unit that coharbored ESBL and the 16S rRNA methyltransferase armA. Conjugation was successful for five selected strains, demonstrating the transferability of a conjugative plasmid of incompatibility group incL/M type. Phylogenetic analysis along with MALDI-TOF clustering likely suggested an outbreak of such isolates in the urology unit. In this study, we report for the first time the co-occurrence of armA methyltransferase with ESBL in S. marcescens clinical isolates in Eastern Algeria. PMID:25884511

  6. Methyltransferase Erm(37) Slips on rRNA to Confer Atypical Resistance in Mycobacterium tuberculosis

    Czech Academy of Sciences Publication Activity Database

    Madsen, Ch. T.; Jakobsen, L.; Buriánková, Karolína; Doucet-Populaire, F.; Perdonet, J. L.; Douthwaite, S.

    2005-01-01

    Roč. 280, č. 47 (2005), s. 38942-38947. ISSN 0021-9258 R&D Projects: GA ČR GA310/03/0292 Institutional research plan: CEZ:AV0Z50200510 Keywords : methyltransferase erm * mycobacterium tuberculosis * rRNA Subject RIV: EE - Microbiology, Virology Impact factor: 5.854, year: 2005

  7. Recognition elements in rRNA for the tylosin resistance methyltransferase RlmA(II)

    DEFF Research Database (Denmark)

    Lebars, Isabelle; Husson, Clotilde; Yoshizawa, Satoko; Douthwaite, Stephen; Fourmy, Dominique

    2007-01-01

    antibiotics. We have previously solved the solution structure of hairpin 35 in the conformation that is recognized by the RlmA(II) methyltransferase from Streptococcus pneumoniae. It was shown that while essential recognition elements are located in hairpin 35, the interactions between RlmA(II) and hairpin 35...

  8. Crystallographic Studies of Two Bacterial AntibioticResistance Enzymes: Aminoglycoside Phosphotransferase (2')-Ic and GES-1\\beta-lactamase

    Energy Technology Data Exchange (ETDEWEB)

    Brynes, Laura; /Rensselaer Poly.

    2007-10-31

    Guiana Extended-Spectrum-1 (GES-1) and Aminoglycoside phosphotransferase (2')-Ic (APH(2')-Ic) are two bacteria-produced enzymes that essentially perform the same task: they provide resistance to an array of antibiotics. Both enzymes are part of a growing resistance problem in the medical world. In order to overcome the ever-growing arsenal of antibiotic-resistance enzymes, it is necessary to understand the molecular basis of their action. Accurate structures of these proteins have become an invaluable tool to do this. Using protein crystallography techniques and X-ray diffraction, the protein structure of GES-1 bound to imipenem (an inhibitor) has been solved. Also, APH(2')-Ic has been successfully crystallized, but its structure was unable to be solved using molecular replacement using APH(2')-Ib as a search model. The structure of GES-1, with bound imipenem was solved to a resolution of 1.89A, and though the inhibitor is bound with only moderate occupancy, the structure shows crucial interactions inside the active site that render the enzyme unable to complete the hydrolysis of the {beta}-lactam ring. The APH(2')-Ic dataset could not be matched to the model, APH(2')-Ib, with which it shares 25% sequence identity. The structural information gained from GES-1, and future studies using isomorphous replacement to solve the APH(2')-Ic structure can aid directly to the creation of novel drugs to combat both of these classes of resistance enzymes.

  9. Purification, crystallization and diffraction studies of the methyltransferases BT-2972 and BVU-3255 from antibiotic-resistant pathogens of the genus Bacteroides from the human intestine

    International Nuclear Information System (INIS)

    The expression, purification, crystallization and diffraction of two methyltransferases BT-2972 and BVU-3255 from two Bacteroides species of antibiotic-resistant pathogens from the human intestine are reported. The methyltransferases BT-2972 and BVU-3255 from two different Bacteroides species that are antibiotic-resistant pathogens from the human intestine were cloned, overexpressed and purified, yielding approximately 120 mg of each protein from 1 l culture. Apo BT-2972 and BVU-3255 and their complexes with S-adenosylmethionine or S-adenosylhomocysteine were crystallized in four different crystal forms using the hanging-drop vapour-diffusion method. These crystals diffracted to resolutions ranging from 2.8 to 2.2 Å. Sequence analysis suggested that the two proteins are homologous small-molecule methyltransferases

  10. 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)

    Kaminska, Katarzyna H; Purta, Elzbieta; Hansen, Lykke H; Bujnicki, Janusz M; Vester, Birte; Long, Katherine S

    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...... 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...

  11. Indigenous and acquired modifications in the aminoglycoside binding sites of Pseudomonas aeruginosa rRNAs

    DEFF Research Database (Denmark)

    Gutierrez, Belen; Douthwaite, Stephen Roger; Gonzalez-Zorn, Bruno

    2013-01-01

    of 16S rRNA helix 44 with a secondary target in 23S rRNA helix 69. Here, we have mapped P. aeruginosa rRNAs using MALDI mass spectrometry and reverse transcriptase primer extension to identify nucleotide modifications that could influence aminoglycoside interactions. Helices 44 and 45 contain...... indigenous (housekeeping) modifications at m (4)Cm1402, m (3)U1498, m (2)G1516, m (6) 2A1518, and m (6) 2A1519; helix 69 is modified at m (3)Ψ1915, with m (5)U1939 and m (5)C1962 modification in adjacent sequences. All modifications were close to stoichiometric, with the exception of m (3)Ψ1915, where about...... 80% of rRNA molecules were methylated. The modification status of a virulent clinical strain expressing the acquired methyltransferase RmtD was altered in two important respects: RmtD stoichiometrically modified m (7)G1405 conferring high resistance to the aminoglycoside tobramycin and, in doing so...

  12. Diversity of enterococcal species and characterization of high-level aminoglycoside resistant enterococci of samples of wastewater and surface water in Tunisia.

    Science.gov (United States)

    Ben Said, Leila; Klibi, Naouel; Lozano, Carmen; Dziri, Raoudha; Ben Slama, Karim; Boudabous, Abdellatif; Torres, Carmen

    2015-10-15

    One hundred-fourteen samples of wastewater (n=64) and surface-water (n=50) were inoculated in Slanetz-Bartley agar plates supplemented or not with gentamicin (SB-Gen and SB plates, respectively) for enterococci recovery. Enterococci were obtained from 75% of tested samples in SB media (72% in wastewater; 78% in surface-water), and 85 enterococcal isolates (one/positive-sample) were obtained. Enterococcus faecium was the most prevalent species (63.5%), followed by Enterococcus faecalis (20%), Enterococcus hirae (9.4%), Enterococcus casseliflavus (4.7%), and Enterococcus gallinarum/Enterococcus durans (2.4%). Antibiotic resistance detected among these enterococci was as follows [percentage/detected gene (number isolates)]: kanamycin [29%/aph(3')-IIIa (n=22)], streptomycin [8%/ant(6)-Ia (n=4)], erythromycin [44%/erm(B) (n=34)], tetracycline [18%/tet(M) (n=6)/tet(M)-tet(L) (n=9)], chloramphenicol [2%/cat(A) (n=1)], ciprofloxacin [7%] and trimethoprim-sulfamethoxazole [94%]. High-level-gentamicin resistant (HLR-G) enterococci were recovered from 15 samples in SB-Gen or SB plates [12/64 samples of wastewater (19%) and 3/50 samples of surface-water (6%)]; HLR-G isolates were identified as E. faecium (n=7), E. faecalis (n=6), and E. casseliflavus (n=2). These HLR-G enterococci carried the aac(6')-Ie-aph(2")-Ia and erm(B) genes, in addition to aph(3')-IIIa (n=10), ant(6)-Ia (n=9), tet(M) (n=13), tet(L) (n=8) and cat(A) genes (n=2). Three HLR-G enterococci carried the esp virulence gene. Sequence-types detected among HLR-G enterococci were as follows: E. faecalis (ST480, ST314, ST202, ST55, and the new ones ST531 and ST532) and E. faecium (ST327, ST12, ST296, and the new ones ST985 and ST986). Thirty-two different PFGE patterns were detected among 36 high-level-aminoglycoside-resistant enterococci recovered in water samples. Diverse genetic lineages of HLR-G enterococci were detected in wastewater and surface-water in Tunisia. Water can represent an important source for the

  13. Development of aminoglycoside and β-lactamase resistance among intestinal microbiota of swine treated with lincomycin, chlortetracycline, and amoxicillin

    OpenAIRE

    Sun, Jian; Liang LI; Liu, Baotao; Xia, Jing; Liao, Xiaoping; Liu, Yahong

    2014-01-01

    Lincomycin, chlortetracycline, and amoxicillin are commonly used antimicrobials for growth promotion and infectious disease prophylaxis in swine production. In this study, we investigated the shifts and resistance development among intestinal microbiota in pregnant sows before and after lincomycin, chlortetracycline, and amoxicillin treatment by using phylogenetic analysis, bacterial enumeration, and PCR. After the antimicrobial treatment, shifts in microbial community, an increased proportio...

  14. A nanoplex PCR assay for the rapid detection of vancomycin and bifunctional aminoglycoside resistance genes in Enterococcus species

    OpenAIRE

    Ravichandran Manickam; Lalitha Pattabhiraman; Yin Lee; Yean Chan

    2007-01-01

    Abstract Background Enterococci have emerged as a significant cause of nosocomial infections in many parts of the world over the last decade. The most common enterococci strains present in clinical isolates are E. faecalis and E. faecium which have acquired resistant to either gentamicin or vancomycin. The conventional culture test takes 2–5 days to yield complete information of the organism and its antibiotic sensitivity pattern. Hence our present study was focused on developing a nanoplex P...

  15. Assessment of hearing loss in multi-drug resistant tuberculosis (MDR-TB) patients undergoing Aminoglycoside treatment

    OpenAIRE

    Sheikh Nizamuddin; Farhan Ahmad Khan; Abdur Rehman Khan; Chand Miyan Kamaal

    2015-01-01

    Background: Incomplete treatments and treatment failures has led to Multi-drug resistant tuberculosis, which has emerged as a significant problem in treating tuberculosis and thus the second line drugs are used with the concomitant increase in the incidence of adverse effects. Methods: This prospective study was carried out from June 2009 to May 2014 in the department of ENT in collaboration with TB and Chest at Teerthanker Mahaveer Medical College and Research Centre. Out of 104, ...

  16. DNA-Aptamers Binding Aminoglycoside Antibiotics

    Directory of Open Access Journals (Sweden)

    Nadia Nikolaus

    2014-02-01

    Full Text Available Aptamers are short, single stranded DNA or RNA oligonucleotides that are able to bind specifically and with high affinity to their non-nucleic acid target molecules. This binding reaction enables their application as biorecognition elements in biosensors and assays. As antibiotic residues pose a problem contributing to the emergence of antibiotic-resistant pathogens and thereby reducing the effectiveness of the drug to fight human infections, we selected aptamers targeted against the aminoglycoside antibiotic kanamycin A with the aim of constructing a robust and functional assay that can be used for water analysis. With this work we show that aptamers that were derived from a Capture-SELEX procedure targeting against kanamycin A also display binding to related aminoglycoside antibiotics. The binding patterns differ among all tested aptamers so that there are highly substance specific aptamers and more group specific aptamers binding to a different variety of aminoglycoside antibiotics. Also the region of the aminoglycoside antibiotics responsible for aptamer binding can be estimated. Affinities of the different aptamers for their target substance, kanamycin A, are measured with different approaches and are in the micromolar range. Finally, the proof of principle of an assay for detection of kanamycin A in a real water sample is given.

  17. Cancer stem cell overexpression of nicotinamide N-methyltransferase enhances cellular radiation resistance

    DEFF Research Database (Denmark)

    D’Andrea, Filippo P.; Safwat, Akmal; Kassem, Moustapha; Gautier, Laurent; Overgaard, Jens; Horsman, Michael R.

    2011-01-01

    BackgroundCancer stem cells are thought to be a radioresistant population and may be the seeds for recurrence after radiotherapy. Using tumorigenic clones of retroviral immortalized human mesenchymal stem cell with small differences in their phenotype, we investigated possible genetic expression...... analysis found the genes involved in cancer, proliferation, DNA repair and cell death. ConclusionsThe higher radiation resistance in clone CE8 is likely due to NNMT overexpression. The higher levels of NNMT could affect the cellular damage resistance through depletion of the accessible amounts of...... nicotinamide, which is a known inhibitor of cellular DNA repair mechanisms....

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

    DEFF Research Database (Denmark)

    Mann, P. A.; Xiong, L.; Mankin, A. S.; Chau, A. S.; Najarian, D. J.; Mendrick, C. A.; Cramer, C. A.; Aarestrup, Frank Møller; Hare, R. S.; Black, T. A.; McNicholas, P. M.

    2001-01-01

    unique to the 23S rRNA extracted from resistant ribosomes. The pause corresponded to methylation of residue G2470 (Escherichia coli numbering). RNA footprinting revealed that G2470 is located within the evernimicin-binding site on the ribosome, thus providing an explanation for the reduced binding of the...

  19. A human tRNA methyltransferase 9-like protein prevents tumour growth by regulating LIN9 and HIF1-α.

    Science.gov (United States)

    Begley, Ulrike; Sosa, Maria Soledad; Avivar-Valderas, Alvaro; Patil, Ashish; Endres, Lauren; Estrada, Yeriel; Chan, Clement T Y; Su, Dan; Dedon, Peter C; Aguirre-Ghiso, Julio A; Begley, Thomas

    2013-03-01

    Emerging evidence points to aberrant regulation of translation as a driver of cell transformation in cancer. Given the direct control of translation by tRNA modifications, tRNA modifying enzymes may function as regulators of cancer progression. Here, we show that a tRNA methyltransferase 9-like (hTRM9L/KIAA1456) mRNA is down-regulated in breast, bladder, colorectal, cervix and testicular carcinomas. In the aggressive SW620 and HCT116 colon carcinoma cell lines, hTRM9L is silenced and its re-expression and methyltransferase activity dramatically suppressed tumour growth in vivo. This growth inhibition was linked to decreased proliferation, senescence-like G0/G1-arrest and up-regulation of the RB interacting protein LIN9. Additionally, SW620 cells re-expressing hTRM9L did not respond to hypoxia via HIF1-α-dependent induction of GLUT1. Importantly, hTRM9L-negative tumours were highly sensitive to aminoglycoside antibiotics and this was associated with altered tRNA modification levels compared to antibiotic resistant hTRM9L-expressing SW620 cells. Our study links hTRM9L and tRNA modifications to inhibition of tumour growth via LIN9 and HIF1-α-dependent mechanisms. It also suggests that aminoglycoside antibiotics may be useful to treat hTRM9L-deficient tumours. PMID:23381944

  20. Characterization of Resistance to Aminoglycosides in Methicillin-Resistant Staphylococcus aureus Strains Isolated From a Tertiary Care Hospital in Tehran, Iran

    OpenAIRE

    Rahimi, Fateh

    2016-01-01

    Background: Methicillin-resistant Staphylococcus aureus (MRSA) is one of the most common nosocomial pathogens which can cause a broad spectrum of infections. Objectives: The current study aimed to describe the frequency and antibiotic susceptibility patterns of clonal groups of gentamicin-resistant strains of MRSA isolated from a tertiary care hospital in Tehran, Iran. Materials and Methods: A total of 301 S. aureus isolates were collected during January to November 2012. All of the isolates ...

  1. Identification of a novel 6'-N-aminoglycoside acetyltransferase, AAC(6')-Iak, from a multidrug-resistant clinical isolate of Stenotrophomonas maltophilia.

    Science.gov (United States)

    Tada, Tatsuya; Miyoshi-Akiyama, Tohru; Dahal, Rajan K; Mishra, Shyam K; Shimada, Kayo; Ohara, Hiroshi; Kirikae, Teruo; Pokhrel, Bharat M

    2014-10-01

    Stenotrophomonas maltophilia IOMTU250 has a novel 6'-N-aminoglycoside acetyltransferase-encoding gene, aac(6')-Iak. The encoded protein, AAC(6')-Iak, consists of 153 amino acids and has 86.3% identity to AAC(6')-Iz. Escherichia coli transformed with a plasmid containing aac(6')-Iak exhibited decreased susceptibility to arbekacin, dibekacin, neomycin, netilmicin, sisomicin, and tobramycin. Thin-layer chromatography showed that AAC(6')-Iak acetylated amikacin, arbekacin, dibekacin, isepamicin, kanamycin, neomycin, netilmicin, sisomicin, and tobramycin but not apramycin, gentamicin, or lividomycin. PMID:25092711

  2. Protein arginine methyltransferase 1 may be involved in pregnane x receptor-activated overexpression of multidrug resistance 1 gene during acquired multidrug resistant

    Science.gov (United States)

    Li, Tingting; Kong, Ah-Ng Tony; Ma, Zhiqiang; Liu, Haiyan; Liu, Pinghua; Xiao, Yu; Jiang, Xuehua; Wang, Ling

    2016-01-01

    Purpose Pregnane x receptor (PXR) - activated overexpression of the multidrug resistance 1 (MDR1) gene is an important way for tumor cells to acquire drug resistance. However, the detailed mechanism still remains unclear. In the present study, we aimed to investigate whether protein arginine methyl transferase 1(PRMT1) is involved in PXR - activated overexpression of MDR1 during acquired multidrug resistant. Experimental Design Arginine methyltransferase inhibitor 1 (AMI-1) was used to pharmacologically block PRMT1 in resistant breast cancer cells (MCF7/adr). The mRNA and protein levels of MDR1 were detected by real-time PCR and western blotting analysis. Immunofluorescence microscopy and co-immunoprecipitation were used to investigate the physical interaction between PXR and PRMT1. Then, 136 candidate compounds were screened for PRMT1 inhibitors. Lastly, luciferase reporter gene and nude mice bearing resistant breast cancer xenografts were adopted to investigate the anti-tumor effect of PRMT1 inhibitors when combined with adriamycin. Results AMI-1 significantly suppressed the expression of MDR1 in MCF7/adr cells and increased cells sensitivity of MCF7/adr to adriamycin. Physical interaction between PRMT1 and PXR exists in MCF7/adr cells, which could be disrupted by AMI-1. Those results suggest that PRMT1 may be involved in PXR-activated overexpression of MDR1 in resistant breast cancer cells, and AMI-1 may suppress MDR1 by disrupting the interaction between PRMT1 and PXR. Then, five compounds including rutin, isoquercitrin, salvianolic acid A, naproxen, and felodipline were identified to be PRMT1 inhibitors. Finally, those PRMT1 inhibitors were observed to significantly decrease MDR1 promoter activity in vitro and enhance the antitumor effect of adriamycin in nude mice that bearing resistant breast cancer xenografts. Conclusions PRMT1 may be an important co-activator of PXR in activating MDR1 gene during acquired resistance, and PRMT1 inhibitor combined with

  3. An integrated epigenetic and genetic analysis of DNA methyltransferase genes (DNMTs) in tumor resistant and susceptible chicken lines

    Science.gov (United States)

    Both epigenetic alterations and genetic variations play essential roles in tumorigenesis. The epigenetic modification of DNA methylation is catalyzed and maintained by the DNA methyltransferases (DNMT3a, DNMT3b and DNMT1). DNA mutations and DNA methylation profiles of DNMTs themselves and their rela...

  4. Kinetic and Structural Analysis of Bisubstrate Inhibition of the Salmonella enterica Aminoglycoside 6′-N-Acetyltransferase†,‡

    OpenAIRE

    Magalhães, Maria L. B.; Vetting, Matthew W.; Gao, Feng; Freiburger, Lee; Auclair, Karine; Blanchard, John S.

    2007-01-01

    Aminoglycosides are antibacterial compounds that act by binding to the A site of the small 30S bacterial ribosomal subunit and inhibiting protein translation. Clinical resistance to aminoglycosides is generally the result of the expression of enzymes that covalently modify the antibiotic, including phosphorylation, adenylylation, and acetylation. Bisubstrate analogs for the aminoglycoside N-acetyl-transferases are nanomolar inhibitors of Enterococcus faecium AAC(6′)-Ii. However, in the case o...

  5. Study of aminoglycoside modifying enzyme associated resistance genes in Enterococcus%产氨基糖苷类修饰酶肠球菌临床分离株相关耐药基因的研究

    Institute of Scientific and Technical Information of China (English)

    姚杰; 徐元宏; 王友梅; 刘灿

    2011-01-01

    目的 了解临床分离的肠球菌对高水平氨基糖苷类抗菌药物的耐药情况并对其氨基糖苷类修饰酶基因进行检测.方法 用琼脂稀释法检测112株粪肠球菌和118株屎肠球菌对高浓度庆大霉素(500 μg/ml)和高浓度链霉素(2 000 μg/ml)的最低抑菌浓度,随机选取氨基糖苷类高水平耐药的粪肠球菌和屎肠球菌各48株进行氨基糖苷类修饰酶基因检测,PCR扩增aac(6')/aph(2″)、aph(3')-Ⅲ和ant(6)-Ⅰ基因,并对其进行测序分析.结果 112株粪肠球菌和118株屎肠球菌对高浓度庆大霉素的耐药率分别为53.6%和78.8%,对高浓度链霉素的耐药率分别为39.3%和64.4%,且粪肠球菌与屎肠球菌的耐药率差异有统计学意义(P<0.01).aac(6')/aph(2″)基因阳性分别为38株和45株,占80.2%和93.8%;aph(3')-Ⅲ基因阳性分别为14株和19株,占29.2%和39.6%;ant(6)-Ⅰ基因阳性分别为15株和21株,占31.3%和43.8%.其中同时检测出两种和两种以上氨基糖苷类修饰酶基因的菌株有40株,高达41.7%.结论 氨基糖苷类高水平耐药肠球菌已成为医院感染的重要耐药菌,AAC(6')/APH(2″)酶的产生是肠球菌最为常见的氨基糖苷类耐药机制.%Objective To investigate enterococci isolated from clinical specimens on the high level of aminoglycoside antibiotic resistance and the detection of aminoglycoside modifying enzyme gene. Methods 112 strains of E.faecalis and 118 strains of E. faecium to the high-level gentamicin(500 μg/ml)and high-level of streptomycin (2 000 μg/ml)minimum inhibitory concentration were detected by agar ditution method. The aac (6')/aph (2"),aph(3')-Ⅲ and ant(6)- Ⅰ genes were amplified by PCR. Finally,their genotype were determined by DNA sequencing. Results 112 strains of E. faecalis and 118 strains of E. faecium HLGR rates were 53. 1% and 73.2%, and HLSR rates were 38. 9% and 59. 8%. The drug resistance of the two species to high-level gentamicin and high

  6. Environmental and genetic factors affecting mutability to aminoglycoside antibiotics among Escherichia coli K12 strains

    Directory of Open Access Journals (Sweden)

    Monteiro A.C.M.

    2003-01-01

    Full Text Available Environmental and genetic factors affecting the in vitro spontaneous mutation frequencies to aminoglycoside resistance in Escherichia coli K12 were investigated. Spontaneous mutation frequencies to kanamycin resistance were at least 100 fold higher on modified Luria agar (L2 plates, when compared to results obtained in experiments carried out with Nutrient agar (NA plates. In contrast to rifampincin, the increased mutability to kanamycin resistance could not be attributed to a mutator phenotype expressed by DNA repair defective strains. Kanamycin mutant selection windows and mutant preventive concentrations on L2 plates were at least fourfold higher than on NA plates, further demonstrating the role of growth medium composition on the mutability to aminoglycosides. Mutability to kanamycin resistance was increased following addition of sorbitol, suggesting that osmolarity is involved on the spontaneous mutability of E. coli K12 strains to aminoglycosides. The spontaneous mutation rates to kanamycin resistance on both L2 and NA plates were strictly associated with the selective antibiotic concentrations. Moreover, mutants selected at different antibiotic concentrations expressed heterogeneous resistance levels to kanamycin and most of them expressing multiple resistance to all tested aminoglycoside antibiotics (gentamicin, neomycin, amykacin and tobramycin. These results will contribute to a better understanding of the complex nature of aminoglycoside resistance and the emergence of spontaneous resistant mutants among E. coli K12 strains.

  7. Minimum inhibitory concentration values and problematic disk break points of tigecycline against vancomycin and/or high-level aminoglycoside-resistant enterococci

    OpenAIRE

    Latife İşeri; Esra Şahin; İştar Dolapçı; Zehra Yürüken

    2016-01-01

    Background: Tigecycline is a new, semisynthetic glycylcycline. It is active against important multidrug resistant pathogens. Aim: The purpose of this study was to investigate the sensitivity of multidrug-resistant enterococci to tigecycline, and to test the correlation between the minimal inhibitory concentration (MIC) and disk diffusion methods. Materials and methods: The antimicrobial sensitivity of 108 multidrug-resistant isolates, which included 52 vancomycin-resistant enterococci (...

  8. Triclosan-Induced Aminoglycoside-Tolerant Listeria monocytogenes Isolates Can Appear as Small-Colony Variants

    DEFF Research Database (Denmark)

    Kastbjerg, Vicky Gaedt; Hein-Kristensen, Line; Gram, Lone

    2014-01-01

    Exposure of the human food-borne pathogen Listeria monocytogenes to sublethal concentrations of triclosan can cause resistance to several aminoglycosides. Aminoglycoside-resistant isolates exhibit two colony morphologies: normal-size and pinpoint colonies. The purposes of the present study were to...... heme gene, and addition of heme caused the pinpoint isolates to revert to normal colony size. Triclosan-induced gentamicin-resistant isolates had mutations in several different genes, and it cannot be directly concluded how the different mutations caused gentamicin resistance. However, since many of...... the mutations affected proteins involved in respiration, it seems likely that the mutations affected the active transport of the antibiotic and thereby caused resistance by decreasing the amount of aminoglycoside that enters the bacterial cell. Our study emphasizes that triclosan likely has more...

  9. An usual approach to treatment of a case of multidrug resistance Pseudomonas aeruginosa peritonitis: parenteral and intraperitoneal aminoglycosides and parenteral colistin

    OpenAIRE

    Ian May; Maha Abu-Khdeir; Roland Alexander Blackwood

    2012-01-01

    Infections caused by Pseudomonas aeruginosa are becoming more common and increasingly more difficult to treat due to the continued development of drug resistance. While sensitivity to colistin (polymyxin E) is well known, it is frequently avoided due to concerns of nephrotoxicity. Reported here is a case of a multi-drug resistance pseudomonal typhlitis, bacteremia and pleural cavity infection that required significant intensive care, and serial abdominal washouts. Intra-peritoneal tobramycin ...

  10. Once-daily aminoglycoside therapy: potential ototoxicity.

    OpenAIRE

    Kirkpatrick, C. M.; Duffull, S. B.; Begg, E J

    1996-01-01

    Current data indicate that once-daily aminoglycoside therapy is as efficacious as traditional multiple daily dosing and equally or less toxic. Our experience with once-daily gentamicin, 6 mg/kg of body weight led to a 10% (3 of 33 patients) occurrence of documented ototoxicity after prolonged aminoglycoside exposure.

  11. An usual approach to treatment of a case of multidrug resistance Pseudomonas aeruginosa peritonitis: parenteral and intraperitoneal aminoglycosides and parenteral colistin

    Directory of Open Access Journals (Sweden)

    Ian May

    2012-09-01

    Full Text Available Infections caused by Pseudomonas aeruginosa are becoming more common and increasingly more difficult to treat due to the continued development of drug resistance. While sensitivity to colistin (polymyxin E is well known, it is frequently avoided due to concerns of nephrotoxicity. Reported here is a case of a multi-drug resistance pseudomonal typhlitis, bacteremia and pleural cavity infection that required significant intensive care, and serial abdominal washouts. Intra-peritoneal tobramycin in combination with broad-spectrum intravenous antibiotics including colistin were used. Several instillations of tobramycin into the abdominal cavity along with concomitant IV administration of colistin, ceftazidime and tobramycin and per os colistin, tobramycin and nystatin resulted in the clearance of the pseudomonal infection without any evidence of toxicity from the treatment. Intra-abdominal tobramycin with parenteral colistin therapy can be used in complicated clinical settings with appropriate nephroprotection.

  12. Cholera in Vietnam: changes in genotypes and emergence of class I integrons containing aminoglycoside resistance gene cassettes in vibrio cholerae O1 strains isolated from 1979 to 1996.

    Science.gov (United States)

    Dalsgaard, A; Forslund, A; Tam, N V; Vinh, D X; Cam, P D

    1999-03-01

    The number of cholera cases and the mortality rates reported from different regions of Vietnam varied considerably in the period from 1979 to 1996, with between 2,500 and 6,000 cases reported annually from 1992 to 1995. Annual mortality rates ranged from 2.0 to 9.6% from 1979 to 1983 to less than 1.8% after 1983. Major cholera outbreaks were reported from the High Plateau region for the first time in 1994 and 1995; this is an area with limited access to health services and safe drinking-water supplies. All cases were associated with Vibrio cholerae O1. Using ribotyping, cholera toxin (CT) genotyping, and characterization of antibiotic susceptibility patterns and antibiotic resistance genes by PCR, we show that strains isolated after 1990 were clearly different from strains isolated before 1991. In contrast to strains isolated before 1991, 94% of 104 strains isolated after 1990 showed an identical ribotype R1, were resistant to sulfamethoxazole and streptomycin, and showed a different CT genotype. Furthermore, PCR analysis revealed that sulfamethoxazole-resistant strains harbored class I integrons containing a gene cassette ant(3")-1a encoding resistance to streptomycin and spectinomycin. This is, to our knowledge, the first report of class I integrons in V. cholerae. The development of cholera and the changes in the phenotypic and genotypic properties of V. cholerae O1 shown in the present study highlight the importance of monitoring V. cholerae O1 in Vietnam as in other parts of the world. In particular, the emergence of the new ribotype R1 strain containing class I integrons should be further studied. PMID:9986842

  13. Resistance to ketolide antibiotics by coordinated expression of rRNA methyltransferases in a bacterial producer of natural ketolides

    DEFF Research Database (Denmark)

    Almutairi, Mashal M; Park, Sung Ryeol; Rose, Simon;

    2015-01-01

    Ketolides are promising new antimicrobials effective against a broad range of Gram-positive pathogens, in part because of the low propensity of these drugs to trigger the expression of resistance genes. A natural ketolide pikromycin and a related compound methymycin are produced by Streptomyces...... activation by ketolide antibiotics. The resistance genes and the induction mechanism remain fully functional when transferred to heterologous bacterial hosts. The anticipated wide use of ketolide antibiotics could promote horizontal transfer of these highly efficient resistance genes to pathogens. Taken...... together, these findings emphasized the need for surveillance of pikR1/pikR2-based bacterial resistance and the preemptive development of drugs that can remain effective against the ketolide-specific resistance mechanism....

  14. Aminoglycoside microarrays to explore interactions of antibiotics with RNAs and proteins.

    Science.gov (United States)

    Disney, Matthew D; Seeberger, Peter H

    2004-07-01

    RNA is an important target for drug discovery efforts. Several clinically used aminoglycoside antibiotics bind to bacterial rRNA and inhibit protein synthesis. Aminoglycosides, however, are losing efficacy due to their inherent toxicity and the increase in antibiotic resistance. Targeting of other RNAs is also becoming more attractive thanks to the discovery of new potential RNA drug targets through genome sequencing and biochemical efforts. Identification of new compounds that target RNA is therefore urgent, and we report here on the development of rapid screening methods to probe binding of low molecular weight ligands to proteins and RNAs. A series of aminoglycosides has been immobilized onto glass microscope slides, and binding to proteins and RNAs has been detected by fluorescence. Construction and analysis of the arrays is completed by standard DNA genechip technology. Binding of immobilized aminoglycosides to proteins that are models for study of aminoglycoside toxicity (DNA polymerase and phospholipase C), small RNA oligonucleotide mimics of aminoglycoside binding sites in the ribosome (rRNA A-site mimics), and a large (approximately 400 nucleotide) group I ribozyme RNA is detected. The ability to screen large RNAs alleviates many complications associated with binding experiments that use isolated truncated regions from larger RNAs. These studies lay the foundation for rapid identification of small organic ligands from combinatorial libraries that exhibit strong and selective RNA binding while displaying decreased affinity to toxicity-causing proteins. PMID:15224340

  15. O6-methylguanine-DNA-methyltransferase and DNA mismatch repair in relation to drug resistance in malignant melanoma

    OpenAIRE

    Ma, Shuhua

    2004-01-01

    Although the survival rate of patients with malignant melanoma has improved, treatment of the disease poses large problems since disseminated melanoma frequently shows primary resistance to chemotherapy. Resistance to chemotherapy is thus a major clinical problem and a cause of failure in the treatment of metastatic malignant melanoma. The primary aim of this thesis was to investigate whether the DNA repair protein 06-methylguanineDNA-metbyltransferase (MGMT) and the post...

  16. Multiple ESBL-Producing Escherichia coli Sequence Types Carrying Quinolone and Aminoglycoside Resistance Genes Circulating in Companion and Domestic Farm Animals in Mwanza, Tanzania, Harbor Commonly Occurring Plasmids.

    Science.gov (United States)

    Seni, Jeremiah; Falgenhauer, Linda; Simeo, Nabina; Mirambo, Mariam M; Imirzalioglu, Can; Matee, Mecky; Rweyemamu, Mark; Chakraborty, Trinad; Mshana, Stephen E

    2016-01-01

    The increased presence of extended-spectrum beta-lactamase (ESBL)-producing bacteria in humans, animals, and their surrounding environments is of global concern. Currently there is limited information on ESBL presence in rural farming communities worldwide. We performed a cross-sectional study in Mwanza, Tanzania, involving 600 companion and domestic farm animals between August/September 2014. Rectal swab/cloaca specimens were processed to identify ESBL-producing Enterobacteriaceae. We detected 130 (21.7%) animals carrying ESBL-producing bacteria, the highest carriage being among dogs and pigs [39.2% (51/130) and 33.1% (43/130), respectively]. The majority of isolates were Escherichia coli [93.3% (125/134)] and exotic breed type [OR (95%CI) = 2.372 (1.460-3.854), p-value animals. Whole-genome sequences of 25 ESBL-producing E. coli were analyzed for phylogenetic relationships using multi-locus sequence typing (MLST) and core genome comparisons. Fourteen different sequence types were detected of which ST617 (7/25), ST2852 (3/25), ST1303 (3/25) were the most abundant. All isolates harbored the bla CTX-M-15 allele, 22/25 carried strA and strB, 12/25 aac(6')-lb-cr, and 11/25 qnrS1. Antibiotic resistance was associated with IncF, IncY, as well as non-typable plasmids. Eleven isolates carried pPGRT46-related plasmids, previously reported from isolates in Nigeria. Five isolates had plasmids exhibiting 85-99% homology to pCA28, previously detected in isolates from the US. Our findings indicate a pan-species distribution of ESBL-producing E. coli clonal groups in farming communities and provide evidence for plasmids harboring antibiotic resistances of regional and international impact. PMID:26904015

  17. 耐药大肠埃希菌β-内酰胺类、氨基糖苷类获得性耐药基因与可移动遗传元件研究%Investigation of acquired resistance genes to β-lactams, aminoglycosides and mobile genetic elements in multidrug-resistant Escherichia coli

    Institute of Scientific and Technical Information of China (English)

    张建明; 万长标; 钱玮和; 程建平; 金跃

    2011-01-01

    目的 调查多药耐药大肠埃希菌β-内酰胺类、氨基糖苷类获得性耐药基因与可移动遗传元件的存在情况.方法 收集医院2007年1-12月患者标本中分离的20株多药耐药大肠埃希菌,采用聚合酶链反应(PCR)的方法,分析42种β-内酰胺类、氨基糖苷类获得性耐药基因以及10种可移动遗传元件的遗传标记;并对上述检测结果做样本聚类分析.结果20 株多药耐药大肠埃希菌检出β-内酰胺类获得性耐药基因TEM-1及CTX-M-55,检出率为80.0%、50.0%,氨基糖苷类获得性耐药基因aac(3)-Ⅱ、aac(6’)-Ⅰb、ant(3")-Ⅰ、aadA5、aph(3’)-Ⅰ,检出率分别为45.0%、5.0%、10.0%、45.0%、5.0%,可移动遗传元件检出intⅠ 1、tnp513、IS26、ISEcp1、traA、trbC,检出率分别为55.0%、25.0%、90.0%、65.0%、80.0%、75.0%;其他39种基因未检出.结论 20株多药耐药大肠埃希菌耐β-内酰胺类、氨基糖苷类药物与细菌产2种β-内酰胺类获得性耐药基因、5种氨基糖苷类获得性耐药基因相关;可移动遗传元件的水平转移使细菌的耐药性在同种细菌菌株之间甚至不同种细菌菌株之间得以快速传播;样本聚类分析提示,该组多药耐药大肠埃希菌尚无克隆传播.%OBJECTIVE To investigate the distribution of acquired resistance genes to [3-lactams, aminoglycosides and mobile genetic elements in multidrug-resistant Escherichia coli (E. Coli). METHODS From Jan 2007 to Dec 2007, 20 strains of E. Coli were collected from The Second People's Hospital of Huaian. Then, 42 kinds of acquired resistance genes to β-lactams, aminoglycosides and 10 kinds of genetic markers of mobile genetic elements were analyzed by PCR. In addition, sample cluster analysis was performed. RESULTS In 20 strains of multidrug-resistant E. Coli, acquired resistance genes to β-actams: TEM-1, CTX-M-55, acquired resistance genes to aminoglycosides! Aac(3)-II , aac(6')-I b, ant(3")-I

  18. The prevalence of aminoglycoside-modifying enzyme genes (aac (6′)-I, aac (6′)-II, ant (2″)-I, aph (3′)-VI) in Pseudomonas aeruginosa

    OpenAIRE

    Farzam Vaziri; Shahin Najar Peerayeh; Qorban Behzadian Nejad; Abbas Farhadian

    2011-01-01

    INTRODUCTION: Pseudomonas aeruginosa (P. aeruginosa) is one of the primary opportunistic pathogens responsible for nosocomial infections. Aminoglycosides are an import ant component of antipseudomonal chemotherapy. The inactivation of drugs by modifying enzymes is the most common mechanism of aminoglycoside resistance. OBJECTIVES: The inactivation of aminoglycosides by modifying enzymes is the primary resistance mechanism employed by P. aeruginosa. The aim of the present study was to investig...

  19. Multiple ESBL-producing Escherichia coli sequence types carrying quinolone and aminoglycoside resistance genes circulating in companion and domestic farm animals in Mwanza, Tanzania, harbor commonly occurring plasmids

    Directory of Open Access Journals (Sweden)

    Jeremiah eSeni

    2016-02-01

    Full Text Available The increased presence of extended-spectrum beta-lactamase (ESBL-producing bacteria in humans, animals and their surrounding environments is of global concern. Currently there is limited information on ESBL presence in rural farming communities worldwide. We performed a cross-sectional study in Mwanza, Tanzania, involving 600 companion and domestic farm animals between August/September 2014. Rectal swab/cloaca specimens were processed to identify ESBL-producing Enterobacteriaceae. We detected 130 (21.7% animals carrying ESBL-producing bacteria, the highest carriage being among dogs and pigs [39.2% (51/130 and 33.1% (43/130, respectively]. The majority of isolates were Escherichia coli [93.3% (125/134] and exotic breed type [OR (95%CI = 2.372 (1.460 - 3.854, p-value <0.001] was found to be a predictor of ESBL carriage among animals. Whole-genome sequences of 25 ESBL-producing E. coli were analyzed for phylogenetic relationships using multi-locus sequence typing (MLST and core genome comparisons. Fourteen different sequence types were detected of which ST617 (7/25, ST2852 (3/25, ST1303 (3/25 were the most abundant. All isolates harbored the blaCTX-M-15 allele, 22/25 carried strA and strB, 12/25 aac(6’Ib-cr and 11/25 qnrS1. Antibiotic resistance was associated with IncF, IncY, as well as non-typable plasmids. Eleven isolates carried pPGRT46-related plasmids, previously reported from isolates in Nigeria. Five isolates had plasmids exhibiting 85-99% homology to pCA28, previously detected in isolates from the United States. Our findings indicate a pan-species distribution of ESBL-producing E. coli clonal groups in farming communities and provide evidence for plasmids harboring antibiotic resistances of regional and international impact.

  20. Structural basis for S-adenosylmethionine binding and methyltransferase activity by mitochondrial transcription factor B1

    OpenAIRE

    Guja, Kip E.; Venkataraman, Krithika; Yakubovskaya, Elena; Hui SHI; Mejia, Edison; Hambardjieva, Elena; Karzai, A. Wali; Garcia-Diaz, Miguel

    2013-01-01

    Eukaryotic transcription factor B (TFB) proteins are homologous to KsgA/Dim1 ribosomal RNA (rRNA) methyltransferases. The mammalian TFB1, mitochondrial (TFB1M) factor is an essential protein necessary for mitochondrial gene expression. TFB1M mediates an rRNA modification in the small ribosomal subunit and thus plays a role analogous to KsgA/Dim1 proteins. This modification has been linked to mitochondrial dysfunctions leading to maternally inherited deafness, aminoglycoside sensitivity and di...

  1. Effects of F171 Mutations in the 6′-N-Acetyltransferase Type Ib [AAC(6′)-Ib] Enzyme on Susceptibility to Aminoglycosides

    OpenAIRE

    Chavideh, Ramona; Sholly, Steven; Panaite, Doina; Tolmasky, Marcelo E.

    1999-01-01

    Substitutions at position F171 of 6′-N-acetyltransferase type Ib cause variable loss of aminoglycoside resistance, indicating that this residue plays an important role in the structure and/or function of the enzyme.

  2. Synthesis of Lysine Methyltransferase Inhibitors

    Science.gov (United States)

    Ye, Tao; Hui, Chunngai

    2015-07-01

    Lysine methyltransferase which catalyze methylation of histone and nonhistone proteins, play a crucial role in diverse biological processes and has emerged as a promising target for the development of various human diseases, including cancer, inflammation, and psychiatric disorders. However, inhibiting Lysine methyltransferases selectively has presented many challenges to medicinal chemists. During the past decade, lysine methyltransferase inhibitors covering many different structural classes have been designed and developed. In this review, we describe the development of selective, small-molecule inhibitors of lysine methyltransferases with an emphasis on their discovery and chemical synthesis. We highlight the current state of lysine methyltransferase inhibitors and discuss future directions and opportunities for lysine methyltransferase inhibitor discovery.

  3. The prevalence of aminoglycoside-modifying enzyme genes (aac (6'-I, aac (6'-II, ant (2"-I, aph (3'-VI in Pseudomonas aeruginosa

    Directory of Open Access Journals (Sweden)

    Farzam Vaziri

    2011-01-01

    Full Text Available INTRODUCTION: Pseudomonas aeruginosa (P. aeruginosa is one of the primary opportunistic pathogens responsible for nosocomial infections. Aminoglycosides are an import ant component of antipseudomonal chemotherapy. The inactivation of drugs by modifying enzymes is the most common mechanism of aminoglycoside resistance. OBJECTIVES: The inactivation of aminoglycosides by modifying enzymes is the primary resistance mechanism employed by P. aeruginosa. The aim of the present study was to investigate the occurrence of aminoglycoside resistance and the prevalence of four import ant modifying enzyme genes (aac (6'-I, aac (6'-II, ant (2"-I, aph (3'-VI in P. aeruginosa in Iran. METHODS: A total of 250 clinical isolates of P. aeruginosa were collected from several hospitals in seven cities in Iran. Antimicrobial susceptibility tests (using the disk diffusion method and E-tests were performed for all 250 isolates. In addition, all isolates were screened for the presence of modifying enzyme genes by polymerase chain reaction. RESULTS: The resistance rates, as determined by the disk diffusion method, were as follows: gentamicin 43%, tobramycin 38%, and amikacin 24%. Of the genes examined, aac (6'-II (36% was the most frequently identified gene in phenotypic resist ant isolates, followed by ant (2"-I, aph (3'-VI, and aac (6'-I. CONCLUSIONS: Aminoglycoside resistance in P. aeruginosa remains a signific ant problem in Iran. Therefore, there is considerable local surveillance of aminoglycoside resistance.

  4. Aminoglycoside Efflux in Pseudomonas aeruginosa: Involvement of Novel Outer Membrane Proteins

    OpenAIRE

    Jo, James T. H.; Brinkman, Fiona S.L.; Hancock, Robert E W

    2003-01-01

    The expression of tripartite multidrug efflux pumps such as MexA-MexB-OprM in Pseudomonas aeruginosa contributes to intrinsic resistance to a wide variety of antimicrobials, including β-lactams, chloramphenicol, macrolides, quinolones, and tetracycline. The MexX-MexY linker-pump combination has been shown to be involved in intrinsic resistance to aminoglycosides, but the identity of the cognate outer membrane channel component remains under debate. Fourteen uncharacterized OprM homologs ident...

  5. Physiological and Molecular Pathology of Aminoglycoside Ototoxicity

    Science.gov (United States)

    Sha, Su-Hua

    2005-01-01

    The problem of aminoglycoside-induced ototoxicity, which was recognized within a year of the discovery of streptomycin to combat tuberculosis in 1944, is still of great concern due to the widespread use of these powerful antibacterial agents. These drugs can damage to varying degrees the cochlea and vestibular system. Their primary targets are the…

  6. Selective condensation of DNA by aminoglycoside antibiotics.

    Science.gov (United States)

    Kopaczynska, M; Schulz, A; Fraczkowska, K; Kraszewski, S; Podbielska, H; Fuhrhop, J H

    2016-05-01

    The condensing effect of aminoglycoside antibiotics on the structure of double-stranded DNA was examined. The selective condensation of DNA by small molecules is an interesting approach in biotechnology. Here, we present the interaction between calf thymus DNA and three types of antibiotic molecules: tobramycin, kanamycin, and neomycin. Several techniques were applied to study this effect. Atomic force microscopy, transmission electron microscopy images, and nuclear magnetic resonance spectra showed that the interaction of tobramycin with double-stranded DNA caused the rod, toroid, and sphere formation and very strong condensation of DNA strands, which was not observed in the case of other aminoglycosides used in the experiment. Studies on the mechanisms by which small molecules interact with DNA are important in understanding their functioning in cells, in designing new and efficient drugs, or in minimizing their adverse side effects. Specific interactions between tobramycin and DNA double helix was modeled using molecular dynamics simulations. Simulation study shows the aminoglycoside specificity to bend DNA double helix, shedding light on the origins of toroid formation. This phenomenon may lighten the ototoxicity or nephrotoxicity issues, but also other adverse reactions of aminoglycoside antibiotics in the human body. PMID:26646261

  7. Modulation of RNA function by aminoglycoside antibiotics.

    Science.gov (United States)

    Schroeder, R; Waldsich, C; Wank, H

    2000-01-01

    One of the most important families of antibiotics are the aminoglycosides, including drugs such as neomycin B, paromomycin, gentamicin and streptomycin. With the discovery of the catalytic potential of RNA, these antibiotics became very popular due to their RNA-binding capacity. They serve for the analysis of RNA function as well as for the study of RNA as a potential therapeutic target. Improvements in RNA structure determination recently provided first insights into the decoding site of the ribosome at high resolution and how aminoglycosides might induce misreading of the genetic code. In addition to inhibiting prokaryotic translation, aminoglycosides inhibit several catalytic RNAs such as self-splicing group I introns, RNase P and small ribozymes in vitro. Furthermore, these antibiotics interfere with human immunodeficiency virus (HIV) replication by disrupting essential RNA-protein contacts. Most exciting is the potential of many RNA-binding antibiotics to stimulate RNA activities, conceiving small-molecule partners for the hypothesis of an ancient RNA world. SELEX (systematic evolution of ligands by exponential enrichment) has been used in this evolutionary game leading to small synthetic RNAs, whose NMR structures gave valuable information on how aminoglycosides interact with RNA, which could possibly be used in applied science. PMID:10619838

  8. Structural Analysis of a Putative Aminoglycoside N-Acetyltransferase from Bacillus anthracis

    Energy Technology Data Exchange (ETDEWEB)

    Klimecka, Maria M.; Chruszcz, Maksymilian; Font, Jose; Skarina, Tatiana; Shumilin, Igor; Onopryienko, Olena; Porebski, Przemyslaw J.; Cymborowski, Marcin; Zimmerman, Matthew D.; Hasseman, Jeremy; Glomski, Ian J.; Lebioda, Lukasz; Savchenko, Alexei; Edwards, Aled; Minor, Wladek (SC); (Toronto); (UV)

    2012-02-15

    For the last decade, worldwide efforts for the treatment of anthrax infection have focused on developing effective vaccines. Patients that are already infected are still treated traditionally using different types of standard antimicrobial agents. The most popular are antibiotics such as tetracyclines and fluoroquinolones. While aminoglycosides appear to be less effective antimicrobial agents than other antibiotics, synthetic aminoglycosides have been shown to act as potent inhibitors of anthrax lethal factor and may have potential application as antitoxins. Here, we present a structural analysis of the BA2930 protein, a putative aminoglycoside acetyltransferase, which may be a component of the bacterium's aminoglycoside resistance mechanism. The determined structures revealed details of a fold characteristic only for one other protein structure in the Protein Data Bank, namely, YokD from Bacillus subtilis. Both BA2930 and YokD are members of the Antibiotic-NAT superfamily (PF02522). Sequential and structural analyses showed that residues conserved throughout the Antibiotic-NAT superfamily are responsible for the binding of the cofactor acetyl coenzyme A. The interaction of BA2930 with cofactors was characterized by both crystallographic and binding studies.

  9. Aminoglycoside 6′-N-Acetyltransferase Variants of the Ib Type with Altered Substrate Profile in Clinical Isolates of Enterobacter cloacae and Citrobacter freundii

    OpenAIRE

    Casin, Isabelle; Bordon, Florence; Bertin, Philippe; Coutrot, Anne; Podglajen, Isabelle; Brasseur, Robert; Collatz, Ekkehard

    1998-01-01

    Three clinical isolates, Enterobacter cloacae EC1562 and EC1563 and Citrobacter freundii CFr564, displayed an aminoglycoside resistance profile evocative of low-level 6′-N acetyltransferase type II [AAC(6′)-II] production, which conferred reduced susceptibility to gentamicin but not to amikacin or isepamicin. Aminoglycoside acetyltransferase assays suggested the synthesis in the three strains of an AAC(6′) which acetylated amikacin practically as well as it acetylated gentamicin in vitro. Bot...

  10. Study of Klebsiella pneumoniae producing extended-spectrum β-lactamases against aminoglycosides

    Institute of Scientific and Technical Information of China (English)

    WEI FENG SHI; SU JIAN WANG; JIAN PING QIN

    2007-01-01

    Klebsiella pneumoniae ( K. pneumoniae) is one of the main gram-negative bacilli in clinical practice. Nosocomial infections caused by K. pneumoniae producing extended-spectrum β-lactamases (ESBLs) are very difficult to treat. This paper investigated the resistant characteristics of K. pneumoniae producing ESBLs and their aminoglycoside-modifying enzyme gene expressions including Nacetyltransferases and O-adenyhransferases. Bacteria identification and ESBLs confirmatory tests were performed by Phoenix TM-100 system. And minimum inhibitory concentrations (MICs) of gentamicin,amikacin, kanamycin, tobramycin, netilmicin and neomycin in 53 K. pneumoniae isolates were detected by agar dilution. In addition, six aminoglycoside-modifying enzyme genes were amplified by polymerase chain reaction (PCR) and verified by DNA sequencer. It was found that imipenem and meropenem against 120 K. pneumoniae isolates produced powerful antimicrobial activities. The resistant rates of gentamicin and amikacin were 55.0% and 46.7%, respectively. Except neomycin,MIC50 and MIC90 of gentamicin, amikacin, kanamycin, tobramycin and netilmicin in 53 K. pneumoniae were all > 128 μg/ml, and the resistant rates were 83.0%, 52.3%, 75.5%, 81. 1% and 69.8%, respectively. However, neomycin was only 39.6%. In addition, five modifying enzyme genes, including aac(3)- Ⅰ , aac(3)-Ⅱ, aac(6′) - Ⅰ b, ant(3″) - Ⅰ, ant(2″) - Ⅰ genes, were found in 53 isoahes except aac (6′)-Ⅱ, and their positive rates were 11.3%, 67.9%, 47.2%,1.9 % and 39.6 %, respectively. It was also confirmed by nucleotide sequence analysis that the above resistant genes shared nearly 100% identities with GenBank published genes. The results obtained in the present study indicated that K. pneumoniae producing ESBLs strains are rapidly spreading in our hospital, and their resistance to aminoglycosides may be associated with aminoglycoside-modifying enzyme gene expressions.

  11. Identification of Genes Coding Aminoglycoside Modifying Enzymes in E. coli of UTI Patients in India

    Directory of Open Access Journals (Sweden)

    Abdul Rouf Mir

    2016-01-01

    Full Text Available This study is to probe the pattern of antibiotic resistance against aminoglycosides and its mechanism in E. coli obtained from patients from Chennai, India. Isolation and identification of pathogens were done on MacConkey agar. Antimicrobial sensitivity testing was done by disc diffusion test. The identification of genes encoding aminoglycoside modifying enzymes was done by Polymerase Chain Reaction (PCR. Out of 98 isolates, 71 (72.45% isolates were identified as E. coli and the remaining 27 (27.55% as other bacteria. Disc diffusion method results showed a resistance level of 72.15% for streptomycin, 73.4% for gentamicin, 63.26% for neomycin, 57.14% for tobramycin, 47.9% for netilmicin, and 8.16% for amikacin in E. coli. PCR screening showed the presence of four genes, namely, rrs, aacC2, aacA-aphD, and aphA3, in their plasmid DNA. The results point towards the novel mechanism of drug resistance in E. coli from UTI patients in India as they confirm the presence of genes encoding enzymes that cause resistance to aminoglycoside drugs. This could be an alarm for drug prescription to UTI patients.

  12. Modulation of RNA function by aminoglycoside antibiotics.

    OpenAIRE

    Schroeder, R; Waldsich, C; Wank, H

    2000-01-01

    One of the most important families of antibiotics are the aminoglycosides, including drugs such as neomycin B, paromomycin, gentamicin and streptomycin. With the discovery of the catalytic potential of RNA, these antibiotics became very popular due to their RNA-binding capacity. They serve for the analysis of RNA function as well as for the study of RNA as a potential therapeutic target. Improvements in RNA structure determination recently provided first insights into the decoding site of the...

  13. Aminoglycoside antibiotics and autism: a speculative hypothesis

    Directory of Open Access Journals (Sweden)

    Manev Hari

    2001-10-01

    Full Text Available Abstract Background Recently, it has been suspected that there is a relationship between therapy with some antibiotics and the onset of autism; but even more curious, some children benefited transiently from a subsequent treatment with a different antibiotic. Here, we speculate how aminoglycoside antibiotics might be associated with autism. Presentation We hypothesize that aminoglycoside antibiotics could a trigger the autism syndrome in susceptible infants by causing the stop codon readthrough, i.e., a misreading of the genetic code of a hypothetical critical gene, and/or b improve autism symptoms by correcting the premature stop codon mutation in a hypothetical polymorphic gene linked to autism. Testing Investigate, retrospectively, whether a link exists between aminoglycoside use (which is not extensive in children and the onset of autism symptoms (hypothesis "a", or between amino glycoside use and improvement of these symptoms (hypothesis "b". Whereas a prospective study to test hypothesis "a" is not ethically justifiable, a study could be designed to test hypothesis "b". Implications It should be stressed that at this stage no direct evidence supports our speculative hypothesis and that its main purpose is to initiate development of new ideas that, eventually, would improve our understanding of the pathobiology of autism.

  14. Chronopharmacokinetics of once daily dosed aminoglycosides in hospitalized infectious patients

    OpenAIRE

    van Maarseveen, Erik; Man, Wai Hong; Proost, Johannes; Neef, Cees; Touw, Daniël

    2015-01-01

    Background hospitalized patients with serious infections treated with aminoglycosides are at risk of developing nephrotoxicity. Previous clinical studies have shown that the pharmacokinetics of aminoglycosides in humans follow a circadian rhythm. Therefore, the time of administration could have important clinical implications with respect to the risk of developing aminoglycoside-associated nephrotoxicity in patients treated with once daily dosing regimens. Objective To examine the effect of t...

  15. Synthesis of 4′-aminopantetheine and derivatives to probe aminoglycoside N-6′-acetyltransferase

    OpenAIRE

    Yan, Xuxu; Akinnusi, T. Olukayode; Larsen, Aaron T.; Auclair, Karine

    2011-01-01

    A convenient synthesis of 4′-aminopantetheine from commercial D-pantethine is reported. The amino group was introduced by reductive amination in order to avoid substitution at a sterically congested position. Derivatives of 4′-aminopantetheine were also prepared to evaluate the effect of O-to-N substitution on inhibitors of the resistance-causing enzyme aminoglycoside N-6′-acetyltransferase. The biological results combined with docking studies indicate that in spite of its reported unusual fl...

  16. Evaluation of Antibacterial Activity of Aminoglycosides and Modulating the Essential Oil of Cymbopogon citratus (DC.) Stapf

    OpenAIRE

    Tintino, Saulo R.; Lucena, Bruno F. F.; Fernando G. Figueredo; Cícera Datiane de M. OLIVEIRA; José J. DOS S. AGUIAR; Edmilson DO N. CARDOSO; Pedro E. A. DE AQUINO; Jacqueline C. ANDRADE; Coutinho, Henrique D. M.; Ednardo F. F. MATIAS

    2014-01-01

     Several works demonstrated the importance of the study of natural products as an alternative source for new antimicrobial drugs or for modulators for these ones. In this point, the aim of this was to investigate the antibacterial activity and the possible interactions between the essential oil of Cymbopogon citratus alone and in association with aminoglycosides against standard and clinically isolated strains of multidrug-resistant bacteria such as S. aureus, E. coli and P. aeruginosa by mic...

  17. Purification, crystallization and preliminary X-ray analysis of the aminoglycoside-6′-acetyltransferase AAC(6′)-Im

    OpenAIRE

    Toth, Marta; Vakulenko, Sergei B.; Smith, Clyde A.

    2012-01-01

    AAC(6′)-Im is an N-acetyltransferase enzyme responsible for aminoglycoside resistance in E. faecium and E. coli isolates. Crystals of the kanamycin complex of this enzyme have been prepared and preliminary X-ray diffraction experiments have been undertaken.

  18. Phytochemical screening and synergistic interactions between aminoglycosides, selected antibiotics and extracts from the bryophyte Octoblepharum albidum Hedw (Calymperaceae

    Directory of Open Access Journals (Sweden)

    Vidal C.A.S.

    2012-01-01

    Full Text Available This work is the first to describe the modulation of antibiotic activity of the bryophyte Octoblepharum albidum Hedw extract. The antibacterial activity of ethanolic extract of O. albidum (EEOa, alone and in association with aminoglycosides, was determined against six bacterial strains by a microdilution test. The results showed a similar inhibitory activity of EEOa against Escherichia coli ATCC 25922 and Klebsiella pneumoniae ATCC 33018 (MICs 512 μg/mL. The synergistic effect of the extracts and aminoglycosides was also verified. The most pronounced effects were obtained with EEOa + gentamicin against E. coli and EEOa + kanamycin against K. pneumoniae with MICs reduction (128 to 32 μg/mL. The data from this study are indicative of the antibacterial activity of the bryophyte O. albidum extracts and its potential in modifying the resistance of aminoglycosides analyzed.

  19. Crystallization and preliminary crystallographic analysis of an aminoglycoside kinase from Legionella pneumophila

    International Nuclear Information System (INIS)

    Two crystal forms of the antibiotic resistance enzyme APH(9)-Ia from L. pneumophila are reported. 9-Aminoglycoside phosphotransferase type Ia [APH(9)-Ia] is a resistance factor in Legionella pneuemophila, the causative agent of legionnaires’ disease. It is responsible for providing intrinsic resistance to the antibiotic spectinomycin. APH(9)-Ia phosphorylates one of the hydroxyl moieties of spectinomycin in an ATP-dependent manner, abolishing the antibiotic properties of this drug. Here, the crystallization and preliminary X-ray studies of this enzyme in two crystal forms is reported. One of the these crystal forms provides diffraction data to a resolution of 1.7 Å

  20. Histone methyltransferases in cancer

    DEFF Research Database (Denmark)

    Albert, Mareike; Helin, Kristian

    2009-01-01

    Cancer is perceived as a heterogeneous group of diseases that is characterized by aberrant patterns of gene expression. In the last decade, an increasing amount of data has pointed to a key role for epigenetic alterations in human cancer. In this review, we focus on a subclass of epigenetic...... regulators, namely histone methyltransferases (HMTs). Several HMTs have been linked to different types of cancer; however, in most cases we only have limited knowledge regarding the molecular mechanisms by which the HMTs contribute to disease development. We summarize the current knowledge regarding some of...

  1. Extracellular DNA Shields against Aminoglycosides in Pseudomonas aeruginosa Biofilms

    DEFF Research Database (Denmark)

    Chiang, Wen-Chi; Nilsson, Martin; Jensen, Peter Østrup;

    2013-01-01

    Within recent years, it has been established that extracellular DNA is a key constituent of the matrix of microbial biofilms. In addition, it has recently been demonstrated that DNA binds positively charged antimicrobials such as aminoglycosides and antimicrobial peptides. In the present study, we...... provide evidence that extracellular DNA shields against aminoglycosides in Pseudomonas aeruginosa biofilms. We show that exogenously supplemented DNA integrates into P. aeruginosa biofilms and increases their tolerance toward aminoglycosides. We provide evidence that biofilms formed by a DNA release......-deficient P. aeruginosa quorum-sensing mutant are more susceptible to aminoglycoside treatment than wild-type biofilms but become rescued from the detrimental action of aminoglycosides upon supplementation with exogenous DNA. Furthermore, we demonstrate that exposure to lysed polymorphonuclear leukocytes...

  2. Structural basis for S-adenosylmethionine binding and methyltransferase activity by mitochondrial transcription factor B1.

    Science.gov (United States)

    Guja, Kip E; Venkataraman, Krithika; Yakubovskaya, Elena; Shi, Hui; Mejia, Edison; Hambardjieva, Elena; Karzai, A Wali; Garcia-Diaz, Miguel

    2013-09-01

    Eukaryotic transcription factor B (TFB) proteins are homologous to KsgA/Dim1 ribosomal RNA (rRNA) methyltransferases. The mammalian TFB1, mitochondrial (TFB1M) factor is an essential protein necessary for mitochondrial gene expression. TFB1M mediates an rRNA modification in the small ribosomal subunit and thus plays a role analogous to KsgA/Dim1 proteins. This modification has been linked to mitochondrial dysfunctions leading to maternally inherited deafness, aminoglycoside sensitivity and diabetes. Here, we present the first structural characterization of the mammalian TFB1 factor. We have solved two X-ray crystallographic structures of TFB1M with (2.1 Å) and without (2.0 Å) its cofactor S-adenosyl-L-methionine. These structures reveal that TFB1M shares a conserved methyltransferase core with other KsgA/Dim1 methyltransferases and shed light on the structural basis of S-adenosyl-L-methionine binding and methyltransferase activity. Together with mutagenesis studies, these data suggest a model for substrate binding and provide insight into the mechanism of methyl transfer, clarifying the role of this factor in an essential process for mitochondrial function. PMID:23804760

  3. Purification, crystallization and preliminary X-ray analysis of the aminoglycoside-6′-acetyltransferase AAC(6′)-Im

    International Nuclear Information System (INIS)

    AAC(6′)-Im is an N-acetyltransferase enzyme responsible for aminoglycoside resistance in E. faecium and E. coli isolates. Crystals of the kanamycin complex of this enzyme have been prepared and preliminary X-ray diffraction experiments have been undertaken. Bacterial resistance to the aminoglycoside antibiotics is primarily the result of enzymatic deactivation of the drugs. The aminoglycoside N-acetyltransferases (AACs) are a large family of bacterial enzymes that are responsible for coenzyme-A-facilitated acetylation of aminoglycosides. The gene encoding one of these enzymes, AAC(6′)-Im, has been cloned and the protein (comprising 178 amino-acid residues) was expressed in Escherichia coli, purified and crystallized as the kanamycin complex. Synchrotron diffraction data to approximately 2.0 Å resolution were collected from a crystal of this complex on beamline BL12-2 at SSRL (Stanford, California, USA). The crystals belonged to the hexagonal space group P65, with approximate unit-cell parameters a = 107.75, c = 37.33 Å, and contained one molecule in the asymmetric unit. Structure determination is under way using molecular replacement

  4. Synergistic Effect of Oleanolic Acid on Aminoglycoside Antibiotics against Acinetobacter baumannii.

    Directory of Open Access Journals (Sweden)

    Bora Shin

    Full Text Available Difficulties involved in treating drug-resistant pathogens have created a need for new therapies. In this study, we investigated the possibility of using oleanolic acid (OA, a natural pentacyclic triterpenoid, as a natural adjuvant for antibiotics against Acinetobacter baumannii. High concentrations of OA can kill cells, partly because it generates reactive oxygen species. Measurement of the fractional inhibitory concentration (FIC for OA and time-kill experiments demonstrated that it only synergizes with aminoglycoside antibiotics (e.g., gentamicin, kanamycin. Other classes of antibiotics (e.g., ampicillin, rifampicin, norfloxacin, chloramphenicol, and tetracycline have no interactions with OA. Microarray and quantitative reverse transcription-PCR analysis indicated that genes involved in ATP synthesis and cell membrane permeability, the gene encoding glycosyltransferase, peptidoglycan-related genes, phage-related genes, and DNA repair genes were upregulated under OA. OA highly induces the expression of adk, which encodes an adenylate kinase, and des6, which encodes a linoleoyl-CoA desaturase, and deletion of these genes increased FICs; these observations indicate that adk and des6 are involved in the synergism of OA with aminoglycosides. Data obtained using 8-anilino-1-naphthalenesulfonic acid, fluorescence-conjugated gentamicin, and membrane fatty acid analysis indicates that adk and des6 are involved in changes in membrane permeability. Proton-motive force and ATP synthesis tests show that those genes are also involved in energy metabolism. Taken together, our data show that OA boosts aminoglycoside uptake by changing membrane permeability and energy metabolism in A. baumannii.

  5. Pediatric fecal microbiota harbor diverse and novel antibiotic resistance genes.

    Directory of Open Access Journals (Sweden)

    Aimée M Moore

    Full Text Available Emerging antibiotic resistance threatens human health. Gut microbes are an epidemiologically important reservoir of resistance genes (resistome, yet prior studies indicate that the true diversity of gut-associated resistomes has been underestimated. To deeply characterize the pediatric gut-associated resistome, we created metagenomic recombinant libraries in an Escherichia coli host using fecal DNA from 22 healthy infants and children (most without recent antibiotic exposure, and performed functional selections for resistance to 18 antibiotics from eight drug classes. Resistance-conferring DNA fragments were sequenced (Illumina HiSeq 2000, and reads assembled and annotated with the PARFuMS computational pipeline. Resistance to 14 of the 18 antibiotics was found in stools of infants and children. Recovered genes included chloramphenicol acetyltransferases, drug-resistant dihydrofolate reductases, rRNA methyltransferases, transcriptional regulators, multidrug efflux pumps, and every major class of beta-lactamase, aminoglycoside-modifying enzyme, and tetracycline resistance protein. Many resistance-conferring sequences were mobilizable; some had low identity to any known organism, emphasizing cryptic organisms as potentially important resistance reservoirs. We functionally confirmed three novel resistance genes, including a 16S rRNA methylase conferring aminoglycoside resistance, and two tetracycline-resistance proteins nearly identical to a bifidobacterial MFS transporter (B. longum s. longum JDM301. We provide the first report to our knowledge of resistance to folate-synthesis inhibitors conferred by a predicted Nudix hydrolase (part of the folate synthesis pathway. This functional metagenomic survey of gut-associated resistomes, the largest of its kind to date, demonstrates that fecal resistomes of healthy children are far more diverse than previously suspected, that clinically relevant resistance genes are present even without recent selective

  6. Microplate phosphocellulose binding assay for aminoglycoside-modifying enzymes.

    OpenAIRE

    Cooksey, R C; Metchock, B G; Thornsberry, C

    1986-01-01

    We modified the phosphocellulose binding assay for aminoglycoside-modifying enzymes (AMEs) by use of microdilution plates and a multichannel micropipette. Batteries of aminoglycoside substrates for screening organisms for the presence of AMEs as well as for subclassifying enzymes were prepared and stored in microdilution plates. When tested in parallel with the conventional tube reaction assay, the microplate assay yielded comparable radioactive counts and therefore equally correct identifica...

  7. Molecular basis of rare aminoglycoside susceptibility and pathogenesis of Burkholderia pseudomallei clinical isolates from Thailand.

    Directory of Open Access Journals (Sweden)

    Lily A Trunck

    Full Text Available BACKGROUND: Burkholderia pseudomallei is intrinsically resistant to aminoglycosides and macrolides, mostly due to AmrAB-OprA efflux pump expression. We investigated the molecular mechanisms of aminoglycoside susceptibility exhibited by Thai strains 708a, 2188a, and 3799a. METHODOLOGY/PRINCIPAL FINDINGS: qRT-PCR revealed absence of amrB transcripts in 708a and greatly reduced levels in 2188a and 3799a. Serial passage on increasing gentamicin concentrations yielded 2188a and 3799a mutants that became simultaneously resistant to other aminoglycosides and macrolides, whereas such mutants could not be obtained with 708a. Transcript analysis showed that the resistance of the 2188a and 3799a mutants was due to upregulation of amrAB-oprA expression by unknown mechanism(s. Use of a PCR walking strategy revealed that the amrAB-oprA operon was missing in 708a and that this loss was associated with deletion of more than 70 kb of genetic material. Rescue of the amrAB-oprB region from a 708a fosmid library and sequencing showed the presence of a large chromosome 1 deletion (131 kb and 141 kb compared to strains K96243 and 1710b, respectively. This deletion not only removed the amrAB-oprA operon, but also the entire gene clusters for malleobactin and cobalamin synthesis. Other genes deleted included the anaerobic arginine deiminase pathway, putative type 1 fimbriae and secreted chitinase. Whole genome sequencing and PCR analysis confirmed absence of these genes from 708a. Despite missing several putative virulence genes, 708a was fully virulent in a murine melioidosis model. CONCLUSIONS/SIGNIFICANCE: Strain 708a may be a natural candidate for genetic manipulation experiments that use Select Agent compliant antibiotics for selection and validates the use of laboratory-constructed Delta(amrAB-oprA mutants in such experiments.

  8. A rapid method for detection of five known mutations associated with aminoglycoside-induced deafness

    Directory of Open Access Journals (Sweden)

    Greinwald John H

    2009-01-01

    Full Text Available Abstract Background South Africa has one of the highest incidences of multidrug-resistant tuberculosis (MDR-TB in the world. Concomitantly, aminoglycosides are commonly used in this country as a treatment against MDR-TB. To date, at least five mutations are known to confer susceptibility to aminoglycoside-induced hearing loss. The aim of the present study was to develop a rapid screening method to determine whether these mutations are present in the South African population. Methods A multiplex method using the SNaPshot technique was used to screen for five mutations in the MT-RNR1 gene: A1555G, C1494T, T1095C, 961delT+C(n and A827G. A total of 204 South African control samples, comprising 98 Mixed ancestry and 106 Black individuals were screened for the presence of the five mutations. Results A robust, cost-effective method was developed that detected the presence of all five sequence variants simultaneously. In this pilot study, the A1555G mutation was identified at a frequency of 0.9% in the Black control samples. The 961delT+C(n variant was present in 6.6% of the Black controls and 2% of the Mixed ancestry controls. The T1095C, C1494T and A827G variants were not identified in any of the study participants. Conclusion The frequency of 0.9% for the A1555G mutation in the Black population in South Africa is of concern given the high incidence of MDR-TB in this particular ethnic group. Future larger studies are warranted to determine the true frequencies of the aminoglycoside deafness mutations in the general South African population. The high frequencies of the 961delT+C(n variant observed in the controls suggest that this change is a common non-pathogenic polymorphism. This genetic method facilitates the identification of individuals at high risk of developing hearing loss prior to the start of aminoglycoside therapy. This is important in a low-resource country like South Africa where, despite their adverse side-effects, aminoglycosides will

  9. Synthesis of 4′-aminopantetheine and derivatives to probe aminoglycoside N-6′-acetyltransferase

    Science.gov (United States)

    Yan, Xuxu; Akinnusi, T. Olukayode; Larsen, Aaron T.; Auclair, Karine

    2011-01-01

    Summary A convenient synthesis of 4′-aminopantetheine from commercial D-pantethine is reported. The amino group was introduced by reductive amination in order to avoid substitution at a sterically congested position. Derivatives of 4′-aminopantetheine were also prepared to evaluate the effect of O-to-N substitution on inhibitors of the resistance-causing enzyme aminoglycoside N-6′-acetyltransferase. The biological results combined with docking studies indicate that in spite of its reported unusual flexibility and ability to adopt different folds, this enzyme is highly specific for AcCoA. PMID:21225062

  10. Synthesis of 4'-aminopantetheine and derivatives to probe aminoglycoside N-6'-acetyltransferase.

    Science.gov (United States)

    Yan, Xuxu; Akinnusi, T Olukayode; Larsen, Aaron T; Auclair, Karine

    2011-03-01

    A convenient synthesis of 4'-aminopantetheine from commercial D-pantethine is reported. The amino group was introduced by reductive amination in order to avoid substitution at a sterically congested position. Derivatives of 4'-aminopantetheine were also prepared to evaluate the effect of O-to-N substitution on inhibitors of the resistance-causing enzyme aminoglycoside N-6'-acetyltransferase. The biological results combined with docking studies indicate that in spite of its reported unusual flexibility and ability to adopt different folds, this enzyme is highly specific for AcCoA. PMID:21225062

  11. Widespread Dissemination of Aminoglycoside Resistance Genes armA and rmtB in Klebsiella pneumoniae Isolates in Taiwan Producing CTX-M-Type Extended-Spectrum β-Lactamases ▿

    OpenAIRE

    Ma, Ling; Lin, Chi-Jan; Chen, Jiun-Han; Fung, Chang-Phone; Chang, Feng-Yee; Lai, Yiu-Kay; Lin, Jung-Chung; Siu, L. K.

    2008-01-01

    Among 235 extended-spectrum β-lactamase-producing Klebsiella pneumoniae (ESBL) isolates collected from a nationwide surveillance performed in Taiwan, 102 (43.4%) were resistant to amikacin. Ninety-two of these 102 (90.2%) isolates were carrying CTX-M-type β-lactamases individually or concomitantly with SHV-type or CMY-2 β-lactamases. The armA and rmtB alleles were individually detected in 44 and 37 of these 92 isolates, respectively. One isolate contained both armA and rmtB. The coexistence o...

  12. 糖尿病足分离的铜绿假单胞菌对氨基糖苷类抗生素耐药机制探讨%Study on aminoglycoside antibiotics resistance of Pseudomonas aeruginosa isolated from diabetic foot infections

    Institute of Scientific and Technical Information of China (English)

    乌洪芳; 孙茜; 李玉珠; 张敏; 孟玲玲; 李代清

    2015-01-01

    Objective To investigate the clinical features, phenotypes and genotypes of Pseudomonas aeruginosa (PA) strains isolated from patients with diabetic foot infection (DFI) resisting to aminoglycosides antibiotics (AmAn). Methods The clinical profiles of 209 DFI patients hospitalized in the Tianjin Metabolic Diseases Hospital were collected and ana⁃lyzed. Forty-one PA strains were identified, and their antibiotic resistance profiles were obtained. The DNAs of PA isolates were extracted and applied to amplifications for several aminoglycosides modifying enzyme genes, including aac(3′)-Ⅰ, aac (3′)-Ⅱ, aac(6′)-Ⅰb, aac(6′)-Ⅱ, ant(2′′)-Ⅰand ant(3′′)-Ⅰby PCR method. Combining with the clinical features and the antibiotic resistance profiles, the relationship between genotypes and phenotypes of the PA strains was analyzed. Results Gram positive bacteria (G+) were the majority of the pathogen with 51.67%detection rate. The total detection rate of PA was 19.62%, listed as the top one pathogenic bacterium among gram negative bacteria (47.67%). There was significant difference in the ratio of ulcer area≥4 cm2 between PA group and non-PA group and G+group. There were significantly higher inci⁃dence rate of ischemic ulcer and osteomyelitis in PA group than those of G+group. There were higher clinical characteristics and ulcer depth (SAD) score, and increased hypersensitive C-reactive protein in PA group than those of G+ group. There were 30 strains of PA being resistant to AmAn (73.17%). The predominant drug resistance gene to AmAn was ant(3′′)-Ⅰ(65.85%), and aac(3′)-Ⅰgene was not found from all PA isolates. Conclusion The detection rate of PA isolated from DFI patients was higher, and patients were with the characteristics of larger, deeper and severe ischemia of ulcer area. The phe⁃nomenon of PA resistant to AmAn was more serious, and ant(3′′)-Ⅰgene identified from PA isolates was the most common resistance gene identified to Am

  13. Purification, crystallization and preliminary X-ray analysis of Enterococcus casseliflavus aminoglycoside-2′′-phosphotransferase-IVa

    International Nuclear Information System (INIS)

    Aminoglycoside-2′′-phosphotransferase-IVa [APH(2′′)-IVa] is an enzyme that is responsible for high-level gentamicin resistance in E. casseliflavus isolates. Three different crystals of wild-type substrate-free APH(2′′)-IVa have been prepared and preliminary X-ray diffraction experiments have been undertaken on all three crystal forms. The deactivation of aminoglycoside antibiotics by chemical modification is one of the major sources of bacterial resistance to this family of therapeutic compounds, which includes the clinically relevant drugs streptomycin, kanamycin and gentamicin. The aminoglycoside phosphotransferases (APHs) form one such family of enzymes responsible for this resistance. The gene encoding one of these enzymes, aminoglycoside-2′′-phosphotransferase-IVa [APH(2′′)-IVa] from Enterococcus casseliflavus, has been cloned and the protein (comprising 306 amino-acid residues) has been expressed in Escherichia coli and purified. The enzyme was crystallized in three substrate-free forms. Two of the crystal forms belonged to the orthorhombic space group P212121 with similar unit-cell parameters, although one of the crystal forms had a unit-cell volume that was approximately 13% smaller than the other and a very low solvent content of around 38%. The third crystal form belonged to the monoclinic space group P21 and preliminary X-ray diffraction analysis was consistent with the presence of two molecules in the asymmetric unit. The orthorhombic crystal forms of apo APH(2′′)-IVa both diffracted to 2.2 Å resolution and the monoclinic crystal form diffracted to 2.4 Å resolution; synchrotron diffraction data were collected from these crystals at SSRL (Stanford, California, USA). Structure determination by molecular replacement using the structure of the related enzyme APH(2′′)-IIa is proceeding

  14. High throughput LSPR and SERS analysis of aminoglycoside antibiotics.

    Science.gov (United States)

    McKeating, Kristy S; Couture, Maxime; Dinel, Marie-Pier; Garneau-Tsodikova, Sylvie; Masson, Jean-Francois

    2016-08-15

    Aminoglycoside antibiotics are used in the treatment of infections caused by Gram-negative bacteria, and are often dispensed only in severe cases due to their adverse side effects. Patients undergoing treatment with these antibiotics are therefore commonly subjected to therapeutic drug monitoring (TDM) to ensure a safe and effective personalised dosage. The ability to detect these antibiotics in a rapid and sensitive manner in human fluids is therefore of the utmost importance in order to provide effective monitoring of these drugs, which could potentially allow for a more widespread use of this class of antibiotics. Herein, we report on the detection of various aminoglycosides, by exploiting their ability to aggregate gold nanoparticles. The number and position of the amino groups of aminoglycoside antibiotics controlled the aggregation process. We investigated the complementary techniques of surface enhanced Raman spectroscopy (SERS) and localized surface plasmon resonance (LSPR) for dual detection of these aminoglycoside antibiotics and performed an in-depth study of the feasibility of carrying out TDM of tobramycin using a platform amenable to high throughput analysis. Herein, we also demonstrate dual detection of tobramycin using both LSPR and SERS in a single platform and within the clinically relevant concentration range needed for TDM of this particular aminoglycoside. Additionally we provide evidence that tobramycin can be detected in spiked human serum using only functionalised nanoparticles and SERS analysis. PMID:27412506

  15. Plant isoflavone and isoflavanone O-methyltransferase genes

    Science.gov (United States)

    Broeckling, Bettina E.; Liu, Chang-Jun; Dixon, Richard A.

    2014-08-19

    The invention provides enzymes that encode O-methyltransferases (OMTs) from Medicago truncatula that allow modification to plant (iso)flavonoid biosynthetic pathways. In certain aspects of the invention, the genes encoding these enzymes are provided. The invention therefore allows the modification of plants for isoflavonoid content. Transgenic plants comprising such enzymes are also provided, as well as methods for improving disease resistance in plants. Methods for producing food and nutraceuticals, and the resulting compositions, are also provided.

  16. Radioenzymatic assays for aminoglycosides with kanamycin 6'- acetyltransferase

    International Nuclear Information System (INIS)

    To facilitate the rapid and accurate quantitation of parenterally administered aminoglycosides, the optimum conditions (pH, duration of incubation, and cofactor concentrations) were defined to permit radioenzymatic assays with kanamycin acetyltransferase. The accuracy in quantitating tobramycin, netilmicin, kanamycin, and amikacin at concentrations in the therapeutic range was greater than 90%, with a mean recovery of 102.8%. The mean of the interassay coefficient of variation was 7.8%. Typical standard curves at six different concentrations resulted in a correlation coefficient (r value) of greater than 0.99 for each aminoglycoside. The radioenzymatic assay correlates well with the bioassay (tobramycin and netilmicin) and radioimmunoassay (amikacin and kanamycin); the correlation coefficient is greater than 0.90 for all. The authors conclude that the radioenzymatic assay utilizing kanamycin 6'-acetyltransferase is feasible for all commercially available parenterally administered aminoglycosides

  17. Study of the Interference between Plectranthus Species Essential Oils from Brazil and Aminoglycosides.

    Science.gov (United States)

    Galvão Rodrigues, Fabíola Fernandes; Costa, José Galberto Martins; Rodrigues, Fábio Fernandes Galvao; Campos, Adriana Rolim

    2013-01-01

    Plectranthus is one of the most representative genera of Lamiaceae family. In this study, the essential oils from Plectranthus amboinicus, Plectranthus ornatus, and Plectranthus barbatus were investigated for their chemical composition and antimicrobial and modulatory activities. The major components found were carvacrol (54.4%-P. amboinicus) and eugenol (22.9%-P. ornatus e 25.1%-P. barbatus). In vitro antimicrobial activity was conducted against Escherichia coli, Proteus vulgaris, Bacillus cereus, Pseudomonas aeruginosa, Staphylococcus aureus, and Staphylococcus aureus (multiresistant) using microdilution method. The results of bioassay showed that all strains were sensitive to the oils, except P. aeruginosa that was resistant to P. amboinicus and P. ornatus. A synergistic effect of all essential oils combined with the aminoglycosides was demonstrated. These results show that P. amboinicus, P. ornatus, and P. barbatus inhibit the growth of pathogenic microorganism, and besides this they present antibiotic modifying activity, providing a new perspective against the problem of bacterial resistance to antibiotics. PMID:23662150

  18. SYNTHESIS AND CYTOTOXIC ACTIVITY OF NEW 5H-INDOLO[2,3-B]QUINOLINE O-AMINOGLYCOSIDES.

    Science.gov (United States)

    Badowska-Rosłonek, Katarzyna; Ciesielska, Agnieszka; Switalska, Marta; Piskozub, Małgorzata; Peczyńska-Czoch, Wanda; Wietrzyk, Joanna; Kaczmarek, Łukasz

    2016-01-01

    Novel 5H-indolo[2,3-b]quinoline O-aminoglycosides were synthesized in order to check the hypothesis that the construction of hybrids composed of the active 5H-indolo[2,3-b]quinoline chromophore and daunosaminyl or acosaminyl moiety may result in the cytotoxic activity of the obtained derivatives that is much higher than the one of the parent DIMIQ (5,11-dimethyl-5H-indolo[2,3-b]quinoline) and 6H-indoloquinoline analogs. Actually, 5H-indolo[2,3-b]indoloquinoline O-aminoglycosides showed the anti-proliferative activity in vitro against human lung adenocarcinoma A549, breast cancer MCF-7, melanoma Hs294T, promyelocytic leukemia HL-60, uterine sarcoma MES-SA and colon cancer LoVo cell lines, which was 10 times higher than that of the 6H-analogs and comparable to the one of the referential DIMIQ. Unexpectedly, it appeared that except for HL-60/MX2 (P-gp-independent and topoisomerase II-dependent resistance), other MDR tumor cell lines (LoVo/DX. P-gp-dependent, MRP-, LRP-dependent multidrug resistance) and MES-SA/DX5 (P-gp-dependent resistance to doxorubicin) are also resistant to the 5H-indolo[2,3-b]indoloquinoline O-aminoglycosides tested. This is surprising because 6H-analogs, in general, 10 times less active against non-MDR tumor cell lines, as well as the DIMIQ itself, are able to overcome drug resistance in all MDR cell lines examined. The cytotoxicity of the tested compounds against tumor cell lines and against normal cells (mice fibroblasts BALB/3T3) was comparable. PMID:27476287

  19. Activation of PI3K signaling prevents aminoglycoside-induced hair cell death in the murine cochlea

    Directory of Open Access Journals (Sweden)

    Azadeh Jadali

    2016-06-01

    Full Text Available Loss of sensory hair cells of the inner ear due to aminoglycoside exposure is a major cause of hearing loss. Using an immortalized multipotent otic progenitor (iMOP cell line, specific signaling pathways that promote otic cell survival were identified. Of the signaling pathways identified, the PI3K pathway emerged as a strong candidate for promoting hair cell survival. In aging animals, components for active PI3K signaling are present but decrease in hair cells. In this study, we determined whether activated PI3K signaling in hair cells promotes survival. To activate PI3K signaling in hair cells, we used a small molecule inhibitor of PTEN or genetically ablated PTEN using a conditional knockout animal. Hair cell survival was challenged by addition of gentamicin to cochlear cultures. Hair cells with activated PI3K signaling were more resistant to aminoglycoside-induced hair cell death. These results indicate that increased PI3K signaling in hair cells promote survival and the PI3K signaling pathway is a target for preventing aminoglycoside-induced hearing loss.

  20. Radioenzymatic assays for aminoglycosides with kanamycin 6'-acetyltransferase.

    OpenAIRE

    Weber, A; Smith, A L; Opheim, K E

    1985-01-01

    To facilitate the rapid and accurate quantitation of parenterally administered aminoglycosides, we defined the optimum conditions (pH, duration of incubation, and cofactor concentrations) to permit radioenzymatic assays with kanamycin acetyltransferase. The accuracy in quantitating tobramycin, netilmicin, kanamycin, and amikacin at concentrations in the therapeutic range was greater than 90%, with a mean recovery of 102.8%. The mean of the interassay coefficient of variation was 7.8%. Typical...

  1. DETERMINATION OF AMINOGLYCOSIDES IN FOOD BY FLUORESCENCE POLARIZATION IMMUNOASSAY

    Directory of Open Access Journals (Sweden)

    FARAFONOVA O.V.

    2015-01-01

    Full Text Available The methodic for quantitative determination of aminoglycoside antibiotics (gentamicin, kanamycin, streptomycin, amikacin, neomycin in food by polarization fluorescent immunoassay (FPIA is developed. The size and structure influence of a fluorescent molecule on a fluorescence polarization degree is analyzed. Affinity constants of antibodies to compounds and tracers were estimated, optimized working concentration of tracers and antibodies that provide the maximum value of analytical signal. Methods were tested in the antibiotics identification in milk, eggs and chicken.

  2. UK31214, a new aminoglycoside and derivative of kanamycin B.

    OpenAIRE

    Wise, R.; Andrews, J. M.; Bedford, K A

    1980-01-01

    The in vitro activity of UK31214, a kanamycin B derivative, was studied against 250 recent isolates and compared with other aminoglycosides. Against the Enterobacteriaceae (with the exception of Proteus mirabilis and Providencia stuartii) UK31214 and amikacin had similar degrees of activity (mode minimum inhibitory concentration [MIC], 1 microgram/ml). Proteus mirabilis and P. stuartii strains were four- to eight-fold more susceptible to amikacin than to UK31214. Pseudomonas aeruginosa strain...

  3. Antifungal amphiphilic aminoglycoside K20: bioactivities and mechanism of action

    OpenAIRE

    Shrestha, Sanjib K.; Cheng-Wei T Chang; Meissner, Nicole; Oblad, John; Shrestha, Jaya P.; Sorensen, Kevin N.; Michelle M. Grilley; Jon Y Takemoto

    2014-01-01

    K20 is a novel amphiphilic antifungal aminoglycoside that is synthetically derived from the antibiotic kanamycin A. Reported here are investigations of K20′s antimicrobial activities, cytotoxicity, and fungicidal mechanism of action. In vitro growth inhibitory activities against a variety of human and plant pathogenic yeasts, filamentous fungi, and bacteria were determined using microbroth dilution assays and time-kill curve analyses, and hemolytic and animal cell cytotoxic activities were de...

  4. Antifungal amphiphilic aminoglycoside K20: bioactivities and mechanism of action

    OpenAIRE

    Shrestha, Sanjib K.; Cheng-Wei Tom Chang; Nicole eMeissner; John eOblad; Shrestha, Jaya P.; Sorensen, Kevin N.; Michelle M. Grilley; Jon Y Takemoto

    2014-01-01

    K20 is a novel amphiphilic antifungal aminoglycoside that is synthetically derived from the antibiotic kanamycin A. Reported here are investigations of K20’s antimicrobial activities, cytotoxicity, and fungicidal mechanism of action. In vitro growth inhibitory activities against a variety of human and plant pathogenic yeasts, filamentous fungi, and bacteria were determined using microbroth dilution assays and time-kill curve analyses, and hemolytic and animal cell cytotoxic activities were d...

  5. The effect of pharmacy intervention on aminoglycoside costs.

    Science.gov (United States)

    Tsuyuki, R T; Nakagawa, R S

    1987-04-01

    Antibiotics constitute a large percentage of every hospital's drug budget. In an effort to control the escalating costs of antimicrobial therapy, we focused on the usage of aminoglycosides at our institution. The aminoglycosides, gentamicin and tobramycin, are similar in terms of antimicrobial spectra and toxicities. Since gentamicin is much less expensive, it was felt that significant cost savings would be realized if gentamicin were to be used preferentially over tobramycin. Specific criteria for the use of tobramycin were developed and approved by the Pharmacy and Therapeutics Committee. All patients prescribed parenteral tobramycin during the five week data collection period were entered into the study. We chose to use direct verbal intervention as our method for altering physician's prescribing patterns. An educational program of intervention was set up such that when a physician prescribed tobramycin, the patient's chart was immediately reviewed. If tobramycin was prescribed for a purpose other than those in the approved criteria for tobramycin use, the physician was contacted personally to discuss the cost-effectiveness of gentamicin use. Utilization figures from the previous six months showed that our interventions would save approximately $32,000 over a one year period. Our interventions on aminoglycoside prescribing represents a highly successful, cost-effective and educational method for altering physician's prescribing patterns. PMID:10282090

  6. In70 of plasmid pAX22, a bla(VIM-1)-containing integron carrying a new aminoglycoside phosphotransferase gene cassette.

    Science.gov (United States)

    Riccio, M L; Pallecchi, L; Fontana, R; Rossolini, G M

    2001-04-01

    An Achromobacter xylosoxydans strain showing broad-spectrum resistance to beta-lactams (including carbapenems) and aminoglycosides was isolated at the University Hospital of Verona (Verona, Italy). This strain was found to produce metallo-beta-lactamase activity and to harbor a 30-kb nonconjugative plasmid, named pAX22, carrying a bla(VIM-1) determinant inserted into a class 1 integron. Characterization of this integron, named In70, revealed an original array of four gene cassettes containing, respectively, the bla(VIM-1) gene and three different aminoglycoside resistance determinants, including an aacA4 allele, a new aph-like gene named aphA15, and an aadA1 allele. The aphA15 gene is the first example of an aph-like gene carried on a mobile gene cassette, and its product exhibits close similarity to the APH(3')-IIa aminoglycoside phosphotransferase encoded by Tn5 (36% amino acid identity) and to an APH(3')-IIb enzyme from Pseudomonas aeruginosa (38% amino acid identity). Expression of the cloned aphA15 gene in Escherichia coli reduced the susceptibility to kanamycin and neomycin as well as (slightly) to amikacin, netilmicin, and streptomycin. Characterization of the 5' and 3' conserved segments of In70 and of their flanking regions showed that In70 belongs to the group of class 1 integrons associated with defective transposon derivatives originating from Tn402-like elements. The structure of the 3' conserved segment indicates the closest ancestry with members of the In0-In2 lineage. In70, with its array of cassette-borne resistance genes, can mediate broad-spectrum resistance to most beta-lactams and aminoglycosides. PMID:11257042

  7. DNA-Aptamers Binding Aminoglycoside Antibiotics

    OpenAIRE

    Nadia Nikolaus; Beate Strehlitz

    2014-01-01

    Aptamers are short, single stranded DNA or RNA oligonucleotides that are able to bind specifically and with high affinity to their non-nucleic acid target molecules. This binding reaction enables their application as biorecognition elements in biosensors and assays. As antibiotic residues pose a problem contributing to the emergence of antibiotic-resistant pathogens and thereby reducing the effectiveness of the drug to fight human infections, we selected aptamers targeted against the aminog...

  8. In vitro activities of aztreonam, piperacillin, and ticarcillin combined with amikacin against amikacin-resistant Pseudomonas aeruginosa and P. cepacia isolates from children with cystic fibrosis.

    OpenAIRE

    Aronoff, S C; Klinger, J D

    1984-01-01

    Amikacin, combined with aztreonam, piperacillin, or ticarcillin, synergistically inhibited amikacin-resistant sputum isolates of Pseudomonas aeruginosa and P. cepacia from children with cystic fibrosis. Ticarcillin-amikacin was the least active combination. Aminoglycoside resistance should not preclude the use of beta-lactam-aminoglycoside combinations in the treatment of pulmonary infections in cystic fibrosis.

  9. Effect of alternative temozolomide schedules on glioblastoma O 6-methylguanine-DNA methyltransferase activity and survival

    OpenAIRE

    Robinson, C G; Palomo, J M; Rahmathulla, G; McGraw, M; Donze, J; L. Liu; Vogelbaum, M A

    2010-01-01

    Background: O 6-methylguanine-DNA methyltransferase (MGMT) expression in glioblastoma correlates with temozolomide resistance. Dose-intense temozolomide schedules deplete MGMT activity in peripheral blood mononuclear cells; however, no published data exist evaluating the effect of temozolomide schedules on intracranial tumour MGMT activity. Methods: Human glioblastoma cells (GBM43) with an unmethylated MGMT promoter were implanted intracranially in immunodeficient rodents. Three weeks later, ...

  10. Infectious drug resistance during an outbreak of salmonellosis

    International Nuclear Information System (INIS)

    The sudden acquisition of aminoglycoside resistance among Salmonella group C1 isolates causing summer diarrhoea raised the possibility of plasmid-mediated resistance. The demonstration of circular DNA species in the resistant, but not in the sensitive salmonellae and the transfer by conjugation of antibiotic resistance to a sensitive strain of Escherichia coli, was consistent with plasmid-mediated resistance

  11. Overexpression of the mitochondrial methyltransferase TFB1M in the mouse does not impact mitoribosomal methylation status or hearing

    DEFF Research Database (Denmark)

    Lee, Seungmin; Rose, Simon; Metodiev, Metodi D;

    2015-01-01

    Mitochondrial dysfunction is a well-established cause of sensorineural deafness, but the pathophysiological events are poorly understood. Non-syndromic deafness and predisposition to aminoglycoside-induced deafness can be caused by specific mutations in the 12S rRNA gene of mtDNA and are thus...... 'hypermethylation' of two conserved adenosines of 12S rRNA in the mitoribosome is of key pathophysiological importance in sensorineural deafness. In support for this concept, it was reported that overexpression of the essential mitochondrial methyltransferase TFB1M in the mouse was sufficient to induce...... mitoribosomal hypermethylation and deafness. At variance with this model, we show here that 12S rRNA is near fully methylated in vivo in the mouse and thus cannot be further methylated to any significant extent. Furthermore, bacterial artificial chromosome transgenic mice overexpressing TFB1M have no increase...

  12. Chaperonins fight aminoglycoside-induced protein misfolding and promote short-term tolerance in Escherichia coli

    DEFF Research Database (Denmark)

    Goltermann, Lise; Good, Liam; Bentin, Thomas

    2013-01-01

    survival, whereas inhibition of chaperonin expression sensitized bacteria. Overexpression of the DnaK/DnaJ/GrpE chaperone system similarly facilitated survival but did not promote growth of aminoglycoside-treated bacteria. Inhibition of chaperonin expression sensitized bacteria to aminoglycosides as...

  13. Unusual regioversatility of acetyltransferase Eis, a cause of drug resistance in XDR-TB

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Wenjing; Biswas, Tapan; Porter, Vanessa R.; Tsodikov, Oleg V.; Garneau-Tsodikova, Sylvie (Michigan)

    2011-09-06

    The emergence of multidrug-resistant and extensively drug-resistant (XDR) tuberculosis (TB) is a serious global threat. Aminoglycoside antibiotics are used as a last resort to treat XDR-TB. Resistance to the aminoglycoside kanamycin is a hallmark of XDR-TB. Here, we reveal the function and structure of the mycobacterial protein Eis responsible for resistance to kanamycin in a significant fraction of kanamycin-resistant Mycobacterium tuberculosis clinical isolates. We demonstrate that Eis has an unprecedented ability to acetylate multiple amines of many aminoglycosides. Structural and mutagenesis studies of Eis indicate that its acetylation mechanism is enabled by a complex tripartite fold that includes two general control non-derepressible 5 (GCN5)-related N-acetyltransferase regions. An intricate negatively charged substrate-binding pocket of Eis is a potential target of new antitubercular drugs expected to overcome aminoglycoside resistance.

  14. Aminoglycoside-derived amphiphilic nanoparticles for molecular delivery.

    Science.gov (United States)

    Miryala, Bhavani; Godeshala, Sudhakar; Grandhi, Taraka Sai Pavan; Christensen, Matthew D; Tian, Yanqing; Rege, Kaushal

    2016-10-01

    The development of effective drug carriers can lead to improved outcomes in a variety of disease conditions. Aminoglycosides have been used as antibacterial therapeutics, and are attractive as monomers for the development of polymeric materials in various applications. Here, we describe the development of novel aminoglycoside-derived amphiphilic nanoparticles for drug delivery, with an eye towards ablation of cancer cells. The aminoglycoside paromomycin was first cross-linked with resorcinol diglycidyl ether leading to the formation of a poly (amino ether), PAE. PAE molecules were further derivatized with methoxy-terminated poly(ethylene glycol) or mPEG resulting in the formation of mPEG-PAE polymer, which self-assembled to form nanoparticles. Formation of the mPEG-PAE amphiphile was characterized using (1)H NMR, (13)C NMR, gel permeation chromatography (GPC) and FTIR spectroscopy. Self-assembly of the polymer into nanoparticles was characterized using dynamic light scattering, zeta potential analyses, atomic force microscopy (AFM) and the pyrene fluorescence assay. mPEG-PAE nanoparticles were able to carry significant amounts of doxorubicin (DOX), presumably by means of hydrophobic interactions between the drug and the core. Cell-based studies indicated that mPEG-PAE nanoparticles, loaded with doxorubicin, were able to induce significant loss in viabilities of PC3 human prostate cancer, MDA-MB-231 human breast cancer, and MB49 murine bladder cancer cells; empty nanoparticles resulted in negligible losses of cell viability under the conditions investigated. Taken together, our results indicate that the mPEG-PAE nanoparticle platform is attractive for drug delivery in different applications, including cancer. PMID:27472455

  15. Caffeine synthase and related methyltransferases in plants.

    Science.gov (United States)

    Misako, Kato; Kouichi, Mizuno

    2004-05-01

    Caffeine (1,3,7-trimethylxanthine) is a purine alkaloid present in high concentrations in tea and coffee and it is also found in a number of beverages such as coca cola. It is necessary to elucidate the caffeine biosynthetic pathway and to clone the genes related to the production of caffeine not only to determine the metabolism of the purine alkaloid but also to control the content of caffeine in tea and coffee. The available data support the operation of a xanthosine-->7-methylxanthosine-->7-methylxanthine-->theobromine-->caffeine pathway as the major route to caffeine. Since the caffeine biosynthetic pathway contains three S-adenosyl-L-methionine (SAM) dependent methylation steps, N-methyltransferases play important roles. This review focuses on the enzymes and genes involved in the methylation of purine ring. Caffeine synthase, the SAM-dependent methyltransferase involved in the last two steps of caffeine biosynthesis, was originally purified from young tea leaves (Camellia sinensis). The isolated cDNA, termed TCS1, consists of 1,483 base pairs and encodes a protein of 369 amino acids. Subsequently, the homologous genes that encode caffeine biosynthetic enzymes from coffee (Coffea arabica) were isolated. The recombinant proteins are classified into the three types on the basis of their substrate specificity i.e. 7-methylxanthosine synthase, theobromine synthase and caffeine synthase. The predicted amino acid sequences of caffeine biosynthetic enzymes derived from C. arabica exhibit more than 80% homology with those of the clones and but show only 40% homology with TCS1 derived from C. sinensis. In addition, they share 40% homology with the amino acid sequences of salicylic carboxyl methyltransferase, benzoic acid carboxyl methyltransferase and jasmonic acid carboxyl methyltransferase which belong to a family of motif B' methyltransferases which are novel plant methyltransferases with motif B' instead of motif B as the conserved region. PMID:14977590

  16. Salmonella enterica Serovar Typhimurium blaPER-1-Carrying Plasmid pSTI1 Encodes an Extended-Spectrum Aminoglycoside 6′-N-Acetyltransferase of Type Ib

    OpenAIRE

    Casin, Isabelle; Hanau-Berçot, Beatrice; Podglajen, Isabelle; Vahaboglu, Haluk; Collatz, Ekkehard

    2003-01-01

    We have studied the aminoglycoside resistance gene, which confers high levels of resistance to both amikacin and gentamicin, that is carried by plasmid pSTI1 in the PER-1 β-lactamase-producing strain of Salmonella enterica serovar Typhimurium previously isolated in Turkey. This gene, called aac(6′)-Ib11, was found in a class 1 integron and codes for a protein of 188 amino acids, a fusion product between the N-terminal moiety (8 amino acids) of the signal peptide of the β-lactamase OXA-1 and t...

  17. Study of the Interference between Plectranthus Species Essential Oils from Brazil and Aminoglycosides

    Directory of Open Access Journals (Sweden)

    Fabíola Fernandes Galvão Rodrigues

    2013-01-01

    Full Text Available Plectranthus is one of the most representative genera of Lamiaceae family. In this study, the essential oils from Plectranthus amboinicus, Plectranthus ornatus, and Plectranthus barbatus were investigated for their chemical composition and antimicrobial and modulatory activities. The major components found were carvacrol (54.4%—P. amboinicus and eugenol (22.9%—P. ornatus e 25.1%—P. barbatus. In vitro antimicrobial activity was conducted against Escherichia coli, Proteus vulgaris, Bacillus cereus, Pseudomonas aeruginosa, Staphylococcus aureus, and Staphylococcus aureus (multiresistant using microdilution method. The results of bioassay showed that all strains were sensitive to the oils, except P. aeruginosa that was resistant to P. amboinicus and P. ornatus. A synergistic effect of all essential oils combined with the aminoglycosides was demonstrated. These results show that P. amboinicus, P. ornatus, and P. barbatus inhibit the growth of pathogenic microorganism, and besides this they present antibiotic modifying activity, providing a new perspective against the problem of bacterial resistance to antibiotics.

  18. Andrographolide: A potent antituberculosis compound that targets Aminoglycoside 2'-N-acetyltransferase in Mycobacterium tuberculosis.

    Science.gov (United States)

    Prabu, Amudha; Hassan, Sameer; Prabuseenivasan; Shainaba, A S; Hanna, L E; Kumar, Vanaja

    2015-09-01

    Tuberculosis (TB) still remains a major challenging infectious disease. The increased rate of emergence of multi-drug resistant and extensively-drug resistant strains of the organism has further complicated the situation, resulting in an urgent need for new anti-TB drugs. Antimycobacterial activity of Andrographis paniculata was evaluated using a rapid LRP assay and the probable targets were identified by docking analysis. The methanolic extract of A. paniculata showed maximum antimycobacterial activity at 250μg/ml against all the tested strains of M. tuberculosis (H37Rv, MDR, and drug sensitive). Based on bioassay guided fractionation, andrographolide was identified as the potent molecule. With the docking analysis, both ICDH (Isocitrate Dehydrogenase) and AAC (Aminoglycoside 2'-N-acetyltransferase) were predicted as targets of andrographolide in M. tuberculosis. Molecular simulation revealed that, ICDH showed low binding affinity to andrographolide. However, for AAC, the andrographolide was observed to be well within the active site after 10ns of molecular simulation. This suggests that ACC (PDB ID 1M4I) could be the probable target for andrographolide. PMID:26245695

  19. Potentiation of aminoglycoside antibiotic activity using the body fat from the snake Boa constrictor

    Directory of Open Access Journals (Sweden)

    Felipe S. Ferreira

    2011-06-01

    Full Text Available Boa constrictor is widely used in traditional communities in many different folk remedies and products derived from it are sold in public markets throughout northeastern Brazil and as its body fat has many different therapeutic indications as a folk remedy. The present work evaluates the antibacterial activity of the body fat from the snake Boa constrictor when employed either alone or in combination with antibiotics and discusses the ecological implications of the use of this traditional remedy. Oil (OBC was extracted from body fat located in the ventral region of B. constrictor using hexane as a solvent. The antibacterial activity of OBC was tested against standard as well as multi-resistant lines, either alone and in combination with antibiotics. OBC did not demonstrate any relevant antibacterial activity against standard or multidrug-resistant bacterial strains. OBC showed synergistic activity when combined with the aminoglycoside antibiotics. Our results indicate that the body fat of Boa constrictor does not possess bactericidal activity, from the clinical point of view, but when combined with an antibiotic, the fat demonstrated a significant synergistic activity.

  20. Directed evolution of improved zinc finger methyltransferases.

    Directory of Open Access Journals (Sweden)

    Brian Chaikind

    Full Text Available The ability to target DNA methylation toward a single, user-designated CpG site in vivo may have wide applicability for basic biological and biomedical research. A tool for targeting methylation toward single sites could be used to study the effects of individual methylation events on transcription, protein recruitment to DNA, and the dynamics of such epigenetic alterations. Although various tools for directing methylation to promoters exist, none offers the ability to localize methylation solely to a single CpG site. In our ongoing research to create such a tool, we have pursued a strategy employing artificially bifurcated DNA methyltransferases; each methyltransferase fragment is fused to zinc finger proteins with affinity for sequences flanking a targeted CpG site for methylation. We sought to improve the targeting of these enzymes by reducing the methyltransferase activity at non-targeted sites while maintaining high levels of activity at a targeted site. Here we demonstrate an in vitro directed evolution selection strategy to improve methyltransferase specificity and use it to optimize an engineered zinc finger methyltransferase derived from M.SssI. The unusual restriction enzyme McrBC is a key component of this strategy and is used to select against methyltransferases that methylate multiple sites on a plasmid. This strategy allowed us to quickly identify mutants with high levels of methylation at the target site (up to ∼80% and nearly unobservable levels of methylation at a off-target sites (<1%, as assessed in E. coli. We also demonstrate that replacing the zinc finger domains with new zinc fingers redirects the methylation to a new target CpG site flanked by the corresponding zinc finger binding sequences.

  1. Deciphering the details of RNA aminoglycoside interactions: from atomistic models to biotechnological applications

    Energy Technology Data Exchange (ETDEWEB)

    Ilgu, Muslum [Iowa State Univ., Ames, IA (United States)

    2012-01-01

    A detailed study was done of the neomycin-B RNA aptamer for determining its selectivity and binding ability to both neomycin– and kanamycin-class aminoglycosides. A novel method to increase drug concentrations in cells for more efficiently killing is described. To test the method, a bacterial model system was adopted and several small RNA molecules interacting with aminoglycosides were cloned downstream of T7 RNA polymerase promoter in an expression vector. Then, the growth analysis of E. coli expressing aptamers was observed for 12-hour period. Our analysis indicated that aptamers helped to increase the intracellular concentration of aminoglycosides thereby increasing their efficacy.

  2. Cp*Rh-based indicator-displacement assays for the identification of amino sugars and aminoglycosides.

    Science.gov (United States)

    Zaubitzer, Friederike; Buryak, Andrey; Severin, Kay

    2006-05-01

    Indicator-displacement assays based on the organometallic complex [{Cp*RhCl2}2] (Cp*=pentamethylcyclopentadienyl) and the dye gallocyanine were used to sense amino sugars and aminoglycosides in buffered aqueous solution by conducting UV-visible spectroscopy. The data of three assays at pH 7.0, 8.0, and 9.0 were sufficient to distinguish between the amino sugars galactosamine, glucosamine, mannosamine and the aminoglycosides kanamycin A, kanamycin B, amikacin, apramycin, paromomycin, and streptomycin. Furthermore, the assays were used to characterize mixtures of aminoglycosides and obtain quantitative information about the respective analytes. PMID:16521137

  3. Potentiation of antibiotic activity of aminoglycosides by natural products from Cordia verbenacea DC.

    Science.gov (United States)

    Matias, Edinardo F F; Alves, Erivania F; Silva, Maria K N; Carvalho, Victoria R A; Medeiros, Cassio R; Santos, Francisco A V; Bitu, Vanessa C N; Souza, Celestina E S; Figueredo, Fernando G; Boligon, Aline A; Athayde, Margareth L; Costa, José G M; Coutinho, Henrique D M

    2016-06-01

    Medicinal plants are often the only therapeutic resource for many communities and ethnic groups. Cordia verbenacea DC., "Erva-baleeira," is one of the species of plants currently used to produce a phytotherapeutic product extracted from its leaves. The present study aimed to establish its chemical profile, antibacterial activity and resistance-modulating potential. The C. verbenacea extracts were prepared from fresh leaves using solvents as methanol and hexane. Ethyl Acetate was used for the preparation of the fraction. Phytochemical screening was carried out using HPLC-DAD for determination and quantification of the secondary metabolites present in the fractions. Antibacterial and resistance-modulation assays were performed to determine minimum inhibitory concentration (MIC) using a microdilution assay. The data were subjected to statistical analysis with two-way ANOVA and Bonferroni posttests. Results of phytochemical prospecting and HPLC analysis of the fractions were in agreement with the literature. The natural products presented moderate antibacterial activity when considering the clinical relevance of a MIC of 256 μg/mL against Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa, and 512 μg/mL against P. aeruginosa. However, when the fractions were combined with antibiotics we observed a synergic effect, as natural products enhanced the antibacterial effect of aminoglycosides, significantly decreasing the MIC of antibiotics at 12.5%-98.4%. We believe that the data obtained from phytochemical analysis and from antibacterial and resistance modulation assays of C. verbenacea extracts new can open perspectives in the search for new alternatives for the treatment of bacterial infections and stimulate the renewed use of antibiotics with reduced effectiveness due to resistance. PMID:27033000

  4. Antifungal amphiphilic aminoglycoside K20: bioactivities and mechanism of action

    Directory of Open Access Journals (Sweden)

    Sanjib K. Shrestha

    2014-12-01

    Full Text Available K20 is a novel amphiphilic antifungal aminoglycoside that is synthetically derived from the antibiotic kanamycin A. Reported here are investigations of K20’s antimicrobial activities, cytotoxicity, and fungicidal mechanism of action. In vitro growth inhibitory activities against a variety of human and plant pathogenic yeasts, filamentous fungi, and bacteria were determined using microbroth dilution assays and time-kill curve analyses, and hemolytic and animal cell cytotoxic activities were determined. Effects on Cryptococcus neoformans H-99 infectivity were determined with a preventive murine lung infection model. The antifungal mechanism of action was studied using intact fungal cells, yeast lipid mutants, and small unilamellar lipid vesicles. K20 exhibited broad-spectrum in vitro antifungal activities but not antibacterial activities. Pulmonary, single dose-administration of K20 reduced C. neoformans lung infection rates 4-fold compared to controls. Hemolysis and half-maximal cytotoxicities of mammalian cells occurred at concentrations that were 10 to 32-fold higher than fungicidal MICs. With fluorescein isothiocyanate, 20 to 25 mg/L K20 caused staining of >95% of C. neoformans and Fusarium graminearum cells and at 31.3 mg/L caused rapid leakage (30 to 80% in 15 min of calcein from preloaded small unilamellar lipid vesicles. K20 appears to be a broad-spectrum fungicide, capable of reducing the infectivity of C. neoformans, and exhibits low hemolytic activity and mammalian cell toxicity. It perturbs the plasma membrane by mechanisms that are lipid modulated. K20 is a novel amphiphilic aminoglycoside amenable to scalable production and a potential lead antifungal for therapeutic and crop protection applications.

  5. Sensitivity of ribosomes of the hyperthermophilic bacterium Aquifex pyrophilus to aminoglycoside antibiotics.

    OpenAIRE

    Bocchetta, M; Huber, R.; Cammarano, P

    1996-01-01

    A poly(U)-programmed cell-free system from the hyperthermophilic bacterium Aquifex pyrophilus has been developed, and the susceptibility of Aquifex ribosomes to the miscoding-inducing and inhibitory actions of all known classes of aminoglycoside antibiotics has been assayed at temperatures (75 to 80 degrees C) close to the physiological optimum for cell growth. Unlike Thermotoga maritima ribosomes, which are systematically refractory to all known classes of aminoglycoside compounds (P. Londei...

  6. Toggled RNA Aptamers Against Aminoglycosides Allowing Facile Detection of Antibiotics Using Gold Nanoparticle Assays

    OpenAIRE

    Derbyshire, Nicola; White, Simon J.; Bunka, David H. J.; Song, Lei; Stead, Sara; Tarbin, Jonathan; Sharman, Matthew; Zhou, Dejian; Stockley, Peter G.

    2012-01-01

    We have used systematic evolution of ligands by exponential enrichment (SELEX) to isolate RNA aptamers against aminoglycoside antibiotics. The SELEX rounds were toggled against four pairs of aminoglycosides with the goal of isolating reagents that recognize conserved structural features. The resulting aptamers bind both of their selection targets with nanomolar affinities. They also bind the less structurally related targets, although they show clear specificity for this class of antibiotics....

  7. Determination of aminoglycoside antibiotics using complex compounds of chromotropic acid bisazoderivatives with rare earth ions

    International Nuclear Information System (INIS)

    Studies of complex formation of bisazo derivatives of chromotropic acid with rare earth ions and aminoglycoside antibiotics have made it possible to choose carboxyarsenazo, orthanyl R and carboxynitrazo as highly sensitive reagents for determining aminoglycoside antibiotics. Conditions have been found for the formation of precipitates of different-ligand complexes containing rare earth ions, bisazo derivatives of chromotropic acid and aminogylcoside antibiotics. A procedure has been worked out of determining the antibiotics in biological samples with carboxyarsenazo

  8. Once Daily Dosing of Aminoglycosides in Pediatric Cystic Fibrosis Patients: A Review of the Literature

    OpenAIRE

    Wassil, Sarah K.; Fox, Kristie M.; White, James W.

    2008-01-01

    Patients with cystic fibrosis receive many courses of antibiotic therapy throughout their lifetime. Dosing aminoglycosides once daily has become common practice in many of these individuals. Due to ease of home administration, decreased nursing time, and improved quality of life, this regimen is being increasingly explored in the cystic fibrosis population. Because patients with cystic fibrosis have increased aminoglycoside clearance, once daily dosing may result in a prolonged time during th...

  9. XBP1 mitigates aminoglycoside-induced endoplasmic reticulum stress and neuronal cell death.

    Science.gov (United States)

    Oishi, N; Duscha, S; Boukari, H; Meyer, M; Xie, J; Wei, G; Schrepfer, T; Roschitzki, B; Boettger, E C; Schacht, J

    2015-01-01

    Here we study links between aminoglycoside-induced mistranslation, protein misfolding and neuropathy. We demonstrate that aminoglycosides induce misreading in mammalian cells and assess endoplasmic reticulum (ER) stress and unfolded protein response (UPR) pathways. Genome-wide transcriptome and proteome analyses revealed upregulation of genes related to protein folding and degradation. Quantitative PCR confirmed induction of UPR markers including C/EBP homologous protein, glucose-regulated protein 94, binding immunoglobulin protein and X-box binding protein-1 (XBP1) mRNA splicing, which is crucial for UPR activation. We studied the effect of a compromised UPR on aminoglycoside ototoxicity in haploinsufficient XBP1 (XBP1(+/-)) mice. Intra-tympanic aminoglycoside treatment caused high-frequency hearing loss in XBP1(+/-) mice but not in wild-type littermates. Densities of spiral ganglion cells and synaptic ribbons were decreased in gentamicin-treated XBP1(+/-) mice, while sensory cells were preserved. Co-injection of the chemical chaperone tauroursodeoxycholic acid attenuated hearing loss. These results suggest that aminoglycoside-induced ER stress and cell death in spiral ganglion neurons is mitigated by XBP1, masking aminoglycoside neurotoxicity at the organismal level. PMID:25973683

  10. Somatic mutations in glioblastoma are associated with methylguanine-DNA methyltransferase methylation

    OpenAIRE

    McDonald, Kerrie L.; Tabone, Tania; Nowak, Anna K; Erber, Wendy N.

    2015-01-01

    The high level of methylguanine-DNA methyltransferase (MGMT) in glioblastoma is responsible for resistance to alkylating agents, such as temozolomide (TMZ). In glioblastomas with a methylated MGMT promoter, MGMT deficiency is presumed, resulting in an enhanced effect of TMZ. The aim of the present study was to investigate whether genomic alterations work synergistically with MGMT methylation status and contribute to the response to treatment and overall prognosis in glioblastoma. The current ...

  11. Serum Aminoglycoside Assay by Enzyme-Mediated Immunoassay (EMIT): Correlation with Radioimmunoassay, Fluoroimmunoassay, and Acetyltransferase and Microbiological Assays

    OpenAIRE

    White, L O; Scammell, L. M.; Reeves, D S

    1981-01-01

    Enzyme-mediated immunoassay (EMIT) serum aminoglycoside assay results were accurate and precise and correlated well with radioimmunoassay, fluoroimmunoassay, and acetyltransferase and microbiological assay determinations.

  12. Multiple antibiotic resistance in Stenotrophomonas maltophilia.

    OpenAIRE

    Alonso, A.; Martínez, J L

    1997-01-01

    A cryptic multidrug resistance (MDR) system in Stenotrophomonas maltophilia, the expression of which is selectable by tetracycline, is described. Tetracycline resistance was the consequence of active efflux of the antibiotic, and it was associated with resistance to quinolones and chloramphenicol, but not to aminoglycosides or beta-lactam antibiotics. MDR is linked to the expression of an outer membrane protein (OMP54) both in a model system and in multidrug-resistant clinical isolates.

  13. Clinical Pharmacokinetics of Penicillins, Cephalosporins and Aminoglycosides in the Neonate: A Review

    Directory of Open Access Journals (Sweden)

    Gian Maria Pacifici

    2010-08-01

    Full Text Available Bacterial infections are common in the neonates and are a major cause of morbidity and mortality. Sixty percent of preterm infants admitted to neonatal intensive care units received at least one antibiotic during the first week of life. Penicillins, aminoglycosides and cephalosporins comprised 53, 43 and 16%, respectively. Kinetic parameters such as the half-life (t1/2, clearance (Cl, and volume of distribution (Vd change with development, so the kinetics of penicillins, cephalosporins and aminoglycosides need to be studied in order to optimise therapy with these drugs. The aim of this study is to review the pharmacokinetics of penicillins, cephalosporins and aminoglycosides in the neonate in a single article in order to provide a critical analysis of the literature and thus provide a useful tool in the hands of physicians. The bibliographic search was performed electronically using PubMed, as the search engine, until February 2nd, 2010. Medline search terms were as follows: pharmacokinetics AND (penicillins OR cephalosporins OR aminoglycosides AND infant, newborn, limiting to humans. Penicillins, cephalosporins and aminoglycosides are fairly water soluble and are mainly eliminated by the kidneys. The maturation of the kidneys governs the pharmacokinetics of penicillins, cephalosporins and aminoglycosides in the neonate. The renal excretory function is reduced in preterms compared to term infants and Cl of these drugs is reduced in premature infants. Gestational and postnatal ages are important factors in the maturation of the neonate and, as these ages proceed, Cl of penicillins, cephalosporins and aminoglycosides increases. Cl and t1/2 are influenced by development and this must be taken into consideration when planning a dosage regimen with these drugs. More pharmacokinetic studies are required to ensure that the dose recommended for the treatment of sepsis in the neonate is evidence based.

  14. Purification, crystallization and preliminary X-ray analysis of aminoglycoside-2′′-phosphotransferase-Ic [APH(2′′)-Ic] from Enterococcus gallinarum

    International Nuclear Information System (INIS)

    APH(2′′)-Ic is an enzyme that is responsible for high-level gentamicin resistance in E. gallinarum isolates. Crystals of the wild-type enzyme and three mutants have been prepared and a complete X-ray diffraction data set was collected to 2.15 Å resolution from an F108L crystal. Bacterial resistance to aminoglycoside antibiotics is primarily the result of deactivation of the drugs. Three families of enzymes are responsible for this activity, with one such family being the aminoglycoside phosphotransferases (APHs). The gene encoding one of these enzymes, aminoglycoside-2′′-phosphotransferase-Ic [APH(2′′)-Ic] from Enterococcus gallinarum, has been cloned and the wild-type protein (comprising 308 amino-acid residues) and three mutants that showed elevated minimum inhibitory concentrations towards gentamicin (F108L, H258L and a double mutant F108L/H258L) were expressed in Escherichia coli and subsequently purified. All APH(2′′)-Ic variants were crystallized in the presence of 14–20%(w/v) PEG 4000, 0.25 M MgCl2, 0.1 M Tris–HCl pH 8.5 and 1 mM Mg2GTP. The crystals belong to the monoclinic space group C2, with one molecule in the asymmetric unit. The approximate unit-cell parameters are a = 82.4, b = 54.2, c = 77.0 Å, β = 108.8°. X-ray diffraction data were collected to approximately 2.15 Å resolution from an F108L crystal at beamline BL9-2 at SSRL, Stanford, California, USA

  15. Catechol-O-Methyltransferase Polymorphism and Endometriosis

    OpenAIRE

    Wieser, Fritz; Wenzl, Rene; Tempfer, Clemens; Worda, Christoph; Huber, Johannes; Schneeberger, Christian

    2002-01-01

    Purpose: Catechol-O-methyltransferase (COMT) inactivates the estradiol metabolites, 2-hydroxy and 4-hydroxy catechols, which have been implicated in the pathogenesis of endometriosis. A COMT valine to methionine polymorphism (G-to-A) in exon 4 of the COMT gene is polymorphic in the human population, with 25% of Caucasians being homozygous for the low-activity allele (COMT-L) of the enzyme. In a case-control study we investigated whether this COMT polymorphism is associated with endometriosis.

  16. A surprising dipolar cycloaddition provides ready access to aminoglycosides.

    Science.gov (United States)

    Dahl, Russell S; Finney, Nathaniel S

    2004-07-14

    This contribution describes the results of a new research effort in our laboratory aimed at the synthesis of novel aminoglycosides and amino-C-glycosides. Despite the importance of such compounds, and the previous development of some methodological solutions, this remains an important area of research. Notable features of our approach, which is distinct from and complementary to previous efforts, are the following: (1) Reliance on a surprising and unprecedented formation of glycal triazolines via an inverse electron demand dipolar cycloaddition of glucal. We believe this desirable transformation has not previously been discovered because of the unusual selection of substrates and solvent required. (2) Very mild reaction conditions. An initial thermal cycloaddition is carried out in an inert solvent, the triazoline generated is photochemically converted to a reactive aziridine, and the crude aziridine undergoes ring opening at room temperature in the presence of a nucleophile and a mild Lewis acid catalyst. (3) Formation of products lacking an N-acyl group, allowing ready synthesis of novel glucosamine derivatives. PMID:15237974

  17. Structural Chemistry of Human RNA Methyltransferases.

    Science.gov (United States)

    Schapira, Matthieu

    2016-03-18

    RNA methyltransferases (RNMTs) play important roles in RNA stability, splicing, and epigenetic mechanisms. They constitute a promising target class that is underexplored by the medicinal chemistry community. Information of relevance to drug design can be extracted from the rich structural coverage of human RNMTs. In this work, the structural chemistry of this protein family is analyzed in depth. Unlike most methyltransferases, RNMTs generally feature a substrate-binding site that is largely open on the cofactor-binding pocket, favoring the design of bisubstrate inhibitors. Substrate purine or pyrimidines are often sandwiched between hydrophobic walls that can accommodate planar ring systems. When the substrate base is laying on a shallow surface, a 5' flanking base is sometimes anchored in a druggable cavity. The cofactor-binding site is structurally more diverse than in protein methyltransferases and more druggable in SPOUT than in Rossman-fold enzymes. Finally, conformational plasticity observed both at the substrate and cofactor binding sites may be a challenge for structure-based drug design. The landscape drawn here may inform ongoing efforts toward the discovery of the first human RNMT inhibitors. PMID:26566070

  18. Mechanistic studies of copper(II)-aminoglycoside mediated DNA damage and magnesium catalyzed nuclease activity of hammerhead ribozyme

    Science.gov (United States)

    Patwardhan, Anjali A.

    The antibacterial activity of aminoglycosides stems from their high affinity binding to the 16S rRNA in bacteria resulting in inhibition of protein synthesis. Used to treat acute bacterial infections these antibiotics have limited applications due to their high dosage requirements and the emergence of resistant strains. We have synthesized and characterized Cu(II) derivatives of the aminoglycosides, kanamycin A, tobramycin, neamine, kanamycin B, neomycin B, and paromomycin. The first three exhibit preferential and tight binding to Cu(II) as against neomycin B and kanamycin B and paromomycin. EPR of frozen solutions and UV-visible spectroscopy suggest a change in geometry around the Cu(II) but the stabilities of the complexes in water differ. These copper derivatives efficiently cleave plasmid DNA at micromolar concentrations (hydrolytic) and at nanomolar concentrations in the presence co-reactants like hydrogen peroxide or ascorbic acid. Hydrolysis is multi turnover and exhibits Michelis-Menten kinetics with enzyme-like behavior whereas oxidative cleavage is highly specific with C-4' H abstraction resulting in characteristic base propenal and nucleotide base products. Hydroxyl radicals generated are copper based and are generated in close proximity of the substrate. Hammerhead ribozymes are selectively hydrolyzed in the presence of divalent ions with Mg2+ being the metal ion of choice in vivo . Our studies with complex ions like cobalt hexaammine and fac-triamminetriaquochromium(III) establish outer sphere interactions of Mg2+ with the hammerhead in the catalytic site. There are two sets of sites, one structural and one catalytic. Complex ions in the catalytic site and divalent ions in the structural site result in a slow but active hammerhead ribozyme suggesting that the complex ions are not inhibitory, contrary to what was suggested previously.

  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. Identification of clinically antibiotic resistant genes Aac(3)-IIa and Aac(6’)-Ib in wastewater samples by multiplex PCR

    OpenAIRE

    Naser Samadi; Rahim Aali; Esrafil Asgari; Hamed Mirhosaeini; Ali Shahriari; Farhad Mahmoodi; Fardin Nouri; Asad Hamdi; Farzad Fanaie; Shahla Yosefi; Saied Dadashi

    2015-01-01

    Background: Aminoglycoside antibiotics are widely used in medical centers, particularly to treat infections. The resistance developed against these agents is a huge concern in health care. A number of researchers have reported that hospital and municipal wastewaters are among the most important dissemination sources of these agent into the environment. Some, however, do not agree with this opinion. In the present study, the prevalence of aminoglycoside resistance genes was investigated in raw...

  1. First report of an OXA-48-producing multidrug-resistant Proteus mirabilis strain from Gaza, Palestine.

    Science.gov (United States)

    Chen, Liang; Al Laham, Nahed; Chavda, Kalyan D; Mediavilla, Jose R; Jacobs, Michael R; Bonomo, Robert A; Kreiswirth, Barry N

    2015-07-01

    We report the first multidrug-resistant Proteus mirabilis strain producing the carbapenemase OXA-48 (Pm-OXA-48) isolated at Al-Shifa hospital in Gaza, Palestine. Draft genome sequencing of Pm-OXA-48 identified 16 antimicrobial resistance genes, encoding resistance to β-lactams, aminoglycosides, fluoroquinolones, phenicols, streptothricin, tetracycline, and trimethoprim-sulfamethoxazole. Complete sequencing of the bla(OXA-48)-harboring plasmid revealed that it is a 72 kb long IncL/M plasmid, harboring carbapenemase gene bla(OXA-48), extended spectrum β-lactamase gene bla(CTX-M-14), and aminoglycoside resistance genes strA, strB, and aph(3')-VIb. PMID:25896692

  2. Draft Genome Sequence for a Clinical Isolate of Vancomycin-Resistant Enterococcus faecalis.

    Science.gov (United States)

    Erickson, Keesha E; Madinger, Nancy E; Chatterjee, Anushree

    2016-01-01

    We report here the draft genome sequence of a multidrug-resistant Enterococcus faecalis strain, isolated from a patient at the University of Colorado Hospital. The genome assembly is 3,040,186 bp in length with 37.6% GC content. This isolate encodes eleven resistance genes, including those for glycopeptide, aminoglycoside, macrolide-lincosamide-streptogramin, and tetracycline resistance. PMID:27340066

  3. Single biosensor immunoassay for the detection of five aminoglycosides in reconstituted skimmed milk

    NARCIS (Netherlands)

    Haasnoot, W.; Cazemier, G.; Koets, M.; Amerongen, van A.

    2003-01-01

    The application of an optical biosensor (Biacore 3000), with four flow channels (Fcs), in combination with a mixture of four specific antibodies resulted in a competitive inhibition biosensor immunoassay (BIA) for the simultaneous detection of the five relevant aminoglycosides in reconstituted skimm

  4. 21 CFR 573.130 - Aminoglycoside 3′-phospho- transferase II.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Aminoglycoside 3â²-phospho- transferase II. 573.130 Section 573.130 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN... genetically modified cotton, oilseed rape, and tomatoes in accordance with the following prescribed...

  5. Natural bizbenzoquinoline derivatives protect zebrafish lateral line sensory hair cells from aminoglycoside toxicity

    Directory of Open Access Journals (Sweden)

    Matthew eKruger

    2016-03-01

    Full Text Available Moderate to severe hearing loss affects 360 million people worldwide and most often results from damage to sensory hair cells. Hair cell damage can result from aging, genetic mutations, excess noise exposure, and certain medications including aminoglycoside antibiotics. Aminoglycosides are effective at treating infections associated with cystic fibrosis and other life-threatening conditions such as sepsis, but cause hearing loss in 20-30% of patients. It is therefore imperative to develop new therapies to combat hearing loss and allow safe use of these potent antibiotics. We approach this drug discovery question using the larval zebrafish lateral line because zebrafish hair cells are structurally and functionally similar to mammalian inner ear hair cells and respond similarly to toxins. We screened a library of 502 natural compounds in order to identify novel hair cell protectants. Our screen identified four bisbenzylisoquinoline derivatives: berbamine, E6 berbamine, hernandezine, and isotetrandrine, each of which robustly protected hair cells from aminoglycoside-induced damage. Using fluorescence microscopy and electrophysiology, we demonstrated that the natural compounds confer protection by reducing antibiotic uptake into hair cells and showed that hair cells remain functional during and after incubation in E6 berbamine. We also determined that these natural compounds do not reduce antibiotic efficacy. Together, these natural compounds represent a novel source of possible otoprotective drugs that may offer therapeutic options for patients receiving aminoglycoside treatment.

  6. 21 CFR 173.170 - Aminoglycoside 3′-phospho-trans-ferase II.

    Science.gov (United States)

    2010-04-01

    ... (CONTINUED) FOOD FOR HUMAN CONSUMPTION (CONTINUED) SECONDARY DIRECT FOOD ADDITIVES PERMITTED IN FOOD FOR HUMAN CONSUMPTION Enzyme Preparations and Microorganisms § 173.170 Aminoglycoside 3′-phospho-trans... development of genetically modified cotton, oilseed rape, and tomatoes in accordance with the...

  7. Natural Bizbenzoquinoline Derivatives Protect Zebrafish Lateral Line Sensory Hair Cells from Aminoglycoside Toxicity.

    Science.gov (United States)

    Kruger, Matthew; Boney, Robert; Ordoobadi, Alexander J; Sommers, Thomas F; Trapani, Josef G; Coffin, Allison B

    2016-01-01

    Moderate to severe hearing loss affects 360 million people worldwide and most often results from damage to sensory hair cells. Hair cell damage can result from aging, genetic mutations, excess noise exposure, and certain medications including aminoglycoside antibiotics. Aminoglycosides are effective at treating infections associated with cystic fibrosis and other life-threatening conditions such as sepsis, but cause hearing loss in 20-30% of patients. It is therefore imperative to develop new therapies to combat hearing loss and allow safe use of these potent antibiotics. We approach this drug discovery question using the larval zebrafish lateral line because zebrafish hair cells are structurally and functionally similar to mammalian inner ear hair cells and respond similarly to toxins. We screened a library of 502 natural compounds in order to identify novel hair cell protectants. Our screen identified four bisbenzylisoquinoline derivatives: berbamine, E6 berbamine, hernandezine, and isotetrandrine, each of which robustly protected hair cells from aminoglycoside-induced damage. Using fluorescence microscopy and electrophysiology, we demonstrated that the natural compounds confer protection by reducing antibiotic uptake into hair cells and showed that hair cells remain functional during and after incubation in E6 berbamine. We also determined that these natural compounds do not reduce antibiotic efficacy. Together, these natural compounds represent a novel source of possible otoprotective drugs that may offer therapeutic options for patients receiving aminoglycoside treatment. PMID:27065807

  8. Evaluation of Aminoglycoside and Non-Aminoglycoside Compounds for Stop-Codon Readthrough Therapy in Four Lysosomal Storage Diseases.

    Directory of Open Access Journals (Sweden)

    Marta Gómez-Grau

    Full Text Available Nonsense mutations are quite prevalent in inherited diseases. Readthrough drugs could provide a therapeutic option for any disease caused by this type of mutation. Geneticin (G418 and gentamicin were among the first to be described. Novel compounds have been generated, but only a few have shown improved results. PTC124 is the only compound to have reached clinical trials. Here we first investigated the readthrough effects of gentamicin on fibroblasts from one patient with Sanfilippo B, one with Sanfilippo C, and one with Maroteaux-Lamy. We found that ARSB activity (Maroteaux-Lamy case resulted in an increase of 2-3 folds and that the amount of this enzyme within the lysosomes was also increased, after treatment. Since the other two cases (Sanfilippo B and Sanfilippo C did not respond to gentamicin, the treatments were extended with the use of geneticin and five non-aminoglycoside (PTC124, RTC13, RTC14, BZ6 and BZ16 readthrough compounds (RTCs. No recovery was observed at the enzyme activity level. However, mRNA recovery was observed in both cases, nearly a two-fold increase for Sanfilippo B fibroblasts with G418 and around 1.5 fold increase for Sanfilippo C cells with RTC14 and PTC124. Afterwards, some of the products were assessed through in vitro analyses for seven mutations in genes responsible for those diseases and, also, for Niemann-Pick A/B. Using the coupled transcription/translation system (TNT, the best results were obtained for SMPD1 mutations with G418, reaching a 35% recovery at 0.25 μg/ml, for the p.W168X mutation. The use of COS cells transfected with mutant cDNAs gave positive results for most of the mutations with some of the drugs, although to a different extent. The higher enzyme activity recovery, of around two-fold increase, was found for gentamicin on the ARSB p.W146X mutation. Our results are promising and consistent with those of other groups. Further studies of novel compounds are necessary to find those with more

  9. Potent Inhibitors of Acetyltransferase Eis Overcome Kanamycin Resistance in Mycobacterium tuberculosis.

    Science.gov (United States)

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

    2016-06-17

    A major cause of tuberculosis (TB) resistance to the aminoglycoside kanamycin (KAN) is the Mycobacterium tuberculosis (Mtb) acetyltransferase Eis. Upregulation of this enzyme is responsible for inactivation of KAN through acetylation of its amino groups. A 123 000-compound high-throughput screen (HTS) yielded several small-molecule Eis inhibitors that share an isothiazole S,S-dioxide heterocyclic core. These were investigated for their structure-activity relationships. Crystal structures of Eis in complex with two potent inhibitors show that these molecules are bound in the conformationally adaptable aminoglycoside binding site of the enzyme, thereby obstructing binding of KAN for acetylation. Importantly, we demonstrate that several Eis inhibitors, when used in combination with KAN against resistant Mtb, efficiently overcome KAN resistance. This approach paves the way toward development of novel combination therapies against aminoglycoside-resistant TB. PMID:27010218

  10. Domain V of 23S rRNA contains all the structural elements necessary for recognition by the ErmE methyltransferase

    DEFF Research Database (Denmark)

    Vester, B; Douthwaite, S

    1994-01-01

    enzyme efficiently modifies 23S rRNA in vitro. Removal of most of the 23S rRNA structure, so that only domain V (nucleotides 2000 to 2624) remains, does not affect the efficiency of modification by the methyltransferase. In addition, modification still occurs after the rRNA tertiary structure has been......The ErmE methyltransferase from the erythromycin-producing actinomycete Saccharopolyspora erythraea dimethylates the N-6 position of adenine 2058 in domain V of 23S rRNA. This modification confers resistance to erythromycin and to other macrolide, lincosamide, and streptogramin B antibiotics. We...

  11. Monolignol 4-O-methyltransferases and uses thereof

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Chang-Jun; Bhuiya, Mohammad-Wadud; Zhang, Kewei

    2014-11-18

    Modified (iso)eugenol 4-O-methyltransferase enzymes having novel capacity for methylation of monolignols and reduction of lignin polymerization in plant cell wall are disclosed. Sequences encoding the modified enzymes are disclosed.

  12. The inheritance of human erythrocyte catechol-O-methyltransferase activity

    International Nuclear Information System (INIS)

    Erythrocyte catechol-O-methyltransferase (COMT) activity was analyzed in 107 individuals from 23 families. The observed activities were consistent with an autosomal codominant inheritance of the gene coding for erythrocyte COMT activity. (author)

  13. Rapid analysis of aminoglycoside antibiotics in bovine tissues using disposable pipette extraction and ultrahigh performance liquid chromatography - tandem mass spectrometry

    Science.gov (United States)

    A high-throughput qualitative screening and identification method for 9 aminoglycosides of regulatory interest has been developed, validated, and implemented for bovine kidney, liver, and muscle tissues. The method involves extraction at previously validated conditions, cleanup using disposable pip...

  14. Hypnotizability and Catechol-O-Methyltransferase (COMT) polymorphysms in Italians

    OpenAIRE

    Alessandro Gialluisi; Serena Barbuti; Giancarlo Carli; Enrica Laura Santarcangelo

    2014-01-01

    Higher brain dopamine content depending on lower activity of Catechol-O-Methyltransferase (COMT) in subjects with high hypnotisability scores (highs) has been considered responsible for their attentional characteristics. However, the results of the previous genetic studies on association between hypnotisability and the Catechol-O-Methyltransferase (COMT) single nucleotide polymorphism (SNP) rs4680 (Val158Met) were inconsistent. Here, we used a selective genotyping approach to re-evaluate the ...

  15. Defining RNA motif–aminoglycoside interactions via two-dimensional combinatorial screening and structure–activity relationships through sequencing

    OpenAIRE

    Velagapudi, Sai Pradeep; Disney, Matthew D.

    2013-01-01

    RNA is an extremely important target for the development of chemical probes of function or small molecule therapeutics. Aminoglycosides are the most well studied class of small molecules to target RNA. However, the RNA motifs outside of the bacterial rRNA A-site that are likely to be bound by these compounds in biological systems is largely unknown. If such information were known, it could allow for aminoglycosides to be exploited to target other RNAs and, in addition, could provide invaluabl...

  16. Assessment on the adverse effects of Aminoglycosides and Flouroquinolone on sperm parameters and male reproductive tissue: A systematic review

    OpenAIRE

    Arash Khaki

    2015-01-01

    Background: Antibiotic therapies used in treatment of many diseases have adverse effects on fertility. This review analyzes previous comparative studies that surveyed the effects of two common groups of antibiotics on male fertility. Objective: To evaluate histo-pathological effects of fluoroquinolones and aminoglycosides on sperm parameters and male reproductive tissue. Materials and Methods: Articles about the effects of aminoglycosides and fluoroquinolones on male infertility, sperm parame...

  17. Directed Evolution of Aminoglycoside Phosphotransferase (3′) Type IIIa Variants That Inactivate Amikacin but Impose Significant Fitness Costs

    OpenAIRE

    Kramer, Joseph R.; Matsumura, Ichiro

    2013-01-01

    The rules that govern adaptive protein evolution remain incompletely understood. Aminoglycoside aminotransferase (3′) type IIIa (hereafter abbreviated APH(3′)-IIIa) is a good model enzyme because it inactivates kanamycin efficiently; it recognizes other aminoglycoside antibiotics, including amikacin, but not nearly as well. Here we direct the evolution of APH(3′)-IIIa variants with increased activity against amikacin. After four rounds of random mutation and selection in Escherichia coli, the...

  18. Cymbopogon citratus protects against the renal injury induced by toxic doses of aminoglycosides in rabbits

    OpenAIRE

    Ullah, N.; Khan, M. A.; Khan, T.; W Ahmad

    2013-01-01

    Renal injury is the most common side-effect of aminoglycosides. These antimicrobial drugs are particularly effective against Gram-negative microorganisms. The present study was conducted to investigate the renal protective activity of Cymbopogon citratus in gentamicin-induced nephrotoxicity. Male rabbits were divided into four groups (n=6) including group 1 (0.9% saline treated), group 2 (80 mg/kg/day gentamicin-treated), group 3 (200 mg/kg/day Cymbopogon citratus treated) and group 4 (80 mg/...

  19. Intrasaccular injection of aminoglycosides: a novel method for temporary damaging fish inner ear hair cells

    OpenAIRE

    Faucher, Karine; Aas-Hansen, Øyvind; Damsgard, Borge; Stenklev, Niels-Christian

    2008-01-01

    Fish models are increasingly being used for hearing research investigations. Aminoglycoside antibiotics that are used for damaging the inner ear hair cells can have systemic side effects leading to death of study animals. This study aimed to compare two methods: i) systemic (intravenous) and ii) local (intrasaccular) gentamicin administration for induction of inner ear hair cell damage in the Atlantic cod, Gadus morhua (L.). Hair cell damage was assessed using scanning electron microscopy; ha...

  20. Effects of salicylates and aminoglycosides on spontaneous otoacoustic emissions in the Tokay gecko

    OpenAIRE

    Stewart, Charles E; Hudspeth, A. James

    2000-01-01

    The high sensitivity and sharp frequency discrimination of hearing depend on mechanical amplification in the cochlea. To explore the basis of this active process, we examined the pharmacological sensitivity of spontaneous otoacoustic emissions (SOAEs) in a lizard, the Tokay gecko. In a quiet environment, each ear produced a complex but stable pattern of emissions. These SOAEs were reversibly modulated by drugs that affect mammalian otoacoustic emissions, the salicylates and the aminoglycoside...

  1. Rescue of non-sense mutated p53 tumor suppressor gene by aminoglycosides

    OpenAIRE

    Floquet, Célia; Deforges, Jules; Rousset, Jean-Pierre; Bidou, Laure

    2010-01-01

    Mutation-based treatments are a new development in genetic medicine, in which the nature of the mutation dictates the therapeutic strategy. Interest has recently focused on diseases caused by premature termination codons (PTCs). Drugs inducing the readthrough of these PTCs restore the production of a full-length protein. In this study, we explored the possibility of using aminoglycoside antibiotics to induce the production of a full-length functional p53 protein from a gene carrying a PTC. We...

  2. Preparation and characterization of dehydration-rehydration vesicles loaded with aminoglycoside and macrolide antibiotics.

    Science.gov (United States)

    Mugabe, Clement; Azghani, Ali O; Omri, Abdelwahab

    2006-01-13

    Enhanced activity of liposomes-encapsulated antibiotics against clinical isolates of Pseudomonas aeruginosa has been documented with liposomes of low encapsulation efficiency. We sought to construct liposomes with high yield entrapment of aminoglycoside and macrolide antibiotics as well as favorable stability in storage and physiological conditions. Liposome-entrapped aminoglycosides (amikacin, gentamicin, tobramycin) and a macrolide (erythromycin) were prepared by a modified dehydration-rehydration vesicles (DRVs) method, and their particle size and entrapment efficiency were determined. We studied in vitro stability of these vesicles over a 48 h period at 4 and 37 degrees C in phosphate-buffered saline (PBS) and in plasma at 37 degrees C. The mean particle size of DRVs loaded with antibiotics varied from 163.37+/-38.44 to 259.83+/-11.80 nm with no significant difference in regard with the type of the antibiotics encapsulated. Encapsulation efficiency of DRVs loaded with amikacin, gentamicin, tobramycin, and erythromycin were 29.27+/-1.17, 33+/-0.76, 22.33+/-1.48 and 32.06+/-0.82% of initial amount of the drug, respectively. These vesicles were stable regardless of the experimental temperature. Indeed, the liposomes retained more than 75% of the initially encapsulated drugs for the study period of 48 h. DRVs incubated in plasma however, released more antibiotics than those incubated in PBS. In conclusion, using this modified DRV method, we obtained small sized vesicles with high yield entrapment for aminoglycoside and macrolide antibiotics. The technique may be utilized to overcome the low encapsulation efficiency associated with aminoglycoside and macrolide antibiotics. PMID:16289986

  3. Sodium-Glucose Transporter-2 (SGLT2; SLC5A2) Enhances Cellular Uptake of Aminoglycosides

    OpenAIRE

    Meiyan Jiang; Qi Wang; Takatoshi Karasawa; Ja-Won Koo; Hongzhe Li; Steyger, Peter S.

    2014-01-01

    Aminoglycoside antibiotics, like gentamicin, continue to be clinically essential worldwide to treat life-threatening bacterial infections. Yet, the ototoxic and nephrotoxic side-effects of these drugs remain serious complications. A major site of gentamicin uptake and toxicity resides within kidney proximal tubules that also heavily express electrogenic sodium-glucose transporter-2 (SGLT2; SLC5A2) in vivo. We hypothesized that SGLT2 traffics gentamicin, and promotes cellular toxicity. We conf...

  4. LC-MS/MS-Methoden zur Rückstandsanalyse von Penicillinen, Cephalosporinen und Aminoglycosid-Antibiotika

    OpenAIRE

    Becker, Matthias

    2014-01-01

    In today’s livestock farming and milk production, therapeutic, metaphylactic and prophylactic use of ß-lactams (penicillins, cephalosporins) and aminoglycosides is inevitable. However, improper use of these antibiotics may lead to residues in milk and edible tissues and can cause human health hazards as well as technological problems in dairy industry. In general, antibiotics are unwanted components in food, and it has to be ensured that the consumer is not exposed to antibioti...

  5. LOWER DOSE OF AMINOGLYCOSIDE OTOTOXIC EXPOSURE CAUSES PRESYNAPTIC ALTERATIONS ASSOICATED WITH HEARING LOSS

    Institute of Scientific and Technical Information of China (English)

    LIU Ke; WANG Xiaoyu; LI Sijun; TANG Siquan; XU Yice; WANG Xuefeng; SUN Jianhe; YANG Weiyan; YANG Shiming

    2014-01-01

    Objective To study presynaptic alternations of cochlear ribbons arising from aminoglycoside ototoxic stimuli in C57BL/6J mice. Methods Animals were injected with low dose gentamicin (100 mg/kg/day) for 14 days, From the 14th to 28th days, the mice were maintained free of gentamicin treatment. Immunohisto-chemistry labeling was employed to trace RIBEYE, a major presynaptic componment of ribbon synapses. RIBEYE/CtBP2 expression levels were assessed and compared with hearing threshold shifts. Auditory func-tion was assessed by auditory brainstem responses. The stereocilia of outer hair cells (OHCs) and IHCs was examined by scanning electron microscopy (SEM). Results Hearing thresholds were elevated with peak hearing loss observed on the 7th day after gentamicin exposure, followed by improvement after the 7th day. RIBEYE/CtBP2 expression directly correlated with observed hearing threshold shifts. Strikingly, we did not see any obvious changes in stereocilia in both OHCs and IHCs until the 28th day. Mild changes in stereocil-ia were only observed in OHCs on the 28th day. Conclusions These findings indicate that presynapse co-chlear ribbons, rather than stereocilia, may be sensitive to aminoglycoside ototoxic exposure in mice cochle-ae. A pattern of RIBEYE/CtBP2 expression changes seems to parallel hearing threshold shifts and suggests presynaptic response properties to lower dosage of aminoglycoside ototoxic stimuli.

  6. [Is it possible to reduce the incidence of aminoglycoside-induced nephrotoxicity?].

    Science.gov (United States)

    Fillastre, J P

    1999-01-01

    The incidence of nephrotoxicity due to aminoglycosides should be sharply reduced. The indications for prescribing these antibiotics should be limited to infectious disorders induced by aerobic Gram-negative bacteria and by some Gram-positive bacteria requiring treatment in specialized hospital units using an association of aminoglycosides and another antibiotic. Daily doses should not exceed those indicated by the manufacturer, and the length of treatment should be as short as possible, with a relay to other antibiotics that are not or are less nephrotoxic. The possibilities for reducing the incidence of nephrotoxicity are few. It is not possible to prevent the antibiotic from entering the renal tubular cell or from producing deleterious effects therein. However, by using short-term intravenous infusion as the administration route, prolonged contact between the antibiotic and its receptors on the brush borders of the proximal tubular cells can be avoided, particularly since the process of cellular absorption is saturable. Essentially, doses should be adapted according to the age and the glomerular filtration of the patient, since renal function usually decreases with age. Volemic and hydroelectrolytic disorders favour nephrotoxicity and should be corrected. Associations with other nephrotoxic drugs should either be avoided or used with increased caution. The same is true in special situations such as endotoxaemia, severe renal parenchymatous infections and cholestasis. In any case, given the well-known insidious onset of nephropathy, aminoglycoside treatment always requires laboratory follow-up consisting of repeated testing of creatinemia during the two weeks of treatment. PMID:10465001

  7. Heparin interferes with the radioenzymatic and homogeneous enzyme immunoassays for aminoglycosides

    International Nuclear Information System (INIS)

    Heparin interferes with measurement of aminoglycosides in serum by biological, radioenzymatic, and homogeneous enzyme immunoassay techniques, but not with radioimmunoassay. At concentrations greater than or equal to 105 and greater than or equal to 3 X 106 USP units/L, respectively, it interferes with the radioenzymatic assay by inhibiting the gentamicin 3-acetyltransferase and kanamycin 6'-acetyltransferase enzymes used in the assay. It interferes with the homogeneous enzyme immunoassays for gentamicin and tobramycin (at concentrations greater than or equal to 105 and greater than or equal to104 USP units/L, respectively), but not with the commercially available homogeneous enzyme immunoassays for other drugs. Heparin interference with the homogeneous enzyme immunoassay for aminoglycosides requires both the heparin polyanion and glucose-6-phosphate dehydrogenase bound to a cationic aminoglycoside. This interference can be reproduced with dextran sulfate (but not dextran), and does not occur with free enzyme (glucose-6-phosphate dehydrogenase) alone. Heparin interference with these two assays and at concentrations that may be present in intravenous infusions or in seriously underfilled blood-collection tubes is described

  8. Human Health Hazards from Antimicrobial-Resistant Escherichia coli of Animal Origin

    DEFF Research Database (Denmark)

    Hammerum, A. M.; Heuer, Ole Eske

    2009-01-01

    antimicrobial agents in food animals may add to the burden of antimicrobial resistance in humans. Bacteria from the animal reservoir that carry resistance to antimicrobial agents that are regarded as highly or critically important in human therapy (e.g., aminoglycosides, fluoroquinolones, and third- and fourth...

  9. Purification and properties of thioether methyltransferase

    International Nuclear Information System (INIS)

    A method to assay activity was developed which measures acceptance of methyl groups from [methyl-3H]-S-adenosylmethionine by dimethyl selenide. The product, [3H]trimethylselenonium ion, is separated by HPLC and quantitated by scintillation counting. Thioether methyltransferase from mouse liver and lung resides primarily in the cytosol. In terms of specific activity the enzyme is most active in the lung and liver. Purification from lung cytosol requires a three-step process of DEAE and gel filtration column chromatographies followed by chromatofocusing. SDS-Polyacrylamide gel electrophoresis shows a single homogeneous band with a molecular mass of 28,000 daltons. Vmax and Km values for dimethyl selenide as a substrate are 15. 7 pmol/min and 0.44 μM, respectively. Our studies have also shown that this purified enzyme is capable of methylating a wide range of compounds. To further test the enzyme's role in detoxification, in vivo studies were performed by injecting mice with substrate and [methyl-3H]methionine and analyzing tissue extracts and urine for [methyl-3H]sulfonium

  10. Isolation of DNA methyltransferase from plants

    International Nuclear Information System (INIS)

    DNA methyltransferases (DMT) were isolated from nuclei of cauliflower, soybean, and pea by extraction with 0.35 M NaCl. Assays were performed on hemimethylated Micrococcus luteus DNA or on M. luteus DNA to test for maintenance or de novo methylase activity, respectively. Fully methylated DNA was used as a substrate to determine background levels of methylation. Based on these tests, yields of maintenance DMT activity in the crude extract from pea hypocotyl, soybean hypocotyl, and cauliflower inflorescence were 2.8, 0.9, and 1.6 units per g wet tissue (one unit equals 1 pmol of methyl from [3H]AdoMet incorporated into acid precipitable material per h at 300). Two peaks of DMT activity were detected in the soybean nuclear extract following phosphocellulose chromatography. One eluted at 0.4 M and the other at 0.8 M KCl. With both fractions maintenance activity was approximately 2 times that of the de novo activity. Using gel filtration the DMT eluted at 220,000 Daltons. The optimal pH for activity was between 6.5 and 7.0, and the optimal temperature was 300

  11. Biochemical and Computational Analysis of the Substrate Specificities of Cfr and RlmN Methyltransferases

    DEFF Research Database (Denmark)

    Ntokou, Eleni; Hansen, Lykke Haastrup; Kongsted, Jacob;

    2015-01-01

    -ray structure of RlmN. We used a trinucleotide as target sequence and assessed its positioning at the active site for methylation. The calculations are in accordance with different poses of the trinucleotide in the two enzymes indicating major evolutionary changes to shift the C2/C8 specificities. To explore......Cfr and RlmN methyltransferases both modify adenine 2503 in 23S rRNA (Escherichia coli numbering). RlmN methylates position C2 of adenine while Cfr methylates position C8, and to a lesser extent C2, conferring antibiotic resistance to peptidyl transferase inhibitors. Cfr and RlmN show high sequence...... interchangeability between Cfr and RlmN we constructed various combinations of their genes. The function of the mixed genes was investigated by RNA primer extension analysis to reveal methylation at 23S rRNA position A2503 and by MIC analysis to reveal antibiotic resistance. The catalytic site is expected to be...

  12. DNA Methyltransferases Inhibitors from Natural Sources.

    Science.gov (United States)

    Zwergel, Clemens; Valente, Sergio; Mai, Antonello

    2016-01-01

    DNA methyltransferases (DNMTs) catalyze the methylation at cytosine-C5 mainly in a CpG dinucleotide context. Although DNA methylation is essential for fundamental processes like embryonic development or differentiation, aberrant expression and/or activities of DNMTs are involved in several pathologies, from neurodegeneration to cancer. DNMTs inhibition can arrest tumor growth, cells invasiveness and induce differentiation, whereas their increased expression is shown in numerous cancer types. Moreover, hypermethylated promoters of tumor suppressor genes lead to their silencing. Hence, the use of specific inhibitors of DNMT might reactivate those genes and stop or even reverse the aberrant cell processes. To date, the only approved DNMTs inhibitors for therapy belong to the nucleoside-based family of drugs, but they display relevant side effects as well as high chemical instability. Thus, there is a keen interest actually exists to develop novel, potent and safe inhibitors possessing a nonnucleoside structure. Increasing literature evidence is highlighting that natural sources could help the researchers to achieve this goal. Indeed, several polyphenols, flavonoids, antraquinones, and others are described able to inhibit DNMTs activity and/or expression, thus decreasing the methylation/silencing of different genes involved in tumorigenesis. These events can lead to re-expression of such genes and to cell death in diverse cancer cell lines. Epigallocatechin-3-gallate (1) and laccaic acid A (11) resulted the most effective DNMT1 inhibitors with submicromolar IC50 values, acting as competitive inhibitors. Compound 1 and 11 both displayed gene demethylation and re-activation in several cancers. However, all of the natural compounds described in this review showed important results, from gene reactivation to cell growth inhibition. Moreover, some of them displayed interesting activity even in rodent cancer models and very recently entered clinical trials. PMID:26303417

  13. Multiple lysine methylation of PCAF by Set9 methyltransferase

    International Nuclear Information System (INIS)

    The molecular functions of several non-histone proteins are regulated through lysine modification by histone methyltransferases. The p300/CBP-associated factor (PCAF) is an acetyltransferase that has been implicated in many cellular processes. Here, we report that PCAF is a novel substrate of Set9 methyltransferase. In vitro mapping experiments revealed six lysine residues could be methylated by Set9. A comparison of amino acid sequences of target sites revealed the novel consensus motif which differs from previously identified Set9-consensus sequence. Further methyltransferase assays focusing on the six lysine residues showed that K78 and K89 are preferentially methylated in full-length PCAF in vitro. Using specific antibodies recognizing mono-methylated K89, in vivo PCAF methylation and its nuclear localization were demonstrated. Our data may lead to a new insight into PCAF functions and provide additional information to identify unknown targets of Set9.

  14. Efflux Pump-mediated Drug Resistance in Burkholderia

    Directory of Open Access Journals (Sweden)

    Nicole L Podnecky

    2015-04-01

    Full Text Available Several members of the genus Burkholderia are prominent pathogens. Infections caused by these bacteria are difficult to treat because of significant antibiotic resistance. Virtually all Burkholderia species are also resistant to polymyxin, prohibiting use of drugs like colistin that are available for treatment of infections caused by most other drug resistant Gram-negative bacteria. Despite clinical significance and antibiotic resistance of Burkholderia species, characterization of efflux pumps lags behind other non-enteric Gram-negative pathogens such as Acinetobacter baumannii and Pseudomonas aeruginosa. Although efflux pumps have been described in several Burkholderia species, they have been best studied in B. cenocepacia and B. pseudomallei. As in other non-enteric Gram-negatives, efflux pumps of the resistance nodulation cell division (RND family are the clinically most significant efflux systems in these two species. Several efflux pumps were described in B. cenocepacia, which when expressed confer resistance to clinically significant antibiotics, including aminoglycosides, chloramphenicol, fluoroquinolones, and tetracyclines. Three RND pumps have been characterized in B. pseudomallei, two of which confer either intrinsic or acquired resistance to aminoglycosides, macrolides, chloramphenicol, fluoroquinolones, tetracyclines, trimethoprim, and in some instances trimethoprim+sulfamethoxazole. Several strains of the host-adapted B. mallei, a clone of B. pseudomallei, lack AmrAB-OprA and are therefore aminoglycoside and macrolide susceptible. B. thailandensis is closely related to B. pseudomallei, but non-pathogenic to humans. Its pump repertoire and ensuing drug resistance profile parallels that of B. pseudomallei. An efflux pump in B. vietnamiensis plays a significant role in acquired aminoglycoside resistance. Summarily, efflux pumps are significant players in Burkholderia drug resistance.

  15. Defining RNA motif-aminoglycoside interactions via two-dimensional combinatorial screening and structure-activity relationships through sequencing.

    Science.gov (United States)

    Velagapudi, Sai Pradeep; Disney, Matthew D

    2013-10-15

    RNA is an extremely important target for the development of chemical probes of function or small molecule therapeutics. Aminoglycosides are the most well studied class of small molecules to target RNA. However, the RNA motifs outside of the bacterial rRNA A-site that are likely to be bound by these compounds in biological systems is largely unknown. If such information were known, it could allow for aminoglycosides to be exploited to target other RNAs and, in addition, could provide invaluable insights into potential bystander targets of these clinically used drugs. We utilized two-dimensional combinatorial screening (2DCS), a library-versus-library screening approach, to select the motifs displayed in a 3×3 nucleotide internal loop library and in a 6-nucleotide hairpin library that bind with high affinity and selectivity to six aminoglycoside derivatives. The selected RNA motifs were then analyzed using structure-activity relationships through sequencing (StARTS), a statistical approach that defines the privileged RNA motif space that binds a small molecule. StARTS allowed for the facile annotation of the selected RNA motif-aminoglycoside interactions in terms of affinity and selectivity. The interactions selected by 2DCS generally have nanomolar affinities, which is higher affinity than the binding of aminoglycosides to a mimic of their therapeutic target, the bacterial rRNA A-site. PMID:23719281

  16. Draft Genome Sequence of a Multidrug-Resistant Klebsiella quasipneumoniae subsp. similipneumoniae Isolate from a Clinical Source

    Science.gov (United States)

    Morris, Andrew R.; Krapp, Fiorella; Henry, Christopher S.; Tyo, Keith E.; Hauser, Alan R.

    2016-01-01

    We report here the draft genome sequence of a multidrug-resistant clinical isolate of Klebsiella quasipneumoniae subsp. similipneumoniae, KP_Z4175. This strain, isolated as part of a hospital infection-control screening program, is resistant to multiple β-lactam antibiotics, aminoglycosides, and trimethoprim-sulfamethoxazole. PMID:27231362

  17. Widespread Transfer of Resistance Genes between Bacterial Species in an Intensive Care Unit: Implications for Hospital Epidemiology

    OpenAIRE

    Naiemi, N.A.; Duim, B.; Savelkoul, P. H. M.; Spanjaard, L.; de Jonge,; Bart, A.; Vandenbroucke-Grauls, C. M. J. E.; Jong, de, M.C.M.

    2005-01-01

    A transferable plasmid encoding SHV-12 extended-spectrum β-lactamase, TEM-116, and aminoglycoside resistance was responsible for two sequential clonal outbreaks of Enterobacter cloacae and Acinetobacter baumannii bacteria. A similar plasmid was present among isolates of four different bacterial species. Recognition of plasmid transfer is crucial for control of outbreaks of multidrug-resistant nosocomial pathogens.

  18. Correction of ATM gene function by aminoglycoside-induced read-through of premature termination codons

    OpenAIRE

    Lai, Chih-Hung; Chun, Helen H.; Nahas, Shareef A.; Mitui, Midori; Gamo, Kristin M.; Du, Liutao; Gatti, Richard A.

    2004-01-01

    Approximately 14% of genetic mutations in patients with ataxia-telangiectsia (A-T) are single-nucleotide changes that result in primary premature termination codons (PTCs), either UAA, UAG, or UGA. The purpose of this study was to explore a potential therapeutic approach for this subset of patients by using aminoglycosides to induce PTC read-through, thereby restoring levels of full-length ATM (A-T mutated) protein. In experiments using a modified in vitro cDNA coupled transcription/translati...

  19. Design of Novel Aminoglycoside Derivatives with Enhanced Suppression of Diseases-Causing Nonsense Mutations.

    Science.gov (United States)

    Sabbavarapu, Narayana Murthy; Shavit, Michal; Degani, Yarden; Smolkin, Boris; Belakhov, Valery; Baasov, Timor

    2016-04-14

    New pseudotrisaccharide derivatives of aminoglycosides that exploit additional interaction on the shallow groove face of the decoding-site rRNA of eukaryotic ribosome were designed, synthesized and biologically evaluated. Novel lead structures (6 and 7 with an additional 7'-OH), exhibiting enhanced specificity to eukaryotic cytoplasmic ribosome, and superior nonsense mutation suppression activity than those of gentamicin, were discovered. The comparative benefit of new leads was demonstrated in four different nonsense DNA-constructs underling the genetic diseases cystic fibrosis, Usher syndrome, and Hurler syndrome. PMID:27096052

  20. Chromosomal replication incompatibility in Dam methyltransferase deficient Escherichia coli cells

    DEFF Research Database (Denmark)

    Freiesleben, Ulrik Von

    1996-01-01

    Dam methyltransferase deficient Escherichia coli cells containing minichromosomes were constructed. Free plasmid DNA could not be detected in these cells and the minichromosomes were found to be integrated in multiple copies in the origin of replication (oriC) region of the host chromosome. The...

  1. Label-free electrochemical detection of human methyltransferase from tumors.

    Science.gov (United States)

    Furst, Ariel L; Muren, Natalie B; Hill, Michael G; Barton, Jacqueline K

    2014-10-21

    The role of abnormal DNA methyltransferase activity in the development and progression of cancer is an essential and rapidly growing area of research, both for improved diagnosis and treatment. However, current technologies for the assessment of methyltransferase activity, particularly from crude tumor samples, limit this work because they rely on radioactivity or fluorescence and require bulky instrumentation. Here, we report an electrochemical platform that overcomes these limitations for the label-free detection of human DNA(cytosine-5)-methyltransferase1 (DNMT1) methyltransferase activity, enabling measurements from crude cultured colorectal cancer cell lysates (HCT116) and biopsied tumor tissues. Our multiplexed detection system involving patterning and detection from a secondary electrode array combines low-density DNA monolayer patterning and electrocatalytically amplified DNA charge transport chemistry to measure selectively and sensitively DNMT1 activity within these complex and congested cellular samples. Based on differences in DNMT1 activity measured with this assay, we distinguish colorectal tumor tissue from healthy adjacent tissue, illustrating the effectiveness of this two-electrode platform for clinical applications. PMID:25288757

  2. Imipenem resistance in nonfermenters causing nosocomial urinary tract infections.

    OpenAIRE

    Taneja N; Maharwal S; Sharma Meera

    2003-01-01

    Nonfermenting gram-negative bacilli (nonfermenters) have emerged as important nosocomial pathogens causing opportunistic infections in immunocompromised hosts. These organisms show high level of resistance to b-lactam agents, fluoroquinolones and aminoglycosides. Imipenem is a carbapenem antibiotic, which can be very useful for treatment of infections caused by nonfermenters. Eighty-five nonfermenters causing nosocomial UTI were tested for MIC to imipenem by agar dilution method. Resistance t...

  3. Structure and Function of Flavivirus NS5 Methyltransferase

    Energy Technology Data Exchange (ETDEWEB)

    Zhou,Y.; Ray, D.; Zhao, Y.; Dong, H.; Ren, S.; Li, Z.; Guo, Y.; Bernard, K.; Shi, P.; Li, H.

    2007-01-01

    The plus-strand RNA genome of flavivirus contains a 5' terminal cap 1 structure (m{sup 7}GpppAmG). The flaviviruses encode one methyltransferase, located at the N-terminal portion of the NS5 protein, to catalyze both guanine N-7 and ribose 2'-OH methylations during viral cap formation. Representative flavivirus methyltransferases from dengue, yellow fever, and West Nile virus (WNV) sequentially generate GpppA {yields} m{sup 7}GpppA {yields} m{sup 7}GpppAm. The 2'-O methylation can be uncoupled from the N-7 methylation, since m{sup 7}GpppA-RNA can be readily methylated to m{sup 7}GpppAm-RNA. Despite exhibiting two distinct methylation activities, the crystal structure of WNV methyltransferase at 2.8 {angstrom} resolution showed a single binding site for S-adenosyl-L-methionine (SAM), the methyl donor. Therefore, substrate GpppA-RNA should be repositioned to accept the N-7 and 2'-O methyl groups from SAM during the sequential reactions. Electrostatic analysis of the WNV methyltransferase structure showed that, adjacent to the SAM-binding pocket, is a highly positively charged surface that could serve as an RNA binding site during cap methylations. Biochemical and mutagenesis analyses show that the N-7 and 2'-O cap methylations require distinct buffer conditions and different side chains within the K{sub 61}-D{sub 146}-K{sub 182}-E{sub 218} motif, suggesting that the two reactions use different mechanisms. In the context of complete virus, defects in both methylations are lethal to WNV; however, viruses defective solely in 2'-O methylation are attenuated and can protect mice from later wild-type WNV challenge. The results demonstrate that the N-7 methylation activity is essential for the WNV life cycle and, thus, methyltransferase represents a novel target for flavivirus therapy.

  4. Engineering Monolignol 4-O-Methyltransferases to Modulate Lignin Biosynthesis

    Energy Technology Data Exchange (ETDEWEB)

    Bhuiya, M.W.; Liu, C.

    2010-01-01

    Lignin is a complex polymer derived from the oxidative coupling of three classical monolignols. Lignin precursors are methylated exclusively at the meta-positions (i.e. 3/5-OH) of their phenyl rings by native O-methyltransferases, and are precluded from substitution of the para-hydroxyl (4-OH) position. Ostensibly, the para-hydroxyls of phenolics are critically important for oxidative coupling of phenoxy radicals to form polymers. Therefore, creating a 4-O-methyltransferase to substitute the para-hydroxyl of monolignols might well interfere with the synthesis of lignin. The phylogeny of plant phenolic O-methyltransferases points to the existence of a batch of evolutionarily 'plastic' amino acid residues. Following one amino acid at a time path of directed evolution, and using the strategy of structure-based iterative site-saturation mutagenesis, we created a novel monolignol 4-O-methyltransferase from the enzyme responsible for methylating phenylpropenes. We show that two plastic residues in the active site of the parental enzyme are vital in dominating substrate discrimination. Mutations at either one of these separate the evolutionarily tightly linked properties of substrate specificity and regioselective methylation of native O-methyltransferase, thereby conferring the ability for para-methylation of the lignin monomeric precursors, primarily monolignols. Beneficial mutations at both sites have an additive effect. By further optimizing enzyme activity, we generated a triple mutant variant that may structurally constitute a novel phenolic substrate binding pocket, leading to its high binding affinity and catalytic efficiency on monolignols. The 4-O-methoxylation of monolignol efficiently impairs oxidative radical coupling in vitro, highlighting the potential for applying this novel enzyme in managing lignin polymerization in planta.

  5. Novel synthesis of O 6-alkylguanine containing oligodeoxyribonucleotides as substrates for the human DNA repair protein, O 6-methylguanine DNA methyltransferase (MGMT)

    OpenAIRE

    Shibata, Takayuki; Glynn, Nicola; McMurry, T. Brian H.; McElhinney, R. Stanley; Margison, Geoffrey P.; David M. Williams

    2006-01-01

    The human DNA repair protein O 6-methylguanine DNA methyltransferase (MGMT) dealkylates mutagenic O 6-alkylguanine lesions within DNA in an irreversible reaction which results in inactivation of the protein. MGMT also provides resistance of tumours to alkylating agents used in cancer chemotherapy and its inactivation is therefore of particular clinical importance. We describe a post-DNA synthesis strategy which exploits the novel, modified base 2-amino-6-methylsulfonylpurine and allows access...

  6. Expression of O6-Methylguanine-DNA Methyltransferase Examined by Alkyl-Transfer Assays, Methylation-Specific PCR and Western Blots in Tumors and Matched Normal Tissue

    OpenAIRE

    Ishiguro, Kimiko; Shyam, Krishnamurthy; Penketh, Philip G.; Baumann, Raymond P.; Sartorelli, Alan C.; Rutherford, Thomas J.; Ratner, Elena S.

    2013-01-01

    The tumor selectivity of alkylating agents that produce guanine O6-chloroethyl (laromustine and carmustine) and O6-methyl (temozolomide) lesions, depends upon O6-methylguanine-DNA methyltransferase (MGMT) activity being lower in tumor than in host tissue. Despite the established role of MGMT as a tumor resistance factor, consensus on how to assess MGMT expression in clinical samples is unsettled. The aim of this study is to examine the relationship between the values derived from distinctive ...

  7. Correlation of O6-methylguanine methyltransferase (MGMT) promoter methylation with clinical outcomes in glioblastoma and clinical strategies to modulate MGMT activity

    OpenAIRE

    Hegi, M E; L. Liu; Herman, J G; Stupp, R; Wick, W.; Weller, M.; Mehta, M P; Gilbert, M. R.

    2008-01-01

    Resistance to alkylating agents via direct DNA repair by O(6)-methylguanine methyltransferase (MGMT) remains a significant barrier to the successful treatment of patients with malignant glioma. The relative expression of MGMT in the tumor may determine response to alkylating agents, and epigenetic silencing of the MGMT gene by promoter methylation plays an important role in regulating MGMT expression in gliomas. MGMT promoter methylation is correlated with improved progression-free and overal...

  8. Cymbopogon citratus protects against the renal injury induced by toxic doses of aminoglycosides in rabbits

    Directory of Open Access Journals (Sweden)

    N Ullah

    2013-01-01

    Full Text Available Renal injury is the most common side-effect of aminoglycosides. These antimicrobial drugs are particularly effective against Gram-negative microorganisms. The present study was conducted to investigate the renal protective activity of Cymbopogon citratus in gentamicin-induced nephrotoxicity. Male rabbits were divided into four groups (n=6 including group 1 (0.9% saline treated, group 2 (80 mg/kg/day gentamicin-treated, group 3 (200 mg/kg/day Cymbopogon citratus treated and group 4 (80 mg/kg/day gentamicin and 200 mg/kg/day Cymbopogon citratus treated. Biochemical kidney functioning parameters, urinary enzymes and histopathological examination were performed. The results of the present study showed that simultaneous administration of Cymbopogon citrates and gentamicin significantly protected alteration in body weight, blood urea nitrogen, serum creatinine, creatinine clearance, serum uric acid, serum electrolytes, urinary volume, urinary protein, urinary lactate dehydrogenase and urinary alkaline phosphatase induced by gentamicin. Histological examination of the kidney also suggested the same. It is concluded from the current study that co-administration of Cymbopogon citratus with gentamicin for 3 weeks successfully prevented renal damage associated with aminoglycosides.

  9. Cymbopogon citratus Protects against the Renal Injury Induced by Toxic Doses of Aminoglycosides in Rabbits.

    Science.gov (United States)

    Ullah, N; Khan, M A; Khan, T; Ahmad, W

    2013-03-01

    Renal injury is the most common side-effect of aminoglycosides. These antimicrobial drugs are particularly effective against Gram-negative microorganisms. The present study was conducted to investigate the renal protective activity of Cymbopogon citratus in gentamicin-induced nephrotoxicity. Male rabbits were divided into four groups (n=6) including group 1 (0.9% saline treated), group 2 (80 mg/kg/day gentamicin-treated), group 3 (200 mg/kg/day Cymbopogon citratus treated) and group 4 (80 mg/kg/day gentamicin and 200 mg/kg/day Cymbopogon citratus treated). Biochemical kidney functioning parameters, urinary enzymes and histopathological examination were performed. The results of the present study showed that simultaneous administration of Cymbopogon citrates and gentamicin significantly protected alteration in body weight, blood urea nitrogen, serum creatinine, creatinine clearance, serum uric acid, serum electrolytes, urinary volume, urinary protein, urinary lactate dehydrogenase and urinary alkaline phosphatase induced by gentamicin. Histological examination of the kidney also suggested the same. It is concluded from the current study that co-administration of Cymbopogon citratus with gentamicin for 3 weeks successfully prevented renal damage associated with aminoglycosides. PMID:24019578

  10. Imatinib causes epigenetic alterations of PTEN gene via upregulation of DNA methyltransferases and polycomb group proteins

    International Nuclear Information System (INIS)

    We have recently reported the possible imatinib-resistant mechanism; long-term exposure of leukemia cells to imatinib downregulated levels of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) via hypermethylation of its promoter region (Leukemia 2010; 24: 1631). The present study explored the molecular mechanisms by which imatinib caused methylation on the promoter region of this tumor suppressor gene in leukemia cells. Real-time reverse transcription PCR found that long-term exposure of chronic eosinophilic leukemia EOL-1 cells expressing FIP1L1/platelet-derived growth factor receptor-α to imatinib induced expression of DNA methyltransferase 3A (DNMT3A) and histone-methyltransferase enhancer of zeste homolog 2 (EZH2), a family of polycomb group, thereby increasing methylation of the gene. Immunoprecipitation assay found the increased complex formation of DNMT3A and EZH2 proteins in these cells. Moreover, chromatin immunoprecipitation assay showed that amounts of both DNMT3A and EZH2 proteins bound around the promoter region of PTEN gene were increased in EOL-1 cells after exposure to imatinib. Furthermore, we found that levels of DNMT3A and EZH2 were strikingly increased in leukemia cells isolated from individuals with chronic myelogenous leukemia (n=1) and Philadelphia chromosome-positive acute lymphoblastic leukemia (n=2), who relapsed after treatment with imatinib compared with those isolated at their initial presentation. Taken together, imatinib could cause drug-resistance via recruitment of polycomb gene complex to the promoter region of the PTEN and downregulation of this gene's transcripts in leukemia patients

  11. Molecular identification of carnosine N-methyltransferase as chicken histamine N-methyltransferase-like protein (hnmt-like.

    Directory of Open Access Journals (Sweden)

    Jakub Drozak

    Full Text Available Anserine (beta-alanyl-N(Pi-methyl-L-histidine, a naturally occurring derivative of carnosine (beta-alanyl-L-histidine, is an abundant constituent of skeletal muscles and brain of many vertebrates. Although it has long been proposed to serve as a proton buffer, radicals scavenger and transglycating agent, its physiological function remains obscure. The formation of anserine is catalyzed by carnosine N-methyltransferase which exhibits unknown molecular identity. In the present investigation, we have purified carnosine N-methyltransferase from chicken pectoral muscle about 640-fold until three major polypeptides of about 23, 26 and 37 kDa coeluting with the enzyme were identified in the preparation. Mass spectrometry analysis of these polypeptides resulted in an identification of histamine N-methyltransferase-like (HNMT-like protein as the only meaningful candidate. Analysis of GenBank database records indicated that the hnmt-like gene might be a paralogue of histamine N-methyltransferase gene, while comparison of their protein sequences suggested that HNMT-like protein might have acquired a new activity. Chicken HNMT-like protein was expressed in COS-7 cells, purified to homogeneity, and shown to catalyze the formation of anserine as confirmed by both chromatographic and mass spectrometry analysis. Both specificity and kinetic studies carried out on the native and recombinant enzyme were in agreement with published data. Particularly, several compounds structurally related to carnosine, including histamine and L-histidine, were tested as potential substrates for the enzyme, and carnosine was the only methyl group acceptor. The identification of the gene encoding carnosine N-methyltransferase might be beneficial for estimation of the biological functions of anserine.

  12. Potentiation of Aminoglycoside Activity in Pseudomonas aeruginosa by Targeting the AmgRS Envelope Stress-Responsive Two-Component System.

    Science.gov (United States)

    Poole, Keith; Gilmour, Christie; Farha, Maya A; Mullen, Erin; Lau, Calvin Ho-Fung; Brown, Eric D

    2016-06-01

    A screen for agents that potentiated the activity of paromomycin (PAR), a 4,5-linked aminoglycoside (AG), against wild-type Pseudomonas aeruginosa identified the RNA polymerase inhibitor rifampin (RIF). RIF potentiated additional 4,5-linked AGs, such as neomycin and ribostamycin, but not the clinically important 4,6-linked AGs amikacin and gentamicin. Potentiation was absent in a mutant lacking the AmgRS envelope stress response two-component system (TCS), which protects the organism from AG-generated membrane-damaging aberrant polypeptides and, thus, promotes AG resistance, an indication that RIF was acting via this TCS in potentiating 4,5-linked AG activity. Potentiation was also absent in a RIF-resistant RNA polymerase mutant, consistent with its potentiation of AG activity being dependent on RNA polymerase perturbation. PAR-inducible expression of the AmgRS-dependent genes htpX and yccA was reduced by RIF, suggesting that AG activation of this TCS was compromised by this agent. Still, RIF did not compromise the membrane-protective activity of AmgRS, an indication that it impacted some other function of this TCS. RIF potentiated the activities of 4,5-linked AGs against several AG-resistant clinical isolates, in two cases also potentiating the activity of the 4,6-linked AGs. These cases were, in one instance, explained by an observed AmgRS-dependent expression of the MexXY multidrug efflux system, which accommodates a range of AGs, with RIF targeting of AmgRS undermining mexXY expression and its promotion of resistance to 4,5- and 4,6-linked AGs. Given this link between AmgRS, MexXY expression, and pan-AG resistance in P. aeruginosa, RIF might be a useful adjuvant in the AG treatment of P. aeruginosa infections. PMID:27021319

  13. Identification of aminoglycoside-acetylating enzymes by high-pressure liquid chromatographic determination of their reaction products.

    OpenAIRE

    Lovering, A M; White, L. O.; Reeves, D S

    1984-01-01

    A method to identify the aminoglycoside-acetyltransferase (AAC) enzymes AAC(3), AAC(2') and AAC(6') by high-pressure liquid chromatographic characterization of their products of reaction with tobramycin or sisomicin is described. Conditions are given for the chromatography of kanamycin A, netilmicin, neomycin, and apramycin, and their products of reaction, if any, with the three AAC enzymes are listed.

  14. Non-derivatization approach to high-performance liquid chromatography-fluorescence detection for aminoglycoside antibiotics based on a ligand displacement reaction.

    Science.gov (United States)

    Yang, M; Tomellini, S A

    2001-12-21

    An indirect fluorescence detection method has been developed for detecting the aminoglycoside antibiotics following chromatographic separation. This approach to detection is based on a displacement reaction between the aminoglycosides and a copper(II)-L-tryptophan (L-Trp) complex, Cu(L-Trp)2. The aminoglycosides, which contain multiple amino groups, have strong affinities for the Cu(II) ion and displace L-Trp from the Cu(L-Trp)2 complex. The resulting increase in L-Trp fluorescence, which is quenched when coordinated to Cu(II), is indicative of the presence of the aminoglycoside. Fluorescence titration data indicate that there is a stoichiometric ratio of 1:1 between the reaction of the aminoglycosides with Cu(L-Trp)2. This HPLC detection scheme is implemented postcolumn by mixing a buffered Cu(L-Trp)2 solution with the column eluent prior to detection. The aminoglycosides were separated with the use of a column packed with a polymeric strong cation-exchanger. Separation and detection variables were optimized and are discussed. The detection limits for the aminoglycosides tested ranged from 4.2 to 14.5 ng injected (S/N=3). A linear working curve was achieved for amikacin in the range of 29-586 ng for a six point linearity test. The developed separation and detection scheme was further tested by analyzing commercial pharmaceutical formulations of these antibiotics. PMID:11806546

  15. 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

  16. Should Moxifloxacin Be Used for the Treatment of Extensively Drug-Resistant Tuberculosis? An Answer from a Murine Model▿

    OpenAIRE

    Poissy, Julien; Aubry, Alexandra; Fernandez, Christine; Lott, Marie-Catherine; Chauffour, Aurelie; Jarlier, Vincent; Farinotti, Robert; Veziris, Nicolas

    2010-01-01

    The prevalence of extensively drug-resistant tuberculosis (XDR-TB), defined as TB that is resistant to isoniazid, rifampin, fluoroquinolones, and aminoglycosides, is rising worldwide. The extent of Mycobacterium tuberculosis resistance to fluoroquinolones depends on the mutation in the DNA gyrase, the only target of fluoroquinolones. The MIC of moxifloxacin, the most active fluoroquinolone against M. tuberculosis, may be lower than its peak serum level for some ofloxacin-resistant strains of ...

  17. Evaluation of Antibacterial Activity of Aminoglycosides and Modulating the Essential Oil of Cymbopogon citratus (DC. Stapf

    Directory of Open Access Journals (Sweden)

    Saulo R. TINTINO

    2014-05-01

    Full Text Available  Several works demonstrated the importance of the study of natural products as an alternative source for new antimicrobial drugs or for modulators for these ones. In this point, the aim of this was to investigate the antibacterial activity and the possible interactions between the essential oil of Cymbopogon citratus alone and in association with aminoglycosides against standard and clinically isolated strains of multidrug-resistant bacteria such as S. aureus, E. coli and P. aeruginosa by microdilution method. The results indicated a synergism between the antibiotics and the essential oil with a subinhibitory concentration (MIC/8, reducing the minimal inhibitory concentration (MIC sixteen times against the multidrug-resistant strains of S. aureus 358, E. coli 27 and P. aeruginosa 143, but none modulatory activity was observed against P. aeruginosa 78 and P. aeruginosa 91 strains. By our results, can be concluded that the essential oil of Cymbopogon citratus can be an interesting source of natural products with antibacterial and/or modulatory antibiotic activitieAVALIAÇÃO DA ATIVIDADE ANTIBACTERIANA E MODULADORA DE AMINOGLICOSÍDEOS DO ÓLEO ESSENCIAL DE Cymbopogon citratus (DC. STAPFVários trabalhos vêm demonstrando a importância do estudo de produtos naturais como fonte alternativa para novos antimicrobianos ou que venham potencializar os já existentes. Neste contexto este trabalho teve como objetivo investigar a atividade antibacteriana e as possíveis interações entre o óleo essencial de Cymbopogon citratus combinados a aminoglicosídeos frente a linhagens padrões e multirresistentes de S. aureus, E. coli e de P. aeruginosa provenientes de isolados clínicos. Um ensaio de microdiluição foi realizado para verificar a atividade antibacteriana e as possíveis interacções entre o produto natural e os antibióticos, utilizando uma concentração sub-inibitória. Através dos resultados foi constatado a interferência sinérgica dos

  18. Development of a miniaturised microarray-based assay for the rapid identification of antimicrobial resistance genes in Gram-negative bacteria

    NARCIS (Netherlands)

    Batchelor, M.; Hopkins, K.L.; Liebana, E.; Slickers, P.; Ehricht, R.; Mafura, M.; Aerestrup, F.; Mevius, D.J.; Clifton-Hadley, F.A.; Woodward, M.; Davies, R.; Threlfall, J.; Anjum, F.M.

    2008-01-01

    We describe the development of a miniaturised microarray for the detection of antimicrobial resistance genes in Gram-negative bacteria. Included on the array are genes encoding resistance to aminoglycosides, trimethoprim, sulphonamides, tetracyclines and ß-lactams, including extended-spectrum ß-lact

  19. Functional Role of G9a Histone Methyltransferase in Cancer

    OpenAIRE

    Casciello, Francesco; Windloch, Karolina; Gannon, Frank; Lee, Jason S.

    2015-01-01

    Post-translational modifications of DNA and histones are epigenetic mechanisms, which affect the chromatin structure, ultimately leading to gene expression changes. A number of different epigenetic enzymes are actively involved in the addition or the removal of various covalent modifications, which include acetylation, methylation, phosphorylation, ubiquitination, and sumoylation. Deregulation of these processes is a hallmark of cancer. For instance, G9a, a histone methyltransferase responsib...

  20. Epigenetic Regulation of Autophagy by the Methyltransferase G9a

    OpenAIRE

    Artal-Martinez de Narvajas, Amaia; Gomez, Timothy S.; Zhang, Jin-San; Mann, Alexander O.; Taoda, Yoshiyuki; Gorman, Jacquelyn A.; Herreros-Villanueva, Marta; Gress, Thomas M; Ellenrieder, Volker; Bujanda, Luis; Kim, Do-Hyung; Kozikowski, Alan P.; Koenig, Alexander; Billadeau, Daniel D.

    2013-01-01

    Macroautophagy is an evolutionarily conserved cellular process involved in the clearance of proteins and organelles. Although the cytoplasmic machinery that orchestrates autophagy induction during starvation, hypoxia, or receptor stimulation has been widely studied, the key epigenetic events that initiate and maintain the autophagy process remain unknown. Here we show that the methyltransferase G9a coordinates the transcriptional activation of key regulators of autophagosome formation by remo...

  1. Histamine N-methyltransferase Modulates Human Bronchial Smooth Muscle Contraction

    OpenAIRE

    Tamaoki, J.; Chiyotani, A.; Tagaya, E; Isono, K; Konno, K

    1994-01-01

    To elucidate the modulatory role of histamine-degrading enzymes in airway constrictor responses, human bronchial strips were studied under isometric conditions in vitro. Pretreatment of tissues with the histamine N-methyltransferase (HMT) inhibitor SKF 91488 specifically potentiated the contractile responses to histamine, causing a leftward displacement of the concentration response curves, whereas the diamine oxidase inhibitor aminoguanidine had no effect. This potentiation was attenuated by...

  2. Betaine-homocysteine methyltransferase: human liver genotype-phenotype correlation

    OpenAIRE

    Feng, Qiping; Kalari, Krishna; Fridley, Brooke L.; Jenkins, Gregory; Ji, Yuan; Abo, Ryan; Hebbring, Scott; Zhang, Jianping; Nye, Monica D.; Leeder, J Steven; Weinshilboum, Richard M.

    2010-01-01

    Betaine-homocysteine methyltransferase (BHMT) catalyzes the remethylation of homocysteine. BHMT is highly expressed in the human liver. In the liver, BHMT catalyzes up to 50% of homocysteine metabolism. Understanding the relationship between BHMT genetic polymorphisms and function might increase our understanding of the role of this reaction in homocysteine remethylation and in S-adenosylmethionine-dependent methylation. To help achieve those goals, we measured levels of BHMT enzyme activity ...

  3. Structural characterization of the mitomycin 7-O-methyltransferase

    Energy Technology Data Exchange (ETDEWEB)

    Singh, Shanteri; Chang, Aram; Goff, Randal D.; Bingman, Craig A.; Grüschow, Sabine; Sherman, David H.; Phillips, Jr., George N.; Thorson, Jon S. (Michigan); (UW)

    2014-10-02

    Mitomycins are quinone-containing antibiotics, widely used as antitumor drugs in chemotherapy. Mitomycin-7-O-methyltransferase (MmcR), a key tailoring enzyme involved in the biosynthesis of mitomycin in Streptomyces lavendulae, catalyzes the 7-O-methylation of both C9{beta}- and C9{alpha}-configured 7-hydroxymitomycins. We have determined the crystal structures of the MmcR-S-adenosylhomocysteine (SAH) binary complex and MmcR-SAH-mitomycin A (MMA) ternary complex at resolutions of 1.9 and 2.3 {angstrom}, respectively. The study revealed MmcR to adopt a common S-adenosyl-L-methionine-dependent O-methyltransferase fold and the presence of a structurally conserved active site general acid-base pair is consistent with a proton-assisted methyltransfer common to most methyltransferases. Given the importance of C7 alkylation to modulate mitomycin redox potential, this study may also present a template toward the future engineering of catalysts to generate uniquely bioactive mitomycins.

  4. DNA repair methyltransferase (Mgmt) knockout mice are sensitive to the lethal effects of chemotherapeutic alkylating agents.

    OpenAIRE

    Glassner, Brian; Weeda, Geert; Allan, James; Broekhof, Jose'; Carls, Nick; Donker, Ingrid; Engelward, Bevin; Hampson, Richard; Hersmus, Remko; Hickman, Mark; Roth, Richard; Warren, Henry; Wu, Mavis; Hoeijmakers, Jan; Samson, Leona

    1999-01-01

    textabstractWe have generated mice deficient in O6-methylguanine DNA methyltransferase activity encoded by the murine Mgmt gene using homologous recombination to delete the region encoding the Mgmt active site cysteine. Tissues from Mgmt null mice displayed very low O6-methylguanine DNA methyltransferase activity, suggesting that Mgmt constitutes the major, if not the only, O6-methylguanine DNA methyltransferase. Primary mouse embryo fibroblasts and bone marrow cells from Mgmt -/- mice were s...

  5. Molecular Mechanism of Action of Plant DRM De Novo DNA Methyltransferases

    OpenAIRE

    Zhong, Xuehua; Du, Jiamu; Hale, Christopher J.; Gallego-Bartolome, Javier; Feng, Suhua; Vashisht, Ajay A.; Chory, Joanne; Wohlschlegel, James A.; Patel, Dinshaw J.; Jacobsen, Steven E.

    2014-01-01

    DNA methylation is a conserved epigenetic gene regulation mechanism. DOMAINS REARRANGED METHYLTRANSFERASE (DRM) is a key de novo methyltransferase in plants, but how DRM acts mechanistically is poorly understood. Here, we report the crystal structure of the methyltransferase domain of tobacco DRM (NtDRM) and reveal a molecular basis for its rearranged structure. NtDRM forms a functional homo-dimer critical for catalytic activity. We also show that Arabidopsis DRM2 exists in complex with the s...

  6. Protective effect of O6-methylguanine-DNA-methyltransferase on mammalian cells

    Institute of Scientific and Technical Information of China (English)

    LI Dong-bo; WANG Ji-shi; FANG Qin; SUN Hai-yang; XU Wei; LI Wei-da

    2007-01-01

    Background O6-methylguanine-DNA-methyltransferase (MGMT) is a specific DNA revising enzyme transferring alkylated groups from DNA to its cysteine residue to avoid the abnormal twisting of DNA. Therefore, it is one of the drug resistant genes targeted in the treatment of cancer. This study explored the protective effect of MGMT gene transferred into mammalian cells.Methods Mammalian expression vector containing the MGMT gene cloned from human hepatocytes by RT-PCR was constructed and transferred into K562 cells and human peripheral blood mononuclear cells (PBMCs) via liposome, then assayed for gene expression at RNA and protein levels. MTT assay was used to check the drug resistance of cells transfected with MGMT gene.Results MGMT gene was successfully cloned. Real-time PCR showed that the mRNA expression in gene transfected groups in K562 cell line and PBMC were 13.4 and 4.0 times that of the empty vector transfected groups respectively.Results of Western blotting showed distinct higher expression of MGMT in gene transfected group than in other two groups. The IC50 values increased to 7 and 2 times that of the original values respectively in stable transfected K562 cells and transient transfected PBMC.Conclusion The alkylating resistance of eukaryotic cells is enhanced after being transfected with MGMT gene which protein product performs the protective function, and may provide the reference for the protective model of peripheral blood cells in cancer chemotherapy.

  7. Determination of stability constants of aminoglycoside antibiotics with their metal complexes

    Science.gov (United States)

    Tiwow, Vanny M. A.

    2014-03-01

    One group of aminoglycoside antibiotics contains aminosugars. The aminosugar neomycin B with its derivate product neamine (2-Deoxy-4-0-(2,6-diamino-2,6-dideoxy-α-D-glucopyranosyl)-D-Streptamine) was identified as a free ligands and metal complexes. In particular, the stability constants of metal complexes by potentiometric titration techniques were investigated. Our previous study had determined the acid dissociation constants of these aminosugars with few metal complexes in fair depth. In this work, the complexation of two pyridine-containing amino alcohols and an amino sugar (neamine) have been measured potentiometrically. For instance, the stability constant of copper(II) complexation were determine and the model system generated an excellent fit. Stability constants with several metals have been determined and will be reported.

  8. Coenzyme A Binding to the Aminoglycoside Acetyltransferase (3)-IIIb Increases Conformational Sampling of Antibiotic Binding Site

    Energy Technology Data Exchange (ETDEWEB)

    Hu, Xiaohu [ORNL; Norris, Adrianne [University of Tennessee, Knoxville (UTK); Baudry, Jerome Y [ORNL; Serpersu, Engin H [University of Tennessee, Knoxville (UTK)

    2011-01-01

    NMR spectroscopy experiments and molecular dynamics simulations were performed to describe the dynamic properties of the aminoglycoside acetyltransferase (3)-IIIb (AAC) in its apo and coenzyme A (CoASH) bound forms. The {sup 15}N-{sup 1}H HSQC spectra indicate a partial structural change and coupling of the CoASH binding site with another region in the protein upon the CoASH titration into the apo enzyme. Molecular dynamics simulations indicate a significant structural and dynamic variation of the long loop in the antibiotic binding domain in the form of a relatively slow (250 ns), concerted opening motion in the CoASH enzyme complex and that binding of the CoASH increases the structural flexibility of the loop, leading to an interchange between several similar equally populated conformations.

  9. Factors impacting the aminoglycoside-induced UGA stop codon readthrough in selenoprotein translation.

    Science.gov (United States)

    Martitz, Janine; Hofmann, Peter Josef; Johannes, Jörg; Köhrle, Josef; Schomburg, Lutz; Renko, Kostja

    2016-09-01

    Aminoglycosides (AG) are oligosaccharide antibiotics that interfere with the small ribosomal subunit in aerobic, Gram-negative bacteria, causing pathogen-destructing error rates in their protein biosynthesis. Aminoglycosides also induce mRNA misinterpretation in eukaryotic cells, especially of the UGA (Opal)-stop codon, albeit to a lower extent. UGA recoding is essentially required for the incorporation of selenocysteine (Sec) into growing selenoproteins during translation. Selenocysteine incorporation requires the presence of a selenoprotein-specific stem-loop structure within the 3'-untranslated region of the mRNA, the so-called Sec-insertion sequence (SECIS) element. Interestingly, selenoprotein genes differ in their SECIS-element sequence and in their UGA base context. We hypothesized that the SECIS-element and the specific codon context synergize in controlling the effects of AG on stop codon readthrough. To this end, the SECIS-elements of glutathione peroxidase 1, glutathione peroxidase 4 and selenoprotein P transcripts were cloned into a reporter system and analyzed in combination with different UGA codon contexts. Our results indicate that a cytosine in position 4 (directly downstream of UGA) confers strongest effects on both the Se- and AG-dependent readthrough. Overall selenoprotein biosynthesis rate depends on the Se-status, AG concentration and the specific SECIS-element present in the transcript. These findings help to get a better understanding for the susceptibility of different transcripts towards AG-mediated interference with the biosynthesis of functional Se-containing selenoproteins, and highlight the importance of the Se-status for successful selenoprotein biosynthesis under antibiotic therapy. PMID:27157664

  10. Investigation on the Mechanism of Exacerbation of Myasthenia Gravis by Aminoglycoside Antibiotics in Mouse Model

    Institute of Scientific and Technical Information of China (English)

    LIU Changqin; HU Fang

    2005-01-01

    Summary: To investigate the underlying mechanism of the exacerbation of myasthenia gravis by aminoglycoside antibiotics. C57/BL6 mice were immunized with acetylcholine receptor (AChR), extracted from electric organ of Narcine timilei according to Xu Haopeng's methods, in complete Fruend's adjuvant (CFA) to establish experimental autoimmune myasthenia gravis (EAMG). EAMG mice were divided randomly into 5 groups: MG group, NS group and three antibiotics groups. The clinical symptom scores of mice were evaluated on d7 after the last immunization and d14 of antibiotics treatment. Repetitive nerve stimulation (RNS) was performed and the levels of anti-AChR antibody (AChR-Ab) were tested at the same time. The mean clinical symptom grades of gentamycin group (1.312, 2.067), amikacin group (1.111, 1.889) and etimicin group (1.263, 1.632) were significantly higher than those of MG group (1.000, 1.200) (P<0.05). The positive rates of RNS of three antibiotics groups were 69.23 %, 58.82 % and 63.16 % respectively, which were significantly higher than those of MG group and NS group (40.00 %, 40.00 %, P<0.05). The AChR-Ab level in serum and the expression of AChR on neuromuscular junction (NMJ) of mice in three antibiotics groups were also higher than those of MG group. Our results indicated that aminoglycoside antibiotics could aggravate the symptom of myasthenia gravis. The exacerbation of myasthenia gravis by these antibiotics probably involves competitively restraining the release of acetylcholine from presynaptic membrane, impairing the depolarization of postsynaptic membrane, depressing the irritability of myocyte membrane around the end-plate membrane and consequently leading to the blockade of neuromuscular junction.

  11. Azobenzene-aminoglycoside: Self-assembled smart amphiphilic nanostructures for drug delivery.

    Science.gov (United States)

    Deka, Smriti Rekha; Yadav, Santosh; Mahato, Manohar; Sharma, Ashwani Kumar

    2015-11-01

    Here, we have designed and synthesized a novel cationic amphiphilic stimuli-responsive azobenzene-aminoglycoside (a small molecule) conjugate, Azo-AG 5, and characterized it by UV and FTIR. Light responsive nature of Azo-AG 5 was assessed under UV-vis light. Self- assembly of Azo-AG 5 in aqueous solutions into nanostructures and their ability to act as drug carrier were also investigated. The nanostructures of Azo-AG 5 showed average hydrodynamic diameter of ∼ 255 nm with aminoglycoside moiety (neomycin) and 4-dimethylaminoazobenzene forming hydrophilic shell and hydrophobic core, respectively. In the hydrophobic core, eosin and aspirin were successfully encapsulated. Dynamic light scattering (DLS) measurements demonstrated that the nanoassemblies showed expansion and contraction on successive UV and visible light irradiations exhibiting reversible on-off switch for controlling the drug release behavior. Similar behavior was observed when these nanostructures were subjected to pH-change. In vitro drug release studies showed a difference in UV and visible light-mediated release pattern. It was observed that the release rate under UV irradiation was comparatively higher than that observed under visible light. Further, azoreductase-mediated cleavage of the azo moiety in Azo-AG 5 nanoassemblies resulted in the dismantling of the structures into aggregated microstructures. Azo-AG 5 nanostructures having positive surface charge (+9.74 mV) successfully interacted with pDNA and retarded its mobility on agarose gel. Stimuli responsiveness of nanostructures and their on-off switch like behavior ensure the great potential as controlled drug delivery systems and in other biomedical applications such as colon-specific delivery and gene delivery. PMID:26255160

  12. Ribozyme-based aminoglycoside switches of gene expression engineered by genetic selection in S. cerevisiae.

    Science.gov (United States)

    Klauser, Benedikt; Atanasov, Janina; Siewert, Lena K; Hartig, Jörg S

    2015-05-15

    Systems for conditional gene expression are powerful tools in basic research as well as in biotechnology. For future applications, it is of great importance to engineer orthogonal genetic switches that function reliably in diverse contexts. RNA-based switches have the advantage that effector molecules interact immediately with regulatory modules inserted into the target RNAs, getting rid of the need of transcription factors usually mediating genetic control. Artificial riboswitches are characterized by their simplicity and small size accompanied by a high degree of modularity. We have recently reported a series of hammerhead ribozyme-based artificial riboswitches that allow for post-transcriptional regulation of gene expression via switching mRNA, tRNA, or rRNA functions. A more widespread application was so far hampered by moderate switching performances and a limited set of effector molecules available. Here, we report the re-engineering of hammerhead ribozymes in order to respond efficiently to aminoglycoside antibiotics. We first established an in vivo selection protocol in Saccharomyces cerevisiae that enabled us to search large sequence spaces for optimized switches. We then envisioned and characterized a novel strategy of attaching the aptamer to the ribozyme catalytic core, increasing the design options for rendering the ribozyme ligand-dependent. These innovations enabled the development of neomycin-dependent RNA modules that switch gene expression up to 25-fold. The presented aminoglycoside-responsive riboswitches belong to the best-performing RNA-based genetic regulators reported so far. The developed in vivo selection protocol should allow for sampling of large sequence spaces for engineering of further optimized riboswitches. PMID:24871672

  13. Aminoglycoside binding to the HIV-1 RNA dimerization initiation site: thermodynamics and effect on the kissing-loop to duplex conversion

    OpenAIRE

    Bernacchi, Serena; Freisz, Séverine; Maechling, Clarisse; Spiess, Bernard; Marquet, Roland; Dumas, Philippe; Ennifar, Eric

    2007-01-01

    Owing to a striking, and most likely fortuitous, structural and sequence similarity with the bacterial 16 S ribosomal A site, the RNA kissing-loop complex formed by the HIV-1 genomic RNA dimerization initiation site (DIS) specifically binds 4,5-disubstituted 2-deoxystreptamine (2-DOS) aminoglycoside antibiotics. We used chemical probing, molecular modeling, isothermal titration calorimetry (ITC) and UV melting to investigate aminoglycoside binding to the DIS loop–loop complex. We showed that ...

  14. Aminoglycoside binding to the HIV-1 RNA dimerization initiation site: thermodynamics and effect on the kissing-loop to duplex conversion.

    Science.gov (United States)

    Bernacchi, Serena; Freisz, Séverine; Maechling, Clarisse; Spiess, Bernard; Marquet, Roland; Dumas, Philippe; Ennifar, Eric

    2007-01-01

    Owing to a striking, and most likely fortuitous, structural and sequence similarity with the bacterial 16 S ribosomal A site, the RNA kissing-loop complex formed by the HIV-1 genomic RNA dimerization initiation site (DIS) specifically binds 4,5-disubstituted 2-deoxystreptamine (2-DOS) aminoglycoside antibiotics. We used chemical probing, molecular modeling, isothermal titration calorimetry (ITC) and UV melting to investigate aminoglycoside binding to the DIS loop-loop complex. We showed that apramycin, an aminoglycoside containing a bicyclic moiety, also binds the DIS, but in a different way than 4,5-disubstituted 2-DOS aminoglycosides. The determination of thermodynamic parameters for various aminoglycosides revealed the role of the different rings in the drug-RNA interaction. Surprisingly, we found that the affinity of lividomycin and neomycin for the DIS (K(d) approximately 30 nM) is significantly higher than that obtained in the same experimental conditions for their natural target, the bacterial A site (K(d) approximately 1.6 microM). In good agreement with their respective affinity, aminoglycoside increase the melting temperature of the loop-loop interaction and also block the conversion from kissing-loop complex to extended duplex. Taken together, our data might be useful for selecting new molecules with improved specificity and affinity toward the HIV-1 DIS RNA. PMID:17942426

  15. Current Epidemiology and Growing Resistance of Gram-Negative Pathogens

    OpenAIRE

    Livermore, David M.

    2012-01-01

    In the 1980s, Gram-negative pathogens appeared to have been beaten by oxyimino-cephalosporins, carbapenems, and fluoroquinolones. Yet these pathogens have fought back, aided by their membrane organization, which promotes the exclusion and efflux of antibiotics, and by a remarkable propensity to recruit, transfer, and modify the expression of resistance genes, including those for extended-spectrum β-lactamases (ESBLs), carbapenemases, aminoglycoside-blocking 16S rRNA methylases, and even a qui...

  16. Draft Whole-Genome Sequence of VIM-1-Producing Multidrug-Resistant Enterobacter cloacae EC_38VIM1

    Science.gov (United States)

    Villa, Jennifer; Viedma, Esther; Otero, Joaquín R.

    2013-01-01

    The VIM-1-producing multidrug-resistant strain Enterobacter cloacae was isolated from blood culture. The strain showed multiple resistances to clinically used antibiotics, including all β-lactams, fluoroquinolones, aminoglycosides, and sulfonamides. Sequence analysis showed the presence of 14 genes associated with resistance to antibiotics, including the metallo-β-lactamase VIM-1 gene, which was located in a class 1 integron. PMID:24009122

  17. Structural Biology of Human H3K9 Methyltransferases

    Energy Technology Data Exchange (ETDEWEB)

    Wu, H.; Min, J; Lunin, V; Antoshenko, T; Dombrovsk, L; Zeng, H; Allali-Hassani, A; Campagna-Slater, V; Vedadi, M; et. al.

    2010-01-01

    SET domain methyltransferases deposit methyl marks on specific histone tail lysine residues and play a major role in epigenetic regulation of gene transcription. We solved the structures of the catalytic domains of GLP, G9a, Suv39H2 and PRDM2, four of the eight known human H3K9 methyltransferases in their apo conformation or in complex with the methyl donating cofactor, and peptide substrates. We analyzed the structural determinants for methylation state specificity, and designed a G9a mutant able to tri-methylate H3K9. We show that the I-SET domain acts as a rigid docking platform, while induced-fit of the Post-SET domain is necessary to achieve a catalytically competent conformation. We also propose a model where long-range electrostatics bring enzyme and histone substrate together, while the presence of an arginine upstream of the target lysine is critical for binding and specificity. Post-translational modifications of histone proteins regulate chromatin compaction, mediate epigenetic regulation of transcription, and control cellular differentiation in health and disease. Methylation of histone tails is one of the fundamental events of epigenetic signaling. Tri-methylation of lysine 9 of histone 3 (H3K9) mediates chromatin recruitment of HP1, heterochromatin condensation and gene silencing. Similarly, methylation of H3K27 and H4K20 are associated with a repressed state of chromatin, whereas expressed genes are methylated at H3K4, H3K36 and H3K79. Histone methyltransferases are divided into protein arginine methyltransferases (PRMTs) and histone lysine methyltransferases (HKMTs). HKMTs catalyze the transfer of a methyl group from the co-factor S-adenosyl-L-methionine (SAM) to a substrate lysine and, with the exception of DOT1L, are all organized around a canonical SET domain. The structures of a number of HKMTs have been reported, including ternary complexes of human orthologs with co-factor and substrate peptides (SETD7-H3K4, SETD8-H4K20 and MLL1-H3K4), as well

  18. Ultrastructural analysis of aminoglycoside-induced hair cell death in the zebrafish lateral line reveals an early mitochondrial response.

    OpenAIRE

    Owens, Kelly,; Cunningham, Dale,; Macdonald, Glen; Rubel, Edwin,; Raible, David,; Pujol, Remy

    2007-01-01

    Loss of the mechanosensory hair cells in the auditory and vestibular organs leads to hearing and balance deficits. To investigate initial, in vivo events in aminoglycoside-induced hair cell damage, we examined hair cells from the lateral line of the zebrafish, Danio rerio. The mechanosensory lateral line is located externally on the animal and therefore allows direct manipulation and observation of hair cells. Labeling with vital dyes revealed a rapid response of hair cells to the aminoglycos...

  19. Screen of FDA-approved drug library reveals compounds that protect hair cells from aminoglycosides and cisplatin

    OpenAIRE

    Vlasits, Anna L.; Simon, Julian A.; Raible, David W.; Rubel, Edwin W; Owens, Kelly N.

    2012-01-01

    Loss of mechanosensory hair cells in the inner ear accounts for many hearing loss and balance disorders. Several beneficial pharmaceutical drugs cause hair cell death as a side effect. These include aminoglycoside antibiotics, such as neomycin, kanamycin and gentamicin, and several cancer chemotherapy drugs, such as cisplatin. Discovering new compounds that protect mammalian hair cells from toxic insults is experimentally difficult because of the inaccessibility of the inner ear. We used the ...

  20. In vitro read-through of phenylalanine hydroxylase (PAH) nonsense mutations using aminoglycosides: a potential therapy for phenylketonuria.

    Science.gov (United States)

    Ho, Gladys; Reichardt, Juergen; Christodoulou, John

    2013-11-01

    Phenylketonuria (PKU, OMIM 261600) is an autosomal recessive inborn error of phenylalanine metabolism, predominantly caused by mutations in the phenylalanine hydroxylase (PAH) gene. Approximately 10% of patients carry a nonsense mutation, which results in an inactive or unstable truncated protein. In some genetic disorders, including cystic fibrosis and Duchenne muscular dystrophy, restoration of full-length protein has been achieved by aminoglycoside antibiotics, such as gentamicin and G-418 (Geneticin). More recently, nonsense read-through has been induced at greater rates using a non-aminoglycoside drug, PTC124 (Ataluren), which has the advantage of being non-toxic in contrast to the antibiotics. The efficacy of read-through induced by three compounds, aminoglycosides G418 and gentamicin, and PTC124 were evaluated for four nonsense mutations of PAH in an in vitro expression system in two mammalian cell lines (COS-7 and HEK293). The production of full-length PAH was investigated using western blotting and the functionality confirmed by enzyme activity. Gentamicin and G-418 induced read-through of nonsense PAH mutations in HEK293 cells. The read-through product partially restored enzymatic activity, which was significantly less than that of wild-type, but comparable to a missense mutation of PAH associated with less severe forms of PKU. Treatment with PTC124 up to 100 μM did not result in full-length PAH polypeptide. Nonsense read-through drugs are a potential form of treatment for PKU, although the high dosage of aminoglycosides used is not appropriate in a clinical setting. In vitro studies with new non-toxic read-through agents as well as in vivo studies would also be essential to determine the extent of read-through required to restore normal phenylalanine levels. PMID:23532445

  1. A SABATH Methyltransferase from the moss Physcomitrella patens catalyzes

    Energy Technology Data Exchange (ETDEWEB)

    Zhao, Nan [ORNL; Ferrer, Jean-Luc [Universite Joseph Fourier, France; Moon, Hong S [Department of Plant Sciences, University of Tennessee; Kapteyn, Jeremy [Institute of Biological Chemistry, Washington State University; Zhuang, Xiaofeng [Department of Plant Sciences, University of Tennessee; Hasebe, Mitsuyasu [Laboratory of Evolutionary Biology, National Institute for Biology, 38 Nishigounaka; Stewart, Neal C. [Department of Plant Sciences, University of Tennessee; Gang, David R. [Institute of Biological Chemistry, Washington State University; Chen, Feng [University of Tennessee, Knoxville (UTK)

    2012-01-01

    Known SABATH methyltransferases, all of which were identified from seed plants, catalyze methylation of either the carboxyl group of a variety of low molecular weight metabolites or the nitrogen moiety of precursors of caffeine. In this study, the SABATH family from the bryophyte Physcomitrella patens was identified and characterized. Four SABATH-like sequences (PpSABATH1, PpSABATH2, PpSABATH3, and PpSABATH4) were identified from the P. patens genome. Only PpSABATH1 and PpSABATH2 showed expression in the leafy gametophyte of P. patens. Full-length cDNAs of PpSABATH1 and PpSABATH2 were cloned and expressed in soluble form in Escherichia coli. Recombinant PpSABATH1 and PpSABATH2 were tested for methyltransferase activity with a total of 75 compounds. While showing no activity with carboxylic acids or nitrogen-containing compounds, PpSABATH1 displayed methyltransferase activity with a number of thiols. PpSABATH2 did not show activity with any of the compounds tested. Among the thiols analyzed, PpSABATH1 showed the highest level of activity with thiobenzoic acid with an apparent Km value of 95.5 lM, which is comparable to those of known SABATHs. Using thiobenzoic acid as substrate, GC MS analysis indicated that the methylation catalyzed by PpSABATH1 is on the sulfur atom. The mechanism for S-methylation of thiols catalyzed by PpSABATH1 was partially revealed by homology-based structural modeling. The expression of PpSABATH1 was induced by the treatment of thiobenzoic acid. Further transgenic studies showed that tobacco plants overexpressing PpSABATH1 exhibited enhanced tolerance to thiobenzoic acid, suggesting that PpSABATH1 have a role in the detoxification of xenobiotic thiols.

  2. Archease from Pyrococcus abyssi improves substrate specificity and solubility of a tRNA m5C methyltransferase

    DEFF Research Database (Denmark)

    Auxilien, Sylvie; El Khadali, Fatima; Rasmussen, Anette;

    2007-01-01

    reading frame (PAB1947), which is shown here to encode a tRNA m(5)C methyltransferase. In vitro, the purified recombinant methyltransferase catalyzes m(5)C formation at several cytosines within tRNAs with preference for C49. The specificity of the methyltransferase is increased by the archease...

  3. Human placental DNA methyltransferase: DNA substrate and DNA binding specificity.

    OpenAIRE

    Wang, R.Y.; Huang, L. H.; Ehrlich, M

    1984-01-01

    We have partially purified a DNA methyltransferase from human placenta using a novel substrate for a highly sensitive assay of methylation of hemimethylated DNA. This substrate was prepared by extensive nick translation of bacteriophage XP12 DNA, which normally has virtually all of its cytosine residues replaced by 5-methylcytosine (m5C). Micrococcus luteus DNA was just as good a substrate if it was first similarly nick translated with m5dCTP instead of dCTP in the polymerization mixture. At ...

  4. Expanding Cofactor Repertoire of Protein Lysine Methyltransferase for Substrate Labeling

    OpenAIRE

    Islam, Kabirul; Zheng, Weihong; Yu, Haiqiang; Deng, Haiteng; Luo, Minkui

    2011-01-01

    Protein lysine methyltransferases (PKMTs) play crucial roles in normal physiology and disease processes. Profiling PKMT targets is an important but challenging task. With cancer-relevant G9a as a target, we have demonstrated the success in developing S-adenosyl-L-methionine (SAM) analogues, particularly (E)-hex-2-en-5-ynyl SAM (Hey-SAM), as cofactors for engineered G9a. Hey-SAM analogue in combination with G9a Y1154A mutant modifies the same set of substrates as their native counterparts with...

  5. Purification and characterization of DNA methyltransferases from Neisseria gonorrhoeae.

    OpenAIRE

    Piekarowicz, A; Yuan, R.; Stein, D C

    1988-01-01

    Three DNA methyltransferases, M.NgoAI, and M.NgoBI and M.NgoBII, free of any nuclease activities were isolated from Neisseria gonorrhoeae strains WR220 and MUG116 respectively. M.NgoAI recognizes the sequence 5' GGCC 3' and methylates the first 5' cytosine on both strands. M.NgoBI and M.NgoBII recognize 5' TCACC 3' and 5' GTAN5CTC 3' respectively. M.NgoBII methylates cytosine on only one strand to produce 5' GTAN5mCTC 3'.

  6. High level expression and purification of HhaI methyltransferase.

    OpenAIRE

    Wu, J. C.; Santi, D V

    1988-01-01

    A cloning system for the DNA-(cytosine-5)-methyltransferase MHhaI and high level expression of the enzyme are described. A parent plasmid was constructed from fragments of the MHhaI gene and synthetic oligonucleotides. The construct permits introduction of various restriction sites for cloning at precise positions near the initiation codon, and beyond the termination codon. The entire MHhaI coding sequence was introduced as a 1042 b.p. NdeI-XbaI fragment into the vector pAR3040 which contains...

  7. The comparative effects of aminoglycoside antibiotics and muscle relaxants on electrical field stimulation response in rat bladder smooth muscle.

    Science.gov (United States)

    Min, Chang Ho; Min, Young Sil; Lee, Sang Joon; Sohn, Uy Dong

    2016-06-01

    It has been reported that several aminoglycoside antibiotics have a potential of prolonging the action of non-depolarizing muscle relaxants by drug interactions acting pre-synaptically to inhibit acetylcholine release, but antibiotics itself also have a strong effect on relaxing the smooth muscle. In this study, four antibiotics of aminoglycosides such as gentamicin, streptomycin, kanamycin and neomycin were compared with skeletal muscle relaxants baclofen, tubocurarine, pancuronium and succinylcholine, and a smooth muscle relaxant, papaverine. The muscle strips isolated from the rat bladder were stimulated with pulse trains of 40 V in amplitude and 10 s in duration, with pulse duration of 1 ms at the frequency of 1-8 Hz, at 1, 2, 4, 6, 8 Hz respectively. To test the effect of four antibiotics on bladder smooth muscle relaxation, each of them was treated cumulatively from 1 μM to 0.1 mM with an interval of 5 min. Among the four antibiotics, gentamicin and neomycin inhibited the EFS response. The skeletal muscle relaxants (baclofen, tubocurarine, pancuronium and succinylcholine) and inhibitory neurotransmitters (GABA and glycine) did not show any significant effect. However, papaverine, had a significant effect in the relaxation of the smooth muscle. It was suggested that the aminoglycoside antibiotics have inhibitory effect on the bladder smooth muscle. PMID:27260628

  8. Catalytic site remodelling of the DOT1L methyltransferase by selective inhibitors

    Energy Technology Data Exchange (ETDEWEB)

    Yu, Wenyu [Univ. of Toronto, ON (Canada); Chory, Emma J. [Dana-Farber Cancer Inst., Boston, MA (United States); Northeastern Univ., Boston, MA (United States); Wernimont, Amy K. [Univ. of Toronto, ON (Canada); Tempel, Wolfram [Univ. of Toronto, ON (Canada); Scopton, Alex [Univ. of Toronto, ON (Canada); Federation, Alexander [Dana-Farber Cancer Inst., Boston, MA (United States); Marineau, Jason J. [Dana-Farber Cancer Inst., Boston, MA (United States); Qi, Jun [Dana-Farber Cancer Inst., Boston, MA (United States); Barsyte-Lovejoy, Dalia [Univ. of Toronto, ON (Canada); Yi, Joanna [Dana-Farber Cancer Inst., Boston, MA (United States); Marcellus, Richard [Ontario Inst. for Cancer Research, Toronto, ON (Canada); Iacob, Roxana E. [Northeastern Univ., Boston, MA (United States); Engen, John R. [Northeastern Univ., Boston, MA (United States); Griffin, Carly [Ontario Inst. for Cancer Research, Toronto, ON (Canada); Aman, Ahmed [Ontario Inst. for Cancer Research, Toronto, ON (Canada); Wienholds, Erno [Univ. of Toronto, ON (Canada); Li, Fengling [Univ. of Toronto, ON (Canada); Pineda, Javier [Dana-Farber Cancer Inst., Boston, MA (United States); Univ. of Notre Dame, IN (United States); Estiu, Guillermina [Univ. of Notre Dame, IN (United States); Shatseva, Tatiana [Univ. of Toronto, ON (Canada); Hajian, Taraneh [Univ. of Toronto, ON (Canada); Al-awar, Rima [Ontario Inst. for Cancer Research, Toronto, ON (Canada); Dick, John E. [Univ. of Toronto, ON (Canada); Vedadi, Masoud [Univ. of Toronto, ON (Canada); Brown, Peter J. [Univ. of Toronto, ON (Canada); Arrowsmith, Cheryl H. [Univ. of Toronto, ON (Canada); Bradner, James E. [Dana-Farber Cancer Inst., Boston, MA (United States); Harvard Medical School, Boston, MA (United States); Schapira, Matthieu [Univ. of Toronto, ON (Canada)

    2012-12-18

    Selective inhibition of protein methyltransferases is a promising new approach to drug discovery. An attractive strategy towards this goal is the development of compounds that selectively inhibit binding of the cofactor, S-adenosylmethionine, within specific protein methyltransferases. Here we report the three-dimensional structure of the protein methyltransferase DOT1L bound toEPZ004777, the first S-adenosylmethionine-competitive inhibitor of a protein methyltransferase with in vivo efficacy. This structure and those of four new analogues reveal remodelling of the catalytic site. EPZ004777 and a brominated analogue, SGC0946, inhibit DOT1L in vitro and selectively kill mixed lineage leukaemia cells, in which DOT1L is aberrantly localized via interaction with an oncogenic MLL fusion protein. These data provide important new insight into mechanisms of cell-active S-adenosylmethionine-competitive protein methyltransferase inhibitors, and establish a foundation for the further development of drug-like inhibitors of DOT1L for cancer therapy.

  9. Regulation of DNA methylation turnover at LTR retrotransposons and imprinted loci by the histone methyltransferase Setdb1.

    Science.gov (United States)

    Leung, Danny; Du, Tingting; Wagner, Ulrich; Xie, Wei; Lee, Ah Young; Goyal, Preeti; Li, Yujing; Szulwach, Keith E; Jin, Peng; Lorincz, Matthew C; Ren, Bing

    2014-05-01

    During mammalian development, DNA methylation patterns need to be reset in primordial germ cells (PGCs) and preimplantation embryos. However, many LTR retrotransposons and imprinted genes are impervious to such global epigenetic reprogramming via hitherto undefined mechanisms. Here, we report that a subset of such genomic regions are resistant to widespread erasure of DNA methylation in mouse embryonic stem cells (mESCs) lacking the de novo DNA methyltransferases (Dnmts) Dnmt3a and Dnmt3b. Intriguingly, these loci are enriched for H3K9me3 in mESCs, implicating this mark in DNA methylation homeostasis. Indeed, deletion of the H3K9 methyltransferase SET domain bifurcated 1 (Setdb1) results in reduced H3K9me3 and DNA methylation levels at specific loci, concomitant with increased 5-hydroxymethylation (5hmC) and ten-eleven translocation 1 binding. Taken together, these data reveal that Setdb1 promotes the persistence of DNA methylation in mESCs, likely reflecting one mechanism by which DNA methylation is maintained at LTR retrotransposons and imprinted genes during developmental stages when DNA methylation is reprogrammed. PMID:24757056

  10. Persistent spread of the rmtB 16S rRNA methyltransferase gene among Escherichia coli isolates from diseased food-producing animals in China.

    Science.gov (United States)

    Xia, Jing; Sun, Jian; Cheng, Ke; Li, Liang; Fang, Liang-Xing; Zou, Meng-Ting; Liao, Xiao-Ping; Liu, Ya-Hong

    2016-05-30

    A total of 963 non-duplicate Escherichia coli strains isolated from food-producing animals between 2002 and 2012 were screened for the presence of the 16S rRNA methyltransferase genes. Among the positive isolates, resistance determinants to extended spectrum β-lactamases, plasmid-mediated quinolone resistance genes as well as floR and fosA/A3/C2 were detected using PCR analysis. These isolates were further subjected to antimicrobial susceptibility testing, molecular typing, PCR-based plasmid replicon typing and plasmid analysis. Of the 963 E. coli isolates, 173 (18.0%), 3 (0.3%) and 2 (0.2%) were rmtB-, armA- and rmtE-positive strains, respectively. All the 16S rRNA methyltransferase gene-positive isolates were multidrug resistant and over 90% of them carried one or more type of resistance gene. IncF (especially IncFII) and non-typeable plasmids played the main role in the dissemination of rmtB, followed by the IncN plasmids. Plasmids that harbored rmtB ranged in size from 20kb to 340kb EcoRI-RFLP testing of the 109 rmtB-positive plasmids from different years and different origins suggested that horizontal (among diverse animals) and vertical transfer of IncF, non-typeable and IncN-type plasmids were responsible for the spread of rmtB gene. In summary, our findings highlight that rmtB was the most prevalent 16S rRNA methyltransferase gene, which present persistent spread in food-producing animals in China and a diverse group of plasmids was responsible for rmtB dissemination. PMID:27139028

  11. Streptococcus suis, an Emerging Drug-Resistant Animal and Human Pathogen

    OpenAIRE

    Palmieri, Claudio; Varaldo, Pietro E.; Facinelli, Bruna

    2011-01-01

    Streptococcus suis, a major porcine pathogen, has been receiving growing attention not only for its role in severe and increasingly reported infections in humans, but also for its involvement in drug resistance. Recent studies and the analysis of sequenced genomes have been providing important insights into the S. suis resistome, and have resulted in the identification of resistance determinants for tetracyclines, macrolides, aminoglycosides, chloramphenicol, antifolate drugs, streptothricin,...

  12. Imipenem resistance in nonfermenters causing nosocomial urinary tract infections.

    Directory of Open Access Journals (Sweden)

    Taneja N

    2003-07-01

    Full Text Available Nonfermenting gram-negative bacilli (nonfermenters have emerged as important nosocomial pathogens causing opportunistic infections in immunocompromised hosts. These organisms show high level of resistance to b-lactam agents, fluoroquinolones and aminoglycosides. Imipenem is a carbapenem antibiotic, which can be very useful for treatment of infections caused by nonfermenters. Eighty-five nonfermenters causing nosocomial UTI were tested for MIC to imipenem by agar dilution method. Resistance to other antimicrobial agents was compared between imipenem sensitive (S and resistance (R groups. Overall 36.4% of nonfermenters were resistant to imipenem. Forty two percent of P. aeruginosa and 18.5% of Acinetobacter baumanii were imipenem resistant. Other nonfermenters showed variable resistance, resistance in Alcaligenes spp. being very high. More than 70% of the nonfermenters were resistant to ceftazidime, gentamicin and ciprofloxacin. Piperacillin and amikacin had the best in vitro susceptibility. No significant difference was found in the antibiotic susceptibility profile among imipenem sensitive (S or resistant (R strains.

  13. An international multicenter study of antimicrobial consumption and resistance in Staphylococcus aureus isolates from 15 hospitals in 14 countries

    DEFF Research Database (Denmark)

    Westh, Henrik Torkil; Zinn, Christina Scheel; Rosdahl, Vibeke Thamdrup

    2004-01-01

    usage of therapeutical subgroups of antimicrobials varied significantly between hospitals. A positive correlation was found between S. aureus resistance to methicillin (MRSA) and consumption of beta-lactam combinations, between resistance to quinolones and consumption of beta-lactam combinations and...... carbapenems and resistance to aminoglycosides and consumption of beta-lactam combinations. The consumption of beta-lactamase-sensitive antibiotics was negatively correlated to resistance to methicillin, quinolones, and aminoglycosides. Usage of the different antimicrobial therapeutical subgroups was also...... correlated. Consumption of beta-lactamase-sensitive antibiotics (penicillin) was positively correlated to consumption of beta-lactamase-resistant penicillins and negatively correlated to consumption of carbapenems, quinolones, and glycopeptides, whereas consumption of cephalosporins was positively correlated...

  14. Lipid substrate specificity of phosphatidylethanolamine N-methyltransferase of Tetrahymena

    International Nuclear Information System (INIS)

    The ciliate protozoan Tetrahymena thermophila forms about 60% of its phosphatidylcholine by methylation of phosphatidylethanolamine with S-adenosylmethionine using the enzyme phosphatidylethanolamine N-methyltransferase. Analogues of ethanolamine or of ethanolamine phosphate are incorporated into the phospholipids of Tetrahymena when cells are cultured in their presence. These compounds, 3-amino-1-propanol, 2-aminoethylphosphonate, 3-aminopropylphosphonate and N,N-dimethylaminoethylphosphonate replace from 50 to 75% of the ethanolamine phosphate in phosphatidylethanolamine. However, analysis of the phospholipids of lipid-altered Tetrahymena showed that none of the phosphatidylethanolamine analogues had been converted to the corresponding phosphatidylcholine analogue. No incorration of [14C-CH3]methionine into the phosphatidylcholine analogues could be demonstrated in vivo, nor was label from [3H-CH3]S-adenosylmethionine incorporated in virto. Thus, only phosphatidylethanolamine and its monomethyl and dimethyl derivatives have been found to be substrates for the phosphatidylethanoiamine N-methyltransferase. The enzyme therefore requires a phospholipid substrate containing an ester linkage between the alkylamine and phosphorus, with the amino group required to be β to the alcohol

  15. Hypnotizability and Catechol-O-Methyltransferase (COMT polymorphysms in Italians

    Directory of Open Access Journals (Sweden)

    Alessandro Gialluisi

    2014-01-01

    Full Text Available Higher brain dopamine content depending on lower activity of Catechol-O-Methyltransferase (COMT in subjects with high hypnotisability scores (highs has been considered responsible for their attentional characteristics. However, the results of the previous genetic studies on association between hypnotisability and the Catechol-O-Methyltransferase (COMT single nucleotide polymorphism (SNP rs4680 (Val158Met were inconsistent. Here, we used a selective genotyping approach to re-evaluate the association between hypnotisability and COMT in the context of a two-SNP haplotype analysis, considering not only the Val158Met polymorphism, but also the closely located rs4818 SNP. An Italian sample of 53 highs, 49 low hypnotizable subjects (lows and 57 controls, were genotyped for a segment of 805 bp of the COMT gene, including Val158Met and the closely located rs4818 SNP. Our selective genotyping approach had 97.1% power to detect the previously reported strongest association at the significance level of 5%. We found no evidence of association at the SNP, haplotype and diplotype levels. Thus, our results challenge the dopamine-based theory of hypnosis and indirectly support recent neuropsychological and neurophysiological findings reporting the lack of any association between hypnotisability and focused attention abilities.

  16. Structural biology of human H3K9 methyltransferases.

    Directory of Open Access Journals (Sweden)

    Hong Wu

    Full Text Available UNLABELLED: SET domain methyltransferases deposit methyl marks on specific histone tail lysine residues and play a major role in epigenetic regulation of gene transcription. We solved the structures of the catalytic domains of GLP, G9a, Suv39H2 and PRDM2, four of the eight known human H3K9 methyltransferases in their apo conformation or in complex with the methyl donating cofactor, and peptide substrates. We analyzed the structural determinants for methylation state specificity, and designed a G9a mutant able to tri-methylate H3K9. We show that the I-SET domain acts as a rigid docking platform, while induced-fit of the Post-SET domain is necessary to achieve a catalytically competent conformation. We also propose a model where long-range electrostatics bring enzyme and histone substrate together, while the presence of an arginine upstream of the target lysine is critical for binding and specificity. ENHANCED VERSION: This article can also be viewed as an enhanced version in which the text of the article is integrated with interactive 3D representations and animated transitions. Please note that a web plugin is required to access this enhanced functionality. Instructions for the installation and use of the web plugin are available in Text S1.

  17. A novel arsenic methyltransferase gene of Westerdykella aurantiaca isolated from arsenic contaminated soil: phylogenetic, physiological, and biochemical studies and its role in arsenic bioremediation.

    Science.gov (United States)

    Verma, Shikha; Verma, Pankaj Kumar; Meher, Alok Kumar; Dwivedi, Sanjay; Bansiwal, Amit Kumar; Pande, Veena; Srivastava, Pankaj Kumar; Verma, Praveen Chandra; Tripathi, Rudra Deo; Chakrabarty, Debasis

    2016-03-01

    Elevated arsenic concentration in the environment and agricultural soil is a serious concern to crop production and human health. Among different detoxification mechanisms, the methylation of arsenic is a widespread phenomenon in nature. A number of microorganisms are able to methylate arsenic, but less is known about the arsenic metabolism in fungi. We identified a novel arsenic methyltransferase (WaarsM) gene from a soil fungus, Westerdykella aurantiaca. WaarsM showed sequence homology with all known arsenic methyltransferases having three conserved SAM binding motifs. The expression of WaarsM enhanced arsenic resistance in E. coli (Δars) and S. cerevisiae (Δacr2) strains by biomethylation and required endogenous reductants, preferably GSH, for methyltransferase activity. The purified WaarsM catalyzes the production of methylated arsenicals from both AsIII and AsV, and also displays AsV reductase activity. It displayed higher methyltransferase activity and lower KM 0.1945 ± 0.021 mM and KM 0.4034 ± 0.078 mM for AsIII and AsV, respectively. S. cerevisiae (Δacr2) cells expressing WaarsM produced 2.2 ppm volatile arsenic and 0.64 ppm DMA(v) with 0.58 ppm volatile arsenicals when exposed to 20 ppm AsV and 2 ppm AsIII, respectively. Arsenic tolerance in rice after co-culture with genetically engineered yeast suggested its potential role in arsenic bioremediation. Thus, characterization of WaarsM provides a potential strategy to reduce arsenic concentration in soil with reduced arsenic accumulation in crops grown in arsenic contaminated areas, and thereby alleviating human health risks. PMID:26776948

  18. Construction of Yeast Vectors with Resistance to Geneticin

    Institute of Scientific and Technical Information of China (English)

    林会兰; 张广; 周全; 陈国强

    2002-01-01

    Two Escherichia coli-Saccharomyces cerevisiae shuttle vectors containing a resistance marker to geneticin (G418) are constructed. Both vectors contain a kanamycin-resistant marker (KanMX4) module coding aminoglycoside 3'-phosphotransferase (APH) that renders E. coli resistant to kanamycin and S. cerevisiae to geneticin. These vectors overcome the shortage of the conventional yeast vectors bearing HIS3, TRP1, LEU2, and URA3 modules as selection markers, which require hosts to be auxotrophic. Green fluorescent protein (GFP) is used as the reporter to examine the functions of the vectors. The vectors are powerful tools for the convenient cloning and controlled expression of genes in most S. cerevisiae strains.

  19. Cationic Amphiphiles Increase Activity of Aminoglycoside Antibiotic Tobramycin in the Presence of Airway Polyelectrolytes

    Energy Technology Data Exchange (ETDEWEB)

    Purdy Drew, Kirstin R.; Sanders, Lori K.; Culumber, Zachary W.; Zribi, Olena; Wong, Gerard C.L.; (UIUC)

    2009-06-17

    It is empirically known that anionic polyelectrolytes present in cystic fibrosis (CF) airways due to bacterial infection significantly decrease the activity of cationic antimicrobials via electrostatic binding. In this work, we use synchrotron small-angle X-ray scattering to investigate the interaction between tobramycin, an aminoglycoside antibiotic commonly administered to CF patients via inhalation, with DNA, which is found in high concentrations in the CF airway. We find that interactions between DNA and tobramycin are significantly modified by the presence of mixtures of amphiphilic molecules. We measure a hierarchy of self-assembled structures formed between tobramycin, DNA, and the amphiphile mixtures and show how interactions between these components can be controlled. Results indicate that mixtures of cationic and negative curvature amphiphiles optimized for DNA binding via charge matching and curvature matching can competitively displace bound tobramycin from DNA and thereby drastically suppress tobramycin-DNA binding and resultant antimicrobial inactivation. Growth inhibition assays confirm the increased activity of tobramycin in the presence of DNA with the addition of the amphiphiles. These results suggest that optimized cationic amphiphile solutions have the potential to enhance antimicrobial function in highly infected environments that contain increased concentrations of anionic inflammatory polymers.

  20. Cationic Amphiphiles Increase Activity of Aminoglycoside Antibiotic Tobramycin in the Presence of Airway Polyelectrolytes

    Energy Technology Data Exchange (ETDEWEB)

    Drew, K.R.Purdy; Sanders, L.K.; Culumber, Z.W.; Zribi, O.; Wong, G.C.L.

    2009-05-21

    It is empirically known that anionic polyelectrolytes present in cystic fibrosis (CF) airways due to bacterial infection significantly decrease the activity of cationic antimicrobials via electrostatic binding. In this work, we use synchrotron small-angle X-ray scattering to investigate the interaction between tobramycin, an aminoglycoside antibiotic commonly administered to CF patients via inhalation, with DNA, which is found in high concentrations in the CF airway. We find that interactions between DNA and tobramycin are significantly modified by the presence of mixtures of amphiphilic molecules. We measure a hierarchy of self-assembled structures formed between tobramycin, DNA, and the amphiphile mixtures and show how interactions between these components can be controlled. Results indicate that mixtures of cationic and negative curvature amphiphiles optimized for DNA binding via charge matching and curvature matching can competitively displace bound tobramycin from DNA and thereby drastically suppress tobramycin-DNA binding and resultant antimicrobial inactivation. Growth inhibition assays confirm the increased activity of tobramycin in the presence of DNA with the addition of the amphiphiles. These results suggest that optimized cationic amphiphile solutions have the potential to enhance antimicrobial function in highly infected environments that contain increased concentrations of anionic inflammatory polymers.

  1. Cationic Amphiphiles Increase Activity of Aminoglycoside Antibiotic Tobramycin in the Presence of Airway Polyelectrolytes

    International Nuclear Information System (INIS)

    It is empirically known that anionic polyelectrolytes present in cystic fibrosis (CF) airways due to bacterial infection significantly decrease the activity of cationic antimicrobials via electrostatic binding. In this work, we use synchrotron small-angle X-ray scattering to investigate the interaction between tobramycin, an aminoglycoside antibiotic commonly administered to CF patients via inhalation, with DNA, which is found in high concentrations in the CF airway. We find that interactions between DNA and tobramycin are significantly modified by the presence of mixtures of amphiphilic molecules. We measure a hierarchy of self-assembled structures formed between tobramycin, DNA, and the amphiphile mixtures and show how interactions between these components can be controlled. Results indicate that mixtures of cationic and negative curvature amphiphiles optimized for DNA binding via charge matching and curvature matching can competitively displace bound tobramycin from DNA and thereby drastically suppress tobramycin-DNA binding and resultant antimicrobial inactivation. Growth inhibition assays confirm the increased activity of tobramycin in the presence of DNA with the addition of the amphiphiles. These results suggest that optimized cationic amphiphile solutions have the potential to enhance antimicrobial function in highly infected environments that contain increased concentrations of anionic inflammatory polymers

  2. Effects of salicylates and aminoglycosides on spontaneous otoacoustic emissions in the Tokay gecko.

    Science.gov (United States)

    Stewart, C E; Hudspeth, A J

    2000-01-01

    The high sensitivity and sharp frequency discrimination of hearing depend on mechanical amplification in the cochlea. To explore the basis of this active process, we examined the pharmacological sensitivity of spontaneous otoacoustic emissions (SOAEs) in a lizard, the Tokay gecko. In a quiet environment, each ear produced a complex but stable pattern of emissions. These SOAEs were reversibly modulated by drugs that affect mammalian otoacoustic emissions, the salicylates and the aminoglycoside antibiotics. The effect of a single i.p. injection of sodium salicylate depended on the initial power of the emissions: ears with strong control SOAEs displayed suppression at all frequencies, whereas those with weak control emissions showed enhancement. Repeated oral administration of acetylsalicylic acid reduced all emissions. Single i.p. doses of gentamicin or kanamycin suppressed SOAEs below 2.6 kHz, while modulating those above 2.6 kHz in either of two ways. For ears whose emission power at 2.6-5.2 kHz encompassed more than half of the total, individual emissions displayed facilitation as great as 35-fold. For the remaining ears, emissions dropped to as little as one-sixth of their initial values. The similarity of the responses of reptilian and mammalian cochleas to pharmacological intervention provides further evidence for a common mechanism of cochlear amplification. PMID:10618439

  3. Hair cell regeneration in the bullfrog vestibular otolith organs following aminoglycoside toxicity

    Science.gov (United States)

    Baird, Richard A.; Torres, M. A.; Schuff, N. R.

    1994-01-01

    Adult bullfrogs were given single intraotic injections of the aminoglycoside antibiotic gentamicin sulfate and sacrificed at postinjection times ranging from 0.5 to 9 days. The saccular and utricular maculae of normal and injected animals were examined in wholemount and cross-section. Intraotic 200 (mu) M gentamicin concentrations resulted in the uniform destruction of the hair bundles and, at later times, the cell bodies of saccular hair cells. In the utriculus, striolar hair cells were selectively damaged while extrastriolar hair cells were relatively unaffected. Regenerating hair cells, identified in sectioned material by their small cell bodies and short, well-formed hair bundles, were seen in the saccular and utricular maculae as early as 24-48 h postinjection. Immature versions of mature hair cell types in both otolith organs were recognized by the presence of absence of a bulbed kinocilia and the relative lengths of their kinocilia and longest sterocilia. Utricular hair cell types with kinocilia longer than their longest stereocilia were observed at earlier times than hair cell types with shorter kinocilia. In the same sacculus, the hair bundles of gentamicin-treated animals, even at 9 days postinjection, were significantly smaller than those of normal animals. The hair bundles of utricular hair cells, on the other hand, reached full maturity within the same time period.

  4. Introducing Aztreonam The First Monobactam Antibiotic, A Suitable Substitution for the Aminoglycosides

    Directory of Open Access Journals (Sweden)

    A. Jahanshahi M.Khajeh - Karamadeni S. Fazli Bazaz

    1992-07-01

    Full Text Available Aztreonam (Azactam for injection, squibb is the first member of a new and unique class of beta - lactam antibiotics designated by researchers at the Squibb Institute for Medical Research as monobactams (monocyclic bacterially produced beta - lactam antibiotics."nIn this research, for the first time, antimicrobial spectrum of aztrenoam was determined by Disk - Agar Diffusion (250 spp. and Macrodilution Broth Methods (150 Spp."nWe also compared this antibiotic with two routine aminoglycoside antibiotics (Amikacin, Gentamicin in Iran. The most active antibiotic in our study was aztreonam which had MIC50 & MIC90 of 4 & 32 ^g/ml specifically against Pseudomonas aeruginosa."nThis rate for the other aerobic gram - negative bacteria (E. coli, Kleb. pneumoniae, Proteus mirabilis, enterobacter spp., Shigella Spp. and Salmonella Spp. was less than 0.5 & 4 g/ml respectively."nAztreonam's MIC90 for kleb pneumoniae was 8/jg/mI our results were Correlated to the other studies"nAll aerobic gram - negative bacteria has been obtained from the Qhaem's Medical Center in Mashhad - IRAN."nThe results of Disk - Agar Diffusion Method determines that all bacteria were 100% susceptible against aztreonam except Pseudomonas aeruginosa with 83% susceptibility.

  5. Pseudomonas Aeruginosa Resistance Phenotypes and Phenotypic Highlighting Methods

    Science.gov (United States)

    BĂLĂŞOIU, MARIA; BĂLĂŞOIU, A.T.; MĂNESCU, RODICA; AVRAMESCU, CARMEN; IONETE, OANA

    2014-01-01

    Pseudomonas aeruginosa genus bacteria are well known for their increased drug resistance (phenotypic ang genotypic resistance). The most important resistance mechanisms are: enzyme production, reduction of pore expression, reduction of the external membrane proteins expression, efflux systems, topoisomerase mutations. These mechanisms often accumulate and lead to multidrug ressitance strains emergence. The most frequent acquired resistance mechanisms are betalactamase-type enzyme production (ESBLs, AmpC, carbapenemases), which determine variable phenotypes of betalactamines resistance, phenotypes which are associated with aminoglycosides and quinolones resistance. The nonenzymatic drug resistance mechanisms are caused by efflux systems, pore reduction and penicillin-binding proteins (PBP) modification, which are often associated to other resistance mechanisms. Phenotypic methods used for testing these mechanisms are based on highlighting these phenotypes using Kirby Bauer antibiogram, clinical breakpoints, and “cut off” values recommended by EUCAST 2013 standard, version 3.1. PMID:25729587

  6. Whole genome sequencing of diverse Shiga toxin-producing and non-producing Escherichia coli strains reveals a variety of virulence and novel antibiotic resistance plasmids

    Science.gov (United States)

    The genomes of a diverse set of Shiga toxin-producing E. coli strains and the presence of 38 plasmids among all the isolates were determined. Among the novel plasmids found, there were eight that encoded resistance genes to antibiotics, including aminoglycosides, carbapenems, penicillins, cephalosp...

  7. Antibiotic resistance pattern in uropathogens

    Directory of Open Access Journals (Sweden)

    Gupta V

    2002-01-01

    Full Text Available Uropathogenic strains from inpatient and outpatient departments were studied from April 1997 to March 1999 for their susceptibility profiles. The various isolates were Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Proteus mirabilis, Acinetobacter baumanii and Enterococcus faecalis. Antibiotic susceptibility pattern of these isolates revealed that for outpatients, first generation cephalosporins, nitrofurantoin, norfloxacin/ciprofloxacin were effective for treatment of urinary tract infection but for inpatients, parenteral therapy with newer aminoglycosides and third generation cephalosporins need to be advocated as the organisms for nosocomial UTI exhibit a high degree of drug resistance. Trimethoprim and sulphamethoxazole combination was not found to be effective for the treatment of urinary tract infections as all the uropathogens from inpatients and outpatients showed high degree of resistance to co-trimoxazole. Culture and sensitivity of the isolates from urine samples should be done as a routine before advocating the therapy.

  8. Identification and characterization of arsenite methyltransferase from an archaeon, Methanosarcina acetivorans C2A.

    Science.gov (United States)

    Wang, Pei-Pei; Sun, Guo-Xin; Zhu, Yong-Guan

    2014-11-01

    Arsenic is a ubiquitous toxic contaminant in the environment. The methylation of arsenic can affect its toxicity and is primarily mediated by biological processes. Few studies have focused on the mechanism of arsenic methylation in archaea although archaea are widespread in the environment. Here, an arsenite [As(III)] methyltransferase (ArsM) was identified and characterized from an archaeon Methanosarcina acetivorans C2A. Heterologous expression of MaarsM was shown to confer As(III) resistance to an arsenic-sensitive strain of E. coli through arsenic methylation and subsequent volatilization. Purified MaArsM protein was further identified the function in catalyzing the formation of various methylated products from As(III) in vitro. Methylation of As(III) by MaArsM is highly dependent on the characteristics of the thiol cofactors used, with some of them (coenzyme M, homocysteine, and dithiothreitol) more efficient than GSH. Site-directed mutagenesis demonstrated that three conserved cysteine (Cys) residues (Cys62, Cys150, and Cys200) in MaArsM were necessary for As(III) methylation, of which only Cys150 and Cys200 were required for the methylation of monomethylarsenic. These results present a molecular pathway for arsenic methylation in archaea and provide some insight into the role of archaea in As biogeochemistry. PMID:25295694

  9. Jasmonic acid carboxyl methyltransferase regulates development and herbivory-induced defense response in rice.

    Science.gov (United States)

    Qi, Jinfeng; Li, Jiancai; Han, Xiu; Li, Ran; Wu, Jianqiang; Yu, Haixin; Hu, Lingfei; Xiao, Yutao; Lu, Jing; Lou, Yonggen

    2016-06-01

    Jasmonic acid (JA) and related metabolites play a key role in plant defense and growth. JA carboxyl methyltransferase (JMT) may be involved in plant defense and development by methylating JA to methyl jasmonate (MeJA) and thus influencing the concentrations of JA and related metabolites. However, no JMT gene has been well characterized in monocotyledon defense and development at the molecular level. After we cloned a rice JMT gene, OsJMT1, whose encoding protein was localized in the cytosol, we found that the recombinant OsJMT1 protein catalyzed JA to MeJA. OsJMT1 is up-regulated in response to infestation with the brown planthopper (BPH; Nilaparvata lugens). Plants in which OsJMT1 had been overexpressed (oe-JMT plants) showed reduced height and yield. These oe-JMT plants also exhibited increased MeJA levels but reduced levels of herbivore-induced JA and jasmonoyl-isoleucine (JA-Ile). The oe-JMT plants were more attractive to BPH female adults but showed increased resistance to BPH nymphs, probably owing to the different responses of BPH female adults and nymphs to the changes in levels of H2 O2 and MeJA in oe-JMT plants. These results indicate that OsJMT1, by altering levels of JA and related metabolites, plays a role in regulating plant development and herbivore-induced defense responses in rice. PMID:26466818

  10. Jasmonic acid carboxyl methyltransferase regulates development and herbivory-induced defense response in rice

    Institute of Scientific and Technical Information of China (English)

    Jinfeng Qi; Yonggen Lou; Jiancai Li; Xiu Han; Ran Li; Jianqiang Wu; Haixin Yu; Lingfei Hu; Yutao Xiao; Jing Lu

    2016-01-01

    Jasmonic acid (JA) and related metabolites play a key role in plant defense and growth. JA carboxyl methyltransferase (JMT) may be involved in plant defense and development by methylating JA to methyl jasmonate (MeJA) and thus influencing the concentrations of JA and related metabolites. However, no JMT gene has been well characterized in monocotyledon defense and development at the molecular level. After we cloned a rice JMT gene, OsJMT1, whose encoding protein was localized in the cytosol, we found that the recombinant OsJMT1 protein catalyzed JA to MeJA. OsJMT1 is up-regulated in response to infestation with the brown planthopper (BPH; Nilaparvata lugens). Plants in which OsJMT1 had been overexpressed (oe-JMT plants) showed reduced height and yield. These oe-JMT plants also exhibited increased MeJA levels but reduced levels of herbivore-induced JA and jasmonoyl-isoleucine (JA-Ile). The oe-JMT plants were more attractive to BPH female adults but showed increased resistance to BPH nymphs, probably owing to the different responses of BPH female adults and nymphs to the changes in levels of H2O2 and MeJA in oe-JMT plants. These results indicate that OsJMT1, by altering levels of JA and related metabolites, plays a role in regulating plant development and herbivore-induced defense responses in rice.

  11. Targeting O⁶-methylguanine-DNA methyltransferase with specific inhibitors as a strategy in cancer therapy.

    Science.gov (United States)

    Kaina, Bernd; Margison, Geoffrey P; Christmann, Markus

    2010-11-01

    O (6)-methylguanine-DNA methyltransferase (MGMT) repairs the cancer chemotherapy-relevant DNA adducts, O (6)-methylguanine and O (6)-chloroethylguanine, induced by methylating and chloroethylating anticancer drugs, respectively. These adducts are cytotoxic, and given the overwhelming evidence that MGMT is a key factor in resistance, strategies for inactivating MGMT have been pursued. A number of drugs have been shown to inactivate MGMT in cells, human tumour models and cancer patients, and O (6)-benzylguanine and O (6)-[4-bromothenyl]guanine have been used in clinical trials. While these agents show no side effects per se, they also inactivate MGMT in normal tissues and hence exacerbate the toxic side effects of the alkylating drugs, requiring dose reduction. This might explain why, in any of the reported trials, the outcome has not been improved by their inclusion. It is, however, anticipated that, with the availability of tumour targeting strategies and hematopoetic stem cell protection, MGMT inactivators hold promise for enhancing the effectiveness of alkylating agent chemotherapy. PMID:20717836

  12. Histone methyltransferase inhibitors are orally bioavailable, fast-acting molecules with activity against different species causing malaria in humans.

    Science.gov (United States)

    Malmquist, Nicholas A; Sundriyal, Sandeep; Caron, Joachim; Chen, Patty; Witkowski, Benoit; Menard, Didier; Suwanarusk, Rossarin; Renia, Laurent; Nosten, Francois; Jiménez-Díaz, María Belén; Angulo-Barturen, Iñigo; Santos Martínez, María; Ferrer, Santiago; Sanz, Laura M; Gamo, Francisco-Javier; Wittlin, Sergio; Duffy, Sandra; Avery, Vicky M; Ruecker, Andrea; Delves, Michael J; Sinden, Robert E; Fuchter, Matthew J; Scherf, Artur

    2015-02-01

    Current antimalarials are under continuous threat due to the relentless development of drug resistance by malaria parasites. We previously reported promising in vitro parasite-killing activity with the histone methyltransferase inhibitor BIX-01294 and its analogue TM2-115. Here, we further characterize these diaminoquinazolines for in vitro and in vivo efficacy and pharmacokinetic properties to prioritize and direct compound development. BIX-01294 and TM2-115 displayed potent in vitro activity, with 50% inhibitory concentrations (IC50s) of ratio analysis confirms a high asexual-stage rate of killing. Both compounds examined displayed oral efficacy in in vivo mouse models of Plasmodium berghei and P. falciparum infection. The discovery of a rapid and broadly acting antimalarial compound class targeting blood stage infection, including transmission stage parasites, and effective against multiple malaria-causing species reveals the diaminoquinazoline scaffold to be a very promising lead for development into greatly needed novel therapies to control malaria. PMID:25421480

  13. Poly(ADP-ribose) polymerase inhibitor ABT-888 potentiates the cytotoxic activity of temozolomide in leukemia cells: influence of mismatch repair status and O6-methylguanine-DNA methyltransferase activity

    OpenAIRE

    Horton, Terzah M.; Jenkins, Gaye; Pati, Debananda; Zhang, Linna; Dolan, M. Eileen; Ribes-Zamora, Albert; Bertuch, Alison A.; Blaney, Susan M.; Delaney, Shannon L.; Hegde, Madhuri; Berg, Stacey L.

    2009-01-01

    The poly(ADP-ribose) polymerase (PARP) inhibitor ABT-888 potentiates the antitumor activity of temozolomide (TMZ). TMZ resistance results from increased O6-methylguanine-DNA methyltransferase (MGMT) activity and from mismatch repair (MMR) system mutations. We evaluated the relative importance of MGMT activity, MMR deficiency, nonhomologous end joining (NHEJ), and PARP activity in ABT-888 potentiation of TMZ. MMR-proficient and MMR-deficient leukemia cells with varying MGMT activity, as well a...

  14. Comparison of ampicillin-sulbactam with vancomycin for treatment of experimental endocarditis due to a beta-lactamase-producing, highly gentamicin-resistant isolate of Enterococcus faecalis.

    OpenAIRE

    Lavoie, S R; Wong, E S; Coudron, P E; Williams, D S; Markowitz, S M

    1993-01-01

    Increasing antibiotic resistance in the enterococci, including the capacity for beta-lactamase production and the development of high-level aminoglycoside resistance, has complicated the treatment of serious enterococcal infections, which often require synergistic antibiotic combinations for cure. We utilized the rabbit model of aortic valve endocarditis to investigate the effects of various antibiotics, alone and in combination, against a multiply antibiotic-resistant isolate of Enterococcus...

  15. High-level amikacin resistance in Pseudomonas aeruginosa associated with a 3'-phosphotransferase with high affinity for amikacin.

    Science.gov (United States)

    Torres, C; Perlin, M H; Baquero, F; Lerner, D L; Lerner, S A

    2000-08-01

    This work describes the characterization of the phosphotransferase enzymatic activity responsible for amikacin resistance in two clinical Pseudomona aeruginosa strains, isolated from a hospital that used amikacin as first-line aminoglycoside. Amikacin-resistant P. aeruginosa PA40 and PA43 (MIC: 128 mg/l) were shown to have APH activity with a substrate profile similar to that of APH(3')-VI. The enzyme from P. aeruginosa PA40 was purified to > 70% homogeneity. The Km of amikacin for this enzyme was 1.4 microM, the Vmax/Km ratio for amikacin was higher than for the other aminoglycosides tested and PCR and DNA sequencing ruled out the presence of aph(3')-IIps. Amikacin resistance in this strain was, therefore, associated with APH(3')-VI and the high affinity of this enzyme for amikacin could explain the high-level resistance that we observed. PMID:10929874

  16. DOWN-STREAM SPATIAL DISTRIBUTION OF ANTIBIOTIC RESISTANCE TRAITS ALONG METAL CONTAMINATED STREAM REACHES

    Energy Technology Data Exchange (ETDEWEB)

    Tuckfield, C; J V Mcarthur (NOEMAIL), J

    2007-04-16

    Sediment bacteria samples were collected from three streams in South Carolina, two contaminated with multiple metals (Four Mile Creek and Castor Creek), one uncontaminated (Meyers Branch), and another metal contaminated stream (Lampert Creek) in northern Washington State. Growth plates inoculated with Four Mile Creek sample extracts show bacteria colony growth after incubation on plates containing either one of two aminoglycosides (kanamycin or streptomycin), tetracycline or chloramphenocol. This study analyzes the spatial pattern of antibiotic resistance in culturable sediment bacteria in all four streams that may be due to metal contamination. We summarize the two aminoglycoside resistance measures and the 10 metals concentrations by Principal Components Analysis. Respectively, 63% and 58% of the variability was explained in the 1st principal component of each variable set. We used the respective multivariate summary metrics (i.e. 1st principal component scores) as input measures for exploring the spatial correlation between antibiotic resistance and metal concentration for each stream reach sampled. Results show a significant and negative correlation between metals scores versus aminoglycoside resistance scores and suggest that selection for metal tolerance among sediment bacteria may influence selection for antibiotic resistance differently than previously supposed.. In addition, we borrow a method from geostatistics (variography) wherein a spatial cross-correlation analysis shows that decreasing metal concentrations scores are associated with increasing aminoglycoside resistance scores as the separation distance between sediment samples decreases, but for contaminated streams only. Since these results were counter to our initial expectation and to other experimental evidence for water column bacteria, we suspect our field results are influenced by metal bioavailability in the sediments and by a contaminant promoted interaction or ''cocktail effect

  17. Low Prevalence of Carbapenem-Resistant Bacteria in River Water: Resistance Is Mostly Related to Intrinsic Mechanisms.

    Science.gov (United States)

    Tacão, Marta; Correia, António; Henriques, Isabel S

    2015-10-01

    Carbapenems are last-resort antibiotics to handle serious infections caused by multiresistant bacteria. The incidence of resistance to these antibiotics has been increasing and new resistance mechanisms have emerged. The dissemination of carbapenem resistance in the environment has been overlooked. The main goal of this research was to assess the prevalence and diversity of carbapenem-resistant bacteria in riverine ecosystems. The presence of frequently reported carbapenemase-encoding genes was inspected. The proportion of imipenem-resistant bacteria was on average 2.24 CFU/ml. Imipenem-resistant strains (n=110) were identified as Pseudomonas spp., Stenotrophomonas maltophilia, Aeromonas spp., Chromobacterium haemolyticum, Shewanella xiamenensis, and members of Enterobacteriaceae. Carbapenem-resistant bacteria were highly resistant to other beta-lactams such as quinolones, aminoglycosides, chloramphenicol, tetracyclines, and sulfamethoxazole/trimethoprim. Carbapenem resistance was mostly associated with intrinsically resistant bacteria. As intrinsic resistance mechanisms, we have identified the blaCphA gene in 77.3% of Aeromonas spp., blaL1 in all S. maltophilia, and blaOXA-48-like in all S. xiamenensis. As acquired resistance mechanisms, we have detected the blaVIM-2 gene in six Pseudomonas spp. (5.45%). Integrons with gene cassettes encoding resistance to aminoglycosides (aacA and aacC genes), trimethoprim (dfrB1b), and carbapenems (blaVIM-2) were found in Pseudomonas spp. Results suggest that carbapenem resistance dissemination in riverine ecosystems is still at an early stage. Nevertheless, monitoring these aquatic compartments for the presence of resistance genes and its host organisms is essential to outline strategies to minimize resistance dissemination. PMID:26430939

  18. Highly stable, protein capped gold nanoparticles as effective drug delivery vehicles for amino-glycosidic antibiotics

    International Nuclear Information System (INIS)

    A method for the production of highly stable gold nanoparticles (Au NP) was optimized using sodium borohydride as reducing agent and bovine serum albumin as capping agent. The synthesized nanoparticles were characterized using UV–visible spectroscopy, transmission electron microscopy, X‐ray diffraction (XRD) and dynamic light scattering techniques. The formation of gold nanoparticles was confirmed from the appearance of pink colour and an absorption maximum at 532 nm. These protein capped nanoparticles exhibited excellent stability towards pH modification and electrolyte addition. The produced nanoparticles were found to be spherical in shape, nearly monodispersed and with an average particle size of 7.8 ± 1.7 nm. Crystalline nature of the nanoparticles in face centered cubic structure is confirmed from the selected‐area electron diffraction and XRD patterns. The nanoparticles were functionalized with various amino-glycosidic antibiotics for utilizing them as drug delivery vehicles. Using Fourier transform infrared spectroscopy, the possible functional groups of antibiotics bound to the nanoparticle surface have been examined. These drug loaded nanoparticle solutions were tested for their antibacterial activity against Gram-negative and Gram-positive bacterial strains, by well diffusion assay. The antibiotic conjugated Au NP exhibited enhanced antibacterial activity, compared to pure antibiotic at the same concentration. Being protein capped and highly stable, these gold nanoparticles can act as effective carriers for drugs and might have considerable applications in the field of infection prevention and therapeutics. - Highlights: ► Method for NaBH4 reduced and BSA capped gold nanoparticle was standardized. ► Nanoparticles were spherical and nearly monodispersed with a size of 7.8 nm. ► Nanoparticles are extremely stable towards pH modification and electrolyte addition. ► Antibiotic conjugated nanoparticles exhibited enhanced antibacterial activity

  19. Characterization of a C3 Deoxygenation Pathway Reveals a Key Branch Point in Aminoglycoside Biosynthesis.

    Science.gov (United States)

    Lv, Meinan; Ji, Xinjian; Zhao, Junfeng; Li, Yongzhen; Zhang, Chen; Su, Li; Ding, Wei; Deng, Zixin; Yu, Yi; Zhang, Qi

    2016-05-25

    Apramycin is a clinically interesting aminoglycoside antibiotic (AGA) containing a highly unique bicyclic octose moiety, and this octose is deoxygenated at the C3 position. Although the biosynthetic pathways for most 2-deoxystreptamine-containing AGAs have been well characterized, the pathway for apramycin biosynthesis, including the C3 deoxygenation process, has long remained unknown. Here we report detailed investigation of apramycin biosynthesis by a series of genetic, biochemical and bioinformatical studies. We show that AprD4 is a novel radical S-adenosyl-l-methionine (SAM) enzyme, which uses a noncanonical CX3CX3C motif for binding of a [4Fe-4S] cluster and catalyzes the dehydration of paromamine, a pseudodisaccharide intermediate in apramycin biosynthesis. We also show that AprD3 is an NADPH-dependent reductase that catalyzes the reduction of the dehydrated product from AprD4-catalyzed reaction to generate lividamine, a C3' deoxygenated product of paromamine. AprD4 and AprD3 do not form a tight catalytic complex, as shown by protein complex immunoprecipitation and other assays. The AprD4/AprD3 enzyme system acts on different pseudodisaccharide substrates but does not catalyze the deoxygenation of oxyapramycin, an apramycin analogue containing a C3 hydroxyl group on the octose moiety, suggesting that oxyapramycin and apramycin are partitioned into two parallel pathways at an early biosynthetic stage. Functional dissection of the C6 dehydrogenase AprQ shows the crosstalk between different AGA biosynthetic gene clusters from the apramycin producer Streptomyces tenebrarius, and reveals the remarkable catalytic versatility of AprQ. Our study highlights the intriguing chemistry in apramycin biosynthesis and nature's ingenuity in combinatorial biosynthesis of natural products. PMID:27120352

  20. Efficacy of an ototoxic aminoglycoside (gentamicin) on the differentiation of the inner ear of cichlid fish

    Science.gov (United States)

    Schönleber, J.; Anken, R. H.

    2004-01-01

    Previous investigations revealed that the growth of fish inner ear otoliths depends on the amplitude and the direction of gravity, thus suggesting the existence of a (negative) feedback mechanism. In the course of these experiments, it was shown that altered gravity both affected otolith size (and thus the provision of the proteinacious matrix) as well as the incorporation of calcium. It is hitherto unknown, as of whether sensory hair cells are involved either in the regulation of otolith growth or in the provision of otolithic material (such as protein or inorganic components) or even both. The ototoxic aminoglycoside gentamicin (GM) damages hair cells in many vertebrates (and is therefore used for the treatment of Meniere's disease in humans). The present study was thus designed to determine as of whether vestibular sensory cells are needed for otolith growth by applying GM in order to induce a (functionally relevant) loss of these cells. Developing cichlid fish Oreochromis mossambicus were therefore immersed in 120 mg/l GM for 10 or 21 days. At the beginning and at the end of the experimental periods, the fish were incubated in the calcium-tracer alizarin complexone (AC). After the experiment, otoliths were dissected and the area grown during GM-exposure (i.e., the area enclosed by the two AC labellings) was determined planimetrically. The results showed that incubating the animals in a GM-solution had no effect on otolith growth, but the development of otolith asymmetry was affected. Ultrastructural examinations of the sensory hair cells revealed that they had obviously not been affected by GM-treatment (no degenerative morphological features observed). Overall, the present results suggest that hair cells are not affected by GM concerning their possible role in (general) otolith growth, but that these cells indeed might have transitionally been impaired by GM resulting in a decreased capacity of regulating otolith symmetry.

  1. Structure of the Antibiotic Resistance Factor Spectinomycin Phosphotransferase from Legionella pneumophila

    Energy Technology Data Exchange (ETDEWEB)

    Fong, D.; Lemke, C; Huang, J; Xiong, B; Berghuis, A

    2010-01-01

    Aminoglycoside phosphotransferases (APHs) constitute a diverse group of enzymes that are often the underlying cause of aminoglycoside resistance in the clinical setting. Several APHs have been extensively characterized, including the elucidation of the three-dimensional structure of two APH(3{prime}) isozymes and an APH(2{double_prime}) enzyme. Although many APHs are plasmid-encoded and are capable of inactivating numerous 2-deoxystreptmaine aminoglycosides with multiple regiospecificity, APH(9)-Ia, isolated from Legionella pneumophila, is an unusual enzyme among the APH family for its chromosomal origin and its specificity for a single non-2-deoxystreptamine aminoglycoside substrate, spectinomycin. We describe here the crystal structures of APH(9)-Ia in its apo form, its binary complex with the nucleotide, AMP, and its ternary complex bound with ADP and spectinomycin. The structures reveal that APH(9)-Ia adopts the bilobal protein kinase-fold, analogous to the APH(3{prime}) and APH(2{double_prime}) enzymes. However, APH(9)-Ia differs significantly from the other two types of APH enzymes in its substrate binding area and that it undergoes a conformation change upon ligand binding. Moreover, kinetic assay experiments indicate that APH(9)-Ia has stringent substrate specificity as it is unable to phosphorylate substrates of choline kinase or methylthioribose kinase despite high structural resemblance. The crystal structures of APH(9)-Ia demonstrate and expand our understanding of the diversity of the APH family, which in turn will facilitate the development of new antibiotics and inhibitors.

  2. Natural antioxidant L-carnosine inhibits LPO intensification in structures of the auditory analyzer under conditions of chronic exposure to aminoglycoside antibiotics.

    Science.gov (United States)

    Zhuravskii, S G; Aleksandrova, L A; Sirot, V S; Ivanov, S A

    2004-10-01

    Intragastric administration of L-carnosine suspension to Wistar-Kyoto rats 3 days before and after 7-day course of intraperitoneal injections of ototoxic aminoglycoside antibiotic kanamycin compensated expenditures of tissue antioxidant systems and significantly eliminated kanamycin-induced intensification of MDA production in tissues of the membrane part of the cochlea and in the auditory cortex of the temporal lobe. L-NAME (competitive NO synthase inhibitor) also inhibited LPO, increased total antioxidant activity, and decreased ototoxicity of kanamycin, which confirms the contribution of NO into LPO intensification under conditions of aminoglycoside treatment. Inhibition of pathological intensification of LPO processes and increase in total antioxidant activity under conditions of induced acute aminoglycoside ototoxicity characterizes L-carnosine as a highly effective otoprotector. PMID:15665945

  3. Molecular characterization of multidrug-resistant extended-spectrum β-lactamase-producing Enterobacteriaceae isolated in Antananarivo, Madagascar.

    OpenAIRE

    Rakotonirina, Hanitra C; Garin, Benoît; Randrianirina, Frédérique; Richard, Vincent; Talarmin, Antoine; Arlet, Guillaume

    2013-01-01

    BACKGROUND: We investigated the molecular characteristics of multidrug-resistant, extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae isolated in community settings and in hospitals in Antananarivo, Madagascar. RESULTS: Forty-nine E. coli, K. pneumoniae, K. oxytoca and E. cloacae ESBL-producing isolates were studied. In antimicrobial susceptibility analyses, many of the isolates exhibited resistance to aminoglycosides, fluoroquinolones and trimethoprim-sulfamethoxazole. Gene amp...

  4. Molecular characterization of multidrug-resistant extended-spectrum β-lactamase-producing Enterobacteriaceae isolated in Antananarivo, Madagascar.

    OpenAIRE

    Rakotonirina, Hanitra C; Garin, Benoît; Randrianirina, Frédérique; Richard, Vincent; Talarmin, Antoine; Arlet, Guillaume

    2013-01-01

    Background We investigated the molecular characteristics of multidrug-resistant, extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae isolated in community settings and in hospitals in Antananarivo, Madagascar. Results Forty-nine E. coli, K. pneumoniae, K. oxytoca and E. cloacae ESBL-producing isolates were studied. In antimicrobial susceptibility analyses, many of the isolates exhibited resistance to aminoglycosides, fluoroquinolones and trimethoprim-sulfamethoxazole. Gene ampli...

  5. An audit of thiopurine methyltransferase genotyping and phenotyping before intended azathioprine treatment for dermatological conditions

    DEFF Research Database (Denmark)

    Vestergaard, T; Bygum, A

    2009-01-01

    Summary Background. Determining thiopurine methyltransferase (TPMT) genotype and phenotype before azathioprine treatment predicts which patients are most likely to develop myelosuppression. Aim. To evaluate the course of azathioprine treatment in people with TPMT heterozygosity and whether this d...

  6. Arsenic (+3 oxidation state) methyltransferase and the methylation of arsenicals in the invertebrate chordate Ciona intestinalis

    Science.gov (United States)

    Biotransformation of inorganic arsenic (iAs) involves methylation catalyzed by arsenic (+3 oxidation state) methyltransferase (As3mt), yielding mono- , di- , and trimethylated arsenicals. To investigate the evolution of molecular mechanisms that mediate arsenic biotransformation,...

  7. Erythrocuyte catechol-O-methyltransferase activity in related families with schizophrenia

    International Nuclear Information System (INIS)

    Erythrocyte catechol-O-methyltransferase (COMT) activity has been analyzed in 72 individuals from 17 related families with 18 schizophrenic members. No association with any of the three COMT genotypes could be found in this pedigree. (author)

  8. “MGMT for pt mgmt”: Is Methylguanine-DNA Methyltransferase Testing Ready for Patient Management?

    OpenAIRE

    Iafrate, A. John; Louis, David N.

    2008-01-01

    This Commentary reports on a robust quantitative assay for the interpretation of O6-methylguanine-DNA methyltransferase (MGMT) promoter methylation assays that should facilitate the comparison and implementation of such assays across laboratories.

  9. Early transcriptional response to aminoglycoside antibiotic suggests alternate pathways leading to apoptosis of sensory hair cells in the mouse inner ear

    Directory of Open Access Journals (Sweden)

    Neil eSegil

    2015-05-01

    Full Text Available Aminoglycoside antibiotics are the drug of choice for treating many bacterial infections, but their administration results in hearing loss in nearly one fourth of the patients who receive them. Several biochemical pathways have been implicated in aminoglycoside antibiotic ototoxicity; however, little is known about how hair cells respond to aminoglycoside antibiotics at the transcriptome level. Here we have investigated the genome-wide response to the aminoglycoside antibiotic gentamicin. Using organotypic cultures of the perinatal organ of Corti, we performed RNA sequencing using cDNA libraries obtained from FACS-purified hair cells. Within 3 hours of gentamicin treatment, the messenger RNA level of more than three thousand genes in hair cells changed significantly. Bioinformatic analysis of these changes highlighted several known signal transduction pathways, including the JNK pathway and the NF-κB pathway, in addition to genes involved in the stress response, apoptosis, cell cycle control, and DNA damage repair. In contrast, only 698 genes, mainly involved in cell cycle and metabolite biosynthetic processes, were significantly affected in the non-hair cell population. The gene expression profiles of hair cells in response to gentamicin share a considerable similarity with those previously observed in gentamicin-induced nephrotoxicity. Our findings suggest that previously observed early responses to gentamicin in hair cells in specific signaling pathways are reflected in changes in gene expression. Additionally, the observed changes in gene expression of cell cycle regulatory genes indicate a disruption of the postmitotic state, which may suggest an alternative pathway regulating gentamicin-induced hair cell death. This work provides a more comprehensive view of aminoglycoside antibiotic ototoxicity, and thus contribute to identifying potential pathways or therapeutic targets to alleviate this important side effect of aminoglycoside

  10. Pioglitazone attenuates hepatic inflammation and fibrosis in phosphatidylethanolamine N-methyltransferase-deficient mice.

    Science.gov (United States)

    van der Veen, Jelske N; Lingrell, Susanne; Gao, Xia; Quiroga, Ariel D; Takawale, Abhijit; Armstrong, Edward A; Yager, Jerome Y; Kassiri, Zamaneh; Lehner, Richard; Vance, Dennis E; Jacobs, René L

    2016-04-01

    Phosphatidylethanolamine N-methyltransferase (PEMT) is an important enzyme in hepatic phosphatidylcholine (PC) biosynthesis. Pemt(-/-) mice are protected against high-fat diet (HFD)-induced obesity and insulin resistance; however, these mice develop nonalcoholic fatty liver disease (NAFLD). We hypothesized that peroxisomal proliferator-activated receptor-γ (PPARγ) activation by pioglitazone might stimulate adipocyte proliferation, thereby directing lipids from the liver toward white adipose tissue. Pioglitazone might also act directly on PPARγ in the liver to improve NAFLD. Pemt(+/+) and Pemt(-/-) mice were fed a HFD with or without pioglitazone (20 mg·kg(-1)·day(-1)) for 10 wk. Pemt(-/-) mice were protected from HFD-induced obesity but developed NAFLD. Treatment with pioglitazone caused an increase in body weight gain in Pemt(-/-) mice that was mainly due to increased adiposity. Moreover, pioglitazone improved NAFLD in Pemt(-/-) mice, as indicated by a 35% reduction in liver weight and a 57% decrease in plasma alanine transaminase levels. Livers from HFD-fed Pemt(-/-) mice were steatotic, inflamed, and fibrotic. Hepatic steatosis was still evident in pioglitazone-treated Pemt(-/-) mice; however, treatment with pioglitazone reduced hepatic fibrosis, as evidenced by reduced Sirius red staining and lowered mRNA levels of collagen type Iα1 (Col1a1), tissue inhibitor of metalloproteinases 1 (Timp1), α-smooth muscle actin (Acta2), and transforming growth factor-β (Tgf-β). Similarly, oxidative stress and inflammation were reduced in livers from Pemt(-/-) mice upon treatment with pioglitazone. Together, these data show that activation of PPARγ in HFD-fed Pemt(-/-) mice improved liver function, while these mice were still protected against diet-induced obesity and insulin resistance. PMID:26797396

  11. Biochemical and Computational Analysis of the Substrate Specificities of Cfr and RlmN Methyltransferases.

    Directory of Open Access Journals (Sweden)

    Eleni Ntokou

    Full Text Available Cfr and RlmN methyltransferases both modify adenine 2503 in 23S rRNA (Escherichia coli numbering. RlmN methylates position C2 of adenine while Cfr methylates position C8, and to a lesser extent C2, conferring antibiotic resistance to peptidyl transferase inhibitors. Cfr and RlmN show high sequence homology and may be evolutionarily linked to a common ancestor. To explore their individual specificity and similarity we performed two sets of experiments. We created a homology model of Cfr and explored the C2/C8 specificity using docking and binding energy calculations on the Cfr homology model and an X-ray structure of RlmN. We used a trinucleotide as target sequence and assessed its positioning at the active site for methylation. The calculations are in accordance with different poses of the trinucleotide in the two enzymes indicating major evolutionary changes to shift the C2/C8 specificities. To explore interchangeability between Cfr and RlmN we constructed various combinations of their genes. The function of the mixed genes was investigated by RNA primer extension analysis to reveal methylation at 23S rRNA position A2503 and by MIC analysis to reveal antibiotic resistance. The catalytic site is expected to be responsible for the C2/C8 specificity and most of the combinations involve interchanging segments at this site. Almost all replacements showed no function in the primer extension assay, apart from a few that had a weak effect. Thus Cfr and RlmN appear to be much less similar than expected from their sequence similarity and common target.

  12. The RNA–Methyltransferase Misu (NSun2) Poises Epidermal Stem Cells to Differentiate

    OpenAIRE

    Sandra Blanco; Agata Kurowski; Jennifer Nichols; Watt, Fiona M.; Salvador Aznar Benitah; Michaela Frye

    2011-01-01

    Homeostasis of most adult tissues is maintained by balancing stem cell self-renewal and differentiation, but whether post-transcriptional mechanisms can regulate this process is unknown. Here, we identify that an RNA methyltransferase (Misu/Nsun2) is required to balance stem cell self-renewal and differentiation in skin. In the epidermis, this methyltransferase is found in a defined sub-population of hair follicle stem cells poised to undergo lineage commitment, and its depletion results in e...

  13. Engineering the DNA cytosine-5 methyltransferase reaction for sequence-specific labeling of DNA

    OpenAIRE

    Lukinavičius, Gražvydas; Lapinaitė, Audronė; Urbanavičiūtė, Giedrė; Gerasimaitė, Rūta; Klimašauskas, Saulius

    2012-01-01

    DNA methyltransferases catalyse the transfer of a methyl group from the ubiquitous cofactor S-adenosyl-L-methionine (AdoMet) onto specific target sites on DNA and play important roles in organisms from bacteria to humans. AdoMet analogs with extended propargylic side chains have been chemically produced for methyltransferase-directed transfer of activated groups (mTAG) onto DNA, although the efficiency of reactions with synthetic analogs remained low. We performed steric engineering of the co...

  14. Structure–function analysis of vaccinia virus mRNA cap (guanine-N7) methyltransferase

    OpenAIRE

    Zheng, Sushuang; Shuman, Stewart

    2008-01-01

    The guanine-N7 methyltransferase domain of vaccinia virus mRNA capping enzyme is a heterodimer composed of a catalytic subunit and a stimulatory subunit. Structure–function analysis of the catalytic subunit by alanine scanning and conservative substitutions (49 mutations at 25 amino acids) identified 12 functional groups essential for methyltransferase activity in vivo, most of which were essential for cap methylation in vitro. Defects in cap binding were demonstrated for a subset of lethal m...

  15. Crystal structure of dengue virus methyltransferase without S-adenosyl-L-methionine.

    Science.gov (United States)

    Noble, Christian G; Li, Shi-Hua; Dong, Hongping; Chew, Sock Hui; Shi, Pei-Yong

    2014-11-01

    Flavivirus methyltransferase is a genetically-validated antiviral target. Crystal structures of almost all available flavivirus methyltransferases contain S-adenosyl-L-methionine (SAM), the methyl donor molecule that co-purifies with the enzymes. This raises a possibility that SAM is an integral structural component required for the folding of dengue virus (DENV) methyltransferase. Here we exclude this possibility by solving the crystal structure of DENV methyltransferase without SAM. The SAM ligand was removed from the enzyme through a urea-mediated denaturation-and-renaturation protocol. The crystal structure of the SAM-depleted enzyme exhibits a vacant SAM-binding pocket, with a conformation identical to that of the SAM-enzyme co-crystal structure. Functionally, equivalent enzymatic activities (N-7 methylation, 2'-O methylation, and GMP-enzyme complex formation) were detected for the SAM-depleted and SAM-containing recombinant proteins. These results clearly indicate that the SAM molecule is not an essential component for the correct folding of DENV methyltransferase. Furthermore, the results imply a potential antiviral approach to search for inhibitors that can bind to the SAM-binding pocket and compete against SAM binding. To demonstrate this potential, we have soaked crystals of DENV methyltransferase without a bound SAM with the natural product Sinefungin and show that preformed crystals are capable of binding ligands in this pocket. PMID:25241250

  16. Molecular Analysis of Cross-Resistance to Capreomycin, Kanamycin, Amikacin, and Viomycin in Mycobacterium tuberculosis

    OpenAIRE

    Maus, Courtney E.; Plikaytis, Bonnie B.; Shinnick, Thomas M

    2005-01-01

    Capreomycin, kanamycin, amikacin, and viomycin are drugs that are used to treat multidrug-resistant tuberculosis. Each inhibits translation, and cross-resistance to them is a concern during therapy. A recent study revealed that mutation of the tlyA gene, encoding a putative rRNA methyltransferase, confers capreomycin and viomycin resistance in Mycobacterium tuberculosis bacteria. Mutations in the 16S rRNA gene (rrs) have been associated with resistance to each of the drugs; however, reports o...

  17. Incidence of High-Level Evernimicin Resistance in Enterococcus faecium among Food Animals and Humans

    OpenAIRE

    Aarestrup, Frank Møller; McNicholas, Paul M.

    2002-01-01

    Six high-level evernimicin-resistant Enterococcus faecium isolates were identified among 304 avilamycin-resistant E. faecium isolates from animals and 404 stool samples from humans with diarrhea. All four animal isolates, and one of the human isolates, were able to transfer resistance to a susceptible E. faecium strain. The resulting transconjugants all tested positive for the presence of emtA, a gene encoding a methyltransferase previously linked with high-level evernimicin resistance. The f...

  18. COMBINATIONAL ADMINISTRATION OF AMINOGLYCOSIDES AND LOOP DIURETICS AS AN EFFICIENT STRATEGY TO ESTABLISH DEAFNESS MODELS IN RATS

    Institute of Scientific and Technical Information of China (English)

    CONG Tao; LIU Riyuan; YUAN Shuolong; XU Liangwei; YANG Shiming

    2014-01-01

    It is known that aminoglycoside antibiotics can damage the vestibular and auditory sensory epithelia, and the loop diuretics can enhance the ototoxic effect of aminoglycosides. Previous studies on the synergistic effect of these two types of drugs have used mice, guinea pigs and cats, but not rats. The aim of this study was to determine this synergistic effects in rat cochleae. Rats received intravenous injections of different doses of furosemide and/or intramuscular injections of kanamycin sulfate. Au-ditory brainstem response (ABR), scanning electron microscopy (SEM) and immunocytochemistry were used to determine the effects of drug administration. In the group receiving combined administration of furosemide and kanamycin, the ABR thresh-old showed significant elevation 3 days after drug administration, greater than single drug administration. The hair cells showed various degrees of injury from the apical turn to the basal turn of the cochlea and from the outer hair cells to the inner hair cells. Neuron fibers of the hair cells showed significant loss 7 days after the drug administration, but the number of spiral ganglia did not decrease and supporting cells showed no signs of injury. Our study suggest that combined administration of fu-rosemide and kanamycin has an synergistic ototoxic effect, and can result in hair cell loss and hearing loss in rats.

  19. Directed evolution of aminoglycoside phosphotransferase (3' type IIIa variants that inactivate amikacin but impose significant fitness costs.

    Directory of Open Access Journals (Sweden)

    Joseph R Kramer

    Full Text Available The rules that govern adaptive protein evolution remain incompletely understood. Aminoglycoside aminotransferase (3' type IIIa (hereafter abbreviated APH(3'-IIIa is a good model enzyme because it inactivates kanamycin efficiently; it recognizes other aminoglycoside antibiotics, including amikacin, but not nearly as well. Here we direct the evolution of APH(3'-IIIa variants with increased activity against amikacin. After four rounds of random mutation and selection in Escherichia coli, the minimum inhibitory concentration of amikacin rose from 18 micrograms/mL (wild-type enzyme to over 1200 micrograms/mL (clone 4.1. The artificially evolved 4.1 APH(3'-IIIa variant exhibited 19-fold greater catalytic efficiency (k cat/K M than did the wild-type enzyme in reactions with amikacin. E. coli expressing the evolved 4.1 APH(3'-IIIa also exhibited a four-fold decrease in fitness (as measured by counting colony forming units in liquid cultures with the same optical density compared with isogenic cells expressing the wild-type protein under non-selective conditions. We speculate that these fitness costs, in combination with the prevalence of other amikacin-modifying enzymes, hinder the evolution of APH(3'-IIIa in clinical settings.

  20. Protein isoaspartate methyltransferase prevents apoptosis induced by oxidative stress in endothelial cells: role of Bcl-Xl deamidation and methylation.

    Directory of Open Access Journals (Sweden)

    Amelia Cimmino

    Full Text Available BACKGROUND: Natural proteins undergo in vivo spontaneous post-biosynthetic deamidation of specific asparagine residues with isoaspartyl formation. Deamidated-isomerized molecules are both structurally and functionally altered. The enzyme isoaspartyl protein carboxyl-O-methyltransferase (PCMT; EC 2.1.1.77 has peculiar substrate specificity towards these deamidated proteins. It catalyzes methyl esterification of the free alpha-carboxyl group at the isoaspartyl site, thus initiating the repair of these abnormal proteins through the conversion of the isopeptide bond into a normal alpha-peptide bond. Deamidation occurs slowly during cellular and molecular aging, being accelerated by physical-chemical stresses brought to the living cells. Previous evidence supports a role of protein deamidation in the acquisition of susceptibility to apoptosis. Aim of this work was to shed a light on the role of PCMT in apoptosis clarifying the relevant mechanism(s. METHODOLOGY/PRINCIPAL FINDINGS: Endothelial cells transiently transfected with various constructs of PCMT, i.e. overexpressing wild type PCMT or negative dominants, were used to investigate the role of protein methylation during apoptosis induced by oxidative stress (H(2O(2; 0.1-0.5 mM range. Results show that A Cells overexpressing "wild type" human PCMT were resistant to apoptosis, whereas overexpression of antisense PCMT induces high sensitivity to apoptosis even at low H(2O(2 concentrations. B PCMT protective effect is specifically due to its methyltransferase activity rather than to any other non-enzymatic interactions. In fact negative dominants, overexpressing PCMT mutants devoid of catalytic activity do not prevent apoptosis. C Cells transfected with antisense PCMT, or overexpressing a PCMT mutant, accumulate isoaspartyl-containing damaged proteins upon H(2O(2 treatment. Proteomics allowed the identification of proteins, which are both PCMT substrates and apoptosis effectors, whose deamidation

  1. A preliminary report on the susceptibility to aminoglycosides of Escherichia coli isolated from the community-acquired urinary tract infections in adults in south-east Poland

    Directory of Open Access Journals (Sweden)

    Fidecka-Skwarzynska Magdalena

    2015-03-01

    Full Text Available World-wide, urinary tract infections (UTIs are an important clinical problem. In such, the most frequently isolated uropathogen is Escherichia coli. In the treatment of uncomplicated UTIs, e.g. cystitis, the widely used antibiotics are nitrofurantoin, trimethoprim/sulfamethoxazole, fosfomycin trometamol or ciprofloxacin, while the treatment of pyelonephritis requires the usage of antibiotics with a broader spectrum of activity, such as cephalosporins of the 3rd and 4th generation, aminoglycosides or even carbapenems. The aim of this study was to assess the susceptibility to aminoglycosides (such as amikacin, gentamicin, netilmicin and tobramycin of E. coli isolated from UTIs in adult community patients living in Lubelszczyzna. We found that all of the 86 strains of E. coli encountered were susceptible to amikacin. Moreover, the prevalence of susceptibility to tobramycin, gentamicin or netilmicin among the tested strains was found to be 89,5%, 90,7% or 94,2%, respectively. The data obtained in the present study shows the high susceptibility to aminoglycosides of E. coli isolated from the community-acquired UTIS in adults. These data, together with that derived from current literature, indicate that aminoglycosides, when employed in combination therapy with other antibiotics, may still be very useful group of antibacterial agents in the treatment of UTI’s in Poland.

  2. Clinical evaluation and mitochondrial DNA sequence analysis in two Chinese families with aminoglycoside-induced and non-syndromic hearing loss

    International Nuclear Information System (INIS)

    We report here the clinical, genetic, and molecular characterization of two Chinese pedigrees with aminoglycoside-induced and non-syndromic hearing impairment. Clinical evaluation revealed the variable phenotype of hearing impairment including audiometric configuration in these subjects. Penetrances of hearing loss in BJ105 and BJ106 pedigrees are 67% and 33%, respectively. In particular, three of 10 affected matrilineal relatives of BJ105 pedigree had aminoglycoside-induced hearing loss, while seven affected matrilineal relatives in BJ105 pedigree and six affected matrilineal relatives in BJ106 pedigree did not have a history of exposure to aminoglycosides. Sequence analysis of the complete mitochondrial genomes in these pedigrees showed the identical homoplasmic A1555G mutation and distinct sets of mtDNA variants belonging to haplogroups F3 and M7b. These variants showed no evolutionary conservation, implying that mitochondrial haplotype may not play a significant role in the phenotypic expression of the A1555G mutation in these Chinese pedigrees. However, aminoglycosides and nuclear backgrounds appear to be major modifier factors for the phenotypic manifestation of the A1555G mutation in these Chinese families

  3. Mitochondrial 12S Ribosomal RNA A1555G Mutation Associated with Cardiomyopathy and Hearing Loss following High-Dose Chemotherapy and Repeated Aminoglycoside Exposure

    DEFF Research Database (Denmark)

    Skou, Anne-Sofie; Tranebjærg, Lisbeth; Jensen, Tim;

    2014-01-01

    A 19-month-old girl with the A1555G mitochondrial mutation in the 12S ribosomal RNA gene and acute myelogenous leukemia developed dilated cardiomyopathy and bilateral sensorineural hearing loss before undergoing allogeneic stem cell transplantation. She had received gentamicin during episodes of ...... febrile neutropenia. Testing for the A1555G mutation is recommended in patients frequently treated with aminoglycosides....

  4. DNA methyltransferase inhibitor CDA-II inhibits myogenic differentiation

    International Nuclear Information System (INIS)

    Highlights: ► CDA-II inhibits myogenic differentiation in a dose-dependent manner. ► CDA-II repressed expression of muscle transcription factors and structural proteins. ► CDA-II inhibited proliferation and migration of C2C12 myoblasts. -- Abstract: CDA-II (cell differentiation agent II), isolated from healthy human urine, is a DNA methyltransferase inhibitor. Previous studies indicated that CDA-II played important roles in the regulation of cell growth and certain differentiation processes. However, it has not been determined whether CDA-II affects skeletal myogenesis. In this study, we investigated effects of CDA-II treatment on skeletal muscle progenitor cell differentiation, migration and proliferation. We found that CDA-II blocked differentiation of murine myoblasts C2C12 in a dose-dependent manner. CDA-II repressed expression of muscle transcription factors, such as Myogenin and Mef2c, and structural proteins, such as myosin heavy chain (Myh3), light chain (Mylpf) and MCK. Moreover, CDA-II inhibited C1C12 cell migration and proliferation. Thus, our data provide the first evidence that CDA-II inhibits growth and differentiation of muscle progenitor cells, suggesting that the use of CDA-II might affect skeletal muscle functions.

  5. Theoretical insights into catalytic mechanism of protein arginine methyltransferase 1.

    Directory of Open Access Journals (Sweden)

    Ruihan Zhang

    Full Text Available Protein arginine methyltransferase 1 (PRMT1, the major arginine asymmetric dimethylation enzyme in mammals, is emerging as a potential drug target for cancer and cardiovascular disease. Understanding the catalytic mechanism of PRMT1 will facilitate inhibitor design. However, detailed mechanisms of the methyl transfer process and substrate deprotonation of PRMT1 remain unclear. In this study, we present a theoretical study on PRMT1 catalyzed arginine dimethylation by employing molecular dynamics (MD simulation and quantum mechanics/molecular mechanics (QM/MM calculation. Ternary complex models, composed of PRMT1, peptide substrate, and S-adenosyl-methionine (AdoMet as cofactor, were constructed and verified by 30-ns MD simulation. The snapshots selected from the MD trajectory were applied for the QM/MM calculation. The typical SN2-favored transition states of the first and second methyl transfers were identified from the potential energy profile. Deprotonation of substrate arginine occurs immediately after methyl transfer, and the carboxylate group of E144 acts as proton acceptor. Furthermore, natural bond orbital analysis and electrostatic potential calculation showed that E144 facilitates the charge redistribution during the reaction and reduces the energy barrier. In this study, we propose the detailed mechanism of PRMT1-catalyzed asymmetric dimethylation, which increases insight on the small-molecule effectors design, and enables further investigations into the physiological function of this family.

  6. Characterization of the phospholipid methyltransferase in RBC ghost preparations

    International Nuclear Information System (INIS)

    The activity of the phospholipid methyltransferase from human RBC ghosts was studied using radio-HPLC techniques to analyze the products. Both monomethyl phosphatidyl ethanolamine (MMPE) and dimethyl phosphatidyl ethanolamine (DMPE) were used as substrated. The reaction rate was linear for 45 min. Apparent K/sub M/s of 24-28 uM and 19-21 uM were measured for these two substrates, respectively. The reaction rate was not linear with protein. It appeared to increase logarithmic. An apparent K/sub M/ for S-adenosylmethionine was 36-45 uM. These K/sub M/ values are similar to those reported by others for liver. As the concentration of MMPE was increased, the ratio of DMPE/PC also increased due largely to a greater increase in DMPE formation. Optimal reaction rates for the formation of DMPE were 0.9-1.3 pmol/mg/min, and an optimal rate of about 1.7-2.4 pmol/min/mg was measured for the conversion of DMPE to phosphatidyl choline (PC). Freezing the ghost preparation did not affect the activity of the enzyme. When no exogenous phospholipid was added to the incubation, the sum of the formation rates of all three methylated products was about 26 pmol/mg/hr. The relative amount of each product was 46% MMPE, 32% DMPE and 22% PC. When either MMPE or DMPE was added as substrate, the formation of MMPE was reduced to less than 1%

  7. Thiopurine methyltransferase activity in red blood cells of dogs.

    Science.gov (United States)

    Kidd, Linda Benjamin; Salavaggione, Oreste E; Szumlanski, Carol L; Miller, Jackie L; Weinshilboum, Richard M; Trepanier, Lauren

    2004-01-01

    Thiopurine methyltransferase (TPMT) is an important enzyme in the metabolism of thiopurine medications such as azathioprine. In humans, activity varies widely among individuals, primarily because of genetic polymorphisms. Low TPMT activity increases the risk of myelosuppression from azathioprine and 6-mercaptopurine, whereas high TPMT activity is associated with poor drug efficacy. The purpose of this study was to determine whether dogs also show a wide range of TPMT activity. Heparinized blood samples were obtained from 177 dogs associated with a veterinary teaching hospital. Red blood cell (RBC) TPMT activity was measured by means of a modification of a radiochemical method as established for use in people. TPMT activity varied across a 9-fold range (7.9-71.8 U of RBC per milliliter; median, 21.7). Variation in TPMT activity was not associated with age, sex, or neutering status. Giant Schnauzers had much lower TPMT activity (7.9-20 U of RBC per milliliter; median, 13.1; P dogs could affect thiopurine drug toxicity and efficacy in canine patients. PMID:15058773

  8. Human erythrocyte thiol methyltransferase: radiochemical microassay and biochemical properties

    International Nuclear Information System (INIS)

    A radiochemical microassay for the measurement of thiol methyltransferase (TMT) activity in human red blood cell (RBC) membranes has been developed. Both 2-mercaptoethanol and dithiothreitol were used as substrates for the enzyme. The pH optimum of the reaction was approximately 9.0 when glycine-NaOH was used as a buffer. The apparent Michaelis-Menten (Ksub(M)) value for the methyl donor for the reaction, S-adenosyl-L-methionine, was 43 μmol/l. Human RBC TMT activity was neither activated nor inhibited by Ca2+, Mg2+, or tropolone, but the enzyme was inhibited by SKF 525A and by reagents that react with sulfhydryl grcups. The mean TMT activity in blood from 289 randomly selected adult white subjects was 10.93 +- 3.22 units per mg protein (mean +- S.D.). The activity was the same in samples from men and women. The results of experiments in which TMT activity was measured in mixtures of RBC membranes with relatively ''low'' and relatively ''high'' activities provided no evidence that individual variations in the enzyme activity were due to variations in endogenous TMT activators or inhibitors. (Auth.)

  9. Identification and functional characterization of lysine methyltransferases of Entamoeba histolytica.

    Science.gov (United States)

    Borbolla-Vázquez, Jessica; Orozco, Esther; Medina-Gómez, Christian; Martínez-Higuera, Aarón; Javier-Reyna, Rosario; Chávez, Bibiana; Betanzos, Abigail; Rodríguez, Mario A

    2016-07-01

    Lysine methylation of histones, a posttranslational modification catalyzed by lysine methyltransferases (HKMTs), plays an important role in the epigenetic regulation of transcription. Lysine methylation of non-histone proteins also impacts the biological function of proteins. Previously it has been shown that lysine methylation of histones of Entamoeba histolytica, the protozoan parasite that infects 50 million people worldwide each year and causing up to 100,000 deaths annually, is implicated in the epigenetic machinery of this microorganism. However, the identification and characterization of HKMTs in this parasite had not yet been determined. In this work we identified four HKMTs in E. histolytica (EhHKMT1 to EhHKMT4) that are expressed by trophozoites. Enzymatic assays indicated that all of them are able to transfer methyl groups to commercial histones. EhHKMT1, EhHKMT2 and EhHKMT4 were detected in nucleus and cytoplasm of trophozoites. In addition EhHKMT2 and EhHKMT4 were located in vesicles containing ingested cells during phagocytosis, and they co-immunoprecipitated with EhADH, a protein involved in the phagocytosis of this parasite. Results suggest that E. histolytica uses its HKMTs to regulate transcription by epigenetic mechanisms, and at least two of them could also be implicated in methylation of proteins that participate in phagocytosis. PMID:27062489

  10. DNA methyltransferase inhibitor CDA-II inhibits myogenic differentiation

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Zirong [State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-Sen University, Guangzhou 510060 (China); Department of Molecular Genetics and Microbiology, Shands Cancer Center, University of Florida, Gainesville, FL 32610 (United States); Jin, Guorong [State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-Sen University, Guangzhou 510060 (China); Lin, Shuibin [Department of Molecular Genetics and Microbiology, Shands Cancer Center, University of Florida, Gainesville, FL 32610 (United States); Lin, Xiumei [Department of Hematology, Guangzhou First Municipal People' s Hospital, Guangzhou 510180 (China); Gu, Yumei [Department of Molecular Genetics and Microbiology, Shands Cancer Center, University of Florida, Gainesville, FL 32610 (United States); Zhu, Yujuan; Hu, Chengbin; Zhang, Qingjiong [State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-Sen University, Guangzhou 510060 (China); Wu, Lizi [Department of Molecular Genetics and Microbiology, Shands Cancer Center, University of Florida, Gainesville, FL 32610 (United States); Shen, Huangxuan, E-mail: shenhx@mail.sysu.edu.cn [State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-Sen University, Guangzhou 510060 (China)

    2012-06-08

    Highlights: Black-Right-Pointing-Pointer CDA-II inhibits myogenic differentiation in a dose-dependent manner. Black-Right-Pointing-Pointer CDA-II repressed expression of muscle transcription factors and structural proteins. Black-Right-Pointing-Pointer CDA-II inhibited proliferation and migration of C2C12 myoblasts. -- Abstract: CDA-II (cell differentiation agent II), isolated from healthy human urine, is a DNA methyltransferase inhibitor. Previous studies indicated that CDA-II played important roles in the regulation of cell growth and certain differentiation processes. However, it has not been determined whether CDA-II affects skeletal myogenesis. In this study, we investigated effects of CDA-II treatment on skeletal muscle progenitor cell differentiation, migration and proliferation. We found that CDA-II blocked differentiation of murine myoblasts C2C12 in a dose-dependent manner. CDA-II repressed expression of muscle transcription factors, such as Myogenin and Mef2c, and structural proteins, such as myosin heavy chain (Myh3), light chain (Mylpf) and MCK. Moreover, CDA-II inhibited C1C12 cell migration and proliferation. Thus, our data provide the first evidence that CDA-II inhibits growth and differentiation of muscle progenitor cells, suggesting that the use of CDA-II might affect skeletal muscle functions.

  11. Anhydrobiosis vs. aging: comparative genomics of protein repair L-isoaspartyl methyltransferases in the sleeping chironomid. .

    Science.gov (United States)

    Gusev, Oleg; Kikawada, Takahiro; Shagimardanova, Elena; Suetsugu, Yoshitaka; Ayupov, Rustam

    Origin of anhydrobiosis in the larvae of the sleeping chironomid Polypedilum vanderplanki represents unique example of set of evolutionary events in a single species, resulted in acquiring new ability allowing survival in extremely changeable environment. Complex comparative analysis of the genome of P. vanderplanki resulted in discovery of a set of features, including existence of the set of unique clusters of genes contributing in desiccation resistance. Surprisingly, in several cases, the genes mainly contributing to the formation of the molecular shield in the larvae are sleeping chironomid-specific and have no homology with genes from other insects, including P. nubifer - a chironomid from the same genus. Protein L-isoaspartyl methyltransferase (PIMT) acts on proteins that have been non-enzymatically damaged due to age, and partially restores aspartic residues, extending life of the polypeptides. PIMT a highly conserved enzyme present in nearly all eukaryotes, and microorganisms mostly in a single copy (or in a few isoforms in certain plants and some bacteria). While conducting a comparative analysis of the genomes of two chironomid midge species different in their ability to stand complete water loss, we have noticed that structure and number of PIMT-coding genes in the desiccation resistant (anhydrobiotic) midge (Polypedilum vanderplanki, Pv) is different from those of the common desiccation-sensitive midge (Polypedilum nubifer, Pn) and the rest of insects. Both species have a clear orthologous PIMT shared by all insects. At the same time, in contrast to Pn which has only one PIMT gene (PnPimt-1), the Pv genome contains 12 additional genes paralogous to Pimt1 (PvPimt-2-12) presumably coding functional PIMT proteins, which are arranged in a single cluster. Remarkably, PvPimt-1 location in the Pv is different from the rest of Pimt-like genes. PvPimt-1 gene is ubiquitously expressed during the life cycle, but expression of the PvPimt2-12 is limited to the eggs

  12. A Picrinine N-Methyltransferase Belongs to a New Family of γ-Tocopherol-Like Methyltransferases Found in Medicinal Plants That Make Biologically Active Monoterpenoid Indole Alkaloids.

    Science.gov (United States)

    Levac, Dylan; Cázares, Paulo; Yu, Fang; De Luca, Vincenzo

    2016-04-01

    Members of the Apocynaceae plant family produce a large number of monoterpenoid indole alkaloids (MIAs) with different substitution patterns that are responsible for their various biological activities. A novel N-methyltransferase involved in the vindoline pathway in Catharanthus roseus showing distinct similarity to γ-tocopherol C-methyltransferases was used in a bioinformatic screen of transcriptomes from Vinca minor, Rauvolfia serpentina, and C. roseus to identify 10 γ-tocopherol-like N-methyltransferases from a large annotated transcriptome database of different MIA-producing plant species (www.phytometasyn.ca). The biochemical function of two members of this group cloned from V. minor (VmPiNMT) and R. serpentina (RsPiNMT) have been characterized by screening their biochemical activities against potential MIA substrates harvested from the leaf surfaces of MIA-accumulating plants. The approach was validated by identifying the MIA picrinine from leaf surfaces of Amsonia hubrichtii as a substrate of VmPiNMT and RsPiNMT. Recombinant proteins were shown to have high substrate specificity and affinity for picrinine, converting it to N-methylpicrinine (ervincine). Developmental studies with V. minor and R. serpentina showed that RsPiNMT and VmPiNMT gene expression and biochemical activities were highest in younger leaf tissues. The assembly of at least 150 known N-methylated MIAs within members of the Apocynaceae family may have occurred as a result of the evolution of the γ-tocopherol-like N-methyltransferase family from γ-tocopherol methyltransferases. PMID:26848097

  13. Efficacy of Ampicillin plus Arbekacin in Experimental Rabbit Endocarditis Caused by an Enterococcus faecalis Strain with High-Level Gentamicin Resistance

    OpenAIRE

    Kak, Vivek; Donabedian, Susan M.; Zervos, Marcus J.; Kariyama, Reiko; Kumon,Hiromi; Chow, Joseph W.

    2000-01-01

    Enterococcus faecalis LC40 is an ampicillin-susceptible clinical isolate with high-level gentamicin resistance due to the aac(6′)-Ie-aph(2")-Ia aminoglycoside resistance gene. The combination of ampicillin plus arbekacin reduced mean bacterial vegetation counts significantly more than ampicillin alone or ampicillin plus gentamicin in a rabbit model of aortic-valve endocarditis caused by E. faecalis LC40.

  14. Isolation, characterization, and cloning of a plasmid-borne gene encoding a phosphotransferase that confers high-level amikacin resistance in enteric bacilli.

    OpenAIRE

    Gaynes, R.; Groisman, E; Nelson, E.; Casadaban, M; Lerner, S A

    1988-01-01

    Clinical isolates of Klebsiella pneumoniae and Serratia marcescens at a hospital that had used amikacin as its principal aminoglycoside for the preceding 42 months demonstrated high-level resistance to amikacin (greater than or equal to 256 micrograms/ml), kanamycin (greater than or equal to 256 micrograms/ml), gentamicin (greater than or equal to 64 micrograms/ml), netilmicin (64 micrograms/ml), and tobramycin (greater than or equal to 16 micrograms/ml). The resistant strains contained an id...

  15. Specificity of the ModA11, ModA12 and ModD1 epigenetic regulator N6-adenine DNA methyltransferases of Neisseria meningitidis

    Science.gov (United States)

    Seib, Kate L.; Jen, Freda E.-C.; Tan, Aimee; Scott, Adeana L.; Kumar, Ritesh; Power, Peter M.; Chen, Li-Tzu; Wu, Hsing-Ju; Wang, Andrew H.-J.; Hill, Dorothea M. C.; Luyten, Yvette A.; Morgan, Richard D.; Roberts, Richard J.; Maiden, Martin C. J.; Boitano, Matthew; Clark, Tyson A.; Korlach, Jonas; Rao, Desirazu N.; Jennings, Michael P.

    2015-01-01

    Phase variation (random ON/OFF switching) of gene expression is a common feature of host-adapted pathogenic bacteria. Phase variably expressed N6-adenine DNA methyltransferases (Mod) alter global methylation patterns resulting in changes in gene expression. These systems constitute phase variable regulons called phasevarions. Neisseria meningitidis phasevarions regulate genes including virulence factors and vaccine candidates, and alter phenotypes including antibiotic resistance. The target site recognized by these Type III N6-adenine DNA methyltransferases is not known. Single molecule, real-time (SMRT) methylome analysis was used to identify the recognition site for three key N. meningitidis methyltransferases: ModA11 (exemplified by M.NmeMC58I) (5′-CGYm6AG-3′), ModA12 (exemplified by M.Nme77I, M.Nme18I and M.Nme579II) (5′-ACm6ACC-3′) and ModD1 (exemplified by M.Nme579I) (5′-CCm6AGC-3′). Restriction inhibition assays and mutagenesis confirmed the SMRT methylome analysis. The ModA11 site is complex and atypical and is dependent on the type of pyrimidine at the central position, in combination with the bases flanking the core recognition sequence 5′-CGYm6AG-3′. The observed efficiency of methylation in the modA11 strain (MC58) genome ranged from 4.6% at 5′-GCGCm6AGG-3′ sites, to 100% at 5′-ACGTm6AGG-3′ sites. Analysis of the distribution of modified sites in the respective genomes shows many cases of association with intergenic regions of genes with altered expression due to phasevarion switching. PMID:25845594

  16. Emerging broad-spectrum resistance in Pseudomonas aeruginosa and Acinetobacter baumannii: Mechanisms and epidemiology.

    Science.gov (United States)

    Potron, Anaïs; Poirel, Laurent; Nordmann, Patrice

    2015-06-01

    Multidrug resistance is quite common among non-fermenting Gram-negative rods, in particular among clinically relevant species including Pseudomonas aeruginosa and Acinetobacter baumannii. These bacterial species, which are mainly nosocomial pathogens, possess a diversity of resistance mechanisms that may lead to multidrug or even pandrug resistance. Extended-spectrum β-lactamases (ESBLs) conferring resistance to broad-spectrum cephalosporins, carbapenemases conferring resistance to carbapenems, and 16S rRNA methylases conferring resistance to all clinically relevant aminoglycosides are the most important causes of concern. Concomitant resistance to fluoroquinolones, polymyxins (colistin) and tigecycline may lead to pandrug resistance. The most important mechanisms of resistance in P. aeruginosa and A. baumannii and their most recent dissemination worldwide are detailed here. PMID:25857949

  17. An enzyme-coupled continuous spectrophotometric assay for S-adenosylmethionine-dependent methyltransferases.

    Science.gov (United States)

    Dorgan, Kathleen M; Wooderchak, Whitney L; Wynn, Donraphael P; Karschner, Erin L; Alfaro, Joshua F; Cui, Yinqiu; Zhou, Zhaohui Sunny; Hevel, Joan M

    2006-03-15

    Modification of small molecules and proteins by methyltransferases affects a wide range of biological processes. Here, we report an enzyme-coupled continuous spectrophotometric assay to quantitatively characterize S-adenosyl-L-methionine (AdoMet/SAM)-dependent methyltransferase activity. In this assay, S-adenosyl-L-homocysteine (AdoHcy/SAH), the transmethylation product of AdoMet-dependent methyltransferases, is hydrolyzed to S-ribosylhomocysteine and adenine by recombinant S-adenosylhomocysteine/5'-methylthioadenosine nucleosidase (SAHN/MTAN, EC 3.2.2.9). Subsequently, adenine generated from AdoHcy is further hydrolyzed to hypoxanthine and ammonia by recombinant adenine deaminase (EC 3.5.4.2). This deamination is associated with a decrease in absorbance at 265 nm that can be monitored continuously. Coupling enzymes are recombinant and easily purified. The utility of this assay was shown using recombinant rat protein arginine N-methyltransferase 1 (PRMT1, EC 2.1.1.125), which catalyzes the mono- and dimethylation of guanidino nitrogens of arginine residues in select proteins. Using this assay, the kinetic parameters of PRMT1 with three synthetic peptides were determined. An advantage of this assay is the destruction of AdoHcy by AdoHcy nucleosidase, which alleviates AdoHcy product feedback inhibition of S-adenosylmethionine-dependent methyltransferases. Finally, this method may be used to assay other enzymes that produce AdoHcy, 5'-methylthioadenosine, or compounds that can be cleaved by AdoHcy nucleosidase. PMID:16460659

  18. Conservation and Functional Importance of Carbon-Oxygen Hydrogen Bonding in AdoMet-Dependent Methyltransferases

    Energy Technology Data Exchange (ETDEWEB)

    Horowitz, Scott; Dirk, Lynnette M.A.; Yesselman, Joseph D.; Nimtz, Jennifer S.; Adhikari, Upendra; Mehl, Ryan A.; Scheiner, Steve; Houtz, Robert L.; Al-Hashimi, Hashim M.; Trievel, Raymond C. [Oregon State U.; (Michigan); (Utah SU); (HHMI); (Kentucky)

    2013-09-06

    S-Adenosylmethionine (AdoMet)-based methylation is integral to metabolism and signaling. AdoMet-dependent methyltransferases belong to multiple distinct classes and share a catalytic mechanism that arose through convergent evolution; however, fundamental determinants underlying this shared methyl transfer mechanism remain undefined. A survey of high-resolution crystal structures reveals that unconventional carbon–oxygen (CH···O) hydrogen bonds coordinate the AdoMet methyl group in different methyltransferases irrespective of their class, active site structure, or cofactor binding conformation. Corroborating these observations, quantum chemistry calculations demonstrate that these charged interactions formed by the AdoMet sulfonium cation are stronger than typical CH···O hydrogen bonds. Biochemical and structural studies using a model lysine methyltransferase and an active site mutant that abolishes CH···O hydrogen bonding to AdoMet illustrate that these interactions are important for high-affinity AdoMet binding and transition-state stabilization. Further, crystallographic and NMR dynamics experiments of the wild-type enzyme demonstrate that the CH···O hydrogen bonds constrain the motion of the AdoMet methyl group, potentially facilitating its alignment during catalysis. Collectively, the experimental findings with the model methyltransferase and structural survey imply that methyl CH···O hydrogen bonding represents a convergent evolutionary feature of AdoMet-dependent methyltransferases, mediating a universal mechanism for methyl transfer.

  19. Mycolic Acid Cyclopropanation is Essential for Viability, Drug Resistance, and Cell Wall Integrity of Mycobacterium tuberculosis

    Energy Technology Data Exchange (ETDEWEB)

    Barkan, Daniel; Liu, Zhen; Sacchettini, James C.; Glickman, Michael S.; (MSKCC); (TAM)

    2009-12-01

    Mycobacterium tuberculosis infection remains a major global health problem complicated by escalating rates of antibiotic resistance. Despite the established role of mycolic acid cyclopropane modification in pathogenesis, the feasibility of targeting this enzyme family for antibiotic development is unknown. We show through genetics and chemical biology that mycolic acid methyltransferases are essential for M. tuberculosis viability, cell wall structure, and intrinsic resistance to antibiotics. The tool compound dioctylamine, which we show acts as a substrate mimic, directly inhibits the function of multiple mycolic acid methyltransferases, resulting in loss of cyclopropanation, cell death, loss of acid fastness, and synergistic killing with isoniazid and ciprofloxacin. These results demonstrate that mycolic acid methyltransferases are a promising antibiotic target and that a family of virulence factors can be chemically inhibited with effects not anticipated from studies of each individual enzyme.

  20. Magnetic isotope effect of magnesium (25)Mg on E. coli resistance to antibiotics.

    Science.gov (United States)

    Letuta, U G; Vekker, A S; Kornilova, T A; Gryaznov, A A; Cheplakov, I A

    2016-07-01

    Effects of synergism and antagonism of antibacterial drugs and magnetic isotope of magnesium (25)Mg on antibiotic resistance of bacteria E. coli were discovered. Fourteen antibiotics from seven different groups were tested. The increase in antibiotic resistance in the presence of the ion (25)Mg(2+) was discovered in E. coli cells incubated with quinolones/fluoroquinolones, indicating the inhibiting effect of the magnetic moments of nuclei (25)Mg on DNA synthesis. The change in antibiotic resistance was also detected in bacteria affected by magnesium (25)Mg and certain antibiotics from aminoglycoside and lincosamide groups. PMID:27599512

  1. Lack of phosphatidylethanolamine N-methyltransferase in mice does not promote fatty acid oxidation in skeletal muscle.

    Science.gov (United States)

    Tasseva, Guergana; van der Veen, Jelske N; Lingrell, Susanne; Jacobs, René L; Vance, Dennis E; Vance, Jean E

    2016-02-01

    Phosphatidylethanolamine N-methyltransferase (PEMT) converts phosphatidylethanolamine (PE) to phosphatidylcholine (PC) in the liver. Mice lacking PEMT are protected from high-fat diet-induced obesity and insulin resistance, and exhibit increased whole-body energy expenditure and oxygen consumption. Since skeletal muscle is a major site of fatty acid oxidation and energy utilization, we determined if rates of fatty acid oxidation/oxygen consumption in muscle are higher in Pemt(-/-) mice than in Pemt(+/+) mice. Although PEMT is abundant in the liver, PEMT protein and activity were undetectable in four types of skeletal muscle. Moreover, amounts of PC and PE in the skeletal muscle were not altered by PEMT deficiency. Thus, we concluded that any influence of PEMT deficiency on skeletal muscle would be an indirect consequence of lack of PEMT in liver. Neither the in vivo rate of fatty acid uptake by muscle nor the rate of fatty acid oxidation in muscle explants and cultured myocytes depended upon Pemt genotype. Nor did PEMT deficiency increase oxygen consumption or respiratory function in skeletal muscle mitochondria. Thus, the increased whole body oxygen consumption in Pemt(-/-) mice, and resistance of these mice to diet-induced weight gain, are not primarily due to increased capacity of skeletal muscle for utilization of fatty acids as an energy source. PMID:26603903

  2. Histone Methyltransferase Inhibitors Are Orally Bioavailable, Fast-Acting Molecules with Activity against Different Species Causing Malaria in Humans

    Science.gov (United States)

    Sundriyal, Sandeep; Caron, Joachim; Chen, Patty; Witkowski, Benoit; Menard, Didier; Suwanarusk, Rossarin; Renia, Laurent; Nosten, Francois; Jiménez-Díaz, María Belén; Angulo-Barturen, Iñigo; Martínez, María Santos; Ferrer, Santiago; Sanz, Laura M.; Gamo, Francisco-Javier; Wittlin, Sergio; Duffy, Sandra; Avery, Vicky M.; Ruecker, Andrea; Delves, Michael J.; Sinden, Robert E.; Fuchter, Matthew J.

    2014-01-01

    Current antimalarials are under continuous threat due to the relentless development of drug resistance by malaria parasites. We previously reported promising in vitro parasite-killing activity with the histone methyltransferase inhibitor BIX-01294 and its analogue TM2-115. Here, we further characterize these diaminoquinazolines for in vitro and in vivo efficacy and pharmacokinetic properties to prioritize and direct compound development. BIX-01294 and TM2-115 displayed potent in vitro activity, with 50% inhibitory concentrations (IC50s) of <50 nM against drug-sensitive laboratory strains and multidrug-resistant field isolates, including artemisinin-refractory Plasmodium falciparum isolates. Activities against ex vivo clinical isolates of both P. falciparum and Plasmodium vivax were similar, with potencies of 300 to 400 nM. Sexual-stage gametocyte inhibition occurs at micromolar levels; however, mature gametocyte progression to gamete formation is inhibited at submicromolar concentrations. Parasite reduction ratio analysis confirms a high asexual-stage rate of killing. Both compounds examined displayed oral efficacy in in vivo mouse models of Plasmodium berghei and P. falciparum infection. The discovery of a rapid and broadly acting antimalarial compound class targeting blood stage infection, including transmission stage parasites, and effective against multiple malaria-causing species reveals the diaminoquinazoline scaffold to be a very promising lead for development into greatly needed novel therapies to control malaria. PMID:25421480

  3. Study of matrix effects for liquid chromatography-electrospray ionization tandem mass spectrometric analysis of 4 aminoglycosides residues in milk.

    Science.gov (United States)

    Wang, Yuan; Li, Shaohui; Zhang, Feifang; Lu, Yifeng; Yang, Bingcheng; Zhang, Feng; Liang, Xinmiao

    2016-03-11

    Matrix effect (ME) is always a major issue for the development of LC-MS/MS method. ME resulting from co-eluting residual matrix components can affect the ionization efficiency of target analytes, leading to quantification errors of the analytes of interest. The present work evaluates MEs of milk samples on simultaneous analysis of four aminoglycosides residues via LC-ESI/MS/MS including streptomycin, dihydrostreptomycin, spectinomycin and kanamycin. Approaches to reduce MEs were examined: optimization of the sample preparation, sample dilution and lower flow rate used. Three commercial sorbents were tested including Oasis MCX, Oasis HLB and Oasis WCX. WCX behaved better for all analytes, but high MEs (80.8-134.9%) were obtained. Therefore, a consecutive SPE of tC18-WCX was found to effectively reduce ME. Milk samples from different manufacturers were analyzed and low MEs (85.6-112.9%) were obtained. PMID:26875117

  4. Effect of mutations in the A site of 16 S rRNA on aminoglycoside antibiotic-ribosome interaction

    DEFF Research Database (Denmark)

    Recht, M I; Douthwaite, S; Dahlquist, K D;

    1999-01-01

    Decoding of genetic information occurs upon interaction of an mRNA codon-tRNA anticodon complex with the small subunit of the ribosome. The ribosomal decoding region is associated with highly conserved sequences near the 3' end of 16 S rRNA. The decoding process is perturbed by the aminoglycoside...... of universally conserved nucleotides at 1406 to 1408 and 1494 to 1495 in the decoding region of plasmid-encoded bacterial 16 S rRNA. Phenotypic changes range from the benign effect of U1406-->A or A1408-->G substitutions, to the highly deleterious 1406G and 1495 mutations that assemble into 30 S subunits...... but are defective in forming functional ribosomes. Changes in the local conformation of the decoding region caused by these mutations were identified by chemical probing of isolated 30 S subunits. Ribosomes containing 16 S rRNA with mutations at positions 1408, 1407+1494, or 1495 had reduced affinity...

  5. Methyltransferases mediate cell memory of a genotoxic insult.

    Science.gov (United States)

    Rugo, R E; Mutamba, J T; Mohan, K N; Yee, T; Chaillet, J R; Greenberger, J S; Engelward, B P

    2011-02-10

    Characterization of the direct effects of DNA-damaging agents shows how DNA lesions lead to specific mutations. Yet, serum from Hiroshima survivors, Chernobyl liquidators and radiotherapy patients can induce a clastogenic effect on naive cells, showing indirect induction of genomic instability that persists years after exposure. Such indirect effects are not restricted to ionizing radiation, as chemical genotoxins also induce heritable and transmissible genomic instability phenotypes. Although such indirect induction of genomic instability is well described, the underlying mechanism has remained enigmatic. Here, we show that mouse embryonic stem cells exposed to γ-radiation bear the effects of the insult for weeks. Specifically, conditioned media from the progeny of exposed cells can induce DNA damage and homologous recombination in naive cells. Notably, cells exposed to conditioned media also elicit a genome-destabilizing effect on their neighbouring cells, thus demonstrating transmission of genomic instability. Moreover, we show that the underlying basis for the memory of an insult is completely dependent on two of the major DNA cytosine methyltransferases, Dnmt1 and Dnmt3a. Targeted disruption of these genes in exposed cells completely eliminates transmission of genomic instability. Furthermore, transient inactivation of Dnmt1, using a tet-suppressible allele, clears the memory of the insult, thus protecting neighbouring cells from indirect induction of genomic instability. We have thus demonstrated that a single exposure can lead to long-term, genome-destabilizing effects that spread from cell to cell, and we provide a specific molecular mechanism for these persistent bystander effects. Collectively, our results impact the current understanding of risks from toxin exposures and suggest modes of intervention for suppressing genomic instability in people exposed to carcinogenic genotoxins. PMID:21057543

  6. Catechol-O-methyltransferase, dopamine, and sleep-wake regulation.

    Science.gov (United States)

    Dauvilliers, Yves; Tafti, Mehdi; Landolt, Hans Peter

    2015-08-01

    Sleep and sleep disorders are complex and highly variable phenotypes regulated by many genes and environment. The catechol-O-methyltransferase (COMT) gene is an interesting candidate, being one of the major mammalian enzymes involved in the catabolism of catecholamines. The activity of COMT enzyme is genetically polymorphic due to a guanine-to-adenine transition at codon 158, resulting in a valine (Val) to methionine (Met) substitution. Individuals homozygous for the Val allele show higher COMT activity, and lower dopaminergic signaling in prefrontal cortex (PFC) than subjects homozygous for the Met allele. Since COMT has a crucial role in metabolising dopamine, it was suggested that the common functional polymorphism in the COMT gene impacts on cognitive function related to PFC, sleep-wake regulation, and potentially on sleep pathologies. The COMT Val158Met polymorphism may predict inter-individual differences in brain electroencephalography (EEG) alpha oscillations and recovery processes resulting from partial sleep loss in healthy individuals. The Val158Met polymorphism also exerts a sexual dimorphism and has a strong effect on objective daytime sleepiness in patients with narcolepsy-cataplexy. Since the COMT enzyme inactivates catecholamines, it was hypothesized that the response to stimulant drugs differs between COMT genotypes. Modafinil maintained executive functioning performance and vigilant attention throughout sleep deprivation in subjects with Val/Val genotype, but less in those with Met/Met genotype. Also, homozygous Met/Met patients with narcolepsy responded to lower doses of modafinil compared to Val/Val carriers. We review here the critical role of the common functional COMT gene polymorphism, COMT enzyme activity, and the prefrontal dopamine levels in the regulation of sleep and wakefulness in normal subjects, in narcolepsy and other sleep-related disorders, and its impact on the response to psychostimulants. PMID:25466290

  7. Demonstration of thiopurine methyltransferase activity in the erythrocytes of cats.

    Science.gov (United States)

    Foster, A P; Shaw, S E; Duley, J A; Shobowale-Bakre, E M; Harbour, D A

    2000-01-01

    Azathioprine is a purine analogue used as an immunosuppressive and immunomodulator agent in various mammals, including cats. Several adverse reactions have been reported and have limited the use of the drug in the cat. Adverse reactions to azathioprine in humans have been correlated with reduced activity of thiopurine methyltransferase (TPMT) in erythrocytes. The purpose of this preliminary study was to determine if cats have TPMT activity in their erythrocytes and to compare the values obtained with the normal range for humans and the normal range for dogs in a preliminary report. Activity of the enzyme was measured in blood samples drawn from 41 cats. Blood also was taken from 5 dogs. The mean erythrocyte TPMT activity in the cats was 2.4 +/- 0.4 nmol (range, 1.2-3.9 nmol) per hour per milliliter of red blood cells (U/mL RBC) or 2-8 nmol per hour per gram of hemoglobin (U/g Hb). This range was far lower than the normal human range (8-15 U/mL RBC; 16-33 U/g Hb) and was of monopolar distribution. This observation apparently precludes any diagnostic purpose in assaying erythrocyte TPMT in this species. Erythrocyte TPMT activity in the 5 dogs ranged from 5.5 to 13.1 U/mL RBC (11-27 U/g Hb), which was comparable with normal and carrier ranges for humans, but proof of TPMT genetic polymorphism in either species will require genotyping studies. PMID:11012121

  8. Adeno-associated virus-mediated Bcl-xL prevents aminoglycoside-induced hearing loss in mice

    Institute of Scientific and Technical Information of China (English)

    LIU Yu-he; KE Xiao-mei; QIN Yong; GU Zhi-ping; XIAO Shui-fang

    2007-01-01

    Background Recent studies showed that aminoglycosides destroyed the cochlear cells and induced ototoxicity by producing reactive oxygen species, including free radicals in the mitochondria, damaging the membrane of mitochondria and resulting in apoptotic cell death. Bcl-xL is a well characterized anti-apoptotic member of the Bcl-2 family. The aim of this study was to determine the potential cochlear protective effect of Bcl-xL as a therapeutic agent in the murine model of aminoglycoside ototoxicity.Methods Serotype 2 of adeno-associated virus (AAV2) as a vector encoding the mouse Bcl-xL gene was injected into mice cochleae prior to injection of kanamycin. Bcl-xL expression in vitro and in vivo was examined with Western blotting and immunohistochemistry separately. Cochlear dissection and auditory steady state responses were checked to evaluate the cochlear structure and function.Results The animals in the AAV2-Bcl-xL/kanamycin group displayed better auditory steady state responses hearing thresholds and cochlear structure than those in the artificial perilymph/kanamycin or AAV2-enhanced humanized green fluorescent protein/kanamycin control group at all tested frequencies. The auditory steady state responses hearing thresholds and cochlear structure in the inoculated side were better than that in the contralateral side.Conclusions AAV2-Bcl-xL afforded significant preservation of the cochlear hair cells against ototoxic insults and protected the cochlear function. AAV2-mediated Bcl-xL might be an approach with respect to potential therapeutic application in the cochlear degeneration.

  9. Two distinct arginine methyltransferases are required for biogenesis of Sm-class ribonucleoproteins

    Science.gov (United States)

    Gonsalvez, Graydon B.; Tian, Liping; Ospina, Jason K.; Boisvert, François-Michel; Lamond, Angus I.; Matera, A. Gregory

    2007-01-01

    Small nuclear ribonucleoproteins (snRNPs) are core components of the spliceosome. The U1, U2, U4, and U5 snRNPs each contain a common set of seven Sm proteins. Three of these Sm proteins are posttranslationally modified to contain symmetric dimethylarginine (sDMA) residues within their C-terminal tails. However, the precise function of this modification in the snRNP biogenesis pathway is unclear. Several lines of evidence suggest that the methyltransferase protein arginine methyltransferase 5 (PRMT5) is responsible for sDMA modification of Sm proteins. We found that in human cells, PRMT5 and a newly discovered type II methyltransferase, PRMT7, are each required for Sm protein sDMA modification. Furthermore, we show that the two enzymes function nonredundantly in Sm protein methylation. Lastly, we provide in vivo evidence demonstrating that Sm protein sDMA modification is required for snRNP biogenesis in human cells. PMID:17709427

  10. Studies of resistance factors against chloroethylnitrosourea drugs in malignant tumor cells

    OpenAIRE

    Egyházi, Suzanne

    1996-01-01

    Drug resistance is a major clinical problem in the chemotherapy of tumor diseases and the identification of factors that make tumor cells resistant to drug treatment is therefore of crucial importance. We have investigated a possible involvement of O6-methylguanine-DNA methyltransferase (MGMT), glutathione transferase (GST) and glutathione (GSH) in the resistance to 1,3- bis(2-chloroethyl)- 1 -nitrosourea (BCNU) in two human lung cancer cell lines. The non-small cell lung ca...

  11. The RNA-methyltransferase Misu (NSun2 poises epidermal stem cells to differentiate.

    Directory of Open Access Journals (Sweden)

    Sandra Blanco

    2011-12-01

    Full Text Available Homeostasis of most adult tissues is maintained by balancing stem cell self-renewal and differentiation, but whether post-transcriptional mechanisms can regulate this process is unknown. Here, we identify that an RNA methyltransferase (Misu/Nsun2 is required to balance stem cell self-renewal and differentiation in skin. In the epidermis, this methyltransferase is found in a defined sub-population of hair follicle stem cells poised to undergo lineage commitment, and its depletion results in enhanced quiescence and aberrant stem cell differentiation. Our results reveal that post-transcriptional RNA methylation can play a previously unappreciated role in controlling stem cell fate.

  12. H3K9 methyltransferase G9a and the related molecule GLP

    OpenAIRE

    Shinkai, Yoichi; Tachibana, Makoto

    2011-01-01

    The discovery of Suv39h1, the first SET domain-containing histone lysine methyltransferase (HKMT), was reported in 2000. Since then, research on histone methylation has progressed rapidly. Among the identified HKMTs in mammals, G9a and GLP are the primary enzymes for mono- and dimethylation at Lys 9 of histone H3 (H3K9me1 and H3K9me2), and exist predominantly as a G9a–GLP heteromeric complex that appears to be a functional H3K9 methyltransferase in vivo. Recently, many important studies have ...

  13. Novel Function of Lysine Methyltransferase G9a in the Regulation of Sox2 Protein Stability

    OpenAIRE

    Lee, Jae-Young; Lee, Se-Hwan; Heo, Sun-Hee; Kim, Kwang-Soo; Kim, Changhoon; Kim, Dae-Kwan; Ko, Jeong-Jae; Park, Kyung-Soon

    2015-01-01

    G9a is a lysine methyltransferase (KMTase) for histone H3 lysine 9 that plays critical roles in a number of biological processes. Emerging evidence suggests that aberrant expression of G9a contributes to tumor metastasis and maintenance of a malignant phenotype in cancer by inducing epigenetic silencing of tumor suppressor genes. Here, we show that G9a regulates Sox2 protein stability in breast cancer cells. When G9a lysine methyltransferase activity was chemically inhibited in the ER(+) brea...

  14. Negative in vitro selection identifies the rRNA recognition motif for ErmE methyltransferase

    DEFF Research Database (Denmark)

    Nielsen, A K; Douthwaite, S; Vester, B

    1999-01-01

    the adjacent single-stranded region around A2058. An RNA transcript of 72 nt that displays this motif functions as an efficient substrate for the ErmE methyltransferase. Pools of degenerate RNAs were formed by doping 34-nt positions that extend over and beyond the putative Erm recognition motif within...... contained substitutions at single sites, and these are confined to 12 nucleotide positions. These nucleotides, corresponding to A2051-A2060, C2611, and A2614 in 23S rRNA, presumably comprise the RNA recognition motif for ErmE methyltransferase. The structure formed by these nucleotides is highly conserved...

  15. The arginine methyltransferase Rmt2 is enriched in the nucleus and co-purifies with the nuclear porins Nup49, Nup57 and Nup100

    International Nuclear Information System (INIS)

    Arginine methylation is a post-translational modification of proteins implicated in RNA processing, protein compartmentalization, signal transduction, transcriptional regulation and DNA repair. In a screen for proteins associated with the nuclear envelope in the yeast Saccharomyces cerevisiae, we have identified the arginine methyltransferase Rmt2, previously shown to methylate the ribosomal protein L12. By indirect immunofluorescence and subcellular fractionations we demonstrate here that Rmt2 has nuclear and cytoplasmic localizations. Biochemical analysis of a fraction enriched in nuclei reveals that nuclear Rmt2 is resistant to extractions with salt and detergent, indicating an association with structural components. This was supported by affinity purification experiments with TAP-tagged Rmt2. Rmt2 was found to co-purify with the nucleoporins Nup49, Nup57 and Nup100, revealing a novel link between arginine methyltransferases and the nuclear pore complex. In addition, a genome-wide transcription study of the rmt2Δ mutant shows significant downregulation of the transcription of MYO1, encoding the Type II myosin heavy chain required for cytokinesis and cell separation

  16. Radiometric assay for phenylethanolamine N-methyltransferase and catechol O-methyltransferase in a single tissue sample: application to rat hypothalamic nuclei, pineal gland, and heart

    International Nuclear Information System (INIS)

    A simple and highly sensitive method for simultaneous assay of phenylethanolamine N-methyltransferase (PNMT) and catechol O-methyltransferase (COMT) is described. These enzymes are determined in a single tissue homogenate using S-[methyl-3H] adenosyl-L-methionine as methyl donor and sequentially incubating with the substrates phenylethanolamine and epinephrine. The radioactive products of the enzymatic reactions, N-methylphenylethanolamine and metanephrine, are extracted and then separated by thin-layer chromatography. The identity of the reaction products has been established chromatographically and the conditions for both enzymatic reactions in the assay procedure have been defined. Measurement of PNMT activity in the rat pineal gland or in minute fragments of other tissues (e.g., brain nuclei) has not been possible using previously described methods. Activities of PNMT and COMT in the rat pineal gland, various hypothalamic nuclei, and the auricular and ventricular myocardia are herein reported

  17. Contribution of Efflux Pumps, Porins, and B-Lactamases to Multidrug Resistance in Clinical Isolates of Acinetobacter baumannii

    OpenAIRE

    Rumbo, C.; Gato, E.; López, M.; Ruiz de Alegría, C.; Fernández-Cuenca, F.; Martínez-Martínez, L.; Vila, J.; Pachón, J.; J. M. Cisneros; Rodríguez-Baño, Jesús; A. Pascual; Bou,G.; Tomás, M.

    2013-01-01

    Weinvestigated the mechanisms of resistance to carbapenems, aminoglycosides, glycylcyclines, tetracyclines, and quinolones in 90 multiresistant clinical strains of Acinetobacter baumannii isolated from two genetically unrelated A. baumannii clones: clone PFGEROC- 1 (53 strains producing the OXA-58B-lactamase enzyme and 18 strains with the OXA-24B-lactamase) and clone PFGE-HUI-1 (19 strains susceptible to carbapenems).Weused real-time reverse transcriptase PCR to correlate antimicrobi...

  18. Antimicrobial sensitivity and frequency of DRUG resistance among bacterial strains isolated from cancer patients

    International Nuclear Information System (INIS)

    Blood stream infections (bacteremia) is potentially life threatening. Concomitant with a change in the incidence and epidemiology of infecting organisms, there has been an increase in resistance to many antibiotic compounds. The widespread emergence of resistance among bacterial pathogens has an impact on our ability to treat patients effectively. The changing spectrum of microbial pathogens and widespread emergence of microbial resistance to antibiotic drugs has emphasized the need to monitor the prevalence of resistance in these strains. In the present study frequency of isolation of clinically significant bacteria and their susceptibility and resistance pattern against a wide range of antimicrobial drugs from positive blood cultures collected during 2001-2003 was studied. A total of 102 consecutive isolates were found with 63% gram positive and 44% gram negative strains. The dominating pathogens were Staphylococcus aureus (51%), Streptococci (31%), Pseudomonas (40%), Proteus (13%), Klebsiella (13%). The isolated strains were tested against a wide range of antibiotics belonging to cephalosporins, aminoglycosides and quinolone derivative group by disk diffusion method. It has been observed that isolated strains among gram positive and negative strains showed different level of resistance against aminoglycosides and cephalosporin group of antibiotics with gram positives showing highest number and frequency of resistance against aminoglycosides (40-50%) and cephalosporins.(35-45%) whereas cephalosporins were found to be more effective against gram negatives with low frequency of resistant strains. Cabapenem and quinolone derivative drugs were found to be most effective among other groups in both gram positive and negative strains with 23-41% strains found sensitive to these two drugs. The frequency of sensitive strains against aminoglycoside and cephalosporin in gram negative and gram positive strains were found to be decreasing yearwise with a trend towards an

  19. Antimicrobial resistance of Escherichia coli isolates from canine urinary tract infections.

    Science.gov (United States)

    Chang, Shao-Kuang; Lo, Dan-Yuan; Wei, Hen-Wei; Kuo, Hung-Chih

    2015-01-01

    This study determined the antimicrobial resistance profiles of Escherichia coli isolates from dogs with a presumptive diagnosis of urinary tract infection (UTI). Urine samples from 201 dogs with UTI diagnosed through clinical examination and urinalysis were processed for isolation of Escherichia coli. Colonies from pure cultures were identified by biochemical reactions (n=114) and were tested for susceptibility to 18 antimicrobials. The two most frequent antimicrobials showing resistance in Urinary E. coli isolates were oxytetracycline and ampicillin. Among the resistant isolates, 17 resistance patterns were observed, with 12 patterns involving multidrug resistance (MDR). Of the 69 tetracycline-resistant E. coli isolates, tet(B) was the predominant resistance determinant and was detected in 50.9% of the isolates, whereas the remaining 25.5% isolates carried the tet(A) determinant. Most ampicillin and/or amoxicillin-resistant E. coli isolates carried blaTEM-1 genes. Class 1 integrons were prevalent (28.9%) and contained previously described gene cassettes that are implicated primarily in resistance to aminoglycosides and trimethoprim (dfrA1, dfrA17-aadA5). Of the 44 quinolone-resistant E. coli isolates, 38 were resistant to nalidixic acid, and 6 were resistant to nalidixic acid, ciprofloxacin and enrofloxacin. Chromosomal point mutations were found in the GyrA (Ser83Leu) and ParC (Ser80Ile) genes. Furthermore, the aminoglycoside resistance gene aacC2, the chloramphenicol resistant gene cmlA and the florfenicol resistant gene floR were also identified. This study revealed an alarming rate of antimicrobial resistance among E. coli isolates from dogs with UTIs. PMID:25720807

  20. Betaine-homocysteine methyltransferase: human liver genotype-phenotype correlation

    Science.gov (United States)

    Feng, Qiping; Kalari, Krishna; Fridley, Brooke L.; Jenkins, Gregory; Ji, Yuan; Abo, Ryan; Hebbring, Scott; Zhang, Jianping; Nye, Monica D.; Leeder, J. Steven; Weinshilboum, Richard. M.

    2010-01-01

    Betaine-homocysteine methyltransferase (BHMT) catalyzes the remethylation of homocysteine. BHMT is highly expressed in the human liver. In the liver, BHMT catalyzes up to 50% of homocysteine metabolism. Understanding the relationship between BHMT genetic polymorphisms and function might increase our understanding of the role of this reaction in homocysteine remethylation and in S-adenosylmethionine-dependent methylation. To help achieve those goals, we measured levels of BHMT enzyme activity and immunoreactive protein in 268 human hepatic surgical biopsy samples from adult subjects as well as 73 fetal hepatic tissue samples obtained at different gestational ages. BHMT protein levels were correlated significantly (p<0.001) with levels of enzyme activity in both fetal and adult tissue, but both were decreased in fetal tissue when compared with levels in the adult hepatic biopsies. To determine possible genotype-phenotype correlations, 12 tag SNPs for BHMT and the closely related BHMT2 gene were selected from SNPs observed during our own gene resequencing studies as well as from HapMap data were used to genotype DNA from the adult hepatic surgical biopsy samples, and genotype-phenotype association analysis was performed. Three SNPs (rs41272270, rs16876512, and rs6875201), located 28 kb upstream, in the 5′-UTR and in intron 1 of BHMT, respectively, were significantly correlated with both BHMT activity (p=3.41E-8, 2.55E-9 and 2.46E-10, respectively) and protein levels (p=5.78E-5, 1.08E-5 and 6.92E-6, respectively). We also imputed 230 additional SNPs across the BHMT and BHMT2 genes, identifying an additional imputed SNP, rs7700790, that was also highly associated with hepatic BHMT enzyme activity and protein. However, none of the 3 genotyped or one imputed SNPs displayed a “shift” during electrophoretic mobility shift assays. These observations may help us to understand individual variation in the regulation of BHMT in the human liver and its possible relationship

  1. Can carbapenem-resistant enterobacteriaceae susceptibility results obtained from surveillance cultures predict the susceptibility of a clinical carbapenem-resistant enterobacteriaceae?

    Science.gov (United States)

    Perez, Leandro Reus Rodrigues; Rodrigues, Diógenes; Dias, Cícero

    2016-08-01

    We evaluated the susceptibility profile of a colonizing carbapenem-resistant enterobacteriaceae to predict its susceptibility when recovered from a clinical specimen. An overall agreement of 88.7% (517 out of 583; 95% confidence interval, 85.8%-91.0%) was observed for the combinations of 11 antibiotics with 53 pairs of Klebsiella pneumoniae carbapenemase-producing K pneumoniae (the only carbapenem-resistant enterobacteriaceae detected). Very major errors were observed mainly for aminoglycoside agents and colistin, limiting the predictability of the susceptibility profile for these clinical isolates. PMID:27021509

  2. Heavy metals in liquid pig manure in light of bacterial antimicrobial resistance

    International Nuclear Information System (INIS)

    Heavy metals are regularly found in liquid pig manure, and might interact with bacterial antimicrobial resistance. Concentrations of heavy metals were determined by atomic spectroscopic methods in 305 pig manure samples and were connected to the phenotypic resistance of Escherichia coli (n=613) against 29 antimicrobial drugs. Concentrations of heavy metals (/kg dry matter) were 0.08–5.30 mg cadmium, 1.1–32.0 mg chrome, 22.4–3387.6 mg copper, <2.0–26.7 mg lead, <0.01–0.11 mg mercury, 3.1–97.3 mg nickel and 93.0–8239.0 mg zinc. Associated with the detection of copper and zinc, resistance rates against β-lactams were significantly elevated. By contrast, the presence of mercury was significantly associated with low antimicrobial resistance rates of Escherichia coli against β-lactams, aminoglycosides and other antibiotics. Effects of subinhibitory concentrations of mercury on bacterial resistance against penicillins, cephalosporins, aminoglycosides and doxycycline were also demonstrated in a laboratory trial. Antimicrobial resistance in the porcine microflora might be increased by copper and zinc. By contrast, the occurrence of mercury in the environment might, due to co-toxicity, act counter-selective against antimicrobial resistant strains.

  3. Heavy metals in liquid pig manure in light of bacterial antimicrobial resistance

    Energy Technology Data Exchange (ETDEWEB)

    Hoelzel, Christina S., E-mail: Christina.Hoelzel@wzw.tum.de [Chair of Animal Hygiene, Technische Universitaet Muenchen, Weihenstephaner Berg 3, 85354 Freising (Germany); Mueller, Christa [Institute for Agroecology, Organic Farming and Soil Protection, Bavarian State Research Center for Agriculture (LfL), Lange Point 12, 85354 Freising (Germany); Harms, Katrin S. [Chair of Animal Hygiene, Technische Universitaet Muenchen, Weihenstephaner Berg 3, 85354 Freising (Germany); Mikolajewski, Sabine [Department for Quality Assurance and Analytics, Bavarian State Research Center for Agriculture (LfL), Lange Point 4, 85354 Freising (Germany); Schaefer, Stefanie; Schwaiger, Karin; Bauer, Johann [Chair of Animal Hygiene, Technische Universitaet Muenchen, Weihenstephaner Berg 3, 85354 Freising (Germany)

    2012-02-15

    Heavy metals are regularly found in liquid pig manure, and might interact with bacterial antimicrobial resistance. Concentrations of heavy metals were determined by atomic spectroscopic methods in 305 pig manure samples and were connected to the phenotypic resistance of Escherichia coli (n=613) against 29 antimicrobial drugs. Concentrations of heavy metals (/kg dry matter) were 0.08-5.30 mg cadmium, 1.1-32.0 mg chrome, 22.4-3387.6 mg copper, <2.0-26.7 mg lead, <0.01-0.11 mg mercury, 3.1-97.3 mg nickel and 93.0-8239.0 mg zinc. Associated with the detection of copper and zinc, resistance rates against {beta}-lactams were significantly elevated. By contrast, the presence of mercury was significantly associated with low antimicrobial resistance rates of Escherichia coli against {beta}-lactams, aminoglycosides and other antibiotics. Effects of subinhibitory concentrations of mercury on bacterial resistance against penicillins, cephalosporins, aminoglycosides and doxycycline were also demonstrated in a laboratory trial. Antimicrobial resistance in the porcine microflora might be increased by copper and zinc. By contrast, the occurrence of mercury in the environment might, due to co-toxicity, act counter-selective against antimicrobial resistant strains.

  4. Wolbachia prophage DNA adenine methyltransferase genes in different Drosophila-Wolbachia associations

    DEFF Research Database (Denmark)

    Saridaki, Aggeliki; Sapountzis, Panagiotis; Harris, Harriet L;

    2011-01-01

    . The importance of DNA methylation in cell fate and biology calls for in depth studying of putative methylation-related genes. We present a molecular and phylogenetic analysis of a putative DNA adenine methyltransferase encoded by a prophage in the Wolbachia genome. Two slightly different copies of the gene, met1...

  5. Suz12 is essential for mouse development and for EZH2 histone methyltransferase activity

    DEFF Research Database (Denmark)

    Pasini, Diego; Bracken, Adrian P; Jensen, Michael R;

    2004-01-01

    SUZ12 is a recently identified Polycomb group (PcG) protein, which together with EZH2 and EED forms different Polycomb repressive complexes (PRC2/3). These complexes contain histone H3 lysine (K) 27/9 and histone H1 K26 methyltransferase activity specified by the EZH2 SET domain. Here we show tha...

  6. Association of Catechol-O-Methyltransferase (COMT) Polymorphism and Academic Achievement in a Chinese Cohort

    Science.gov (United States)

    Yeh, Ting-Kuang; Chang, Chun-Yen; Hu, Chung-Yi; Yeh, Ting-Chi; Lin, Ming-Yeh

    2009-01-01

    Catechol-O-methyltransferase (COMT) is a methylation enzyme that catalyzes the degradation pathway and inactivation of dopamine. It is accepted widely as being involved in the modulation of dopaminergic physiology and prefrontal cortex (PFC) function. The COMT Val158Met polymorphism is associated with variation in COMT activity. COMT 158Met allele…

  7. Catechol-O-methyltransferase gene methylation and substance use in adolescents: The TRAILS study

    NARCIS (Netherlands)

    L.J. van der Knaap (Lisette); J.M. Schäfer (Johanna); I.H.A. Franken (Ingmar); F.C. Verhulst (Frank); F.V.A. van Oort (Floor); H. Riese (Harriëtte)

    2014-01-01

    textabstractSubstance use often starts in adolescence and poses a major problem for society and individual health. The dopamine system plays a role in substance use, and catechol-O-methyltransferase (COMT) is an important enzyme that degrades dopamine. The Val108/158Met polymorphism modul

  8. Clinical Pharmacogenetics Implementation Consortium guidelines for thiopurine methyltransferase genotype and thiopurine dosing

    DEFF Research Database (Denmark)

    Relling, M V; Gardner, E E; Sandborn, W J;

    2011-01-01

    Thiopurine methyltransferase (TPMT) activity exhibits monogenic co-dominant inheritance, with ethnic differences in the frequency of occurrence of variant alleles. With conventional thiopurine doses, homozygous TPMT-deficient patients (~1 in 178 to 1 in 3,736 individuals with two nonfunctional TP...

  9. SABATH Methyltransferases from White Spruce (Picea glauca [Moench] Voss): Gene Cloning, Functional Characterization and Structural Analysis

    Science.gov (United States)

    Known members of the plant SABATH family of methyltransferases have important biological functions by methylating hormones, signaling molecules and other metabolites. While all previously characterized SABATH genes were isolated from angiosperms, in this article, we report on the isolation and funct...

  10. DNA repair methyltransferase (Mgmt) knockout mice are sensitive to the lethal effects of chemotherapeutic alkylating agents.

    NARCIS (Netherlands)

    B.J. Glassner (Brian); G. Weeda (Geert); J.M. Allan (James); J.L.M. Broekhof (Jose'); N.H.E. Carls (Nick); I. Donker (Ingrid); B.P. Engelward (Bevin); R.J. Hampson (Richard); R. Hersmus (Remko); M.J. Hickman (Mark); R.B. Roth (Richard); H.B. Warren (Henry); M.M. Wu (Mavis); J.H.J. Hoeijmakers (Jan); L.D. Samson (Leona)

    1999-01-01

    textabstractWe have generated mice deficient in O6-methylguanine DNA methyltransferase activity encoded by the murine Mgmt gene using homologous recombination to delete the region encoding the Mgmt active site cysteine. Tissues from Mgmt null mice displayed very low O6-methylguanine DNA methyltransf

  11. Functional characterization of cinnamyl alcohol dehydrogenase and caffeic acid O-methyltransferase in Brachypodium distachyon.

    Science.gov (United States)

    Lignin is a significant recalcitrant in the conversion of plant biomass to bioethanol. Cinnamyl alcohol dehydrogenase (CAD) and caffeic acid O-methyltransferase (COMT) catalyze key steps in the pathway of lignin monomer biosynthesis. Brown midrib mutants in Zea mays and Sorghum bicolor with impaired...

  12. Mutations in the DNA methyltransferase gene DNMT3A cause an overgrowth syndrome with intellectual disability

    DEFF Research Database (Denmark)

    Tatton-Brown, Katrina; Seal, Sheila; Ruark, Elise; Harmer, Jenny; Ramsay, Emma; Del Vecchio Duarte, Silvana; Zachariou, Anna; Hanks, Sandra; O'Brien, Eleanor; Aksglaede, Lise; Baralle, Diana; Dabir, Tabib; Gener, Blanca; Goudie, David; Homfray, Tessa; Kumar, Ajith; Pilz, Daniela T; Selicorni, Angelo; Temple, I Karen; Van Maldergem, Lionel; Yachelevich, Naomi; van Montfort, Robert; Rahman, Nazneen

    2014-01-01

    and histone binding. Similar mutations were not present in 1,000 UK population controls (13/152 cases versus 0/1,000 controls; P < 0.0001). Mutation carriers had a distinctive facial appearance, intellectual disability and greater height. DNMT3A encodes a DNA methyltransferase essential for...

  13. Local chromatin microenvironment determines DNMT activity : from DNA methyltransferase to DNA demethylase or DNA dehydroxymethylase

    NARCIS (Netherlands)

    van der Wijst, Monique G. P.; Venkiteswaran, Muralidhar; Chen, Hui; Xu, Guo-Liang; Plosch, Torsten; Rots, Marianne G.

    2015-01-01

    Insights on active DNA demethylation disproved the original assumption that DNA methylation is a stable epigenetic modification. Interestingly, mammalian DNA methyltransferases 3A and 3B (DNMT-3A and -3B) have also been reported to induce active DNA demethylation, in addition to their well-known fun

  14. Clinical Pharmacogenetics Implementation Consortium guidelines for thiopurine methyltransferase genotype and thiopurine dosing

    DEFF Research Database (Denmark)

    Relling, M V; Gardner, E E; Sandborn, W J; Schmiegelow, K; Pui, C-H; Yee, S W; Stein, Paul C.; Carrillo, Maria Berrocal; Evans, W E; Klein, T E

    2011-01-01

    Thiopurine methyltransferase (TPMT) activity exhibits monogenic co-dominant inheritance, with ethnic differences in the frequency of occurrence of variant alleles. With conventional thiopurine doses, homozygous TPMT-deficient patients (~1 in 178 to 1 in 3,736 individuals with two nonfunctional TP...... recommendations (updates at http://www.pharmgkb.org) for azathioprine, mercaptopurine (MP), and thioguanine based on TPMT genotype....

  15. Tyrosine 87 is vital for the activity of human protein arginine methyltransferase 3 (PRMT3)

    Czech Academy of Sciences Publication Activity Database

    Handrková, H.; Petrák, J.; Halada, Petr; Pospíšilová, D.; Čmejla, R.

    2011-01-01

    Roč. 1814, č. 2 (2011), s. 277-282. ISSN 1570-9639 R&D Projects: GA MŠk LC07017 Institutional research plan: CEZ:AV0Z50200510 Keywords : DIAMOND-BLACKFAN ANEMIA * SUBSTRATE-SPECIFICITY * N-METHYLTRANSFERASE Subject RIV: CE - Biochemistry Impact factor: 3.635, year: 2011

  16. A mouse speciation gene encodes a meiotic histone H3 methyltransferase

    Czech Academy of Sciences Publication Activity Database

    Mihola, Ondřej; Trachtulec, Zdeněk; Vlček, Čestmír; Schimenti, J.C.; Forejt, Jiří

    2009-01-01

    Roč. 323, č. 5912 (2009), s. 373-375. ISSN 0036-8075 Institutional research plan: CEZ:AV0Z50520514 Keywords : hybrid sterility * histone H3K4 methyltransferase * Prdm9 Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 29.747, year: 2009

  17. Aminoglycoside antibiotics and the sensory hair cells of inner ear and lateral line system in the Atlantic cod, Gadus morhua: effects on fish hearing threshold

    OpenAIRE

    Faucher, Karine; Aas-Hansen, Øyvind; Damsgard, Borge; Bégout, Marie-Laure; Fuhr, Torgrim; Laukli, Einar; Stenklev, Niels-Christian

    2007-01-01

    The aims of the present study were to investigate: a) the potential involvement of the fish lateral line system in hearing at 250 Hz and b) the possible regeneration of the inner ear hair cells in the Atlantic cod (Gadus morhua). The inner ear and lateral line system of the Atlantic cod were inactivated using ototoxic aminoglycoside antibiotics by injection (gentamicin) or bath (gentamicin and streptomycin), respectively. Hearing thresholds were measured in the fish using the Auditory Brainst...

  18. Mechanisms of antimicrobial resistance in Gram-negative bacilli.

    Science.gov (United States)

    Ruppé, Étienne; Woerther, Paul-Louis; Barbier, François

    2015-12-01

    The burden of multidrug resistance in Gram-negative bacilli (GNB) now represents a daily issue for the management of antimicrobial therapy in intensive care unit (ICU) patients. In Enterobacteriaceae, the dramatic increase in the rates of resistance to third-generation cephalosporins mainly results from the spread of plasmid-borne extended-spectrum beta-lactamase (ESBL), especially those belonging to the CTX-M family. The efficacy of beta-lactam/beta-lactamase inhibitor associations for severe infections due to ESBL-producing Enterobacteriaceae has not been adequately evaluated in critically ill patients, and carbapenems still stands as the first-line choice in this situation. However, carbapenemase-producing strains have emerged worldwide over the past decade. VIM- and NDM-type metallo-beta-lactamases, OXA-48 and KPC appear as the most successful enzymes and may threaten the efficacy of carbapenems in the near future. ESBL- and carbapenemase-encoding plasmids frequently bear resistance determinants for other antimicrobial classes, including aminoglycosides (aminoglycoside-modifying enzymes or 16S rRNA methylases) and fluoroquinolones (Qnr, AAC(6')-Ib-cr or efflux pumps), a key feature that fosters the spread of multidrug resistance in Enterobacteriaceae. In non-fermenting GNB such as Pseudomonas aeruginosa, Acinetobacter baumannii and Stenotrophomonas maltophilia, multidrug resistance may emerge following the sole occurrence of sequential chromosomal mutations, which may lead to the overproduction of intrinsic beta-lactamases, hyper-expression of efflux pumps, target modifications and permeability alterations. P. aeruginosa and A. baumannii also have the ability to acquire mobile genetic elements encoding resistance determinants, including carbapenemases. Available options for the treatment of ICU-acquired infections due to carbapenem-resistant GNB are currently scarce, and recent reports emphasizing the spread of colistin resistance in environments with high

  19. Physiology and Posttranscriptional Regulation of Methanol:Coenzyme M Methyltransferase Isozymes in Methanosarcina acetivorans C2A ▿ §

    OpenAIRE

    Opulencia, Rina B.; Bose, Arpita; Metcalf, William W.

    2009-01-01

    Methanosarcina species possess three operons (mtaCB1, mtaCB2, and mtaCB3) encoding methanol-specific methyltransferase 1 (MT1) isozymes and two genes (mtaA1 and mtaA2) with the potential to encode a methanol-specific methyltransferase 2 (MT2). Previous genetic studies showed that these genes are differentially regulated and encode enzymes with distinct levels of methyltransferase activity. Here, the effects of promoter strength on growth and on the rate of methane production were examined by ...

  20. Therapy and progression--induced O6-methylguanine-DNA methyltransferase and mismatch repair alterations in recurrent glioblastoma multiforme.

    Science.gov (United States)

    Agarwal, S; Suri, V; Sharma, M C; Sarkar, C

    2015-01-01

    Despite multimodality treatment protocol including surgical resection, radiotherapy, and chemotherapy in patients with glioblastoma multiforme (GBM), most suffer from treatment failure and tumor recurrence within a few months of initial surgery. The effectiveness of temozolomide (TMZ), the most commonly used chemotherapeutic agent, is largely dependent on the methylation status of the promoter of the gene O6-methylguanine-DNA methyltransferase (MGMT) and the integrity of the mismatch repair (MMR) system. Changes in these regulatory mechanisms at the time of recurrence may influence response to therapy. Deciphering the molecular mechanisms of resistance to these drugs may in future lead to improvised patient management. In this article, we provide an update of the spectrum of molecular changes that occur in recurrent GBMs, and thus may have an impact on patient survival and treatment response. For review, electronic search for the keywords "Recurrent GBM", "Recurrent GBM AND MGMT" "Recurrent glioma AND MGMT", "Recurrent GBM AND MMR" and "Recurrent glioma AND MMR", "Recurrent GBM AND MMR" and "Recurrent glioma AND MMR" was done on PubMed and relevant citations were screened including cross-references. PMID:26960480

  1. Therapy and progression – induced O6-methylguanine-DNA methyltransferase and mismatch repair alterations in recurrent glioblastoma multiforme

    Directory of Open Access Journals (Sweden)

    S Agarwal

    2015-01-01

    Full Text Available Despite multimodality treatment protocol including surgical resection, radiotherapy, and chemotherapy in patients with glioblastoma multiforme (GBM, most suffer from treatment failure and tumor recurrence within a few months of initial surgery. The effectiveness of temozolomide (TMZ, the most commonly used chemotherapeutic agent, is largely dependent on the methylation status of the promoter of the gene O6-methylguanine-DNA methyltransferase (MGMT and the integrity of the mismatch repair (MMR system. Changes in these regulatory mechanisms at the time of recurrence may influence response to therapy. Deciphering the molecular mechanisms of resistance to these drugs may in future lead to improvised patient management. In this article, we provide an update of the spectrum of molecular changes that occur in recurrent GBMs, and thus may have an impact on patient survival and treatment response. For review, electronic search for the keywords “Recurrent GBM”, “Recurrent GBM AND MGMT” “Recurrent glioma AND MGMT”, “Recurrent GBM AND MMR” and “Recurrent glioma AND MMR”, “Recurrent GBM AND MMR” and “Recurrent glioma AND MMR” was done on PubMed and relevant citations were screened including cross-references.

  2. Posttranslational Regulation of O(6)-Methylguanine-DNA Methyltransferase (MGMT) and New Opportunities for Treatment of Brain Cancers.

    Science.gov (United States)

    Srivenugopal, Kalkunte S; Rawat, Amit; Niture, Suryakant K; Paranjpe, Ameya; Velu, Chinavenmani; Venugopal, Sanjay N; Madala, Hanumantha Rao; Basak, Debasish; Punganuru, Surendra R

    2016-01-01

    O(6)-Methylguanine-DNA-methyltransferase (MGMT) is an antimutagenic DNA repair protein highly expressed in human brain tumors. Because MGMT repairs the mutagenic, carcinogenic and cytotoxic O(6)-alkylguanine adducts, including those generated by the clinically used anticancer alkylating agents, it has emerged as a central and rational target for overcoming tumor resistance to alkylating agents. Although the pseudosubstrates for MGMT [O(6)-benzylguanine, O(6)-(4- bromothenyl)guanine] have gained attention as powerful and clinically-relevant inhibitors, bone marrow suppression due to excessive alkylation damage has diminished this strategy. Our laboratory has been working on various posttranslational modifications of MGMT that affect its protein stability, DNA repair activity and response to oxidative stress. While these modifications greatly impact the physiological regulation of MGMT, they also highlight the opportunities for inactivating DNA repair and new drug discovery in this specific area. This review briefly describes the newer aspects of MGMT posttranslational regulation by ubiquitination, sumoylation and glutathionylation and reveals how the reactivity of the active site Cys145 can be exploited for potent inhibition and depletion of MGMT by thiol-reacting drugs such as the disulfiram and various dithiocarbamate derivatives. The possible repurposing of these nontoxic and safe drugs for improved therapy of pediatric and adult brain tumors is discussed. PMID:26202203

  3. Status of O 6 -methylguanine-DNA methyltransferase [MGMT] gene promoter methylation among patients with glioblastomas from India

    Directory of Open Access Journals (Sweden)

    Gopal Arun Nehru

    2012-01-01

    Full Text Available Background: O 6 -methylguanine DNA methyltransferase [MGMT] gene promoter methylation has emerged as a promising marker in determining resistance to temozolomide, used in the treatment of patients with glioblastomas. Aim: To determine the frequency of MGMT promoter methylation among patients with glioblastomas using methylation-specific polymerase chain reaction (MSP and compare it to the results obtained by bisulfite sequencing of a subset of samples. Materials and Methods: DNA obtained from the frozen tissue of 27 samples of glioblastomas and three other gliomas, were analyzed for MGMT promoter methylation using a nested MSP assay. Sixteen samples were also subjected to bisulfite sequencing to determine the methylation status of 27 CpG sites within the sequenced region of the MGMT promoter. Data with respect to radiation, chemotherapy and survival outcome was also collected. Results: MGMT promoter methylation was seen in 67% of the cases included in the study using frozen tissues by MSP analysis, while 62% were methylated among glioblastomas alone. There was a 100% concordance between the results obtained by MSP analysis and bisulfite sequencing. Clinical outcome was known among 67% of cases and methylation was higher among those patients who had no recurrence, though it was not statistically significant [P=0.44]. Conclusion: The frequency of methylation seen in this study concurs with that reported earlier from the country. MSP was easy to perform and interpret. However, the utility of this testing system in a routine diagnostic setting is still being debated.

  4. MALDI-TOF MS as a Tool To Detect a Nosocomial Outbreak of Extended-Spectrum-β-Lactamase- and ArmA Methyltransferase-Producing Enterobacter cloacae Clinical Isolates in Algeria

    Science.gov (United States)

    Khennouchi, Nour Chems el Houda; Loucif, Lotfi; Boutefnouchet, Nafissa; Allag, Hamoudi

    2015-01-01

    Enterobacter cloacae is among the most important pathogens responsible for nosocomial infections and outbreaks. In this study, 77 Enterobacter isolates were collected: 27 isolates from Algerian hospitals (in Constantine, Annaba, and Skikda) and 50 isolates from Marseille, France. All strains were identified by matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS). Antibiotic susceptibility testing was performed by the disk diffusion method. PCR was used to detect extended-spectrum-beta-lactamase (ESBL)-encoding, fluoroquinolone resistance-encoding, and aminoglycoside-modifying enzyme (AME) genes. Epidemiological typing was performed using MALDI-TOF MS with data mining approaches, along with multilocus sequence typing (MLST). Sixty-eight isolates (27 from Algeria, 41 from Marseille) were identified by MALDI-TOF MS as E. cloacae. Resistance to antibiotics in the Algerian isolates was significantly higher than that in the strains from Marseille, especially for beta-lactams and aminoglycosides. Eighteen of the 27 Algerian isolates and 11 of the 41 Marseille isolates possessed at least one ESBL-encoding gene: blaCTX-M and/or blaTEM. AME genes were detected in 20 of the 27 Algerian isolates and 8 of the 41 Marseille isolates [ant(2″)-Ia, aac(6′)-Ib-cr, aadA1, aadA2, and armA]. Conjugation experiments showed that armA was carried on a transferable plasmid. MALDI-TOF typing showed three separate clusters according to the geographical distribution and species level. An MLST-based phylogenetic tree showed a clade of 14 E. cloacae isolates from a urology unit clustering together in the MALDI-TOF dendrogram, suggesting the occurrence of an outbreak in this unit. In conclusion, the ability of MALDI-TOF to biotype strains was confirmed, and surveillance measures should be implemented, especially for Algerian patients hospitalized in France. PMID:26239991

  5. MALDI-TOF MS as a Tool To Detect a Nosocomial Outbreak of Extended-Spectrum-β-Lactamase- and ArmA Methyltransferase-Producing Enterobacter cloacae Clinical Isolates in Algeria.

    Science.gov (United States)

    Khennouchi, Nour Chems el Houda; Loucif, Lotfi; Boutefnouchet, Nafissa; Allag, Hamoudi; Rolain, Jean-Marc

    2015-10-01

    Enterobacter cloacae is among the most important pathogens responsible for nosocomial infections and outbreaks. In this study, 77 Enterobacter isolates were collected: 27 isolates from Algerian hospitals (in Constantine, Annaba, and Skikda) and 50 isolates from Marseille, France. All strains were identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Antibiotic susceptibility testing was performed by the disk diffusion method. PCR was used to detect extended-spectrum-beta-lactamase (ESBL)-encoding, fluoroquinolone resistance-encoding, and aminoglycoside-modifying enzyme (AME) genes. Epidemiological typing was performed using MALDI-TOF MS with data mining approaches, along with multilocus sequence typing (MLST). Sixty-eight isolates (27 from Algeria, 41 from Marseille) were identified by MALDI-TOF MS as E. cloacae. Resistance to antibiotics in the Algerian isolates was significantly higher than that in the strains from Marseille, especially for beta-lactams and aminoglycosides. Eighteen of the 27 Algerian isolates and 11 of the 41 Marseille isolates possessed at least one ESBL-encoding gene: blaCTX-M and/or blaTEM. AME genes were detected in 20 of the 27 Algerian isolates and 8 of the 41 Marseille isolates [ant(2″)-Ia, aac(6')-Ib-cr, aadA1, aadA2, and armA]. Conjugation experiments showed that armA was carried on a transferable plasmid. MALDI-TOF typing showed three separate clusters according to the geographical distribution and species level. An MLST-based phylogenetic tree showed a clade of 14 E. cloacae isolates from a urology unit clustering together in the MALDI-TOF dendrogram, suggesting the occurrence of an outbreak in this unit. In conclusion, the ability of MALDI-TOF to biotype strains was confirmed, and surveillance measures should be implemented, especially for Algerian patients hospitalized in France. PMID:26239991

  6. Development of a miniaturised microarray-based assay for the rapid identification of antimicrobial resistance genes in Gram-negative bacteria

    DEFF Research Database (Denmark)

    Batchelor, Miranda; Hopkins, Katie L; Liebana, Ernesto;

    2008-01-01

    We describe the development of a miniaturised microarray for the detection of antimicrobial resistance genes in Gram-negative bacteria. Included on the array are genes encoding resistance to aminoglycosides, trimethoprim, sulphonamides, tetracyclines and beta-lactams, including extended...... also seen in the number and type of resistance genes harboured by E. coli and Salmonella strains. The array provides an effective, fast and simple method for detection of resistance genes in clinical isolates suitable for use in diagnostic laboratories, which in future will help to understand the...... epidemiology of isolates and to detect gene linkage in bacterial populations. (C) 2008 Published by Elsevier B.V. and the International Society of Chemotherapy....

  7. Drug-resistant genes carried by Acinetobacter baumanii isolated from patients with lower respiratory tract infection

    Institute of Scientific and Technical Information of China (English)

    DAI Ning; ZHANG Wei; LI Jia-shu; YU Qin; WAN Huan-ying; MU Lan; ZHONG Xiao-ning; WEI Li-ping; MA Jian-jun; WANG Qiu-yue; HU Ke; LI De-zhi; TIAN Gui-zhen; CAI Shao-xi; WANG Rui-qin; HE Bei; WANG Si-qin; WANG Zhan-wei; ZHAO Su-rui; GAO Zhan-cheng; CHEN Ji-chao; CHEN Yu-sheng; GENG Rong; HU Ying-hui; YANG Jing-ping; DU Juan; HU Cheng-ping

    2010-01-01

    Background Acinetobacter baumanii (A. baumanii) remains an important microbial pathogen resulting in nosocomial acquired infections with significant morbidity and mortality. The mechanism by which nosocomial bacteria, like A.baumanii, attain multidrug resistance to antibiotics is of considerable interest. The aim in this study was to investigate the spread status of antibiotic resistance genes, such as multiple β-lactamase genes and aminoglycoside-modifying enzyme genes, from A. baumanii strains isolated from patients with lower respiratory tract infections (LRTIs).Methods Two thousand six hundred and ninety-eight sputum or the bronchoalveolar lavage samples from inpatients with LRTIs were collected in 21 hospitals in the mainland of China from November 2007 to February 2009. All samples were routinely inoculated. The isolated bacterial strains and their susceptibility were analyzed via VITEK-2 expert system.Several kinds of antibiotic resistant genes were further differentiated via polymerase chain reaction and sequencing methods.Results Totally, 39 A. baumanii strains were isolated from 2698 sputum or bronchoalveolar lavage samples. There was not only a high resistant rate of the isolated A. baumanii strains to ampicillin and first- and second-generation cephalosporins (94.87%, 100% and 97.44%, respectively), but also to the third-generation cephalosporins (ceftriaxone at 92.31%, ceftazidine at 51.28%) and imipenem (43.59%) as well. The lowest antibiotic resistance rate of 20.51% was found to amikacin. The OXA-23 gene was identified in 17 strains of A. baumanii, and the AmpC gene in 23 strains. The TEM-1 gene was carried in 15 strains. PER-1 and SHV-2 genes were detected in two different strains.Aminoglycoside-modifying enzyme gene aac-3-la was found in 23 strains, and the aac-6'-lb gene in 19 strains, aac-3-la and aac-6'-lb genes hibernated in three A. baumanii strains that showed no drug-resistant phenotype.Conclusions A. baumaniican carry multiple drug-resistant

  8. Temozolomide Resistance in Glioblastoma Cell Lines: Implication of MGMT, MMR, P-Glycoprotein and CD133 Expression

    OpenAIRE

    Gloria Perazzoli; Jose Prados; Raul Ortiz; Octavio Caba; Laura Cabeza; Maria Berdasco; Beatriz Gónzalez; Consolación Melguizo

    2015-01-01

    Background The use of temozolomide (TMZ) has improved the prognosis for glioblastoma multiforme patients. However, TMZ resistance may be one of the main reasons why treatment fails. Although this resistance has frequently been linked to the expression of O6-methylguanine-DNA methyltransferase (MGMT) it seems that this enzyme is not the only molecular mechanism that may account for the appearance of drug resistance in glioblastoma multiforme patients as the mismatch repair (MMR) complex, P-gly...

  9. Hair cell stereociliary bundle regeneration by espin gene transduction after aminoglycoside damage and hair cell induction by Notch inhibition.

    Science.gov (United States)

    Taura, A; Taura, K; Koyama, Y; Yamamoto, N; Nakagawa, T; Ito, J; Ryan, A F

    2016-05-01

    Once inner ear hair cells (HCs) are damaged by drugs, noise or aging, their apical structures including the stereociliary arrays are frequently the first cellular feature to be lost. Although this can be followed by progressive loss of HC somata, a significant number of HC bodies often remain even after stereociliary loss. However, in the absence of stereocilia they are nonfunctional. HCs can sometimes be regenerated by Atoh1 transduction or Notch inhibition, but they also may lack stereociliary bundles. It is therefore important to develop methods for the regeneration of stereocilia, in order to achieve HC functional recovery. Espin is an actin-bundling protein known to participate in sterociliary elongation during development. We evaluated stereociliary array regeneration in damaged vestibular sensory epithelia in tissue culture, using viral vector transduction of two espin isoforms. Utricular HCs were damaged with aminoglycosides. The utricles were then treated with a γ-secretase inhibitor, followed by espin or control transduction and histochemistry. Although γ-secretase inhibition increased the number of HCs, few had stereociliary arrays. In contrast, 46 h after espin1 transduction, a significant increase in hair-bundle-like structures was observed. These were confirmed to be immature stereociliary arrays by scanning electron microscopy. Increased uptake of FM1-43 uptake provided evidence of stereociliary function. Espin4 transduction had no effect. The results demonstrate that espin1 gene therapy can restore stereocilia on damaged or regenerated HCs. PMID:26886463

  10. Determination of aminoglycosides in honey by capillary electrophoresis tandem mass spectrometry and extraction with molecularly imprinted polymers.

    Science.gov (United States)

    Moreno-González, David; Lara, Francisco J; Jurgovská, Nikola; Gámiz-Gracia, Laura; García-Campaña, Ana M

    2015-09-01

    A new analytical method based on capillary zone electrophoresis-tandem mass spectrometry is proposed and validated for the identification and simultaneous quantification of nine aminoglycosides in honey samples. Detection using an ion trap mass analyzer operating in the multiple reaction monitoring mode was used. Different parameters were optimized in order to obtain an adequate separation combined with the highest sensitivity. In order to achieve high selectivity in the sample treatment, a commercially-available molecularly imprinted polymer has been used for the solid phase extraction of the analytes. Under optimum conditions, recoveries for fortified samples ranged from 88.2 to 99.8%, with relative standard deviations lower than 8%. The limits of detection ranged from 0.4 to 28.5 μg kg(-1). Furthermore, the decision limit and the detection capability were evaluated, ranging from 3.5 to 60.5 μg kg(-1) and from 6.0 to 103.1 μg kg(-1), respectively, demonstrating the sensitivity and applicability of this fast and simple method. PMID:26388393

  11. YccW is the m5C methyltransferase specific for 23S rRNA nucleotide 1962

    DEFF Research Database (Denmark)

    Purta, Elzbieta; O'Connor, Michelle; Bujnicki, Janusz M;

    2008-01-01

    . However, YccW does not methylate assembled 50S subunits, and this is somewhat surprising as the published crystal structures show nucleotide C1962 to be fully accessible at the subunit interface. YccW-directed methylation at nucleotide C1962 is conserved in bacteria, and loss of this methylation in E....... coli marginally reduces its growth rate. YccW had previously eluded identification because it displays only limited sequence similarity to the m(5)C methyltransferases RsmB and RsmF and is in fact more similar to known m(5)U (5-methyluridine) RNA methyltransferases. In keeping with the previously...... proposed nomenclature system for bacterial rRNA methyltransferases, yccW is now designated as the rRNA large subunit methyltransferase gene rlmI....

  12. Detection of Multi-drug Resistant Acinetobacter Lwoffii Isolated from Soil of Mink Farm.

    Science.gov (United States)

    Sun, Na; Wen, Yong Jun; Zhang, Shu Qin; Zhu, Hong Wei; Guo, Li; Wang, Feng Xue; Chen, Qiang; Ma, Hong Xia; Cheng, Shi Peng

    2016-07-01

    There were 4 Acinetobacter lwoffii obtained from soil samples. The antimicrobial susceptibility of the strains to 16 antimicrobial agents was investigated using K-B method. Three isolates showed the multi-drug resistance. The presence of resistance genes and integrons was determined using PCR. The aadA1, aac(3')-IIc, aph(3')-VII, aac(6')-Ib, sul2, cat2, floR, and tet(K) genes were detected, respectively. Three class 1 integrons were obtained. The arr-3-aacA4 and blaPSE-1 gene cassette, which cause resistance to aminoglycoside and beta-lactamase antibiotics. Our results reported the detection of multi-drug resistant and carried resistant genes Acinetobacter lwoffii from soil. The findings suggested that we should pay close attention to the prevalence of multi-drug resistant bacterial species of environment. PMID:27554122

  13. N-6-Adenine-Specific DNA Methyltransferase 1 (N6AMT1) Polymorphisms and Arsenic Methylation in Andean Women

    OpenAIRE

    Harari, Florencia; Engström, Karin; Concha, Gabriela; Colque, Graciela; Vahter, Marie; Broberg, Karin

    2013-01-01

    BACKGROUND: In humans, inorganic arsenic is metabolized to methylated metabolites mainly by arsenic (+3 oxidation state) methyltransferase (AS3MT). AS3MT polymorphisms are associated with arsenic metabolism efficiency. Recently, a putative N-6-adenine-specific DNA methyltransferase 1 (N6AMT1) was found to methylate arsenic in vitro. OBJECTIVE: We evaluated the role of N6AMT1 polymorphisms in arsenic methylation efficiency in humans. METHODS: We assessed arsenic methylation efficiency in 188 w...

  14. MOLECULAR AND FUNCTIONAL CHARACTERIZATION OF A JUVENILE HORMONE ACID METHYLTRANSFERASE EXPRESSED IN THE CORPORA ALLATA OF MOSQUITOES

    OpenAIRE

    Mayoral, Jaime G.; Nouzova, Marcela; Yoshiyama, Michiyo; Shinoda, Tetsuro; Hernandez-Martinez, Salvador; Dolghih, Elena; Turjanski, Adrian G; Roitberg, Adrian E.; Priestap, Horacio; Perez, Mario; Mackenzie, Lucy; Li, Yiping; Noriega, Fernando G.

    2008-01-01

    A juvenile hormone acid methyltransferase (JHAMT) was isolated as an abundant EST in a library of the corpora allata of the adult female mosquito Aedes aegypti. Its full-length cDNA encodes a 278-aa protein that has 43 % amino acid identity with BmJHAMT, a juvenile hormone acid methyltransferase previously cloned from Bombyx mori. Heterologous expression produced a recombinant protein that metabolizes farnesoic acid (FA) into methyl farnesoate, as well as juvenile hormone acid into juvenile h...

  15. The Histone Methyltransferase Inhibitor A-366 Uncovers a Role for G9a/GLP in the Epigenetics of Leukemia

    OpenAIRE

    Pappano, William N.; Guo, Jun; He, Yupeng; Ferguson, Debra; Jagadeeswaran, Sujatha; Osterling, Donald J.; Gao, Wenqing; Spence, Julie K.; Pliushchev, Marina; Sweis, Ramzi F.; Buchanan, Fritz G.; Michaelides, Michael R.; Shoemaker, Alexander R.; Tse, Chris; Chiang, Gary G.

    2015-01-01

    Histone methyltransferases are epigenetic regulators that modify key lysine and arginine residues on histones and are believed to play an important role in cancer development and maintenance. These epigenetic modifications are potentially reversible and as a result this class of enzymes has drawn great interest as potential therapeutic targets of small molecule inhibitors. Previous studies have suggested that the histone lysine methyltransferase G9a (EHMT2) is required to perpetuate malignant...

  16. Inhibition of G9a Histone Methyltransferase Converts Bone Marrow Mesenchymal Stem Cells to Cardiac Competent Progenitors

    OpenAIRE

    Jinpu Yang; Keerat Kaur; Li Lin Ong; Eisenberg, Carol A; Leonard M. Eisenberg

    2015-01-01

    The G9a histone methyltransferase inhibitor BIX01294 was examined for its ability to expand the cardiac capacity of bone marrow cells. Inhibition of G9a histone methyltransferase by gene specific knockdown or BIX01294 treatment was sufficient to induce expression of precardiac markers Mesp1 and brachyury in bone marrow cells. BIX01294 treatment also allowed bone marrow mesenchymal stem cells (MSCs) to express the cardiac transcription factors Nkx2.5, GATA4, and myocardin when subsequently exp...

  17. Paromomycin Affects Translation and Vesicle-Mediated Trafficking as Revealed by Proteomics of Paromomycin –Susceptible –Resistant Leishmania donovani

    OpenAIRE

    Bhavna Chawla; Anupam Jhingran; Aswini Panigrahi; Stuart, Kenneth D.; Rentala Madhubala

    2011-01-01

    Leishmania donovani is a protozoan parasite that causes visceral leishmaniasis (VL) and is responsible for significant mortality and morbidity. Increasing resistance towards antimonial drugs poses a great challenge in chemotherapy of VL. Paromomycin is an aminoglycosidic antibiotic and is one of the drugs currently being used in the chemotherapy of cutaneous and visceral leishmaniasis. To understand the mode of action of this antibiotic at the molecular level, we have investigated the global ...

  18. Identification of clinically antibiotic resistant genes Aac(3-IIa and Aac(6’-Ib in wastewater samples by multiplex PCR

    Directory of Open Access Journals (Sweden)

    Naser Samadi

    2015-06-01

    Full Text Available Background: Aminoglycoside antibiotics are widely used in medical centers, particularly to treat infections. The resistance developed against these agents is a huge concern in health care. A number of researchers have reported that hospital and municipal wastewaters are among the most important dissemination sources of these agent into the environment. Some, however, do not agree with this opinion. In the present study, the prevalence of aminoglycoside resistance genes was investigated in raw and effluent wastewater from hospital and municipal wastewater treatment plants. Methods: To conduct this descriptive-analytical study, 30 samples were taken according to sampling principles and cold cycle and transferred to the molecular laboratory. DNA was extracted by the freeze-thaw method using a kit (Promega. The genes aac(3-IIa and aac(6’-Ib which code aminoglycoside resistance were examined in this study. Results: The results indicated that the studied genes are present in 35% of urban and hospital wastewaters, and their frequency percentage is higher in hospital wastewater (52% than urban wastewater (48%. The studied genes were identified in 61% of raw hospital wastewater samples; however, they were not detected in the output wastewater from the studied treatment plants. Conclusion: Although, the studied genes were not detected in the final effluent, there is a high potential for their release into the environment. The current study demonstrated that the coding genes of aminoglycoside antibiotic resistance are present in raw urban and hospital wastewaters. In the case of improper exploitation of wastewater treatment plants, the output water can contaminate other environmental sections, such as soil and water resources, and result in the emission of these contaminants.

  19. Mutation of Salmonella paratyphi A conferring cross-resistance to several groups of antibiotics by decreased permeability and loss of invasiveness.

    OpenAIRE

    Gutmann, L; Billot-Klein, D; Williamson, R.; Goldstein, F W; Mounier, J; Acar, J F; Collatz, E

    1988-01-01

    A spontaneous one-step mutant of Salmonella paratyphi A selected on ampicillin showed cross-resistance to all beta-lactam antibiotics except imipenem and to aminoglycosides, chloramphenicol, tetracycline, trimethoprim, and quinolones. It also grew as small colonies. Examination of the cell envelope of the mutant showed a quantitative decrease in three major outer membrane proteins of 40.6, 39.6 (presumably porins), and 24 kilodaltons and quantitative as well as qualitative modifications in th...

  20. Multidrug-resistant tuberculosis in the United Kingdom and Lithuania.

    Science.gov (United States)

    Gonzalo, X; Hutchison, D C S; Drobniewski, F A; Pimkina, E; Davidaviciene, E

    2014-06-01

    Rates of resistance to first- and second-line drugs in multidrug-resistant tuberculosis (MDR-TB) cases in the United Kingdom were studied during 2010-2012. The highest rates for ethambutol, pyrazinamide and aminoglycosides occurred among patients originating in Eastern Europe, of whom 47% were Lithuanian. Rates of resistance to kanamycin were significantly lower (P < 0.0001) in the Lithuanian National TB Register than among Lithuanian patients resident in the United Kingdom (5% vs. 78%). In 2010, the majority of UK patients of Eastern European origin were located within the London region, whereas in 2011 the majority were located outside this region, a significant change (P = 0.01). PMID:24903935

  1. Epigenetic modulation of the drug resistance genes MGMT, ABCB1 and ABCG2 in glioblastoma multiforme

    OpenAIRE

    Oberstadt, Moritz C.; Bien-Möller, Sandra; Weitmann, Kerstin; Herzog, Susann; Hentschel, Katharina; Rimmbach, Christian; Vogelgesang, Silke; Balz, Ellen; Fink, Matthias; Michael, Heike; Zeden, Jan-Philip; Bruckmüller, Henrike; Werk, Anneke N.; Cascorbi, Ingolf; Hoffmann, Wolfgang

    2013-01-01

    Background Resistance of the highly aggressive glioblastoma multiforme (GBM) to drug therapy is a major clinical problem resulting in a poor patient’s prognosis. Beside promoter methylation of the O 6 -methylguanine-DNA-methyltransferase (MGMT) gene the efflux transporters ABCB1 and ABCG2 have been suggested as pivotal factors contributing to drug resistance, but the methylation of ABCB1 and ABCG2 has not been assessed before in GBM. Methods Therefore, we evaluated the proportion and pr...

  2. Enhanced MGMT expression contributes to temozolomide resistance in glioma stem-like cells

    OpenAIRE

    Zhi-Kun Qiu; Dong Shen; Yin-Sheng Chen; Qun-Ying Yang; Cheng-Cheng Guo; Bing-Hong Feng; Zhong-Ping Chen

    2014-01-01

    O6-methylguanine DNA methyltransferase (MGMT) can remove DNA alkylation adducts, thereby repairing damaged DNA and contributing to the drug resistance of gliomas to alkylating agents. In addition, glioma stem-like cells (GSCs) have been demonstrated to be involved in the recurrence and treatment resistance of gliomas. In this study, we aimed to investigate MGMT expression and regulatory mechanisms in GSCs and the association of MGMT with temozolomide (TMZ) sensitivity. GSC...

  3. Induction of MGMT expression is associated with temozolomide resistance in glioblastoma xenografts

    OpenAIRE

    Kitange, Gaspar J.; Carlson, Brett L.; Schroeder, Mark A.; Grogan, Patrick T.; Lamont, Jeff D.; Decker, Paul A.; Wu, Wenting; James, C. David; Sarkaria, Jann N.

    2009-01-01

    Temozolomide (TMZ)-based therapy is the standard of care for patients with glioblastoma multiforme (GBM), and resistance to this drug in GBM is modulated by the DNA repair protein O6-methylguanine-DNA methyl-transferase (MGMT). Expression of MGMT is silenced by promoter methylation in approximately half of GBM tumors, and clinical studies have shown that elevated MGMT protein levels or lack of MGMT promoter methylation is associated with TMZ resistance in some, but not all, GBM tumors. In thi...

  4. Discovery of sphingosine 1-O-methyltransferase in rat kidney and liver homogenates

    Institute of Scientific and Technical Information of China (English)

    Santosh J SACKET; Dong-soon IM

    2008-01-01

    Aim:To characterize sphingosine methyltransferase in rat tissues.Methods:By using S-adenosyl-L-(methyl-3H) methionine,enzymatic activity was measured in the rat liver and kidney homogenates.Results:The optimum pH and reaction time for the enzyme assay were pH 7.8 and 1 h.ZnCl2 inhibited the activity,but not MgCl2,CaCl2,CoCl2,or NiCl2.In the kidney homogenate,enzymatic activity was detectable in the cytosol and all membrane fractions from the plasma membrane and other organelles; however,in the liver homogenate,enzymatic activity was detectable in all membrane fractions,but not in the cytosol.We also tested the enzymatic activity with structurally-modified sphingosine derivatives.Conclusion:We found sphingosine l-O-methyltransferase activity in the rat liver and kidney homogenates.

  5. Super-resolution optical DNA Mapping via DNA methyltransferase-directed click chemistry

    DEFF Research Database (Denmark)

    Vranken, Charlotte; Deen, Jochem; Dirix, Lieve;

    2014-01-01

    We demonstrate an approach to optical DNA mapping, which enables near single-molecule characterization of whole bacteriophage genomes. Our approach uses a DNA methyltransferase enzyme to target labelling to specific sites and copper-catalysed azide-alkyne cycloaddition to couple a fluorophore...... to the DNA. We achieve a labelling efficiency of ∼70% with an average labelling density approaching one site every 500 bp. Such labelling density bridges the gap between the output of a typical DNA sequencing experiment and the long-range information derived from traditional optical DNA mapping. We lay...... the foundations for a wider-scale adoption of DNA mapping by screening 11 methyltransferases for their ability to direct sequence-specific DNA transalkylation; the first step of the DNA labelling process and by optimizing reaction conditions for fluorophore coupling via a click reaction. Three of 11 enzymes...

  6. Crystallization and preliminary X-ray diffraction studies of a catechol-O-methyltransferase/inhibitor complex

    International Nuclear Information System (INIS)

    Catechol-O-methyltransferase has been co-crystallized with a novel inhibitor, which has potential therapeutic application in the Parkinson’s disease therapy. Inhibitors of the enzyme catechol-O-methyltransferase (COMT) are used as co-adjuvants in the therapy of Parkinson’s disease. A recombinant form of the soluble cytosolic COMT from rat has been co-crystallized with a new potent inhibitor, BIA 8-176 [(3,4-dihydroxy-2-nitrophenyl)phenylmethanone], by the vapour-diffusion method using PEG 6K as precipitant. Crystals diffract to 1.6 Å resolution on a synchrotron-radiation source and belong to the monoclinic space group P21, with unit-cell parameters a = 52.77, b = 79.63, c = 61.54 Å, β = 91.14°

  7. Crystallization and preliminary X-ray diffraction studies of a catechol-O-methyltransferase/inhibitor complex

    Energy Technology Data Exchange (ETDEWEB)

    Rodrigues, M. L. [Instituto de Tecnologia Química e Biológica (ITQB), Universidade Nova de Lisboa, Av. República, Apt. 127, 2781-901 Oeiras (Portugal); Bonifácio, M. J.; Soares-da-Silva, P. [Department of Research and Development, BIAL, 4785 S. Mamede do Coronado (Portugal); Carrondo, M. A.; Archer, M., E-mail: archer@itqb.unl.pt [Instituto de Tecnologia Química e Biológica (ITQB), Universidade Nova de Lisboa, Av. República, Apt. 127, 2781-901 Oeiras (Portugal)

    2005-01-01

    Catechol-O-methyltransferase has been co-crystallized with a novel inhibitor, which has potential therapeutic application in the Parkinson’s disease therapy. Inhibitors of the enzyme catechol-O-methyltransferase (COMT) are used as co-adjuvants in the therapy of Parkinson’s disease. A recombinant form of the soluble cytosolic COMT from rat has been co-crystallized with a new potent inhibitor, BIA 8-176 [(3,4-dihydroxy-2-nitrophenyl)phenylmethanone], by the vapour-diffusion method using PEG 6K as precipitant. Crystals diffract to 1.6 Å resolution on a synchrotron-radiation source and belong to the monoclinic space group P2{sub 1}, with unit-cell parameters a = 52.77, b = 79.63, c = 61.54 Å, β = 91.14°.

  8. Targeting histone methyltransferases and demethylases in clinical trials for cancer therapy.

    Science.gov (United States)

    Morera, Ludovica; Lübbert, Michael; Jung, Manfred

    2016-01-01

    The term epigenetics is defined as heritable changes in gene expression that are not due to alterations of the DNA sequence. In the last years, it has become more and more evident that dysregulated epigenetic regulatory processes have a central role in cancer onset and progression. In contrast to DNA mutations, epigenetic modifications are reversible and, hence, suitable for pharmacological interventions. Reversible histone methylation is an important process within epigenetic regulation, and the investigation of its role in cancer has led to the identification of lysine methyltransferases and demethylases as promising targets for new anticancer drugs. In this review, we describe those enzymes and their inhibitors that have already reached the first stages of clinical trials in cancer therapy, namely the histone methyltransferases DOT1L and EZH2 as well as the demethylase LSD1. PMID:27222667

  9. Aflatoxin B1 induced upregulation of protein arginine methyltransferase 5 in human cell lines.

    Science.gov (United States)

    Ghufran, Md Sajid; Ghosh, Krishna; Kanade, Santosh R

    2016-09-01

    The exposure of naturally occurring mycotoxins affects human health and play a vital role in cancer initiation and progression. Aflatoxin B1 is a difuranocoumarin mycotoxin, classified as a group I carcinogen. The present study was conducted to assess the effect of aflatoxin B1 on epigenetic regulatory proteins. The protein arginine methyltransferase 5 expression was induced upon aflatoxin B1 treatment in a dose and time dependent manner. Further global arginine methylation was also increased in the same manner. This is the first report showing the induction of epigenetic regulatory protein, protein arginine methyltransferase 5 upon aflatoxin B1 treatment. Further study is required to establish the detailed pathway of PRMT5 induction. PMID:27242039

  10. Optimising dosing strategies of antibacterials utilising pharmacodynamic principles: impact on the development of resistance.

    Science.gov (United States)

    DeRyke, C Andrew; Lee, Su Young; Kuti, Joseph L; Nicolau, David P

    2006-01-01

    Evolving antimicrobial resistance is of global concern. The impact of decreased susceptibility to current antibacterials coupled with the decline in the marketing of new agents with novel mechanisms of action places a tremendous burden on clinicians to appropriately use available agents. Optimising antibacterial dose administration through the use of pharmacodynamic principles can aid clinicians in accomplishing this task more effectively. Methods to achieve this include: continuous or prolonged infusion, or the use of smaller doses administered more frequently for the time-dependent beta-lactam agents; or higher, less frequent dose administration of the concentration-dependent aminoglycosides and fluoroquinolones. Pharmacodynamic breakpoints, which are predictive of clinical and/or microbiological success in the treatment of infection, have been determined for many classes of antibacterials, including the fluoroquinolones, aminoglycosides and beta-lactams. Although surpassing these values may predict efficacy, it may not prevent the development of resistance. Recent studies seek to determine the pharmacodynamic breakpoints that prevent the development of resistance. Numerous studies to this point have determined these values in fluoroquinolones in both Gram-positive and Gram-negative bacteria. However, among the other antibacterial classes, there is a lack of sufficient data. Additionally, a new term, the mutant prevention concentration, has been based on the concentrations above which resistance is unlikely to occur. Future work is needed to fully characterise these target concentrations that prevent resistance. PMID:16398565

  11. Structure and possible mechanism of the CcbJ methyltransferase from Streptomyces caelestis

    Czech Academy of Sciences Publication Activity Database

    Bauer, J.; Ondrovičová, G.; Najmanová, Lucie; Pevala, V.; Kameník, Zdeněk; Koštan, J.; Janata, Jiří; Kutejová, Eva

    2014-01-01

    Roč. 70, APR 2014 (2014), s. 943-957. ISSN 0907-4449 R&D Projects: GA MŠk(CZ) EE2.3.30.0003; GA MŠk(CZ) ED1.1.00/02.0109 Institutional support: RVO:61388971 Keywords : CATECHOL-O-METHYLTRANSFERASE * SN2-LIKE TRANSITION-STATE * CRYSTAL-STRUCTURES Subject RIV: CE - Biochemistry Impact factor: 7.232, year: 2013

  12. Selenium induced selenocysteine methyltransferase gene expression and antioxidant enzyme activities in Astragalus chrysochlorus

    OpenAIRE

    Çakir, Özgür; Turgut-Kara, Neslihan; Ari, Şule

    2016-01-01

    Astragalus sp. are used in folk medicine because of their biological activities and are known for the ability to accumulate high levels of selenium (Se). The purpose of this study was to explore gene expression of selenocysteine methyltransferase (SMT), responsible for forming MeSeCys, and activities of ascorbate peroxidase (APX), peroxidase (POX), catalase (CAT) and glutathione reductase (GR) enzymes in callus tissues of Astragalus chrysochlorus growing in different Se-containing media. Quan...

  13. Recombinant rat liver guanidinoacetate methyltransferase: Reactivity and function of sulfhydryl groups

    International Nuclear Information System (INIS)

    Rat liver guanidinoacetate methyltransferase, produced in Escherichia coli by recombinant DNA technique, possesses five cysteine residues per molecule. No disulfide bond is present. Analysis of the chymotryptic peptides derived from the iodo[14C]acetate-modified enzyme shows that Cys-90, Cys-15, Cys-219, and Cys-207 are alkylated by the reagent in order of decreasing reactivity. Incubation of the enzyme with excess 5,5'-dithiobis(2-nitrobenzoate) (DTNB) in the absence and presence of cystamine [2,2'-dithiobis(ethylamine)] causes the appearance of 4 and 5 mol of 2-nitro-5-mercaptobenzoate/mol of enzyme, respectively. Reaction of the methyltransferase with an equimolar amount of DTNB results in an almost quantitative disulfide cross-linking of Cys-15 and Cys-90 with loss of a large portion of the activity. The methyltransferase is completely inactivated by iodoacetate following nonlinear kinetics. Comparison of the extent of inactivation with that of modification of cysteine residues and the experiment with the enzyme whose Cys-15 and Cys-90 are cross-linked suggest that alkylation of Cys-15 and Cys-90 results in a partially active enzyme and that carboxymethylation of Cys-219 completely eliminates enzyme activity. The inactivation of guanidinoacetate methyltransferase by iodoacetate or DTNB is not protected by substrates. Furthermore, disulfide cross-linking of Cys-15 and Cys-90 or carboxymethylation of Cys-219 does not impair the enzyme's capacity to bind S-adenosylmethionine. Thus, these cysteine residues appear to occur outside the active-site region, but their integrity is crucial for the expression of enzyme activity

  14. Vector design for expression of O6-methylguanine-DNA methyltransferase in hematopoietic cells

    OpenAIRE

    Schambach, Axel; Baum, Christopher

    2007-01-01

    Enhancing DNA repair activity of hematopoietic cells by stably integrating gene vectors that express O(6)-methylguanine-DNA-methyltransferase (MGMT) is of major interest for innovative approaches in tumor chemotherapy and for the control of hematopoietic chimerism in the treatment of multiple other acquired or inherited disorders. Crucial determinants of this selection principle are the stringency of treatment with O(6)-alkylating agents and the level of transgenic MGMT expression. Attempts t...

  15. Identification, Characterization, and Ontogenic Study of a Catechol O-methyltransferase from Zebrafish

    OpenAIRE

    Alazizi, Adnan; Liu, Ming-Yih; Williams, Frederick E.; Kurogi, Katsuhisa; Sakakibara, Yoichi; Suiko, Masahito; Liu, Ming-Cheh

    2010-01-01

    To establish the zebrafish as a model for investigating the methylation pathway of drug metabolism, we embarked on the molecular cloning of the zebrafish catechol O-methyltransferase (COMT). By searching the GenBank database, a zebrafish nucleotide sequence encoding a putative COMT was identified. Based on the sequence information, we designed and synthesized oligonucleotides corresponding to its 5’- and 3’-coding regions of this zebrafish COMT. Using the first-strand cDNA reverse-transcribed...

  16. Expression of O6-methylguanine DNA methyltransferase (MGMT) and its clinical significance in gastroenteropancreatic neuroendocrine neoplasm

    OpenAIRE

    Yang, Qiu-chen; Wang, Yu-Hong; Lin, Yuan; Xue, Ling; Chen, Yuan-Jia; Chen, Min-hu; Chen, Jie

    2014-01-01

    O6-methylguanine-DNA methyltransferase (MGMT) is a widespread DNA repair enzyme defending against mutation caused by guanine O6-alkylating agents. Until now, we know only little about the expression of MGMT in gastroenteropancreatic neuroendocrine neoplasm (GEP-NEN). To study the expression of MGMT and its clinical significance in GEP-NEN, 174 specimens of GEP-NEN were examined, of which 152 specimens came from The First Affiliated Hospital, Sun Yat-sen University during October 1995 to Novem...

  17. Survival and tumorigenesis in O6-methylguanine DNA methyltransferase-deficient mice following cyclophosphamide exposure

    OpenAIRE

    Nagasubramanian, Ramamoorthy; Hansen, Ryan J.; Delaney, Shannon M.; Cherian, Mathew M.; Samson, Leona D.; Kogan, Scott C.; Dolan, M. Eileen

    2008-01-01

    O6-methylguanine DNA methyltransferase (MGMT) deficiency is associated with an increased susceptibility to alkylating agent toxicity. To understand the contribution of MGMT in protecting against cyclophosphamide (CP)-induced toxicity, mutagenesis and tumorigenesis, we compared the biological effects of this agent in transgenic Mgmt knockout and wild-type mice. In addition, neurofibromin (Nf1)+/− background was used to increase the likelihood of CP-induced tumorigenesis. Cohorts of Mgmt-profic...

  18. Deficiency of Glycine N-Methyltransferase Aggravates Atherosclerosis in Apolipoprotein E–Null Mice

    OpenAIRE

    Chen, Chien-Yu; Ching, Li-Chieh; Liao, Yi-Jen; Yu, Yuan-Bin; Tsou, Chia-Yuan; Shyue, Song-Kun; Chen, Yi-Ming Arthur; Lee, Tzong-Shyuan

    2012-01-01

    The mechanism underlying the dysregulation of cholesterol metabolism and inflammation in atherogenesis is not understood fully. Glycine N-methyltransferase (GNMT) has been implicated in hepatic lipid metabolism and the pathogenesis of liver diseases. However, little is known about the significance of GNMT in atherosclerosis. We showed the predominant expression of GNMT in foamy macrophages of mouse atherosclerotic aortas. Genetic deletion of GNMT exacerbated the hyperlipidemia, inflammation a...

  19. Nicotinamide N-methyltransferase knockdown protects against diet-induced obesity

    OpenAIRE

    Kraus, Daniel; Yang, Qin; Kong, Dong; Banks, Alexander S.; Zhang, Lin; Rodgers, Joseph T; Pirinen, Eija; Pulinilkunnil, Thomas C.; Gong, Fengying; Wang, Ya-chin; Cen, Yana; Sauve, Anthony A.; Asara, John M.; Peroni, Odile D.; Monia, Brett P.

    2014-01-01

    In obesity and type 2 diabetes, Glut4 glucose transporter expression is decreased selectively in adipocytes1. Adipose-specific knockout or overexpression of Glut4 alters systemic insulin sensitivity2. Here we show, using DNA array analyses, that nicotinamide N-methyltransferase (Nnmt) is the most strongly reciprocally regulated gene when comparing gene expression in white adipose tissue (WAT) from adipose-specific Glut4-knockout or adipose-specific Glut4-overexpressing mice with their respect...

  20. Homocysteine Induces Collagen I Expression by Downregulating Histone Methyltransferase G9a

    OpenAIRE

    Lei, Wenjing; Long, Yanjun; LI, Shuang; Liu, Ze; Zhu, Fengxin; Hou, Fan Fan; Nie, Jing

    2015-01-01

    Hyperhomocysteinemia (HHcy) leads to several clinical manifestations including hepatic fibrosis. Excess deposition of extracellular matrix (ECM) components including collagen is the eponymous lesion of liver fibrosis. In this study, we demonstrated that elevated concentration of Hcy induced the expression of collagen type I in cultured human liver cells as well as in liver tissue of HHcy mice. Meanwhile, Hcy inhibited the expression of histone methyltransferase G9a. Mechanistically, silencing...

  1. Histone H3K9 methyltransferase G9a represses PPARγ expression and adipogenesis

    OpenAIRE

    Wang, Lifeng; Xu, Shiliyang; Lee, Ji-Eun; Baldridge, Anne; Grullon, Sean; Peng, Weiqun; Ge, Kai

    2012-01-01

    PPARγ promotes adipogenesis while Wnt proteins inhibit adipogenesis. However, the mechanisms that control expression of these positive and negative master regulators of adipogenesis remain incompletely understood. By genome-wide histone methylation profiling in preadipocytes, we find that among gene loci encoding adipogenesis regulators, histone methyltransferase (HMT) G9a-mediated repressive epigenetic mark H3K9me2 is selectively enriched on the entire PPARγ locus. H3K9me2 and G9a levels dec...

  2. HISTONE H3 LYSINE 9 METHYLTRANSFERASE G9a IS A TRANSCRIPTIONAL COACTIVATOR FOR NUCLEAR RECEPTORS*

    OpenAIRE

    David Y. Lee; Northrop, Jeffrey P.; Kuo, Min-Hao; Stallcup, Michael R.

    2006-01-01

    Methylation of Lys-9 of histone H3 has been associated with repression of transcription. G9a is a histone H3 Lys-9 methyltransferase localized in euchromatin and acts as a corepressor for specific transcription factors. Here we demonstrate that G9a also functions as a coactivator for nuclear receptors, cooperating synergistically with nuclear receptor coactivators GRIP1, CARM1, and p300 in transient transfection assays. This synergy depends strongly on the arginine-specific protein methyltran...

  3. The methyltransferase G9a regulates HoxA9-dependent transcription in AML

    OpenAIRE

    Lehnertz, Bernhard; Pabst, Caroline; Su, Le; Miller, Michelle; Liu, Feng; Yi, Lin; Zhang, Regan; Krosl, Jana; Yung, Eric; Kirschner, Jeanette; Rosten, Patty; Underhill, T. Michael; Jin, Jian; Hébert, Josée; Sauvageau, Guy

    2014-01-01

    Lehnertz et al. identify the histone methyltransferase G9a as a selective regulator of fast proliferating myeloid progenitors with no discernible function in hematopoietic stem cells. Loss of G9a significantly delays disease progression and reduces leukemia stem cell frequency in mouse models of acute myeloid leukemia. G9a interacts with the leukemogenic transcription factor HoxA9 and regulates HoxA9-dependent transcription. These results highlight G9a inhibition as a means to counteract the ...

  4. CCAAT displacement protein/cut homolog recruits G9a histone lysine methyltransferase to repress transcription

    OpenAIRE

    Nishio, Hitomi; Walsh, Martin J.

    2004-01-01

    CCAAT displacement protein/cut homolog (CDP/cut) is a highly conserved homeodomain protein that contains three cut repeat sequences. CDP/cut is a transcriptional factor for many diverse cellular and viral genes that are involved in most cellular processes, including differentiation, development, and proliferation. Here, we report that CDP/cut interacts with a histone lysine methyltransferase (HKMT), G9a, in vivo and in vitro. The deletion of the cut repeats within CDP/cut abrogates the intera...

  5. Negative Regulation of JAK2 by H3K9 Methyltransferase G9a in Leukemia

    OpenAIRE

    Son, Hye-Ju; Kim, Ji-Young; Hahn, Yoonsoo; Seo, Sang-Beom

    2012-01-01

    Histone methylation at specific lysine residues is a crucial regulatory process in transcriptional regulation. Using chromatin immunoprecipitation with microarray technology (ChIP-chip) analysis, we found that the H3K9-me2 target gene JAK2 was an important factor during differentiation of the HL-60 promyelocytic leukemia cell line by all-trans-retinoic acid (ATRA) treatment. Here, we report that the H3K9 methyltransferase G9a negatively regulated JAK2 transcription in histone methyltransferas...

  6. Three-dimensional culture sensitizes epithelial ovarian cancer cells to EZH2 methyltransferase inhibition

    Science.gov (United States)

    Amatangelo, Michael D.; Garipov, Azat; Li, Hua; Conejo-Garcia, Jose R.; Speicher, David W.; Zhang, Rugang

    2013-01-01

    Inhibitors of EZH2 methyltransferase activity have been demonstrated to selectively suppress the growth of diffused large B cell lymphoma (DLBCL) cells with gain-of-function mutations in EZH2, while exhibiting very limited effects on the growth of DLBCL cells with wild-type EZH2. Given that EZH2 is often overexpressed but not mutated in solid tumors, it is important to investigate the determinants of sensitivity of solid tumor cells to EZH2 inhibitors. In the current study, we show that three-dimensional (3D) culture of epithelial ovarian cancer (EOC) cells that overexpress EZH2 sensitizes these cells to EZH2 methyltransferase inhibition. Treatment of EOC cells with GSK343, a specific inhibitor of EZH2 methyltransferase, decreases the level of H3K27Me3, the product of EZH2’s enzymatic activity. However, GSK343 exhibited limited effects on the growth of EOC cells in conventional two-dimensional (2D) culture. In contrast, GSK343 significantly suppressed the growth of EOC cells cultured in 3D matrigel extracellular matrix (ECM), which more closely mimics the tumor microenvironment in vivo. Notably, GSK343 induces apoptosis of EOC cells in 3D but not 2D culture. In addition, GSK343 significantly inhibited the invasion of EOC cells. In summary, we show that the 3D ECM sensitizes EOC cells to EZH2 methyltransferase inhibition, which suppresses cell growth, induces apoptosis and inhibits invasion. Our findings imply that in EZH2 wild-type solid tumors, the ECM tumor microenvironment plays an important role in determining sensitivity to EZH2 inhibition and suggest that targeting the ECM represents a novel strategy for enhancing EZH2 inhibitor efficacy. PMID:23759589

  7. Kupplung der DNA-Methyltransferase M.SssI mit Triplehelix-bildenden Oligodesoxynucleotiden

    OpenAIRE

    Monami, Amélie Joséphine

    2007-01-01

    DNA modifying enzymes, like DNA methyltransferases (DNA MTases) and restriction endonucleases (REases), could in principle be used to map or manipulate genomes. However, these enzymes are inappropriate for that purpose because of their generally short recognition sequences. An increase of their sequence specificity would therefore be desirable. One strategy to produce megaspecific DNA modifying enzymes is to couple them with triple helix forming oligodeoxynucleotide (TFO). These additional DN...

  8. Catecholamine-o-methyltransferase polymorphisms are associated with postoperative pain intensity.

    LENUS (Irish Health Repository)

    Lee, Peter J

    2011-02-01

    single nucleotide polymorphisms (SNPs) in the genes for catecholamine-O-methyltransferase (COMT), μ-opioid receptor and GTP cyclohydrolase (GCH1) have been linked to acute and chronic pain states. COMT polymorphisms are associated with experimental pain sensitivity and a chronic pain state. No such association has been identified perioperatively. We carried out a prospective observational clinical trial to examine associations between these parameters and the development of postoperative pain in patients undergoing third molar (M3) extraction.

  9. Identification and Characterization of a Highly Conserved Crenarchaeal Protein Lysine Methyltransferase with Broad Substrate Specificity

    OpenAIRE

    Chu, Yindi; Zhang, Zhenfeng; Wang, Qian; Luo, Yuanming; Huang, Li

    2012-01-01

    Protein lysine methylation occurs extensively in the Crenarchaeota, a major kingdom in the Archaea. However, the enzymes responsible for this type of posttranslational modification have not been found. Here we report the identification and characterization of the first crenarchaeal protein lysine methyltransferase, designated aKMT, from the hyperthermophilic crenarchaeon Sulfolobus islandicus. The enzyme was capable of transferring methyl groups to selected lysine residues in a substrate prot...

  10. Genetic, physiological and biochemical characterization of multiple methanol methyltransferase isozymes in Methanosarcina acetivorans C2A.

    Science.gov (United States)

    Pritchett, Matthew A; Metcalf, William W

    2005-06-01

    Biochemical evidence suggests that methanol catabolism in Methanosarcina species requires the concerted effort of methanol:5-hydroxybenzimidazolylcobamide methyltransferase (MtaB), a corrinoid-containing methyl-accepting protein (MtaC) and Co-methyl-5-hydroxybenzimidazolylcobamide:2-mercapto-ethanesulphonic acid methyltransferase (MtaA). Here we show that Methanosarcina acetivorans possesses three operons encoding putative methanol-specific MtaB and corrinoid proteins: mtaCB1, mtaCB2 and mtaCB3. Deletion mutants lacking the three operons, in all possible combinations, were constructed and characterized. Strains deleted for any two of the operons grew on methanol, whereas strains lacking all three did not. Therefore, each operon encodes a bona fide methanol-utilizing MtaB/corrinoid protein pair. Most of the mutants were similar to the wild-type strain, with the exception of the DeltamtaCB1 DeltamtaCB2 double mutant, which grew more slowly and had reduced cell yields on methanol medium. However, all mutants displayed significantly longer lag times when switching from growth on trimethylamine to growth on methanol. This indicates that all three operons are required for wild-type growth on methanol and suggests that each operon has a distinct role in the metabolism of this substrate. The combined methanol:CoM methyltransferase activity of strains carrying only mtaCB1 was twofold higher than strains carrying only mtaCB2 and fourfold higher than strains carrying only mtaCB3. Interestingly, the presence of the mtaCB2 and mtaCB3 operons, in addition to the mtaCB1 operon, did not increase the overall methyltransferase activity, suggesting that these strains may be limited by MtaA availability. All deletion mutants were unaffected with respect to growth on trimethylamine and acetate corroborating biochemical evidence indicating that each methanogenic substrate has specific methyltransfer enzymes. PMID:15882413

  11. The histone methyltransferase and putative oncoprotein MMSET is overexpressed in a large variety of human tumors

    DEFF Research Database (Denmark)

    Hudlebusch, Heidi Rye; Santoni-Rugiu, Eric; Simon, Ronald;

    2011-01-01

    Multiple myeloma SET (Suppressor of variegation, Enhancer of zeste, and Trithorax) domain (MMSET) is a histone lysine methyltransferase deregulated in a subgroup of multiple myelomas with the t(4;14)(p16;q32) translocation and poor prognosis. With the aim of understanding, if MMSET can be involve...... in other types of cancer we investigated the expression of MMSET protein in different types of human tumors....

  12. Is catechol-o-methyltransferase gene polymorphism a risk factor in the development of premenstrual syndrome?

    OpenAIRE

    Deveci, Esma Ozturk; Incebiyik, Adnan; Selek, Salih; Camuzcuoglu, Aysun; Hilali, Nese Gul; Camuzcuoglu, Hakan; Erdal, Mehmet Emin; Vural, Mehmet

    2014-01-01

    Objective The objective of this study was to investigate whether there was a correlation between catechol-o-methyltransferase (COMT) gene polymorphism, which is believed to play a role in the etiology of psychotic disorders, and premenstrual syndrome (PMS). Methods Fifty-three women with regular menstrual cycles, aged between 18 and 46 years and diagnosed with PMS according to the American Congress of Obstetrics and Gynecology criteria were included in this study as the study group, and 53 he...

  13. Sensorimotor gating of schizophrenia patients depends on catechol O-methyltransferase Val158Met polymorphism

    OpenAIRE

    Quednow, B B; Wagner, M.; Mössner, R; Maier, W; Kühn, K U

    2010-01-01

    It has been recently shown that Catechol O-methyltransferase (COMT) Val(158)Met polymorphism strongly influences prepulse inhibition (PPI) of the acoustic startle response (ASR) in healthy human volunteers. Given that schizophrenia patients exhibit impairment in PPI and that COMT is a putative susceptibility gene for schizophrenia, we investigated the impact of the COMT Val(158)Met polymorphisms on PPI in schizophrenic inpatients. We analyzed COMT Val(158)Met polymorphisms and assessed startl...

  14. Genetic Contribution of Catechol-O-methyltransferase Polymorphism in Patients with Migraine without Aura

    OpenAIRE

    Park, Jeong Wook; Lee, Kwang Soo; Kim, Joong Seok; Kim, Yeong In; Shin, Hae Eun

    2007-01-01

    Background Recent genetic association studies have investigated the possible genetic role of the dopaminergic system in migraine. Catechol-O-methyltransferase (COMT) is an enzyme that plays a crucial role in the metabolism of dopamine and its genetic polymorphism is associated with three- to fourfold variation of enzymatic activity. Objectives The objective of this study was to elucidate the role of the COMT polymorphism in the genetic susceptibility to migraine and its phenotypic expression ...

  15. Polymorphism of the catechol-O-methyltransferase gene in Han Chinese patients with psoriasis vulgaris

    OpenAIRE

    Lin Gao; Hong Li; Kai Li; Zhu Shen; Ling Liu; Chunying Li; Zhengdong Zhang; Yufeng Liu

    2009-01-01

    Psoriasis vulgaris is defined by a series of linked cellular changes in the skin: hyperplasia of epidermal keratinocytes, vascular hyperplasia and ectasia, and infiltration of T lymphocytes, neutrophils and other types of leukocytes in the affected skin. Catechol-O-methyltransferase ( COMT ) 158 polymorphism can reduce the activity of the COMT enzyme that may trigger defective differentiation of keratinocytes in psoriasis. Immunocytes can degrade and inactivate catecholamines via monamine oxi...

  16. Catechol-O-methyltransferase (COMT) influences the connectivity of the prefrontal cortex at rest

    OpenAIRE

    Tunbridge, Elizabeth M.; Farrell, Sarah M.; Harrison, Paul J; Mackay, Clare E

    2013-01-01

    Catechol-O-methyltransferase (COMT) modulates dopamine in the prefrontal cortex (PFC) and influences PFC dopamine-dependent cognitive task performance. A human COMT polymorphism (Val158Met) alters enzyme activity and is associated with both the activation and functional connectivity of the PFC during task performance, particularly working memory. Here, we used functional magnetic resonance imaging and a data-driven, independent components analysis (ICA) approach to compare resting state funct...

  17. Polymorphism of the catechol-O-methyltransferase gene in Han Chinese patients with psoriasis vulgaris

    OpenAIRE

    Gao, Lin; Li, Hong; Li, Kai; Shen, Zhu; Liu, Ling; Li, Chunying; Zhang, Zhengdong; Liu, Yufeng

    2009-01-01

    Psoriasis vulgaris is defined by a series of linked cellular changes in the skin: hyperplasia of epidermal keratinocytes, vascular hyperplasia and ectasia, and infiltration of T lymphocytes, neutrophils and other types of leukocytes in the affected skin. Catechol-O-methyltransferase (COMT) 158 polymorphism can reduce the activity of the COMT enzyme that may trigger defective differentiation of keratinocytes in psoriasis. Immunocytes can degrade and inactivate catecholamines via monamine oxida...

  18. Towards more specific O6-Methylguanine-DNA methyltransferase (MGMT) inactivators

    OpenAIRE

    CORDEIRO MACHADO, ALESSANDRA; MC MURRY, THOMAS; Rozas, Isabel

    2011-01-01

    PUBLISHED Searching for a novel family of inactivators of the human DNA repair protein O6?methylguanine?DNA methyltransferase (MGMT) which is known to bind to the DNA minor groove, we have computationally modelled and synthesised two series of 2?amino?6?aryloxy?5?nitropyrimidines with morpholino or aminodiaryl substituents (potential minor groove binders) at the 4?position. Synthesis of these compounds was achieved by successive substitution of each of the two Cl atoms of 2?amino?4,6?dichl...

  19. miR-221/222 Target the DNA Methyltransferase MGMT in Glioma Cells

    OpenAIRE

    Cristina Quintavalle; Davide Mangani; Giuseppina Roscigno; Giulia Romano; Angel Diaz-Lagares; Margherita Iaboni; Elvira Donnarumma; Danilo Fiore; Pasqualino De Marinis; Ylermi Soini; Manel Esteller; Gerolama Condorelli

    2013-01-01

    Glioblastoma multiforme (GBM) is one of the most deadly types of cancer. To date, the best clinical approach for treatment is based on administration of temozolomide (TMZ) in combination with radiotherapy. Much evidence suggests that the intracellular level of the alkylating enzyme O(6)-methylguanine-DNA methyltransferase (MGMT) impacts response to TMZ in GBM patients. MGMT expression is regulated by the methylation of its promoter. However, evidence indicates that this is not the only regula...

  20. O6-methylguanine-DNA methyltransferase (MGMT): can function explain a suicidal mechanism?

    OpenAIRE

    Gouws, Chrisna; Pretorius, Petrus Jacobus

    2011-01-01

    Why does O6-methylguanine-DNA methyltransferase (MGMT), an indispensable DNA repair enzyme, have a mechanism which seems to run counter to its importance? This enzyme is key to the removal of detrimental alkyl adducts from guanine bases. Although the mechanism is well known, an unusual feature surrounds its mode of action, which is its so-called suicidal endpoint. In addition, induction of MGMT is highly variable and its kinetics is atypical. These features raise some questions on the seeming...

  1. Arsenic (+3 oxidation state) methyltransferase and the inorganic arsenic methylation phenotype

    International Nuclear Information System (INIS)

    Inorganic arsenic is enzymatically methylated; hence, its ingestion results in exposure to the parent compound and various methylated arsenicals. Both experimental and epidemiological evidences suggest that some of the adverse health effects associated with chronic exposure to inorganic arsenic may be mediated by these methylated metabolites. If i As methylation is an activation process, then the phenotype for inorganic arsenic methylation may determine risk associated with exposure to this metalloid. We examined inorganic arsenic methylation phenotypes and arsenic (+3 oxidation state) methyltransferase genotypes in four species: three that methylate inorganic arsenic (human (Homo sapiens), rat (Rattus norwegicus), and mouse (Mus musculus)) and one that does not methylate inorganic arsenic (chimpanzee, Pan troglodytes). The predicted protein products from arsenic (+3 oxidation state) methyltransferase are similar in size for rat (369 amino acid residues), mouse (376 residues), and human (375 residues). By comparison, a 275-nucleotide deletion beginning at nucleotide 612 in the chimpanzee gene sequence causes a frameshift that leads to a nonsense mutation for a premature stop codon after amino acid 205. The null phenotype for inorganic arsenic methylation in the chimpanzee is likely due to the deletion in the gene for arsenic (+3 oxidation state) methyltransferase that yields an inactive truncated protein. This lineage-specific loss of function caused by the deletion event must have occurred in the Pan lineage after Homo-Pan divergence about 5 million years ago

  2. Chromatin Targeting of de Novo DNA Methyltransferases by the PWWP Domain

    Institute of Scientific and Technical Information of China (English)

    Ying-ZiGe; Min-TiePu; HumairaGowher; Hai-PingWu; Jian-PingDing; AlbertJeltsch; Guo-LiangXu

    2005-01-01

    DNA methylation patterns of mammalian genomes are generated in gametogenesis and early embryonic development. Two de novo DNA methyltransferases, Dnmt3a and Dnmt3b, are responsible for the process. Both en-zymes contain a long N-terminal regulatory region linked to a conserved C-terminal domain responsible forthe catalytic activity. Although a PWWP domain in the N-terminal region has been shown to bind DNA in vitro, it is unclear how the DNA methyltransferases access their substrate in chromatin in vivo. We show here that the two proteins are associated with chromatin including mitotic chromosomes in mammalian cells, and the PWWP domain is essential for the chromatin targeting of the enzymes. The functional significance of PWWPmediated chromatin targeting is suggested by the fact that a missense mutation in this domain of human DNMT3B causes immunodeficiency, centromeric heterochromatin instability, facial anomalies (ICF) syndrome, which is characterized by loss of methylation insatellite DNA, pericentromeric instability, and immunodeficiency. We demonstrate that the mutant protein completely loses its chromatin targeting capacity. Our data establish the PWWP domain as a novel chromatin/chromosome-targeting module and suggest that the PWWP-mediated chromatin association is essential for the function of the de novo methyltransferases during development.

  3. Transcriptome profiling of Set5 and Set1 methyltransferases: Tools for visualization of gene expression

    Directory of Open Access Journals (Sweden)

    Glòria Mas Martín

    2014-12-01

    Full Text Available Cells regulate transcription by coordinating the activities of multiple histone modifying complexes. We recently identified the yeast histone H4 methyltransferase Set5 and discovered functional overlap with the histone H3 methyltransferase Set1 in gene expression. Specifically, using next-generation RNA sequencing (RNA-Seq, we found that Set5 and Set1 function synergistically to regulate specific transcriptional programs at subtelomeres and transposable elements. Here we provide a comprehensive description of the methodology and analysis tools corresponding to the data deposited in NCBI's Gene Expression Omnibus (GEO under the accession number GSE52086. This data complements the experimental methods described in Mas Martín G et al. (2014 and provides the means to explore the cooperative functions of histone H3 and H4 methyltransferases in the regulation of transcription. Furthermore, a fully annotated R code is included to enable researchers to use the following computational tools: comparison of significant differential expression (SDE profiles; gene ontology enrichment of SDE; and enrichment of SDE relative to chromosomal features, such as centromeres, telomeres, and transposable elements. Overall, we present a bioinformatics platform that can be generally implemented for similar analyses with different datasets and in different organisms.

  4. Rapid restriction enzyme free detection of DNA methyltransferase activity based on DNA-templated silver nanoclusters.

    Science.gov (United States)

    Kermani, Hanie Ahmadzade; Hosseini, Morteza; Dadmehr, Mehdi; Ganjali, Mohammad Reza

    2016-06-01

    DNA methylation has significant roles in gene regulation. DNA methyltransferase (MTase) enzyme characterizes DNA methylation and also induces an aberrant methylation pattern that is related to many diseases, especially cancers. Thus, it is required to develop a method to detect the DNA MTase activity. In this study, we developed a new sensitive and reliable method for methyltransferase activity assay by employing DNA-templated silver nanoclusters (DNA/Ag NCs) without using restriction enzymes. The Ag NCs have been utilized for the determination of M.SssI MTase activity and its inhibition. We designed an oligonucleotide probe which contained an inserted six-cytosine loop as Ag NCs formation template. The changes in fluorescence intensity were monitored to quantify the M.SssI activity. The fluorescence spectra showed a linear decrease in the range of 0.4 to 20 U/ml with a detection limit of 0.1 U/ml, which was significant compared with previous reports. The proposed method was applied successfully for demonstrating the Gentamicin effect as MTase inhibitor. The proposed method showed convenient reproducibility and sensitivity indicating its potential for the determination of methyltransferase activity. PMID:27052776

  5. miR-29 Represses the Activities of DNA Methyltransferases and DNA Demethylases

    Directory of Open Access Journals (Sweden)

    Izuho Hatada

    2013-07-01

    Full Text Available Members of the microRNA-29 (miR-29 family directly target the DNA methyltransferases, DNMT3A and DNMT3B. Disturbances in the expression levels of miR-29 have been linked to tumorigenesis and tumor aggressiveness. Members of the miR-29 family are currently thought to repress DNA methylation and suppress tumorigenesis by protecting against de novo methylation. Here, we report that members of the miR-29 family repress the activities of DNA methyltransferases and DNA demethylases, which have opposing roles in control of DNA methylation status. Members of the miR-29 family directly inhibited DNA methyltransferases and two major factors involved in DNA demethylation, namely tet methylcytosine dioxygenase 1 (TET1 and thymine DNA glycosylase (TDG. Overexpression of miR-29 upregulated the global DNA methylation level in some cancer cells and downregulated DNA methylation in other cancer cells, suggesting that miR-29 suppresses tumorigenesis by protecting against changes in the existing DNA methylation status rather than by preventing de novo methylation of DNA.

  6. Purification of homologous protein carboxyl methyltransferase isozymes from human and bovine erythrocytes

    International Nuclear Information System (INIS)

    The authors have purified the two major isozymes of the L-isoaspartyl/D-aspartyl protein methyltransferase from both human and bovine erythrocytes. These four enzymes all have polypeptide molecular weights of approximately 26,500 and appear to be monomers in solution. Each of these enzymes cross-reacts with antibodies directed against protein carboxyl methyltransferase I from bovine brain. Their structures also appear to be similar when analyzed by dodecyl sulfate gel electrophoresis for the large fragments produced by digestion with Staphylococcus aureus protease V8 or when analyzed by high-performance liquid chromatography (HPLC) for tryptic peptides. The structural relatedness of these enzymes was confirmed by sequence analysis of a total of 433 residues in 32 tryptic fragments of the human erythrocyte isozymes I and II and of the bovine erythrocyte isozyme II. They found sequence identity or probable identity in 111 out of 112 residues when they compared the human isozymes I and II and identities in 127 out of 134 residues when the human and bovine isozymes II were compared. These results suggest that the erythrocyte isozymes from both organisms may have nearly identical structures and confirm the similarities in the function of these methyltransferases that have been previously demonstrated

  7. Preliminary characterization of (nucleoside-2′-O-)-methyltransferase crystals from Meaban and Yokose flaviviruses

    Energy Technology Data Exchange (ETDEWEB)

    Mastrangelo, Eloise; Bollati, Michela; Milani, Mario [Department of Biomolecular Sciences and Biotechnology, CNR-INFM, University of Milano, Via Celoria 26, 20133 Milano (Italy); Lamballeire, Xavier de; Brisbare, Nadege [Unité des Virus Emergents, Faculté de Médecine, 27 Boulevard Jean Moulin, 13005 Marseille (France); Dalle, Karen; Lantez, Violaine; Egloff, Marie-Pierre; Coutard, Bruno; Canard, Bruno [Laboratoire Architecture et Fonction des Macromolécules Biologiques, UMR 6098 CNRS ESIL, Case 932, 163 Avenue de Luminy, 13288 Marseille CEDEX 9 (France); Gould, Ernest; Forrester, Naomi [CEH Oxford, Mansfield Road, Oxford OX1 3SR (United Kingdom); Bolognesi, Martino, E-mail: martino.bolognesi@unimi.it [Department of Biomolecular Sciences and Biotechnology, CNR-INFM, University of Milano, Via Celoria 26, 20133 Milano (Italy)

    2006-08-01

    Two methyltransferases from flaviviruses (Meaban and Yokose viruses) have been overexpressed and crystallized. Diffraction data and characterization of the two crystal forms are presented, together with a preliminary molecular-replacement solution for both enzymes. Viral methyltranferases (MTase) are involved in the third step of the mRNA-capping process, transferring a methyl group from S-adenosyl-l-methionine (SAM) to the capped mRNA. MTases are classified into two groups: (guanine-N7)-methyltransferases (N7MTases), which add a methyl group onto the N7 atom of guanine, and (nucleoside-2′-O-)-methyltransferases (2′OMTases), which add a methyl group to a ribose hydroxyl. The MTases of two flaviviruses, Meaban and Yokose viruses, have been overexpressed, purified and crystallized in complex with SAM. Characterization of the crystals together with details of preliminary X-ray diffraction data collection (at 2.8 and 2.7 Å resolution, respectively) are reported here. The sequence homology relative to Dengue virus 2′OMTase and the structural conservation of specific residues in the putative active sites suggest that both enzymes belong to the 2′OMTase subgroup.

  8. MtrA of the sodium ion pumping methyltransferase binds cobalamin in a unique mode.

    Science.gov (United States)

    Wagner, Tristan; Ermler, Ulrich; Shima, Seigo

    2016-01-01

    In the three domains of life, vitamin B12 (cobalamin) is primarily used in methyltransferase and isomerase reactions. The methyltransferase complex MtrA-H of methanogenic archaea has a key function in energy conservation by catalysing the methyl transfer from methyl-tetrahydromethanopterin to coenzyme M and its coupling with sodium-ion translocation. The cobalamin-binding subunit MtrA is not homologous to any known B12-binding proteins and is proposed as the motor of the sodium-ion pump. Here, we present crystal structures of the soluble domain of the membrane-associated MtrA from Methanocaldococcus jannaschii and the cytoplasmic MtrA homologue/cobalamin complex from Methanothermus fervidus. The MtrA fold corresponds to the Rossmann-type α/β fold, which is also found in many cobalamin-containing proteins. Surprisingly, the cobalamin-binding site of MtrA differed greatly from all the other cobalamin-binding sites. Nevertheless, the hydrogen-bond linkage at the lower axial-ligand site of cobalt was equivalently constructed to that found in other methyltransferases and mutases. A distinct polypeptide segment fixed through the hydrogen-bond linkage in the relaxed Co(III) state might be involved in propagating the energy released upon corrinoid demethylation to the sodium-translocation site by a conformational change. PMID:27324530

  9. Special characteristics of fluorescence and resonance Rayleigh scattering for cadmium telluride nanocrystal aqueous solution and its interactions with aminoglycoside antibiotics

    Institute of Scientific and Technical Information of China (English)

    LI TaiShan; LIU ShaoPu; LIU ZhongFang; HU XiaoLi; ZHANG LiPing

    2009-01-01

    CdTe nanocrystals (CdTe NCs) were achieved by reaction of CdCl2 with KHTe solution and were capped with sodium mercaptoacetate. The product was detected by transmission electron microscopy (TEM), high-resolution transmission electron microscopy (HRTEM), energy dispersive spectroscopy (EDS), fluorescence spectra, ultraviolet-visible spectra and X-ray diffraction (XRD). The CdTe NCs are of cubic structure and the average size is about 5 nm. The fluorescence quantum yield of CdTe NCs aqueous solution increased from 37% to 97% after 20 d under room light. The maximum λem of fluorescence changed from 543 nm to 510 nm and the blue shift was 33 nm. CdTe NCs aqueous solution can be steady for at least 10 months at 4℃ in a refrigerator. The resonance Rayleigh scattering (RRS) of CdTe NCs in the aqueous solution was investigated. The maximum scattering peak was located at about 554 nm. The interactions of CdTe NCs with amikacin sulfate (AS) and micronomicin sulfate (MS) were in-vestigated respectively. The effects of AS and MS on fluorescence and RRS of CdTe NCs were analyzed. It was found that AS and MS quenched the photoluminescence of CdTe NCs and enhanced RRS of CdTe NCs. Under optimum conditions, there are linear relationships between quenching intensity (F0-F), intensity of RRS (1-10) and concentration of AS and MS. The detection limits (3σ) of AS and MS are re-spectively 3.4 ng.mL-1 and 2.6 ng.mL-1 by the fluorescence quenching method, and 15.2 ng.mL-1 and 14.0 ng.mL-1 by the RRS method. The methods have high sensitivity, thus CdTe NCs may be used as fluorescence probes and RRS probes for the detection of aminoglycoside antibiotics.

  10. Kinetics of kill of bacterial conjunctivitis isolates with moxifloxacin, a fluoroquinolone, compared with the aminoglycosides tobramycin and gentamicin

    Directory of Open Access Journals (Sweden)

    Rudolph S Wagner

    2010-01-01

    Full Text Available Rudolph S Wagner1, David B Granet2, Steven J Lichtenstein3, Tiffany Jamison4, Joseph J Dajcs4, Robert D Gross5, Paul Cockrum41New Jersey Medical School, Newark, NJ, USA; 2Ratner Children’s Eye Center, University of California – San Diego, La Jolla, CA, USA; 3University of Illinois College of Medicine at Peoria, Peoria, Illinois, USA; 4Alcon Research, Ltd, Fort Worth, TX, USA; 5Department of Ophthalmology, University of Texas Southwestern Medical Center, Dallas, TX, USAPurpose: To compare the kinetics and speed of kill of Streptococcus pneumoniae and Haemophilus influenzae on exposure to three topical ophthalmic antibiotic solutions.Materials and methods: Bacterial conjunctivitis isolates of S. pneumoniae and H. influenzae were exposed to 1:1000 dilutions of moxifloxacin 0.5%, tobramycin 0.3%, gentamicin 0.3%, and water (control. At 15, 30, 60, 120, and 180 minutes after exposure, aliquots were collected, cells were cultured, and viable cell counts were determined using standard microbiological methods.Results: Moxifloxacin achieved 99.9% kill (3-log reduction at approximately 2 hours for S. pneumoniae and at 15 minutes for H. influenzae. Tobramycin and gentamicin did not achieve 3-log reduction of S. pneumoniae during the 180-minute study period. An increase in bacterial growth was noted for these isolates. Gentamicin took more than 120 minutes to achieve the 3-log reduction of H. influenzae and tobramycin did not reach the 3-log reduction of this pathogen during the 180-minute study period.Conclusion: Moxifloxacin killed S. pneumoniae and H. influenzae in vitro faster than tobramycin and gentamicin, suggesting its potential clinical benefit as a first-line treatment for bacterial conjunctivitis to minimize patient symptoms and to limit the contagiousness of the disease.Keywords: kinetics of kill, bacterial conjunctivitis, in vitro, Streptococcus pneumoniae, Haemophilus influenzae, fluoroquinolones, aminoglycosides

  11. Ex vivo treatment with a novel synthetic aminoglycoside NB54 in primary fibroblasts from Rett syndrome patients suppresses MECP2 nonsense mutations.

    Directory of Open Access Journals (Sweden)

    Manuela Vecsler

    Full Text Available BACKGROUND: Nonsense mutations in the X-linked methyl CpG-binding protein 2 (MECP2 comprise a significant proportion of causative MECP2 mutations in Rett syndrome (RTT. Naturally occurring aminoglycosides, such as gentamicin, have been shown to enable partial suppression of nonsense mutations related to several human genetic disorders, however, their clinical applicability has been compromised by parallel findings of severe toxic effects. Recently developed synthetic NB aminoglycosides have demonstrated significantly improved effects compared to gentamicin evident in substantially higher suppression and reduced acute toxicity in vitro. RESULTS: We performed comparative study of suppression effects of the novel NB54 and gentamicin on three MECP2 nonsense mutations (R294X, R270X and R168X common in RTT, using ex vivo treatment of primary fibroblasts from RTT patients harboring these mutations and testing for the C-terminal containing full-length MeCP2. We observed that NB54 induces dose-dependent suppression of MECP2 nonsense mutations more efficiently than gentamicin, which was evident at concentrations as low as 50 µg/ml. NB54 read-through activity was mutation specific, with maximal full-length MeCP2 recovery in R168X (38%, R270X (27% and R294X (18%. In addition, the recovered MeCP2 was translocated to the cell nucleus and moreover led to parallel increase in one of the most important MeCP2 downstream effectors, the brain derived neurotrophic factor (BDNF. CONCLUSION: Our findings suggest that NB54 may induce restoration of the potentially functional MeCP2 in primary RTT fibroblasts and encourage further studies of NB54 and other rationally designed aminoglycoside derivatives as potential therapeutic agents for nonsense MECP2 mutations in RTT.

  12. Transcriptome Profiling of Antimicrobial Resistance in Pseudomonas aeruginosa.

    Science.gov (United States)

    Khaledi, Ariane; Schniederjans, Monika; Pohl, Sarah; Rainer, Roman; Bodenhofer, Ulrich; Xia, Boyang; Klawonn, Frank; Bruchmann, Sebastian; Preusse, Matthias; Eckweiler, Denitsa; Dötsch, Andreas; Häussler, Susanne

    2016-08-01

    Emerging resistance to antimicrobials and the lack of new antibiotic drug candidates underscore the need for optimization of current diagnostics and therapies to diminish the evolution and spread of multidrug resistance. As the antibiotic resistance status of a bacterial pathogen is defined by its genome, resistance profiling by applying next-generation sequencing (NGS) technologies may in the future accomplish pathogen identification, prompt initiation of targeted individualized treatment, and the implementation of optimized infection control measures. In this study, qualitative RNA sequencing was used to identify key genetic determinants of antibiotic resistance in 135 clinical Pseudomonas aeruginosa isolates from diverse geographic and infection site origins. By applying transcriptome-wide association studies, adaptive variations associated with resistance to the antibiotic classes fluoroquinolones, aminoglycosides, and β-lactams were identified. Besides potential novel biomarkers with a direct correlation to resistance, global patterns of phenotype-associated gene expression and sequence variations were identified by predictive machine learning approaches. Our research serves to establish genotype-based molecular diagnostic tools for the identification of the current resistance profiles of bacterial pathogens and paves the way for faster diagnostics for more efficient, targeted treatment strategies to also mitigate the future potential for resistance evolution. PMID:27216077

  13. Structural Basis for Dual Functionality of Isoflavonoid O-Methyltransferases in the Evolution of Plant Defense Responses

    Energy Technology Data Exchange (ETDEWEB)

    Liu, C.-J.; Deavours, B.E.; Richard, S.B.; Ferrer, J.-L.; Blount, J.W.; Huhman, D.; Dixon, R.A.; Noel, J.

    2007-07-10

    In leguminous plants such as pea (Pisum sativum), alfalfa (Medicago sativa), barrel medic (Medicago truncatula), and chickpea (Cicer arietinum), 4'-O-methylation of isoflavonoid natural products occurs early in the biosynthesis of defense chemicals known as phytoalexins. However, among these four species, only pea catalyzes 3-O-methylation that converts the pterocarpanoid isoflavonoid 6a-hydroxymaackiain to pisatin. In pea, pisatin is important for chemical resistance to the pathogenic fungus Nectria hematococca. While barrel medic does not biosynthesize 6a-hydroxymaackiain, when cell suspension cultures are fed 6a-hydroxymaackiain, they accumulate pisatin. In vitro, hydroxyisoflavanone 4'-O-methyltransferase (HI4'OMT) from barrel medic exhibits nearly identical steady state kinetic parameters for the 4'-O-methylation of the isoflavonoid intermediate 2,7,4'-trihydroxyisoflavanone and for the 3-O-methylation of the 6a-hydroxymaackiain isoflavonoid-derived pterocarpanoid intermediate found in pea. Protein x-ray crystal structures of HI4'OMT substrate complexes revealed identically bound conformations for the 2S,3R-stereoisomer of 2,7,4'-trihydroxyisoflavanone and the 6aR,11aR-stereoisomer of 6a-hydroxymaackiain. These results suggest how similar conformations intrinsic to seemingly distinct chemical substrates allowed leguminous plants to use homologous enzymes for two different biosynthetic reactions. The three-dimensional similarity of natural small molecules represents one explanation for how plants may rapidly recruit enzymes for new biosynthetic reactions in response to changing physiological and ecological pressures.

  14. Structural Basis for Dual Functionality of Isoflavonoid O-Methyltransferases in the Evolution of Plant Defense Responses

    Energy Technology Data Exchange (ETDEWEB)

    Liu, C.; Deavours, B; Richard, S; Ferrer, J; Blount, J; Huhman, D; Dixon, R; Noel, J

    2006-01-01

    In leguminous plants such as pea (Pisum sativum), alfalfa (Medicago sativa), barrel medic (Medicago truncatula), and chickpea (Cicer arietinum), 4'-O-methylation of isoflavonoid natural products occurs early in the biosynthesis of defense chemicals known as phytoalexins. However, among these four species, only pea catalyzes 3-O-methylation that converts the pterocarpanoid isoflavonoid 6a-hydroxymaackiain to pisatin. In pea, pisatin is important for chemical resistance to the pathogenic fungus Nectria hematococca. While barrel medic does not biosynthesize 6a-hydroxymaackiain, when cell suspension cultures are fed 6a-hydroxymaackiain, they accumulate pisatin. In vitro, hydroxyisoflavanone 4'-O-methyltransferase (HI4'OMT) from barrel medic exhibits nearly identical steady state kinetic parameters for the 4'-O-methylation of the isoflavonoid intermediate 2,7,4'-trihydroxyisoflavanone and for the 3-O-methylation of the 6a-hydroxymaackiain isoflavonoid-derived pterocarpanoid intermediate found in pea. Protein x-ray crystal structures of HI4'OMT substrate complexes revealed identically bound conformations for the 2S,3R-stereoisomer of 2,7,4'-trihydroxyisoflavanone and the 6aR,11aR-stereoisomer of 6a-hydroxymaackiain. These results suggest how similar conformations intrinsic to seemingly distinct chemical substrates allowed leguminous plants to use homologous enzymes for two different biosynthetic reactions. The three-dimensional similarity of natural small molecules represents one explanation for how plants may rapidly recruit enzymes for new biosynthetic reactions in response to changing physiological and ecological pressures.

  15. Marker-free plasmids for gene therapeutic applications--lack of antibiotic resistance gene substantially improves the manufacturing process.

    Science.gov (United States)

    Mairhofer, Jürgen; Cserjan-Puschmann, Monika; Striedner, Gerald; Nöbauer, Katharina; Razzazi-Fazeli, Ebrahim; Grabherr, Reingard

    2010-04-01

    Plasmid DNA is being considered as a promising alternative to traditional protein vaccines or viral delivery methods for gene therapeutic applications. DNA-based products are highly flexible, stable, are easily stored and can be manufactured on a large scale. Although, much safer than viral approaches, issues have been raised with regard to safety due to possible integration of plasmid DNA into cellular DNA or spread of antibiotic resistance genes to intestinal bacteria by horizontal gene transfer. Accordingly, there is interest in methods for the production of plasmid DNA that lacks the antibiotic resistance gene to further improve their safety profile. Here, we report for the first time the gram-scale manufacturing of a minimized plasmid that is devoid of any additional sequence elements on the plasmid backbone, and merely consists of the target expression cassette and the bacterial origin of replication. Three different host/vector combinations were cultivated in a fed-batch fermentation process, comparing the progenitor strain JM108 to modified strains JM108murselect, hosting a plasmid either containing the aminoglycoside phosphotransferase which provides kanamycin resistance, or a marker-free variant of the same plasmid. The metabolic load exerted by expression of the aminoglycoside phosphotransferase was monitored by measuring ppGpp- and cAMP-levels. Moreover, we revealed that JM108 is deficient of the Lon protease and thereby refined the genotype of JM108. The main consequences of Lon-deficiency with regard to plasmid DNA production are discussed herein. Additionally, we found that the expression of the aminoglycoside phosphotransferase, conferring resistance to kanamycin, was very high in plasmid DNA producing processes that actually inclusion bodies were formed. Thereby, a severe metabolic load on the host cell was imposed, detrimental for overall plasmid yield. Hence, deleting the antibiotic resistance gene from the vector backbone is not only beneficial

  16. [Multidrug resistance in Klebsiella pneumoniae: multicenter study].

    Science.gov (United States)

    Boutiba-Ben Boubaker, Ilhem; Ben Salah, Dorra; Besbes, Makram; Mahjoubi, Faouzia; Ghozzi, Rafiaa; Ben Redjeb, Saida; Ben Hassen, Assia; Hammami, Adnène

    2002-01-01

    The extensive use of broad spectrum antibiotics, especially the third generation cephalosporins (C3G), was followed by the emergence of newer plasmid mediated betalactamases called extended spectrum betalactamases (ESBLs). To assess the impact of K. pneumoniae resistant to 3GC in Tunisia, this study was conducted in 3 teaching hospitals. A total of 1110 strains of K pneumoniae was collected. The antibiotics susceptibilities were tested by diffusion method using Mueller-Hinton agar. The quality control was regularly performed. I ESBLs producing solates were detected using the double-disc synergy test. Data analysis was done using the Whonet 4 software. 23.6% K. pneumoniae isolates showed phenotype pattern of ESBLs producers. The double-disc synergy test was positive in 75% of the cases. These isolates were recovered from hospitalized patients in different wards but mainly from pediatrics (23.6%), medicine (23.2%), surgery (22.9%), intensive care units (11%) and neonatology (11%). 54% were isolated from urines, 22% from blood cultures. These isolates remained susceptible to imipenem (100%) and most of them to cefoxitin (96.4%) but all had associated resistance to aminoglycosides, quinolones and trimethoprim-sulfamethoxazole. The prevalence of multidrug resistant K. pneumoniae is high. This resistance can be minimized by the implementation of infection control measures including handwashing and isolation procedures. PMID:12071040

  17. Mechanisms of antibiotic resistance of Enterobacteriaceae family representatives

    Directory of Open Access Journals (Sweden)

    K. R. Kotsyuba

    2014-04-01

    Full Text Available The paper deals with the basic medical scheme of antibiotics use for treatment of lesions caused by enterobacteria and mechanisms of resistance of Enterobacteriaceae to different classes of antibiotics. It is known that the main mechanisms of resistance to antibiotics are enzymatic inactivation, modification of the target, efflux, violation of conduct through the membrane and formation of metabolic shunt. The most common cases of resistance to beta-lactams among Enterobacteriaceae relate to production of plasmid and chromosomal beta-lactamases, violation of the permeability of the outer membrane, and modification of target penicillin binding proteins. Active release of antibiotics from the cell, or efflux, in Enterobacteriaceae is used for maintaining resistance to tetracyclines, macrolides, carbapenems. Genes of efflux system are localized on plasmids and contribute to rapid spreading among Enterobacteriaceae. Mutations are the basis of resistance to novobiocinum and rifampicinum. Enzymatic inactivation by modifying is typical for resistance to aminoglycosides. Three groups of enzymes are engaged in the process, by adding the molecule of acetic acid, phosphate or adenine. Joining of these groups is irreversible and leads to complete loss of biological activity of the antibiotic. Resistance to aminoglycosides appears also due to inhibition of drug penetration, that is associated with genetically determined mechanisms of electron transport through the membrane. Resistance to quinolones and fluoroquinolones is associated with the modification of topoisomerase II and IV which are targets of these groups of antibiotics. Resistance is possible as a result of changes in the structure of the target, breaching of penetration into the cell, and active release from the cell. The highest level of resistance is develope in the case of two- or three-stage mutations in one or the other, or both, subunits in different genes. At the same time, for breaching of

  18. The catechol-O-methyltransferase inhibitory potential of Z-vallesiachotamine by in silicoand in vitro approaches

    Directory of Open Access Journals (Sweden)

    Carolina dos Santos Passos

    2015-08-01

    Full Text Available AbstractZ-Vallesiachotamine is a monoterpene indole alkaloid that has a β-N-acrylate group in its structure. This class of compounds has already been described in different Psychotriaspecies. Our research group observed that E/Z-vallesiachotamine exhibits a multifunctional feature, being able to inhibit targets related to neurodegeneration, such as monoamine oxidase A, sirtuins 1 and 2, and butyrylcholinesterase enzymes. Aiming at better characterizing the multifunctional profile of this compound, its effect on cathecol-O-methyltransferase activity was investigated. The cathecol-O-methyltransferase activity was evaluated in vitro by a fluorescence-based method, using S-(5′-adenosyl-l-methionine as methyl donor and aesculetin as substrate. The assay optimization was performed varying the concentrations of methyl donor (S-(5′-adenosyl-l-methionine and enzyme. It was observed that the highest concentrations of both factors (2.25 U of the enzyme and 100 µM of S-(5′-adenosyl-l-methionine afforded the more reproducible results. The in vitro assay demonstrated that Z-vallesiachotamine was able to inhibit the cathecol-O-methyltransferase activity with an IC50 close to 200 µM. Molecular docking studies indicated that Z-vallesiachotamine can bind the catechol pocket of catechol-O-methyltransferase enzyme. The present work demonstrated for the first time the inhibitory properties of Z-vallesiachotamine on cathecol-O-methyltransferase enzyme, affording additional evidence regarding its multifunctional effects in targets related to neurodegenerative diseases.

  19. Overexpression of the mitochondrial methyltransferase TFB1M in the mouse does not impact mitoribosomal methylation status or hearing

    OpenAIRE

    Lee, Seungmin; Rose, Simon; Metodiev, Metodi D.; Becker, Lore; Vernaleken, Alexandra; Klopstock, Thomas; Gailus-Durner, Valerie; Fuchs, Helmut; Hrabě de Angelis, Martin; Douthwaite, Stephen; Larsson, Nils-Göran

    2015-01-01

    Mitochondrial dysfunction is a well-established cause of sensorineural deafness, but the pathophysiological events are poorly understood. Non-syndromic deafness and predisposition to aminoglycoside-induced deafness can be caused by specific mutations in the 12S rRNA gene of mtDNA and are thus maternally inherited traits. The pathophysiology induced by mtDNA mutations has traditionally been attributed to deficient oxidative phosphorylation, which causes energy crisis with functional impairment...

  20. Analysis of 76 veterinary pharmaceuticals from 13 classes including aminoglycosides in bovine muscle by hydrophilic interaction liquid chromatography-tandem mass spectrometry.

    Science.gov (United States)

    Dasenaki, Marilena E; Michali, Christina S; Thomaidis, Nikolaos S

    2016-06-24

    A multiresidue/multiclass method for the simultaneous determination of 76 veterinary drugs and pharmaceuticals in bovine muscle tissue has been developed and validated according to the requirements of European Commission Decision 2002/657/EC. The analytes belong in 13 different classes, including aminoglycoside antibiotics, whose different physicochemical properties (extremely polar character) render their simultaneous determination with other veterinary drugs quite problematic. The method combines a two-step extraction procedure (extraction with acetonitrile followed by an acidic aqueous buffer extraction) with hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS) determination, allowing confirmation and quantification in a single chromatographic run. Further cleanup with solid phase extraction was performed using polymeric SPE cartridges. A thorough ionization study of aminoglycosides was performed in order to increase their sensitivity and significant differences in the abundance of the precursor ions of the analytes were revealed, depending on the composition of the mobile phase tested. Further gradient elution optimization and injection solvent optimization were performed for all target analytes.The method was validated according to the European Commission Decision 2002/657. Quantitative analysis was performed by means of standard addition calibration. Recoveries varied from 37.4% (bromhexine) to 106% (kanamycin) in the lowest validation level and 82% of the compounds showed recovery >70%. Detection capability (CCβ) varied from 2.4 (salinomycin) to 1302 (apramycin) μgkg(-1). PMID:27215463

  1. Whole genome sequencing-based characterization of extensively drug resistant (XDR) strains of Mycobacterium tuberculosis from Pakistan

    KAUST Repository

    Hasan, Zahra

    2015-03-01

    Objectives: The global increase in drug resistance in Mycobacterium tuberculosis (MTB) strains increases the focus on improved molecular diagnostics for MTB. Extensively drug-resistant (XDR) - TB is caused by MTB strains resistant to rifampicin, isoniazid, fluoroquinolone and aminoglycoside antibiotics. Resistance to anti-tuberculous drugs has been associated with single nucleotide polymorphisms (SNPs), in particular MTB genes. However, there is regional variation between MTB lineages and the SNPs associated with resistance. Therefore, there is a need to identify common resistance conferring SNPs so that effective molecular-based diagnostic tests for MTB can be developed. This study investigated used whole genome sequencing (WGS) to characterize 37 XDR MTB isolates from Pakistan and investigated SNPs related to drug resistance. Methods: XDR-TB strains were selected. DNA was extracted from MTB strains, and samples underwent WGS with 76-base-paired end fragment sizes using Illumina paired end HiSeq2000 technology. Raw sequence data were mapped uniquely to H37Rv reference genome. The mappings allowed SNPs and small indels to be called using SAMtools/BCFtools. Results: This study found that in all XDR strains, rifampicin resistance was attributable to SNPs in the rpoB RDR region. Isoniazid resistance-associated mutations were primarily related to katG codon 315 followed by inhA S94A. Fluoroquinolone resistance was attributable to gyrA 91-94 codons in most strains, while one did not have SNPs in either gyrA or gyrB. Aminoglycoside resistance was mostly associated with SNPs in rrs, except in 6 strains. Ethambutol resistant strains had embB codon 306 mutations, but many strains did not have this present. The SNPs were compared with those present in commercial assays such as LiPA Hain MDRTBsl, and the sensitivity of the assays for these strains was evaluated. Conclusions: If common drug resistance associated with SNPs evaluated the concordance between phenotypic and

  2. Antimicrobial resistance and genetic characterization of fluoroquinolone resistance of Pseudomonas aeruginosa isolated from canine infections.

    Science.gov (United States)

    Rubin, J; Walker, R D; Blickenstaff, K; Bodeis-Jones, S; Zhao, S

    2008-09-18

    Infections with antimicrobial-resistant bacteria are a great challenge in both human and veterinary medicine. The purpose of this study was to determine antimicrobial susceptibility of 106 strains of Pseudomonas aeruginosa isolated from dogs with otitis and pyoderma from 2003 to 2006 in the United States. Three antimicrobial panels, including 6 classes and 32 antimicrobial agents, were used. A wide range of susceptibility patterns were noted with some isolates being resistant to between 8 and 28 (mean 16) of the antimicrobials tested. Among the beta-lactams, all isolates were resistant to ampicillin, cefoxitin, cefpodoxime, cephalothin and cefazolin followed by amoxicillin/clavulanic acid (99%), ceftiofur (97%), ceftriaxone (39%), cefotaxime (26%), and cefotaxime/clavulanic acid (20%), whereas less than 7% of isolates were resistant to ceftazidime/clavulanic acid, ceftazidime, piperacillin/tazobactam or cefepime. Two isolates were resistant to the carbapenems. Among the quinolones and fluoroquinolones, the most isolates were resistant to naladixic acid (96%), followed by orbifloxacin (52%), difloxacin (43%), enrofloxacin (31%), marbofloxacin (27%), gatifloxacin (23%), levofloxacin (21%), and ciprofloxacin (16%). Among the aminoglycosides, the most resistance was seen to kanamycin (90%), followed by streptomycin (69%), gentamicin (7%), and amikacin (3%). Of the remaining antimicrobials 100% of the isolates were resistant to chloramphenicol followed by tetracycline (98%), trimethoprim/sulfamethoxazole (57%), and sulfisoxazole (51%). Point mutations were present in gyrA, gyrB, parC, and/or parE genes among 34 of the 102 naladixic acid-resistant isolates. Two isolates contained class 1 integrons carrying aadA gene conferring streptomycin and spectinomycin resistance. The findings suggest that many antimicrobial agents commonly used in companion animals may not constitute appropriate therapy for canine pseudomonas infections. PMID:18395369

  3. Special characteristics of fluorescence and resonance Rayleigh scattering for cadmium telluride nanocrystal aqueous solution and its interactions with aminoglycoside antibiotics

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

    CdTe nanocrystals(CdTe NCs) were achieved by reaction of CdCl2 with KHTe solution and were capped with sodium mercaptoacetate.The product was detected by transmission electron microscopy(TEM),high-resolution transmission electron microscopy(HRTEM),energy dispersive spectroscopy(EDS),fluorescence spectra,ultraviolet-visible spectra and X-ray diffraction(XRD).The CdTe NCs are of cubic structure and the average size is about 5 nm.The fluorescence quantum yield of CdTe NCs aqueous solution increased from 37% to 97% after 20 d under room light.The maximum λem of fluorescence changed from 543 nm to 510 nm and the blue shift was 33 nm.CdTe NCs aqueous solution can be steady for at least 10 months at 4℃ in a refrigerator.The resonance Rayleigh scattering(RRS) of CdTe NCs in the aqueous solution was investigated.The maximum scattering peak was located at about 554 nm.The interactions of CdTe NCs with amikacin sulfate(AS) and micronomicin sulfate(MS) were investigated respectively.The effects of AS and MS on fluorescence and RRS of CdTe NCs were analyzed.It was found that AS and MS quenched the photoluminescence of CdTe NCs and enhanced RRS of CdTe NCs.Under optimum conditions,there are linear relationships between quenching intensity(F0-F),intensity of RRS(I-I0) and concentration of AS and MS.The detection limits(3б) of AS and MS are respectively 3.4 ng·mL-1 and 2.6 ng·mL-1 by the fluorescence quenching method,and 15.2 ng·mL-1 and 14.0 ng·mL-1 by the RRS method.The methods have high sensitivity,thus CdTe NCs may be used as fluorescence probes and RRS probes for the detection of aminoglycoside antibiotics.

  4. Current epidemiology and growing resistance of gram-negative pathogens.

    Science.gov (United States)

    Livermore, David M

    2012-06-01

    In the 1980s, gram-negative pathogens appeared to have been beaten by oxyimino-cephalosporins, carbapenems, and fluoroquinolones. Yet these pathogens have fought back, aided by their membrane organization, which promotes the exclusion and efflux of antibiotics, and by a remarkable propensity to recruit, transfer, and modify the expression of resistance genes, including those for extended-spectrum β-lactamases (ESBLs), carbapenemases, aminoglycoside-blocking 16S rRNA methylases, and even a quinolone-modifying variant of an aminoglycoside-modifying enzyme. Gram-negative isolates--both fermenters and non-fermenters--susceptible only to colistin and, more variably, fosfomycin and tigecycline, are encountered with increasing frequency, including in Korea. Some ESBLs and carbapenemases have become associated with strains that have great epidemic potential, spreading across countries and continents; examples include Escherichia coli sequence type (ST)131 with CTX-M-15 ESBL and Klebsiella pneumoniae ST258 with KPC carbapenemases. Both of these high-risk lineages have reached Korea. In other cases, notably New Delhi Metallo carbapenemase, the relevant gene is carried by promiscuous plasmids that readily transfer among strains and species. Unless antibiotic stewardship is reinforced, microbiological diagnosis accelerated, and antibiotic development reinvigorated, there is a real prospect that the antibiotic revolution of the 20th century will crumble. PMID:22707882

  5. Reduction of MLH1 and PMS2 confers temozolomide resistance and is associated with recurrence of glioblastoma

    OpenAIRE

    Shinsato, Yoshinari; Furukawa, Tatsuhiko; Yunoue, Shunji; Yonezawa, Hajime; Minami, Kentarou; Nishizawa, Yukihiko; IKEDA, Ryuji; Kawahara, Kohichi; YAMAMOTO, Masatatsu; Hirano, Hirofumi; Tokimura, Hiroshi; Arita, Kazunori

    2013-01-01

    Although there is a relationship between DNA repair deficiency and temozolomide (TMZ) resistance in glioblastoma (GBM), it remains unclear which molecule is associated with GBM recurrence. We isolated three TMZ-resistant human GBM cell lines and examined the expression of O6-methylguanine-DNA methyltransferase (MGMT) and mismatch repair (MMR) components. We used immunohistochemical analysis to compare MutL homolog 1 (MLH1), postmeiotic segregation increased 2 (PMS2) and MGMT expression in pri...

  6. Structure and Function of the Glycopeptide N-methyltransferase MtfA, a Tool for the Biosynthesis of Modified Glycopeptide Antibiotics

    Energy Technology Data Exchange (ETDEWEB)

    Shi, Rong; Lamb, Sherry S.; Zakeri, Bijan; Proteau, Ariane; Cui, Qizhi; Sulea, Traian; Matte, Allan; Wright, Gerard D.; Cygler, Miroslaw; (NRCC); (McGill); (McMaster U.)

    2009-06-01

    There is a considerable interest in the modification of existing antibiotics to generate new antimicrobials. Glycopeptide antibiotics (GPAs) are effective against serious Gram-positive bacterial pathogens including methicillin-resistant Staphylococcus aureus. However, resistance to these antibiotics is becoming a serious problem requiring new strategies. We show that the Amycolatopsis orientalis (S)-adenosyl-L-methionine-dependent methyltransferase MtfA, from the vancomycin-class GPA chloroeremomycin biosynthetic pathway, catalyzes in vivo and in vitro methyl transfer to generate methylated GPA derivatives of the teicoplanin class. The crystal structure of MtfA complexed with (S)-adenosyl-L-methionine, (S)-adenosylhomocysteine, or sinefungin inhibitor, coupled with mutagenesis, identified His228 as a likely general base required for methyl transfer to the N terminus of the glycopeptide. Computational docking and molecular dynamics simulations were used to model binding of demethyl-vancomycin aglycone to MtfA. These results demonstrate its utility as a tool for engineering methylated analogs of GPAs.

  7. O6-Methylguanine-Methyltransferase (MGMT Promoter Methylation Status in Glioma Stem-Like Cells is Correlated to Temozolomide Sensitivity Under Differentiation-Promoting Conditions

    Directory of Open Access Journals (Sweden)

    Lucie Karayan-Tapon

    2012-06-01

    Full Text Available Glioblastoma (GBM is the most malignant type of primary brain tumor with a very poor prognosis. The actual standard protocol of treatment for GBM patients consists of radiotherapy and concomitant temozolomide (TMZ. However, the therapeutic efficacy of this treatment is limited due to tumor recurrence and TMZ resistance. Recently isolated, glioma stem-like cells (GSCs are thought to represent the population of tumorigenic cells responsible for GBM resistance and recurrence following surgery and chemotherapy. In addition, MGMT (O6-methylguanine-methyltransferase methylation is considered as one of the principal mechanisms contributing to TMZ sensitivity of GBM. In this study we have isolated GSCs from 10 adult GBM patients and investigated the relationship between MGMT methylation status and Temozolomide (TMZ sensitivity of these lines grown either in stem-like or differentiation promoting conditions. Sensitivity to TMZ was significantly associated with MGMT methylation status in cells committed to differentiation but not in stem-like cells. In addition, patients harboring highly methylated MGMT promoters had a longer overall survival. These results reveal the importance of the differentiation process when considering the predictive value of MGMT status in GSCs for clinical response to TMZ.

  8. Genetic examination of SETD7 and SUV39H1/H2 methyltransferases and the risk of diabetes complications in patients with type 1 diabetes

    DEFF Research Database (Denmark)

    Syreeni, Anna; El-Osta, Assam; Forsblom, Carol;

    2011-01-01

    episodes of hyperglycemia. Epigenetic modifications mediated by histone methyltransferases are associated with gene-activating events that promote enhanced expression of key proinflammatory molecules implicated in vascular injury. In this study, we investigated genetic polymorphisms of the SETD7, SUV39H1......, and SUV39H2 methyltransferases as predictors of risk for micro- and macrovascular complications in type 1 diabetes....

  9. Epigenetic changes of Arabidopsis genome associated with altered DNA methyltransferase and demethylase expressions after gamma irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Ji Eun; Cho, Eun Ju; Kim, Ji Hong; Chung, Byung Yeoup; Kim, Jin Hong [Korea Atomic Energy Research Institute, Daejeon (Korea, Republic of)

    2012-05-15

    DNA methylation at carbon 5 of cytosines is a hall mark of epigenetic inactivation and heterochromatin in both plants and mammals. In Arabidopsis, DNA methylation has two roles that protect the genome from selfish DNA elements and regulate gene expression. Plant genome has three types of DNA methyltransferase, METHYLTRANSFERASE 1 (MET1), DOMAINREARRANGED METHYLASE (DRM) and CHROMOMETHYLASE 3 (CMT3) that are capable of methylating CG, CHG (where H is A, T, or C) and CHH sites, respectively. MET1 is a maintenance DNA methyltransferase that controls CG methylation. Two members of the DRM family, DRM1 and DRM2, are responsible for de novo methylation of CG, CHG, and CHH sites but show a preference for CHH sites. Finally, CMT3 principally carries out CHG methylation and is involved in both de novo methylation and maintenance. Alternatively, active DNA demethylation may occur through the glycosylase activity by removing the methylcytosines from DNA. It may have essential roles in preventing transcriptional silencing of transgenes and endogenous genes and in activating the expression of imprinted genes. DNA demetylation in Arabidopsis is mediated by the DEMETER (DME) family of bifunctional DNA glycosylase. Three targets of DME are MEA (MEDEA), FWA (FLOWERING WAGENINGEN), and FIS2 (FERTILIZATION INDEPENDENT SEED 2). The DME family contains DEMETER-LIKE 2 (DML2), DML3, and REPRESSOR OF SILENING 1 (ROS1). DNA demetylation by ROS1, DML2, and DML3 protect the hypermethylation of specific genome loci. ROS1 is necessary to suppress the promoter methylation and the silencing of endogenous genes. In contrast, the function of DML2 and DML3 has not been reported. Several recent studies have suggested that epigenetic alterations such as change in DNA methylation and histone modification should be caused in plant genomes upon exposure to ionizing radiation. However, there is a lack of data exploring the underlying mechanisms. Therefore, the present study aims to characterize and

  10. Epigenetic changes of Arabidopsis genome associated with altered DNA methyltransferase and demethylase expressions after gamma irradiation

    International Nuclear Information System (INIS)

    DNA methylation at carbon 5 of cytosines is a hall mark of epigenetic inactivation and heterochromatin in both plants and mammals. In Arabidopsis, DNA methylation has two roles that protect the genome from selfish DNA elements and regulate gene expression. Plant genome has three types of DNA methyltransferase, METHYLTRANSFERASE 1 (MET1), DOMAINREARRANGED METHYLASE (DRM) and CHROMOMETHYLASE 3 (CMT3) that are capable of methylating CG, CHG (where H is A, T, or C) and CHH sites, respectively. MET1 is a maintenance DNA methyltransferase that controls CG methylation. Two members of the DRM family, DRM1 and DRM2, are responsible for de novo methylation of CG, CHG, and CHH sites but show a preference for CHH sites. Finally, CMT3 principally carries out CHG methylation and is involved in both de novo methylation and maintenance. Alternatively, active DNA demethylation may occur through the glycosylase activity by removing the methylcytosines from DNA. It may have essential roles in preventing transcriptional silencing of transgenes and endogenous genes and in activating the expression of imprinted genes. DNA demetylation in Arabidopsis is mediated by the DEMETER (DME) family of bifunctional DNA glycosylase. Three targets of DME are MEA (MEDEA), FWA (FLOWERING WAGENINGEN), and FIS2 (FERTILIZATION INDEPENDENT SEED 2). The DME family contains DEMETER-LIKE 2 (DML2), DML3, and REPRESSOR OF SILENING 1 (ROS1). DNA demetylation by ROS1, DML2, and DML3 protect the hypermethylation of specific genome loci. ROS1 is necessary to suppress the promoter methylation and the silencing of endogenous genes. In contrast, the function of DML2 and DML3 has not been reported. Several recent studies have suggested that epigenetic alterations such as change in DNA methylation and histone modification should be caused in plant genomes upon exposure to ionizing radiation. However, there is a lack of data exploring the underlying mechanisms. Therefore, the present study aims to characterize and

  11. 2'-O methylation of internal adenosine by flavivirus NS5 methyltransferase.

    Directory of Open Access Journals (Sweden)

    Hongping Dong

    Full Text Available RNA modification plays an important role in modulating host-pathogen interaction. Flavivirus NS5 protein encodes N-7 and 2'-O methyltransferase activities that are required for the formation of 5' type I cap (m(7GpppAm of viral RNA genome. Here we reported, for the first time, that flavivirus NS5 has a novel internal RNA methylation activity. Recombinant NS5 proteins of West Nile virus and Dengue virus (serotype 4; DENV-4 specifically methylates polyA, but not polyG, polyC, or polyU, indicating that the methylation occurs at adenosine residue. RNAs with internal adenosines substituted with 2'-O-methyladenosines are not active substrates for internal methylation, whereas RNAs with adenosines substituted with N⁶-methyladenosines can be efficiently methylated, suggesting that the internal methylation occurs at the 2'-OH position of adenosine. Mass spectroscopic analysis further demonstrated that the internal methylation product is 2'-O-methyladenosine. Importantly, genomic RNA purified from DENV virion contains 2'-O-methyladenosine. The 2'-O methylation of internal adenosine does not require specific RNA sequence since recombinant methyltransferase of DENV-4 can efficiently methylate RNAs spanning different regions of viral genome, host ribosomal RNAs, and polyA. Structure-based mutagenesis results indicate that K61-D146-K181-E217 tetrad of DENV-4 methyltransferase forms the active site of internal methylation activity; in addition, distinct residues within the methyl donor (S-adenosyl-L-methionine pocket, GTP pocket, and RNA-binding site are critical for the internal methylation activity. Functional analysis using flavivirus replicon and genome-length RNAs showed that internal methylation attenuated viral RNA translation and replication. Polymerase assay revealed that internal 2'-O-methyladenosine reduces the efficiency of RNA elongation. Collectively, our results demonstrate that flavivirus NS5 performs 2'-O methylation of internal adenosine of

  12. Molecular Evolution of the Substrate Specificity of Chloroplastic Aldolases/Rubisco Lysine Methyltransferases in Plants.

    Science.gov (United States)

    Ma, Sheng; Martin-Laffon, Jacqueline; Mininno, Morgane; Gigarel, Océane; Brugière, Sabine; Bastien, Olivier; Tardif, Marianne; Ravanel, Stéphane; Alban, Claude

    2016-04-01

    Rubisco and fructose-1,6-bisphosphate aldolases (FBAs) are involved in CO2 fixation in chloroplasts. Both enzymes are trimethylated at a specific lysine residue by the chloroplastic protein methyltransferase LSMT. Genes coding LSMT are present in all plant genomes but the methylation status of the substrates varies in a species-specific manner. For example, chloroplastic FBAs are naturally trimethylated in both Pisum sativum and Arabidopsis thaliana, whereas the Rubisco large subunit is trimethylated only in the former species. The in vivo methylation status of aldolases and Rubisco matches the catalytic properties of AtLSMT and PsLSMT, which are able to trimethylate FBAs or FBAs and Rubisco, respectively. Here, we created chimera and site-directed mutants of monofunctional AtLSMT and bifunctional PsLSMT to identify the molecular determinants responsible for substrate specificity. Our results indicate that the His-Ala/Pro-Trp triad located in the central part of LSMT enzymes is the key motif to confer the capacity to trimethylate Rubisco. Two of the critical residues are located on a surface loop outside the methyltransferase catalytic site. We observed a strict correlation between the presence of the triad motif and the in vivo methylation status of Rubisco. The distribution of the motif into a phylogenetic tree further suggests that the ancestral function of LSMT was FBA trimethylation. In a recent event during higher plant evolution, this function evolved in ancestors of Fabaceae, Cucurbitaceae, and Rosaceae to include Rubisco as an additional substrate to the archetypal enzyme. Our study provides insight into mechanisms by which SET-domain protein methyltransferases evolve new substrate specificity. PMID:26785049

  13. Human erythrocyte phenol O-methyltransferase: Radiochemical microassay and biochemical properties

    International Nuclear Information System (INIS)

    A radiochemical microassay for the determination of phenol O-methyltransferase (PMT) activity in human red blood cell membranes has been developed. Acetaminophen was used as the substrate. The apparent Michaelis-Menten Ksub(M) value for acetaminophen was 21.2 X 10-3M. The apparent Ksub(M) value for S-adenosyl-L-methionine, a co-substrate for the reaction, was 4.8 X 10-6 M, and the pH optimum of the reaction was approximately 9.0 with four different buffer systems. Phenol was also tested as a substrate and had an apparent Ksub(M) value of 2.0 X 10-3 M. Human erythrocyte (RBC) membrane PMT activity did not have the biochemical characteristics of catechol O-methyltransferase, another RBC membrane methyltransferase enzyme activity. Blood samples obtained from 212 randomly selected adult white subjects had a mean activity of 134.5 +41.5 pmol of p-acetanisidide formed per mg protein per hour (mean +- S.D.). Activities varied from 44 to 282 units. There were no differences in the mean activities of samples from men and women. Experiments in which mixtures of 'low' and 'high' activity RBC membrane preparations were assayed for PMT provided no evidence that the variations in enzyme activity were due to the presence of endogenous PMT activators or inhibitors. RBC membrane PMT activity in blood from 9 patients with renal failure, a pathological state in which there are elevated circulating levels of phenols, was found to be significantly decreased with average activity of 76.2+-9.7 (mean +- S.E.M., P <0.001). (Auth.)

  14. Arabidopsis DNA methyltransferase AtDNMT2 associates with histone deacetylase AtHD2s activity

    International Nuclear Information System (INIS)

    DNA methyltransferase2 (DNMT2) is always deemed to be enigmatic, because it contains highly conserved DNA methyltransferase motifs but lacks the DNA methylation catalytic capability. Here we show that Arabidopsis DNA methyltransferase2 (AtDNMT2) is localized in nucleus and associates with histone deacetylation. Bimolecular fluorescence complementation and pull-down assays show AtDNMT2 interacts with type-2 histone deacetylases (AtHD2s), a unique type of histone deacetylase family in plants. Through analyzing the expression of AtDNMT2: ss-glucuronidase (GUS) fusion protein, we demonstrate that AtDNMT2 has the ability to repress gene expression at transcription level. Meanwhile, the expression of AtDNMT2 gene is altered in athd2c mutant plants. We propose that AtDNMT2 possibly involves in the activity of histone deacetylation and plant epigenetic regulatory network.

  15. Arabidopsis DNA methyltransferase AtDNMT2 associates with histone deacetylase AtHD2s activity

    Energy Technology Data Exchange (ETDEWEB)

    Song, Yuan [Key Laboratory of Arid and Grassland Agroecology, Ministry of Education, School of Life Science, Lanzhou University, Lanzhou 730000 (China); Southern Crop Protection and Food Research Centre, Agriculture and Agri-Food Canada, 1391 Sandford Street, London, ON, Canada N5V4T3 (Canada); Wu, Keqiang [Institute of Plant Biology, National Taiwan University, Taipei 106, Taiwan (China); Dhaubhadel, Sangeeta [Southern Crop Protection and Food Research Centre, Agriculture and Agri-Food Canada, 1391 Sandford Street, London, ON, Canada N5V4T3 (Canada); An, Lizhe, E-mail: lizhean@lzu.edu.cn [Key Laboratory of Arid and Grassland Agroecology, Ministry of Education, School of Life Science, Lanzhou University, Lanzhou 730000 (China); Tian, Lining, E-mail: tianl@agr.gc.ca [Southern Crop Protection and Food Research Centre, Agriculture and Agri-Food Canada, 1391 Sandford Street, London, ON, Canada N5V4T3 (Canada)

    2010-05-28

    DNA methyltransferase2 (DNMT2) is always deemed to be enigmatic, because it contains highly conserved DNA methyltransferase motifs but lacks the DNA methylation catalytic capability. Here we show that Arabidopsis DNA methyltransferase2 (AtDNMT2) is localized in nucleus and associates with histone deacetylation. Bimolecular fluorescence complementation and pull-down assays show AtDNMT2 interacts with type-2 histone deacetylases (AtHD2s), a unique type of histone deacetylase family in plants. Through analyzing the expression of AtDNMT2: ss-glucuronidase (GUS) fusion protein, we demonstrate that AtDNMT2 has the ability to repress gene expression at transcription level. Meanwhile, the expression of AtDNMT2 gene is altered in athd2c mutant plants. We propose that AtDNMT2 possibly involves in the activity of histone deacetylation and plant epigenetic regulatory network.

  16. Diversity of Plasmids and Antimicrobial Resistance Genes in Multidrug-Resistant Escherichia coli Isolated from Healthy Companion Animals.

    Science.gov (United States)

    Jackson, C R; Davis, J A; Frye, J G; Barrett, J B; Hiott, L M

    2015-09-01

    The presence and transfer of antimicrobial resistance genes from commensal bacteria in companion animals to more pathogenic bacteria may contribute to dissemination of antimicrobial resistance. The purpose of this study was to determine antimicrobial resistance gene content and the presence of genetic elements in antimicrobial resistant Escherichia coli from healthy companion animals. In our previous study, from May to August, 2007, healthy companion animals (155 dogs and 121 cats) from three veterinary clinics in the Athens, GA, USA area were sampled and multidrug-resistant E. coli (n = 36; MDR, resistance to ≥ 2 antimicrobial classes) were obtained. Of the 25 different plasmid replicon types tested by PCR, at least one plasmid replicon type was detected in 94% (34/36) of the MDR E. coli; four isolates contained as many as five different plasmid replicons. Nine replicon types (FIA, FIB, FII, I2, A/C, U, P, I1 and HI2) were identified with FIB, FII, I2 as the most common pattern. The presence of class I integrons (intI) was detected in 61% (22/36) of the isolates with eight isolates containing aminoglycoside- and/or trimethoprim-resistance genes in the variable cassette region of intI. Microarray analysis of a subset of the MDR E. coli (n = 9) identified the presence of genes conferring resistance to aminoglycosides (aac, aad, aph and strA/B), β-lactams (ampC, cmy, tem and vim), chloramphenicol (cat), sulfonamides (sulI and sulII), tetracycline [tet(A), tet(B), tet(C), tet(D) and regulator, tetR] and trimethoprim (dfrA). Antimicrobial resistance to eight antimicrobials (ampicillin, cefoxitin, ceftiofur, amoxicillin/clavulanic acid, streptomycin, gentamicin, sulfisoxazole and trimethoprim-sulfamethoxazole) and five plasmid replicons (FIA, FIB, FII, I1 and I2) were transferred via conjugation. The presence of antimicrobial resistance genes, intI and transferable plasmid replicons indicate that E. coli from companion animals may play an important role in the

  17. Development of fed-batch profiles for efficient biosynthesis of catechol-O-methyltransferase

    OpenAIRE

    G.M. Espírito Santo; A.Q. Pedro; D. Oppolzer; Bonifácio, M J; Queiroz, J. A.; Silva, F.; L.A. Passarinha

    2014-01-01

    Catechol-O-methyltransferase (COMT, EC 2.1.1.6) plays a crucial role in dopamine metabolism which has intimately linked this enzyme to some neurodegenerative diseases, such as Parkinson's disease. In recent years, in the attempt of developing new therapeutic strategies for Parkinson's disease, there has been a growing interest in the search for effective COMT inhibitors. In order to do so, large amounts of COMT in an active form are needed, and the best way to achieve this is by up-scaling it...

  18. Structural Basis for Binding of RNA and Cofactor by a KsgA Methyltransferase

    OpenAIRE

    Tu, Chao; Tropea, Joseph E.; Austin, Brian P; Court, Donald L.; Waugh, David S.; Ji, Xinhua

    2009-01-01

    Among methyltransferases, KsgA and the reaction it catalyzes are conserved throughout evolution. However, the specifics of substrate recognition by the enzyme remain unknown. Here, we report structures of Aquifex aeolicus KsgA, in its ligand-free form, in complex with RNA and in complex with both RNA and S-adenosylhomocysteine (SAH, reaction product of cofactor S-adenosylmethionine), providing the first pieces of structural information on KsgA-RNA and KsgA-SAH interactions. Moreover, the stru...

  19. Inhibition of SUV39H1 Methyltransferase Activity by DBC1*

    OpenAIRE

    Li, Zhenyu; Chen, Lihong; Kabra, Neha; Wang, Chuangui; Fang, Jia; Chen, Jiandong

    2009-01-01

    SUV39H1 is a histone H3K9-specific methyltransferase important for heterochromatin formation, regulation of gene expression, and induction of senescence in premalignant cells. SUV39H1 forms a complex with SirT1, and its activity is stimulated by SirT1 binding. Here we present evidence that the product of the DBC1 (deleted in breast cancer 1) gene disrupts the SUV39H1-SirT1 complex. Furthermore, DBC1 binds to the SUV39H1 catalytic domain and inhibits its ability to ...

  20. Histone methyltransferase Suv39h1 represses MyoD-stimulated myogenic differentiation

    OpenAIRE

    Mal, Asoke K.

    2006-01-01

    Suv39h1 is a histone H3 lysine-9 (H3-K9) specific methyltransferase (HMT) that is associated with gene silencing through chromatin modification. The transition from proliferation into differentiation of muscle cell is accompanied by transcriptional activation of previously silent muscle genes. I report Suv39h1 interaction with myogenic regulator MyoD in proliferating muscle cells and its HMT activity, which is associated with MyoD, diminishes as differentiation proceeds. The Suv39h1–MyoD comp...

  1. The DmtA methyltransferase contributes to Aspergillus flavus conidiation, sclerotial production, aflatoxin biosynthesis and virulence

    OpenAIRE

    Kunlong Yang; Linlin Liang; Fanlei Ran; Yinghang Liu; Zhenguo Li; Huahui Lan; Peili Gao; Zhenhong Zhuang; Feng Zhang; Xinyi Nie; Shimuye Kalayu Yirga; Shihua Wang

    2016-01-01

    DNA methylation is essential for epigenetic regulation of gene transcription and development in many animals, plants and fungi. We investigated whether DNA methylation plays a role in the development and secondary metabolism of Aspergillus flavus, identified the DmtA methyltransferase from A. flavus, and produced a dmtA knock-out mutant by replacing the dmtA coding sequence with the pyrG selectable marker. The A. flavus dmtA null mutant lines produced white fluffy mycelium in liquid medium, a...

  2. Histone methyltransferase G9a contributes to H3K27 methylation in vivo

    Institute of Scientific and Technical Information of China (English)

    Hui Wu; Bing Zhu; Xiuzhen Chen; Jun Xiong; Yingfeng Li; Hong Li; Xiaojun Ding; Sheng Liu; She Chen; Shaorong Gao

    2011-01-01

    @@ Dear Editor, Histone modifications play a vital role in the conformation and function of their associated chromatin templates[1].Histone H3K27 methylation mediated by the PRC2 complex is critical for transcriptional regulation,Polycomb silencing,Drosophila segmentation,mammalian X inactivation and cancer[1].Interestingly,H3K27me1(H3 mono-methylated at residue K27)levels in vivo remain unaffected after PRC2 disruption[2,3],which is an indication for the existence of other contributing histone methyltransferase(s)to H3K27me1.

  3. Juvenile hormone acid methyltransferase: A key regulatory enzyme for insect metamorphosis

    OpenAIRE

    Shinoda, Tetsuro; Itoyama, Kyo

    2003-01-01

    Juvenile hormone (JH) acid methyltransferase (JHAMT) is an enzyme that converts JH acids or inactive precursors of JHs to active JHs at the final step of JH biosynthesis pathway in insects. By fluorescent mRNA differential display, we have cloned a cDNA encoding JHAMT from the corpora allata (CA) of the silkworm, Bombyx mori (BmJHAMT). The BmJHAMT cDNA encodes an ORF of 278 aa with a calculated molecular mass of 32,544 Da. The predicted amino acid sequence contains a conserved S-adenosyl-l-me...

  4. Crystal structure of phosphoethanolamine methyltransferase from Plasmodium falciparum in complex with amodiaquine

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Soon Goo; Alpert, Tara D.; Jez, Joseph M. (WU)

    2012-07-17

    Phosphoethanolamine N-methyltransferase (PMT) is essential for phospholipid biogenesis in the malarial parasite Plasmodium falciparum. PfPMT catalyzes the triple methylation of phosphoethanolamine to produce phosphocholine, which is then used for phosphatidylcholine synthesis. Here we describe the 2.0 {angstrom} resolution X-ray crystal structure of PfPMT in complex with amodiaquine. To better characterize inhibition of PfPMT by amodiaquine, we determined the IC{sub 50} values of a series of aminoquinolines using a direct radiochemical assay. Both structural and functional analyses provide a possible approach for the development of new small molecule inhibitors of PfPMT.

  5. O6-Methylguanine-DNA methyltransferase deficiency in developing brain: Implications for brain tumorigenesis

    OpenAIRE

    Bobola, Michael S.; Blank, A.; Berger, Mitchel S.; Silber, John R

    2007-01-01

    The DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT) is a cardinal defense against the mutagenic and carcinogenic effects of alkylating agents. We have reported evidence that absence of detectable MGMT activity (MGMT− phenotype) in human brain is a predisposing factor for primary brain tumors that affects ca. 12% of individuals [J.R. Silber, et. al. Proc. Natl. Acad. Sci. USA 93 (1996) 6941–6946]. We report here that MGMT− phenotype in the brain of children and adults, and the...

  6. Verminderte Expression von O6-Methylguanin-DNA-Methyltransferase bei Gliomen durch Promotormethylierung

    OpenAIRE

    Nickolay, Carla

    2009-01-01

    Pro Jahr erkranken 10 von 100 000 Menschen an einem malignen Hirntumor. Die Prognose ist schlecht. Die Therapie schließt Chirurgie, Bestrahlung und Chemotherapie ein. Das DNA-Reparaturenzym O6-Methylguanin-DNA-Methyltransferase, MGMT, erkennt DNA-Schäden, die durch alkylierende Substanzen wie Temozolomid entstanden sind. Übermäßige Methylierung der Promotorregion von MGMT führt zum Funktionsverlust des Enzyms. Dieser kann zu einer erhöhten Sensibilität für Alkylanzien führen. In der Therapie ...

  7. DNA Methyltransferase Gene dDnmt2 and Longevity of Drosophila

    Institute of Scientific and Technical Information of China (English)

    Meng-JauLin; Lin-YaTang; M.NarsaReddy; C.K.JamesShen

    2005-01-01

    The DNA methylation program of the fruit fly Drosophila melanogaster is carried out by the single DNA methyltransferase gene dDnmt2, the function of which is unknown before. We present evidence that intactness of the gene is required for maintenance of the normal life span of the fruit flies. In contrast, overexpression of dDnmt2 could extend Drosophila life span. The study links the Drosophila DNA methylation program with the small heatshock proteins and longevity/aging and has interesting implication on the eukaryotic DNA methyl-ation programs in general.

  8. Discovery of an in vivo Chemical Probe of the Lysine Methyltransferases G9a and GLP

    OpenAIRE

    Liu, Feng; Barsyte-Lovejoy, Dalia; Li, Fengling; Xiong, Yan; Korboukh, Victoria; Huang, Xi-Ping; Allali-Hassani, Abdellah; Janzen, William P.; Roth, Bryan L.; Frye, Stephen V.; Arrowsmith, Cheryl H.; Brown, Peter J.; Vedadi, Masoud; Jin, Jian

    2013-01-01

    Among epigenetic “writers”, “readers”, and “erasers”, the lysine methyltransferases G9a and GLP, which catalyze mono- and dimethylation of histone H3 lysine 9 (H3K9me2) and non-histone proteins, have been implicated in a variety of human diseases. A “toolkit” of well-characterized chemical probes will allow biological and disease hypotheses concerning these proteins to be tested in cell-based and animal models with high confidence. We previously discovered potent and selective G9a/GLP inhibit...

  9. Protein lysine methyltransferase G9a acts on non-histone targets

    OpenAIRE

    Rathert, Philipp; Dhayalan, Arunkumar; Murakami, Marie; Zhang, Xing; Tamas, Raluca; Jurkowska, Renata; Komatsu, Yasuhiko; Shinkai, Yoichi; Cheng, Xiaodong; Jeltsch, Albert

    2008-01-01

    By methylation of peptide arrays, we determined the specificity profile of the protein methyltransferase G9a. We show that it mostly recognizes an Arg-Lys sequence and that its activity is inhibited by methylation of the arginine residue. Using the specificity profile, we identified new non-histone protein targets of G9a, including CDYL1, WIZ, ACINUS and G9a (automethylation), as well as peptides derived from CSB. We demonstrate potential downstream signaling pathways for methylation of non-h...

  10. A chemical probe selectively inhibits G9a and GLP methyltransferase activity in cells

    OpenAIRE

    Vedadi, Masoud; Barsyte-Lovejoy, Dalia; Liu, Feng; Rival-Gervier, Sylvie; Allali-Hassani, Abdellah; Labrie, Viviane; Wigle, Tim J.; DiMaggio, Peter A.; Wasney, Gregory A.; Siarheyeva, Alena; Dong, Aiping; Tempel, Wolfram; Wang, Sun-Chong; Chen, Xin; Chau, Irene

    2011-01-01

    Protein lysine methyltransferases G9a and GLP modulate the transcriptional repression of a variety of genes via dimethylation of Lys9 on histone H3 (H3K9me2) as well as dimethylation of non-histone targets. Here we report the discovery of UNC0638, an inhibitor of G9a and GLP with excellent potency and selectivity over a wide range of epigenetic and non-epigenetic targets. UNC0638 treatment of a variety of cell lines resulted in lower global H3K9me2 levels, equivalent to levels observed for sm...

  11. Quantitative proteomics reveals direct and indirect alterations in the histone code following methyltransferase knockdown

    OpenAIRE

    Plazas-Mayorca, Mariana D.; Bloom, Joshua S.; Zeissler, Ulrike; LeRoy, Gary; Young, Nicolas L.; DiMaggio, Peter A.; Krugylak, Leonid; Schneider, Robert; Garcia, Benjamin A

    2010-01-01

    Histones are highly conserved proteins that organize cellular DNA. These proteins, especially their N-terminal domains, are adorned with many post-translational modifications (PTMs) such as lysine methylation, which are associated with active or repressed transcriptional states. The lysine methyltransferase G9a and its interaction partner Glp1 can mono- or dimethylate histone H3 on lysine (H3K9me1 or me2); possible cross-talk between these modifications and other PTMs on the same or other his...

  12. A fluorescence resonance energy transfer-based method for histone methyltransferases

    DEFF Research Database (Denmark)

    Devkota, Kanchan; Lohse, Brian; Nyby Jakobsen, Camilla;

    2015-01-01

    A simple dye–quencher fluorescence resonance energy transfer (FRET)-based assay for methyltransferases was developed and used to determine kinetic parameters and inhibitory activity at EHMT1 and EHMT2. Peptides mimicking the truncated histone H3 tail were functionalized in each end with a dye and a...... quencher, respectively. When lysine-9 residues in the peptides were methylated, they were protected from cleavage by endoproteinase–EndoLysC, whereas unmethylated peptides were cleaved, resulting in an increase in fluorescent intensity....

  13. Regioselectivity of catechol O-methyltransferase confers enhancement of catalytic activity

    Science.gov (United States)

    Tsao, Douglas; Liu, Shubin; Dokholyan, Nikolay V.

    2011-04-01

    Catechol O-methyltransferase (COMT) metabolizes catechol moieties by methylating a single hydroxyl group at the meta- or para- hydroxyl position. Hydrophobic amino acids near the active site of COMT influence the regioselectivity of this reaction. Our sequence analysis highlights their importance by showing that these residues are highly conserved throughout evolution. Reaction barriers calculated in the gas phase reveal a lower barrier during methylation at the meta- position, suggesting that the observed meta-regioselectivity of COMT can be attributed to the substrate itself, and that COMT has evolved residues to orient the substrate in a manner that increases the rate of catalysis.

  14. Small RNA-mediated DNA (cytosine-5) methyltransferase 1 inhibition leads to aberrant DNA methylation

    OpenAIRE

    Zhang, Guoqiang; Estève, Pierre-Olivier; Chin, Hang Gyeong; Terragni, Jolyon; Dai, Nan; Corrêa, Ivan R.; Pradhan, Sriharsa

    2015-01-01

    Mammalian cells contain copious amounts of RNA including both coding and noncoding RNA (ncRNA). Generally the ncRNAs function to regulate gene expression at the transcriptional and post-transcriptional level. Among ncRNA, the long ncRNA and small ncRNA can affect histone modification, DNA methylation targeting and gene silencing. Here we show that endogenous DNA methyltransferase 1 (DNMT1) co-purifies with inhibitory ncRNAs. MicroRNAs (miRNAs) bind directly to DNMT1 with high affinity. The bi...

  15. Queen pheromones modulate DNA methyltransferase activity in bee and ant workers.

    Science.gov (United States)

    Holman, Luke; Trontti, Kalevi; Helanterä, Heikki

    2016-01-01

    DNA methylation is emerging as an important regulator of polyphenism in the social insects. Research has concentrated on differences in methylation between queens and workers, though we hypothesized that methylation is involved in mediating other flexible phenotypes, including pheromone-dependent changes in worker behaviour and physiology. Here, we find that exposure to queen pheromone affects the expression of two DNA methyltransferase genes in Apis mellifera honeybees and in two species of Lasius ants, but not in Bombus terrestris bumblebees. These results suggest that queen pheromones influence the worker methylome, pointing to a novel proximate mechanism for these key social signals. PMID:26814223

  16. DNA methyltransferase-dependent transcription of the phage Mu mom gene.

    OpenAIRE

    Hattman, S

    1982-01-01

    The phage Mu mom gene controls an unusual DNA modification. Expression of the mom function requires an active host (dam+) DNA adenine methylase [S-adenosyl-L-methionine:DNA (6-aminopurine)-methyltransferase]; in dam- hosts, Mu development is normal except that the viral DNA does not undergo the mom modification. The present communication compares transcription of the mom gene in dam+ versus dam- cells. 32P-labeled probes were prepared by nick-translation of a purified mom gene-containing rest...

  17. New erythromycin derivatives from Saccharopolyspora erythraea using sugar O-methyltransferases from the spinosyn biosynthetic gene cluster.

    Science.gov (United States)

    Gaisser, S; Lill, R; Wirtz, G; Grolle, F; Staunton, J; Leadlay, P F

    2001-09-01

    Using a previously developed expression system based on the erythromycin-producing strain of Saccharopolyspora erythraea, O-methyltransferases from the spinosyn biosynthetic gene cluster of Saccharopolyspora spinosa have been shown to modify a rhamnosyl sugar attached to a 14-membered polyketide macrolactone. The spnI, spnK and spnH methyltransferase genes were expressed individually in the S. erythraea mutant SGT2, which is blocked both in endogenous macrolide biosynthesis and in ery glycosyltransferases eryBV and eryCIII. Exogenous 3-O-rhamnosyl-erythronolide B was efficiently converted into 3-O-(2'-O-methylrhamnosyl)-erythronolide B by the S. erythraea SGT2 (spnI) strain only. When 3-O-(2'-O-methylrhamnosyl)-erythronolide B was, in turn, fed to a culture of S. erythraea SGT2 (spnK), 3-O-(2',3'-bis-O-methylrhamnosyl)-erythronolide B was identified in the culture supernatant, whereas S. erythraea SGT2 (spnH) was without effect. These results confirm the identity of the 2'- and 3'-O-methyltransferases, and the specific sequence in which they act, and they demonstrate that these methyltransferases may be used to methylate rhamnose units in other polyketide natural products with the same specificity as in the spinosyn pathway. In contrast, 3-O-(2',3'-bis-O-methylrhamnosyl)-erythronolide B was found not to be a substrate for the 4'-O-methyltransferase SpnH. Although rhamnosylerythromycins did not serve directly as substrates for the spinosyn methyltransferases, methylrhamnosyl-erythromycins were obtained by subsequent conversion of the corresponding methylrhamnosyl-erythronolide precursors using the S. erythraea strain SGT2 housing EryCIII, the desosaminyltransferase of the erythromycin pathway. 3-O-(2'-O-methylrhamnosyl)-erythromycin D was tested and found to be significantly active against a strain of erythromycin-sensitive Bacillus subtilis. PMID:11555300

  18. IN VITRO STUDY ON THE CLONING AND TRANSDUCTION OF HUMAN O6-METHYLGUANINE-DNA-METHYLTRANSFERASE CDNA INTO HUMAN UMBILICAL CORD BLOOD CD34+ CELLS

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Objective: To explore whether human umbilical cord blood hematopoietic progenitor cells transduced with human O6-methylguanine-DNA-methyltransferase (MGMT) gene could increase resistance to 1,3-Bis(2-Chloroethyl)-1-Nitrosourea (BCNU). Methods: The cDNA encoding the MGMT was isolated by using RT-PCR method from total RNA of fresh human liver, the fragment was cloned into pGEM-T vector and further subcloned into G1Na retrovirus vector. Then the G1Na-MGMT was transduced into the packaging cell lines GP+E86 and PA317 by LipofectAMINE. By using the medium containing BCNU for cloning selection and ping-ponging supernatant infection between ecotropic producer clone and amphotropic producer clone, high titer amphotropic PA317 producer clone with the highest titer up to 5.8′ 105 CFU/ml was obtained. Cord blood CD34+ cells were transfected repeatedly with supernatant of retrovirus containing human MGMT-cDNA under stimulation of hemopoietic growth factors. Results: The retrovirus vector construction was verified by restriction endonuclease analysis and DNA sequencing. PCR, RT-PCR, Southern Blot, Western Blot and MTT analyses showed that MGMT drug resistance gene has been integrated into the genomic DNA of cord blood CD34+ cells and expressed efficiently. The transgene cord blood CD34+ cells conferred 4-folds stronger resistance to BCNU than untransduced cells. Conclusion: The retrovirus vector-mediated transfer of MGMT drug resistance gene into human cord blood CD34+ cells and its expression provided an experimental foundation for gene therapy in clinical trial.

  19. O6-methylguanine-DNA methyltransferase activity is associated with response to alkylating agent therapy and with MGMT promoter methylation in glioblastoma and anaplastic glioma

    Science.gov (United States)

    Bobola, Michael S.; Alnoor, Mohammad; Chen, John Y.-S.; Kolstoe, Douglas D.; Silbergeld, Daniel L.; Rostomily, Robert C.; Blank, A.; Chamberlain, Marc C.; Silber, John R.

    2014-01-01

    Background CpG methylation in the O6-methylguanine-DNA methyltransferase (MGMT) promoter is associated with better outcome following alkylating agent chemotherapy in glioblastoma (GBM) and anaplastic glioma (AG). To what extent improved response reflects low or absent MGMT activity in glioma tissue has not been unequivocally assessed. This information is central to developing anti-resistance therapies. Methods We examined the relationship of MGMT activity in 91 GBMs and 84 AGs with progression-free survival (PFS) following alkylator therapy and with promoter methylation status determined by methylation-specific PCR (MSP). Results Cox regression analysis revealed that GBMs with high activity had a significantly greater risk for progression in dichotomous (P ≤ 0.001) and continuous (P ≤ 0.003) models, an association observed for different alkylator regimens, including concurrent chemo-radiation with temozolomide. Analysis of MGMT promoter methylation status in 47 of the GBMs revealed that methylated tumors had significantly lower activity (P ≤ 0.005) and longer PFS (P ≤ 0.036) compared to unmethylated tumors, despite overlapping activities. PFS was also significantly greater in methylated vs. unmethylated GBMs with comparable activity (P ≤ 0.005), and among unmethylated tumors with less than median activity (P ≤ 0.026), suggesting that mechanisms in addition to MGMT promote alkylator resistance. Similar associations of MGMT activity with PFS and promoter methylation status were observed for AGs. Conclusions Our results provide strong support for the hypotheses that MGMT activity promotes alkylator resistance and reflects promoter methylation status in malignant gliomas. General significance MGMT activity is an attractive target for anti-resistance therapy regardless of methylation status. PMID:25558448

  20. Clinical and molecular analysis of a four-generation Chinese family with aminoglycoside-induced and nonsyndromic hearing loss associated with the mitochondrial 12S rRNA C1494T mutation

    International Nuclear Information System (INIS)

    We report here the clinical, genetic, and molecular characterization of a four-generation Chinese family with aminoglycoside-induced and nonsyndromic hearing loss. Five of nine matrilineal relatives had aminoglycoside-induced hearing loss. These matrilineal relatives exhibited variable severity and audiometric configuration of hearing impairment, despite sharing some common features: being bilateral and having sensorineural hearing impairment. Sequence analysis of mitochondrial DNA (mtDNA) in the pedigree identified 16 variants and the homoplasmic 12S rRNA C1494T mutation, which was associated with hearing loss in the other large Chinese family. In fact, the occurrence of the C1494T mutation in these genetically unrelated pedigrees affected by hearing impairment strongly indicated that this mutation is involved in the pathogenesis of aminoglycoside-induced and nonsyndromic hearing loss. However, incomplete penetrance of hearing loss indicated that the C1494T mutation itself is not sufficient to produce a clinical phenotype but requires the involvement of modifier factors for the phenotypic expression. Those mtDNA variants, showing no evolutional conservation, may not have a potential modifying role in the pathogenesis of the C1494T mutation. However, nuclear background seems to contribute to the phenotypic variability of matrilineal relatives in this family. Furthermore, aminoglycosides modulate the expressivity and penetrance of deafness associated with the C1494T mutation in this family