Cloning and characterization of a glutathione S-transferase homologue from the plant pathogenic fungus Botrytis cinerea

NARCIS (Netherlands)

Prins, T.W.; Wagemakers, L.; Schouten, A.; Kan, van J.A.L.

2000-01-01

A gene was cloned from Botrytis cinerea that encodes a protein homologous to glutathione S-transferase (GST). The gene, denominated Bcgst1, is present in a single copy and represents the first example of such a gene from a filamentous fungus. The biochemical function of GSTs is to conjugate toxic

Involvement of the VDE homing endonuclease and rapamycin in regulation of the Saccharomyces cerevisiae GSH11 gene encoding the high affinity glutathione transporter.

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Miyake, Tsuyoshi; Hiraishi, Hiroyuki; Sammoto, Hiroyuki; Ono, Bun-Ichiro

2003-10-10

The Saccharomyces cerevisiae gene HGT1/GSH11 encodes the high affinity glutathione transporter and is repressed by cysteine added to the culture medium. It has been found previously that a 5'-upstream cis-element, CCGCCACAC, is responsible for regulating GSH11 expression and that several proteins bind to this element (Miyake, T., Kanayama, M., Sammoto, H., and Ono, B. (2002) Mol. Genet. Genomics 266, 1004-1011). In this report we present evidence that the most prominent of these proteins is VDE, known previously as the homing endonuclease encoded by VMA1. We show also that GSH11 is not expressed in a VDE-deleted strain and that inability to express the GSH11 of this strain is overcome by introduction of the coding region of VDE or the entire VMA1 gene. It is also found that VDE does not cut DNA in the vicinity of the GSH11 cis-element. Rapamycin, an inhibitor of the target of rapamycin (TOR) signal-transduction system, is found to enhance expression of GSH11 in a VDE-dependent manner under conditions of sulfur starvation. These results indicate that GSH11 is regulated by a system sensitive to sulfur starvation (presumably via cysteine depletion) and a more general system involving the nutritional starvation signal mediated by the TOR system. Both systems need to be operational (inhibition of TOR and sulfur starvation) for full expression of GSH11.

Endoplasmic reticulum transport of glutathione by Sec61 is regulated by Ero1 and Bip

DEFF Research Database (Denmark)

Ponsero, Alise J.; Igbaria, Aeid; Darch, Maxwell A.

2017-01-01

In the endoplasmic reticulum (ER), Ero1 catalyzes disulfide bond formation and promotes glutathione (GSH) oxidation to GSSG. Since GSSG cannot be reduced in the ER, maintenance of the ER glutathione redox state and levels likely depends on ER glutathione import and GSSG export. We used quantitative...... oxidation through Ero1 reductive activation, which inhibits glutathione import in a negative regulatory loop. During ER stress, transport is activated by UPR-dependent Ero1 induction, and cytosolic glutathione levels increase. Thus, the ER redox poise is tuned by reciprocal control of glutathione import...... by reduction, causing Bip oxidation and inhibition of glutathione transport. Coupling of glutathione ER import to Ero1 activation provides a basis for glutathione ER redox poise maintenance....

Membrane Transporters for Bilirubin and Its Conjugates: A Systematic Review

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Jovana Čvorović

2017-12-01

Full Text Available Background: Bilirubin is a highly-hydrophobic tetrapyrrole which binds to plasma albumin. It is conjugated in the liver to glucuronic acid, and the water-soluble glucuronides are excreted in urine and bile. The membrane transporters of bilirubin diglucuronide are well-known. Still undefined are however the transporters performing the uptake of bilirubin from the blood into the liver, a process known to be fast and not rate-limited. The biological importance of this process may be appraised by considering that in normal adults 200–300 mg of bilirubin are produced daily, as a result of the physiologic turnover of hemoglobin and cellular cytochromes. Nevertheless, research in this field has yielded controversial and contradicting results. We have undertaken a systematic review of the literature, believing in its utility to improve the existing knowledge and promote further advancements.Methods: We have sourced the PubMed database until 30 June 2017 by applying 5 sequential searches. Screening and eligibility criteria were applied to retain research articles reporting results obtained by using bilirubin molecules in membrane transport assays in vitro or by assessing serum bilirubin levels in in vivo experiments.Results: We have identified 311 articles, retaining 44, reporting data on experimental models having 6 incremental increases of complexity (isolated proteins, membrane vesicles, cells, organ fragments, in vivo rodents, and human studies, demonstrating the function of 19 membrane transporters, encoded by either SLCO or ABC genes. Three other bilirubin transporters have no gene, though one, i.e., bilitranslocase, is annotated in the Transporter Classification Database.Conclusions: This is the first review that has systematically examined the membrane transporters for bilirubin and its conjugates. Paradoxically, the remarkable advancements in the field of membrane transport of bilirubin have pointed to the elusive mechanism(s enabling

Glutathione role in gallium induced toxicity

African Journals Online (AJOL)

Asim

2012-01-26

GSH) present in tissues. It is very important and interesting to study the reaction of gallium nitrate and glutathione as biomarker of glutathione role in detoxification and conjugation in whole blood components (plasma and ...

Upconversion luminescent logic gates and turn-on sensing of glutathione based on two-photon excited quantum dots conjugated with dopamine.

Science.gov (United States)

Gui, Rijun; Jin, Hui; Liu, Xifeng; Wang, Zonghua; Zhang, Feifei; Xia, Jianfei; Yang, Min; Bi, Sai

2014-12-07

Under the two-photon excitation, upconversion luminescent "INHIBIT" and "OR" logic gates of water-dispersed CdTe quantum dots (QDs) were constituted by conjugating the QDs with dopamine. This facilitated the development of a novel QDs-based upconversion luminescent probe for efficient turn-on sensing of glutathione.

A glutathione conjugate of hepoxilin A3: Formation and action in the rat central nervous system

International Nuclear Information System (INIS)

Pace-Asciak, C.R.; Laneuville, O.; Su, W.G.; Corey, E.J.; Gurevich, N.; Wu, P.; Carlen, P.L.

1990-01-01

Incubation of (8R)- and (8S)-[1-14C]hepoxilin A3 [where hepoxilin A3 is 8-hydroxy-11,12-epoxyeicosa-(5Z,9E,14Z)-trienoic acid] and glutathione with homogenates of rat brain hippocampus resulted in a product that was identified as the (8R) and (8S) diastereomers of 11-glutathionyl hepoxilin A3 by reversed-phase high performance liquid chromatographic comparison with the authentic standard made by total synthesis. Identity was further confirmed by cleavage of the isolated product with gamma-glutamyltranspeptidase to yield the corresponding cysteinylglycinyl conjugate that was identical by reversed-phase high performance liquid chromatographic analysis with the enzymic cleavage product derived from the synthetic glutathionyl conjugate. The glutathionyl and cysteinylglycinyl conjugate are referred to as hepoxilin A3-C and hepoxilin A3-D, respectively, by analogy with the established leukotriene nomenclature. Formation of hepoxilin A3-C was greatly enhanced with a concomitant decrease in formation of the epoxide hydrolase product, trioxilin A3, when the epoxide hydrolase inhibitor trichloropropene oxide was added to the incubation mixture demonstrating the presence of a dual metabolic pathway in this tissue involving hepoxilin epoxide hydrolase and glutathione S-transferase processes. Hepoxilin A3-C was tested using intracellular electrophysiological techniques on hippocampal CA1 neurons and found to be active at concentrations as low as 16 nM in causing membrane hyperpolarization, enhanced amplitude and duration of the post-spike train afterhyperpolarization, a marked increase in the inhibitory postsynaptic potential, and a decrease in the spike threshold. These findings suggest that these products in the hepoxilin pathway of arachidonic acid metabolism formed by the rat brain may function as neuromodulators

Formation of glutathione conjugates by reactive metabolites of vinylidene chloride in microsomes and isolated hepatocytes

International Nuclear Information System (INIS)

Liebler, D.C.; Meredith, M.J.; Guengerich, F.P.

1985-01-01

Oxidation of the vinyl halide carcinogen and hepatotoxin vinylidene chloride (VDC) by microsomal cytochrome P-450 yields 2,2-dichloroacetaldehyde, 2-chloroacetyl chloride, 2-chloroacetic acid, and 1,1-dichloroethylene oxide. The roles of these metabolites in covalent modification of proteins and reduced glutathione (GSH) were examined. 2-Chloroacetyl chloride reacted with model thiols at least 10(3)-fold faster than did 1,1-dichloroethylene oxide and at least 10(5)-fold faster than did 2,2-dichloroacetaldehyde or 2-chloroacetic acid. Microsomal covalent binding of [ 14 C]VDC was inhibited by GSH but not by lysine, suggesting that protein thiols, rather than amino groups, are major targets. Liver microsomes catalyzed the formation of three GSH:VDC metabolite conjugates, identified as S-(2,2-dichloro-1-hydroxy)ethylglutathione, 2-(S-glutathionyl)acetate, and S-(2-glutathionyl)acetylglutathione, a novel conjugate containing both stable (thioether) and labile (thioester) linkages. The latter two conjugates also were formed in isolated rat hepatocytes and measurable amounts of 2-(S-glutathionyl)acetate were released into the incubation medium. Both 2-(S-glutathionyl)acetate and S-(2-glutathionyl)acetylglutathione were formed with [ 35 S]GSH added to the hepatic medium, indicating that reactive VDC metabolites are capable of crossing the plasma membrane to react with extracellular targets. Unlabeled S-(2-glutathionyl)-acetylglutathione underwent carbonyl substitution with added [ 35 S]GSH, suggesting that this conjugate may participate in modification of protein thiols. This conjugate also underwent hydrolysis with a half-life of approximately 3 hr. GSH:VDC metabolite conjugates may serve as accessible models for labile covalent adducts formed between VDC metabolites and protein thiols

Glutathione-binding site of a bombyx mori theta-class glutathione transferase.

Directory of Open Access Journals (Sweden)

M D Tofazzal Hossain

Full Text Available The glutathione transferase (GST superfamily plays key roles in the detoxification of various xenobiotics. Here, we report the isolation and characterization of a silkworm protein belonging to a previously reported theta-class GST family. The enzyme (bmGSTT catalyzes the reaction of glutathione with 1-chloro-2,4-dinitrobenzene, 1,2-epoxy-3-(4-nitrophenoxy-propane, and 4-nitrophenethyl bromide. Mutagenesis of highly conserved residues in the catalytic site revealed that Glu66 and Ser67 are important for enzymatic function. These results provide insights into the catalysis of glutathione conjugation in silkworm by bmGSTT and into the metabolism of exogenous chemical agents.

Water-mediated green synthesis of PbS quantum dot and its glutathione and biotin conjugates for non-invasive live cell imaging

Science.gov (United States)

Vijaya Bharathi, M.; Maiti, Santanu; Sarkar, Bidisha; Ghosh, Kaustab; Paira, Priyankar

2018-03-01

This study addresses the cellular uptake of nanomaterials in the field of bio-applications. In the present study, we have synthesized water-soluble lead sulfide quantum dot (PbS QD) with glutathione and 3-MPA (mercaptopropionic acid) as the stabilizing ligand using a green approach. 3-MPA-capped QDs were further modified with streptavidin and then bound to biotin because of its high conjugation efficiency. Labelling and bio-imaging of cells with these bio-conjugated QDs were evaluated. The bright red fluorescence from these types of QDs in HeLa cells makes these materials suitable for deep tissue imaging.

Metabolism of acetaminophen (paracetamol) in plants--two independent pathways result in the formation of a glutathione and a glucose conjugate.

Science.gov (United States)

Huber, Christian; Bartha, Bernadett; Harpaintner, Rudolf; Schröder, Peter

2009-03-01

Pharmaceuticals and their metabolites are detected in the aquatic environment and our drinking water supplies. The need for high quality drinking water is one of the most challenging problems of our times, but still only little knowledge exists on the impact of these compounds on ecosystems, animals, and man. Biological waste water treatment in constructed wetlands is an effective and low-cost alternative, especially for the treatment of non-industrial, municipal waste water. In this situation, plants get in contact with pharmaceutical compounds and have to tackle their detoxification. The mechanisms for the detoxification of xenobiotics in plants are closely related to the mammalian system. An activation reaction (phase I) is followed by a conjugation (phase II) with hydrophilic molecules like glutathione or glucose. Phase III reactions can be summarized as storage, degradation, and transport of the xenobiotic conjugate. Until now, there is no information available on the fate of pharmaceuticals in plants. In this study, we want to investigate the fate and metabolism of N-acetyl-4-aminophenol (paracetamol) in plant tissues using the cell culture of Armoracia rusticana L. as a model system. A hairy root culture of A. rusticana was treated with acetaminophen in a liquid culture. The formation and identification of metabolites over time were analyzed using HPLC-DAD and LC-MSn techniques. With LC-MS technique, we were able to detect paracetamol and identify three of its metabolites in root cells of A. rusticana. Six hours after incubation with 1 mM of acetaminophen, the distribution of acetaminophen and related metabolites in the cells resulted in 18% paracetamol, 64% paracetamol-glucoside, 17% paracetamol glutathione, and 1% of the corresponding cysteine conjugate. The formation of two independently formed metabolites in plant root cells again revealed strong similarities between plant and mammalian detoxification systems. The detoxification mechanism of

Spray-dried mucoadhesives for intravesical drug delivery using N-acetylcysteine- and glutathione-glycol chitosan conjugates.

Science.gov (United States)

Denora, Nunzio; Lopedota, Angela; Perrone, Mara; Laquintana, Valentino; Iacobazzi, Rosa M; Milella, Antonella; Fanizza, Elisabetta; Depalo, Nicoletta; Cutrignelli, Annalisa; Lopalco, Antonio; Franco, Massimo

2016-10-01

This work describes N-acetylcysteine (NAC)- and glutathione (GSH)-glycol chitosan (GC) polymer conjugates engineered as potential platform useful to formulate micro-(MP) and nano-(NP) particles via spray-drying techniques. These conjugates are mucoadhesive over the range of urine pH, 5.0-7.0, which makes them advantageous for intravesical drug delivery and treatment of local bladder diseases. NAC- and GSH-GC conjugates were generated with a synthetic approach optimizing reaction times and purification in order to minimize the oxidation of thiol groups. In this way, the resulting amount of free thiol groups immobilized per gram of NAC- and GSH-GC conjugates was 6.3 and 3.6mmol, respectively. These polymers were completely characterized by molecular weight, surface sulfur content, solubility at different pH values, substitution and swelling degree. Mucoadhesion properties were evaluated in artificial urine by turbidimetric and zeta (ζ)-potential measurements demonstrating good mucoadhesion properties, in particular for NAC-GC at pH 5.0. Starting from the thiolated polymers, MP and NP were prepared using both the Büchi B-191 and Nano Büchi B-90 spray dryers, respectively. The resulting two formulations were evaluated for yield, size, oxidation of thiol groups and ex-vivo mucoadhesion. The new spray drying technique provided NP of suitable size (polymers, avoiding thiolic oxidation during the formulation. MP with acceptable size produced by spray-dryer Büchi B-191 were compared with NP made with the apparatus Nano Büchi B-90. Copyright © 2016 Acta Materialia Inc. All rights reserved.

[Role of proton-motive force in the conjugative DNA transport in Staphylococci].

Science.gov (United States)

Gavriliuk, V G; Vinnikov, A I

1997-01-01

Sensitivity of the conjugative process in staphylococci to the action of uncouplers of oxidative phosphorylation and inhibitors of electron transport systems have been proved, that testifies to the energy-dependent character of conjugative transport of DNA. Proceeding of the conjugation process depends upon the generation of delta microH+ on the membrane of both the donor and recipient cells. contribution of protonmotive forces to providing for the transfer of plasmids during conjugation to staphylococci has been defined.

Characterization of inter-tissue and inter-strain variability of TCE glutathione conjugation metabolites DCVG, DCVC, and NAcDCVC in the mouse.

Science.gov (United States)

Luo, Yu-Syuan; Furuya, Shinji; Chiu, Weihsueh; Rusyn, Ivan

2018-01-01

Trichloroethylene (TCE) is a ubiquitous environmental toxicant that is a liver and kidney carcinogen. Conjugation of TCE with glutathione (GSH) leads to formation of nepthrotoxic and mutagenic metabolites postulated to be critical for kidney cancerdevelopment; however, relatively little is known regarding their tissue levels as previous analytical methods for their detection lacked sensitivity. Here, an LC-MS/MS-based method for simultaneous detection of S-(1,2-dichlorovinyl)-glutathione (DCVG), S-(1,2-dichlorovinyl)-L-cysteine (DCVC), and N-acetyl-S-(1,2-dichlorovinyl)-L-cysteine (NAcDCVC) in multiple mouse tissues was developed. This analytical method is rapid, sensitive (limits of detection (LOD) 3-30 fmol across metabolites and tissues), and robust to quantify all three metabolites in liver, kidneys, and serum. The method was used to characterize inter-tissue and inter-strain variability in formation of conjugative metabolites of TCE. Single oral dose of TCE (24, 240 or 800 mg/kg) was administered to male mice from 20 inbred strains of Collaborative Cross. Inter-strain variability in the levels of DCVG, DCVC, and NAcDCVC (GSD = 1.6-2.9) was observed. Whereas NAcDCVC was distributed equally among analyzed tissues, highest levels of DCVG were detected in liver and DCVC in kidneys. Evidence indicated that inter-strain variability in conjugative metabolite formation of TCE might affect susceptibility to adverse health effects and that this method might aid in filling data gaps in human health assessment of TCE.

Glutathione.

Science.gov (United States)

Noctor, Graham; Queval, Guillaume; Mhamdi, Amna; Chaouch, Sejir; Foyer, Christine H

2011-01-01

Glutathione is a simple sulfur compound composed of three amino acids and the major non-protein thiol in many organisms, including plants. The functions of glutathione are manifold but notably include redox-homeostatic buffering. Glutathione status is modulated by oxidants as well as by nutritional and other factors, and can influence protein structure and activity through changes in thiol-disulfide balance. For these reasons, glutathione is a transducer that integrates environmental information into the cellular network. While the mechanistic details of this function remain to be fully elucidated, accumulating evidence points to important roles for glutathione and glutathione-dependent proteins in phytohormone signaling and in defense against biotic stress. Work in Arabidopsis is beginning to identify the processes that govern glutathione status and that link it to signaling pathways. As well as providing an overview of the components that regulate glutathione homeostasis (synthesis, degradation, transport, and redox turnover), the present discussion considers the roles of this metabolite in physiological processes such as light signaling, cell death, and defense against microbial pathogen and herbivores.

The Bacillus subtilis Conjugative Plasmid pLS20 Encodes Two Ribbon-Helix-Helix Type Auxiliary Relaxosome Proteins That Are Essential for Conjugation

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Andrés Miguel-Arribas

2017-11-01

Full Text Available Bacterial conjugation is the process by which a conjugative element (CE is transferred horizontally from a donor to a recipient cell via a connecting pore. One of the first steps in the conjugation process is the formation of a nucleoprotein complex at the origin of transfer (oriT, where one of the components of the nucleoprotein complex, the relaxase, introduces a site- and strand specific nick to initiate the transfer of a single DNA strand into the recipient cell. In most cases, the nucleoprotein complex involves, besides the relaxase, one or more additional proteins, named auxiliary proteins, which are encoded by the CE and/or the host. The conjugative plasmid pLS20 replicates in the Gram-positive Firmicute bacterium Bacillus subtilis. We have recently identified the relaxase gene and the oriT of pLS20, which are separated by a region of almost 1 kb. Here we show that this region contains two auxiliary genes that we name aux1LS20 and aux2LS20, and which we show are essential for conjugation. Both Aux1LS20 and Aux2LS20 are predicted to contain a Ribbon-Helix-Helix DNA binding motif near their N-terminus. Analyses of the purified proteins show that Aux1LS20 and Aux2LS20 form tetramers and hexamers in solution, respectively, and that they both bind preferentially to oriTLS20, although with different characteristics and specificities. In silico analyses revealed that genes encoding homologs of Aux1LS20 and/or Aux2LS20 are located upstream of almost 400 relaxase genes of the RelLS20 family (MOBL of relaxases. Thus, Aux1LS20 and Aux2LS20 of pLS20 constitute the founding member of the first two families of auxiliary proteins described for CEs of Gram-positive origin.

The Bacillus subtilis Conjugative Plasmid pLS20 Encodes Two Ribbon-Helix-Helix Type Auxiliary Relaxosome Proteins That Are Essential for Conjugation.

Science.gov (United States)

Miguel-Arribas, Andrés; Hao, Jian-An; Luque-Ortega, Juan R; Ramachandran, Gayetri; Val-Calvo, Jorge; Gago-Córdoba, César; González-Álvarez, Daniel; Abia, David; Alfonso, Carlos; Wu, Ling J; Meijer, Wilfried J J

2017-01-01

Bacterial conjugation is the process by which a conjugative element (CE) is transferred horizontally from a donor to a recipient cell via a connecting pore. One of the first steps in the conjugation process is the formation of a nucleoprotein complex at the origin of transfer ( oriT ), where one of the components of the nucleoprotein complex, the relaxase, introduces a site- and strand specific nick to initiate the transfer of a single DNA strand into the recipient cell. In most cases, the nucleoprotein complex involves, besides the relaxase, one or more additional proteins, named auxiliary proteins, which are encoded by the CE and/or the host. The conjugative plasmid pLS20 replicates in the Gram-positive Firmicute bacterium Bacillus subtilis . We have recently identified the relaxase gene and the oriT of pLS20, which are separated by a region of almost 1 kb. Here we show that this region contains two auxiliary genes that we name aux1 LS20 and aux2 LS20 , and which we show are essential for conjugation. Both Aux1 LS20 and Aux2 LS20 are predicted to contain a Ribbon-Helix-Helix DNA binding motif near their N-terminus. Analyses of the purified proteins show that Aux1 LS20 and Aux2 LS20 form tetramers and hexamers in solution, respectively, and that they both bind preferentially to oriT LS20 , although with different characteristics and specificities. In silico analyses revealed that genes encoding homologs of Aux1 LS20 and/or Aux2 LS20 are located upstream of almost 400 relaxase genes of the Rel LS20 family (MOB L ) of relaxases. Thus, Aux1 LS20 and Aux2 LS20 of pLS20 constitute the founding member of the first two families of auxiliary proteins described for CEs of Gram-positive origin.

Comparison of preconditioned generalized conjugate gradient methods to two-dimensional neutron and photon transport equation

International Nuclear Information System (INIS)

Chen, G.S.

1997-01-01

We apply and compare the preconditioned generalized conjugate gradient methods to solve the linear system equation that arises in the two-dimensional neutron and photon transport equation in this paper. Several subroutines are developed on the basis of preconditioned generalized conjugate gradient methods for time-independent, two-dimensional neutron and photon transport equation in the transport theory. These generalized conjugate gradient methods are used. TFQMR (transpose free quasi-minimal residual algorithm), CGS (conjuage gradient square algorithm), Bi-CGSTAB (bi-conjugate gradient stabilized algorithm) and QMRCGSTAB (quasi-minimal residual variant of bi-conjugate gradient stabilized algorithm). These sub-routines are connected to computer program DORT. Several problems are tested on a personal computer with Intel Pentium CPU. (author)

SXT/R391 Integrative and Conjugative Elements (ICEs) Encode a Novel 'Trap-Door' Strategy for Mobile Element Escape.

Science.gov (United States)

Ryan, Michael P; Armshaw, Patricia; Pembroke, J Tony

2016-01-01

Integrative conjugative elements (ICEs) are a class of bacterial mobile elements that have the ability to mediate their own integration, excision, and transfer from one host genome to another by a mechanism of site-specific recombination, self-circularisation, and conjugative transfer. Members of the SXT/R391 ICE family of enterobacterial mobile genetic elements display an unusual UV-inducible sensitization function which results in stress induced killing of bacterial cells harboring the ICE. This sensitization has been shown to be associated with a stress induced overexpression of a mobile element encoded conjugative transfer gene, orf43, a traV homolog. This results in cell lysis and release of a circular form of the ICE. Induction of this novel system may allow transfer of an ICE, enhancing its survival potential under conditions not conducive to conjugative transfer.

Metabolic fate of endogenous molecular damage: Urinary glutathione conjugates of DNA-derived base propenals as markers of inflammation.

Science.gov (United States)

Jumpathong, Watthanachai; Chan, Wan; Taghizadeh, Koli; Babu, I Ramesh; Dedon, Peter C

2015-09-01

Although mechanistically linked to disease, cellular molecules damaged by endogenous processes have not emerged as significant biomarkers of inflammation and disease risk, due in part to poor understanding of their pharmacokinetic fate from tissue to excretion. Here, we use systematic metabolite profiling to define the fate of a common DNA oxidation product, base propenals, to discover such a biomarker. Based on known chemical reactivity and metabolism in liver cell extracts, 15 candidate metabolites were identified for liquid chromatography-coupled tandem mass spectrometry (LC-MS/MS) quantification in urine and bile of rats treated with thymine propenal (Tp). Analysis of urine revealed three metabolites (6% of Tp dose): thymine propenoate and two mercapturate derivatives of glutathione conjugates. Bile contained an additional four metabolites (22% of Tp dose): cysteinylglycine and cysteine derivatives of glutathione adducts. A bis-mercapturate was observed in urine of untreated rats and increased approximately three- to fourfold following CCl4-induced oxidative stress or treatment with the DNA-cleaving antitumor agent, bleomycin. Systematic metabolite profiling thus provides evidence for a metabolized DNA damage product as a candidate biomarker of inflammation and oxidative stress in humans.

A mathematical modeling approach to assessing the reliabilty of biomarkers of glutathione metabolism.

NARCIS (Netherlands)

Geenen, S.; du Preez, F.B.; Reed, M.; Nijhout, H.F.; Kenna, J.G.; Wilson, I.D.; Westerhoff, H.V.; Snoep, J.L.

2012-01-01

One of the main pathways for the detoxification of reactive metabolites in the liver involves glutathione conjugation. Metabolic profiling studies have shown paradoxical responses in glutathione-related biochemical pathways. One of these is the increase in 5-oxoproline and ophthalmic acid

Novel type of ornithine-glutathione double conjugate excreted as a major metabolite into the bile of rats administered clebopride

International Nuclear Information System (INIS)

Ishizuka, T.; Komiya, I.; Hiratsuka, A.; Watabe, T.

1990-01-01

Rats orally given radioactive Clebopride [[14C]CP; N-(1'-benzyl-4'-piperidyl)-2-[14C]methoxy-4-amino-5-chlorobenzamide++ +], an antiulcer agent, excreted a novel type of ornithine (Orn)-GSH double conjugate in the bile as a major metabolite [(14C]BMCP), corresponding to 18% of the dose. The present study provides the first evidence for Orn conjugation of a xenobiotic in mammals and demonstrates that the structure of the radioactive conjugate differs fundamentally from those known in birds and reptiles. The structure of the biliary metabolite, [14C]BMCP, purified to homogeneity by silica gel thin layer and reverse phase high pressure liquid chromatography, was elucidated as S-[2-ornithylamino-4-[14C]methoxy-5-(1'-methyl-4'-piperidylamin o) carboxyphenyl]glutathione, based mainly on the following facts: (1) BMCP showed a protonated molecular ion (M + H)+ peak at m/z 683 in the secondary ion mass spectrum and (2) [14C]BMCP afforded Orn, glutamic acid, glycine, S-(2-amino-4-[14C]methoxy-5-carboxyphenyl)cysteine [( 14C]AMCC), and 1-methyl-4-aminopiperidine (MAP) quantitatively, in an equal molar ratio, by complete hydrolysis with peptidase. Thus, BMCP was a metabolite with three enzymatically hydrolyzable amide bonds in addition to the one existing originally in the parent structure of the drug, which produces MAP by peptic digestion. Of the three additional amide bonds of BMCP, one was a novel type of bond formed by condensation of the alpha-carboxylic acid group of Orn with the primary aromatic amino group of the drug and the other two were in the S-glutathionyl residue, substituted for the chlorine atom vicinal to the Orn-conjugating primary amino group in the aromatic ring and affording glutamic acid, glycine, and the S-cysteine conjugate AMCC by hydrolysis of BMCP with the peptidase

Role of glutathione transport processes in kidney function

International Nuclear Information System (INIS)

Lash, Lawrence H.

2005-01-01

The kidneys are highly dependent on an adequate supply of glutathione (GSH) to maintain normal function. This is due, in part, to high rates of aerobic metabolism, particularly in the proximal tubules. Additionally, the kidneys are potentially exposed to high concentrations of oxidants and reactive electrophiles. Renal cellular concentrations of GSH are maintained by both intracellular synthesis and transport from outside the cell. Although function of specific carriers has not been definitively demonstrated, it is likely that multiple carriers are responsible for plasma membrane transport of GSH. Data suggest that the organic anion transporters OAT1 and OAT3 and the sodium-dicarboxylate 2 exchanger (SDCT2 or NaDC3) mediate uptake across the basolateral plasma membrane (BLM) and that the organic anion transporting polypeptide OATP1 and at least one of the multidrug resistance proteins mediate efflux across the brush-border plasma membrane (BBM). BLM transport may be used pharmacologically to provide renal proximal tubular cells with exogenous GSH to protect against oxidative stress whereas BBM transport functions physiologically in turnover of cellular GSH. The mitochondrial GSH pool is derived from cytoplasmic GSH by transport into the mitochondrial matrix and is mediated by the dicarboxylate and 2-oxoglutarate exchangers. Maintenance of the mitochondrial GSH pool is critical for cellular and mitochondrial redox homeostasis and is important in determining susceptibility to chemically induced apoptosis. Hence, membrane transport processes are critical to regulation of renal cellular and subcellular GSH pools and are determinants of susceptibility to cytotoxicity induced by oxidants and electrophiles

Antioxidant role of glutathione S-transferases: 4-Hydroxynonenal, a key molecule in stress-mediated signaling

International Nuclear Information System (INIS)

Singhal, Sharad S.; Singh, Sharda P.; Singhal, Preeti; Horne, David; Singhal, Jyotsana; Awasthi, Sanjay

2015-01-01

4-Hydroxy-2-trans-nonenal (4HNE), one of the major end products of lipid peroxidation (LPO), has been shown to induce apoptosis in a variety of cell lines. It appears to modulate signaling processes in more than one way because it has been suggested to have a role in signaling for differentiation and proliferation. It has been known that glutathione S-transferases (GSTs) can reduce lipid hydroperoxides through their Se-independent glutathione-peroxidase activity and that these enzymes can also detoxify LPO end-products such as 4HNE. Available evidence from earlier studies together with results of recent studies in our laboratories strongly suggests that LPO products, particularly hydroperoxides and 4HNE, are involved in the mechanisms of stress-mediated signaling and that it can be modulated by the alpha-class GSTs through the regulation of the intracellular concentrations of 4HNE. We demonstrate that 4HNE induced apoptosis in various cell lines is accompanied with c-Jun-N-terminal kinase (JNK) and caspase-3 activation. Cells exposed to mild, transient heat or oxidative stress acquire the capacity to exclude intracellular 4HNE at a faster rate by inducing GSTA4-4 which conjugates 4HNE to glutathione (GSH), and RLIP76 which mediates the ATP-dependent transport of the GSH-conjugate of 4HNE (GS-HNE). The balance between formation and exclusion promotes different cellular processes — higher concentrations of 4HNE promote apoptosis; whereas, lower concentrations promote proliferation. In this article, we provide a brief summary of the cellular effects of 4HNE, followed by a review of its GST-catalyzed detoxification, with an emphasis on the structural attributes that play an important role in the interactions with alpha-class GSTA4-4. Taken together, 4HNE is a key signaling molecule and that GSTs being determinants of its intracellular concentrations, can regulate stress-mediated signaling, are reviewed in this article. - Highlights: • GSTs are the major

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

NARCIS (Netherlands)

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

2003-01-01

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

[The activity of glutathione antioxidant system at melaksen and valdoxan action under experimental hyperthyroidism in rats].

Science.gov (United States)

Gorbenko, M V; Popova, T N; Shul'gin, K K; Popov, S S

2013-01-01

Investigation of glutathione antioxidant system activity and diene conjugates content in rats liver and blood serum at the influence of melaksen and valdoxan under experimental hyperthyroidism (EG) has been revealed. It has been established that the activities of glutathione reductase (GR), glutathione peroxidase (GP) and glutathione transferase (GT), growing at pathological conditions, change to the side of control value at these substunces introduction. Reduced glutathione content (GSH) at melaxen and valdoxan action increased compared with values under the pathology, that, obviously, could be associated with a reduction of its spending on the detoxication of free radical oxidation (FRO) toxic products. Diene conjugates level in rats liver and blood serum, increasing at experimental hyperthyroidism conditions, under introduction of melatonin level correcting drugs, also approached to the control meaning. Results of the study indicate on positive effect of melaxen and valdoxan on free radical homeostasis, that appears to be accompanied by decrease of load on the glutathione antioxidant system in comparison with the pathology.

Mechanism-based biomarker gene sets for glutathione depletion-related hepatotoxicity in rats

International Nuclear Information System (INIS)

Gao Weihua; Mizukawa, Yumiko; Nakatsu, Noriyuki; Minowa, Yosuke; Yamada, Hiroshi; Ohno, Yasuo; Urushidani, Tetsuro

2010-01-01

Chemical-induced glutathione depletion is thought to be caused by two types of toxicological mechanisms: PHO-type glutathione depletion [glutathione conjugated with chemicals such as phorone (PHO) or diethyl maleate (DEM)], and BSO-type glutathione depletion [i.e., glutathione synthesis inhibited by chemicals such as L-buthionine-sulfoximine (BSO)]. In order to identify mechanism-based biomarker gene sets for glutathione depletion in rat liver, male SD rats were treated with various chemicals including PHO (40, 120 and 400 mg/kg), DEM (80, 240 and 800 mg/kg), BSO (150, 450 and 1500 mg/kg), and bromobenzene (BBZ, 10, 100 and 300 mg/kg). Liver samples were taken 3, 6, 9 and 24 h after administration and examined for hepatic glutathione content, physiological and pathological changes, and gene expression changes using Affymetrix GeneChip Arrays. To identify differentially expressed probe sets in response to glutathione depletion, we focused on the following two courses of events for the two types of mechanisms of glutathione depletion: a) gene expression changes occurring simultaneously in response to glutathione depletion, and b) gene expression changes after glutathione was depleted. The gene expression profiles of the identified probe sets for the two types of glutathione depletion differed markedly at times during and after glutathione depletion, whereas Srxn1 was markedly increased for both types as glutathione was depleted, suggesting that Srxn1 is a key molecule in oxidative stress related to glutathione. The extracted probe sets were refined and verified using various compounds including 13 additional positive or negative compounds, and they established two useful marker sets. One contained three probe sets (Akr7a3, Trib3 and Gstp1) that could detect conjugation-type glutathione depletors any time within 24 h after dosing, and the other contained 14 probe sets that could detect glutathione depletors by any mechanism. These two sets, with appropriate scoring

Transport of S-cysteine conjugates in LL-PK1 cells and its role in toxicity

International Nuclear Information System (INIS)

Schaeffer, V.; Stevens, J.

1986-01-01

In order to study its role in S-cysteine conjugate toxicity, the transport of the nephrotoxic cysteine conjugate, S-1,2-dichlorovinyl-L-cysteine (L-DCVC), was investigated in the kidney cell line, LLC-PK1. When incubated with [ 35 S]-DCVC, accumulation of radioactivity within the cells was linear for at least 5 min with 92% of the 35 S present as unmetabolized L-DCVC. Kinetic analysis indicated two saturable uptake systems; Km = 6.8 μM and 1.6 mM; V/sub max/ = .048 and 1.4 nmol/min/mg protein. Both systems were Na + -independent and were inhibited by amino acid transport system L substrates but not substrates of systems A, ASC or organic anion transport. L-DCVC uptake at 5 μM and 500 μM was significantly greater in subconfluent cells than in confluent cultures by 5 and 2.8 fold, respectively. The presence of the non-toxic conjugates S-methyl-(SMC), S-ethyl-(SEC), S-benzyl-L- cysteine (SBC) and the D isomer of DCVC blocked the toxicity and inhibited the transport of L-DCVC in LLC-PK1 cells in the rank order of SBC > SEC congruent to D-DCVC > SMC. The metabolism of L-DCVC, previously shown to be required for cytotoxicity, was only slightly inhibited by these conjugates and did not correlate with their relative protection against toxicity. This study suggests that L-DCVC is transported into these cells by the system L transporter and that protection against toxicity by non-toxic S-cysteine conjugates is due to the inhibition of transport

Expression of the glutathione enzyme system of human colon mucosa by localisation, gender and age.

NARCIS (Netherlands)

Hoensch, H.; Peters, W.H.M.; Roelofs, H.M.J.; Kirch, W.

2006-01-01

BACKGROUND: The glutathione S-transferases (GST) can metabolise endogenous and exogenous toxins and carcinogens by catalysing the conjugation of diverse electrophiles with reduced glutathione (GSH). Variations of GST enzyme activity could influence the susceptibility of developing cancers in certain

Two-dimensional charge transport in self-organized, high-mobility conjugated polymers

DEFF Research Database (Denmark)

Sirringhaus, H.; Brown, P.J.; Friend, R.H.

1999-01-01

Self-organization in many solution-processed, semiconducting conjugated polymers results in complex microstructures, in which ordered microcrystalline domains are embedded in an amorphous matrix(I). This has important consequences for electrical properties of these materials: charge transport...... of the ordered microcrystalline domains in the conjugated polymer poly(3-hexylthiophene), P3HT, Self-organization in P3HT results in a lamella structure with two-dimensional conjugated sheets formed by interchain stacking. We find that, depending on processing conditions, the lamellae can adopt two different...... of polymer transistors in logic circuits(5) and active-matrix displays(4,6)....

Type 3 fimbriae, encoded by the conjugative plasmid pOLA52, enhance biofilm formation and transfer frequencies in Enterobacteriaceae strains

DEFF Research Database (Denmark)

Burmølle, Mette; Bahl, Martin Iain; Jensen, Lars Bogø

2008-01-01

pathogenic, members of the family Enterobacteriaceae, including Klebsiella pneumoniae, Salmonella Typhimurium, Kluyvera sp. and Enterobacter aerogenes, pOLA52 facilitated increased biofilm formation. pOLA52 is believed to represent the first example of a conjugative plasmid encoding type 3 fimbriae...

The glutathione cycle: Glutathione metabolism beyond the γ-glutamyl cycle.

Science.gov (United States)

Bachhawat, Anand Kumar; Yadav, Shambhu

2018-04-17

Glutathione was discovered in 1888, over 125 years ago. Since then, our understanding of various functions and metabolism of this important molecule has grown over these years. But it is only now, in the last decade, that a somewhat complete picture of its metabolism has emerged. Glutathione metabolism has till now been largely depicted and understood by the γ-glutamyl cycle that was proposed in 1970. However, new findings and knowledge particularly on the transport and degradation of glutathione have revealed that many aspects of the γ-glutamyl cycle are incorrect. Despite this, an integrated critical analysis of the cycle has never been undertaken and this has led to the cycle and its errors perpetuating in the literature. This review takes a careful look at the γ-glutamyl cycle and its shortcomings and presents a "glutathione cycle" that captures the current understanding of glutathione metabolism. © 2018 IUBMB Life, 2018. © 2018 International Union of Biochemistry and Molecular Biology.

Novel type of ornithine-glutathione double conjugate excreted as a major metabolite into the bile of rats administered clebopride.

Science.gov (United States)

Ishizuka, T; Komiya, I; Hiratsuka, A; Watabe, T

1990-06-01

Rats orally given radioactive Clebopride [[14C]CP; N-(1'-benzyl-4'-piperidyl)-2-[14C]methoxy-4-amino-5-chlorobenzamide++ +], an antiulcer agent, excreted a novel type of ornithine (Orn)-GSH double conjugate in the bile as a major metabolite [( 14C]BMCP), corresponding to 18% of the dose. The present study provides the first evidence for Orn conjugation of a xenobiotic in mammals and demonstrates that the structure of the radioactive conjugate differs fundamentally from those known in birds and reptiles. The structure of the biliary metabolite, [14C]BMCP, purified to homogeneity by silica gel thin layer and reverse phase high pressure liquid chromatography, was elucidated as S-[2-ornithylamino-4-[14C]methoxy-5-(1'-methyl-4'-piperidylamin o) carboxyphenyl]glutathione, based mainly on the following facts: 1) BMCP showed a protonated molecular ion (M + H)+ peak at m/z 683 in the secondary ion mass spectrum and 2) [14C]BMCP afforded Orn, glutamic acid, glycine, S-(2-amino-4-[14C]methoxy-5-carboxyphenyl)cysteine [( 14C]AMCC), and 1-methyl-4-aminopiperidine (MAP) quantitatively, in an equal molar ratio, by complete hydrolysis with peptidase. Thus, BMCP was a metabolite with three enzymatically hydrolyzable amide bonds in addition to the one existing originally in the parent structure of the drug, which produces MAP by peptic digestion. Of the three additional amide bonds of BMCP, one was a novel type of bond formed by condensation of the alpha-carboxylic acid group of Orn with the primary aromatic amino group of the drug and the other two were in the S-glutathionyl residue, substituted for the chlorine atom vicinal to the Orn-conjugating primary amino group in the aromatic ring and affording glutamic acid, glycine, and the S-cysteine conjugate AMCC by hydrolysis of BMCP with the peptidase. Substitution of a methyl group for the benzyl group at the piperidine ring nitrogen atom, leading to the formation of MAP by peptic digestion, also occurred during metabolism of CP to

Preparation of bioconjugates by solid-phase conjugation to ion exchange matrix-adsorbed carrier proteins

DEFF Research Database (Denmark)

Houen, G.; Olsen, D.T.; Hansen, P.R.

2003-01-01

A solid-phase conjugation method utilizing carrier protein bound to an ion exchange matrix was developed. Ovalbumin was adsorbed to an anion exchange matrix using a batch procedure, and the immobilized protein was then derivatized with iodoacetic acid N-hydroxysuccinimid ester. The activated......, and immunization experiments with the eluted conjugates showed that the more substituted conjugates gave rise to the highest titers of glutathione antibodies. Direct immunization with the conjugates adsorbed to the ion exchange matrix was possible and gave rise to high titers of glutathione antibodies. Conjugates...... of ovalbumin and various peptides were prepared in a similar manner and used for production of peptide antisera by direct immunization with the conjugates bound to the ion exchanger. Advantages of the method are its solid-phase nature, allowing fast and efficient reactions and intermediate washings...

Upregulation of cellular glutathione levels in human ABCB5- and murine Abcb5-transfected cells.

Science.gov (United States)

Kondo, Shingo; Hongama, Keita; Hanaya, Kengo; Yoshida, Ryota; Kawanobe, Takaaki; Katayama, Kazuhiro; Noguchi, Kohji; Sugimoto, Yoshikazu

2015-12-15

Previously, we have demonstrated that human ABCB5 is a full-sized ATP-binding cassette transporter that shares strong homology with ABCB1/P-glycoprotein. ABCB5-transfected cells showed resistance to taxanes and anthracyclines. Herein, we further screened ABCB5 substrates, and explored the mechanism of resistance. Sensitivity of the cells to test compounds was evaluated using cell growth inhibition assay. Cellular levels of buthionine sulfoximine (BSO), glutathione and amino acids were measured using HPLC and an enzyme-based assay. Cellular and vesicular transport of glutathione was evaluated by a radiolabeled substrate. Expression levels of glutathione-metabolizing enzymes were assessed by RT-PCR. Human ABCB5-transfected 293/B5-11 cells and murine Abcb5-transfected 293/mb5-8 cells showed 6.5- and 14-fold higher resistance to BSO than the mock-transfected 293/mock cells, respectively. BSO is an inhibitor of gamma-glutamylcysteine ligase (GCL), which is a key enzyme of glutathione synthesis. 293/B5-11 and 293/mb5-8 cells also showed resistance to methionine sulfoximine, another GCL inhibitor. A cellular uptake experiment revealed that BSO accumulation in 293/B5-11 and 293/mb5-8 cells was similar to that in 293/mock cells, suggesting that BSO is not an ABCB5 substrate. The cellular glutathione content in 293/B5-11 and 293/mb5-8 cells was significantly higher than that in 293/mock cells. Evaluation of the BSO effect on the cellular glutathione content showed that compared with 293/mock cells the BSO concentration required for a 50 % reduction in glutathione content in 293/B5-11 and 293/mb5-8 cells was approximately 2- to 3-fold higher. This result suggests that the BSO resistance of the ABCB5- and Abcb5-transfected cells can be attributed to the reduced effect of BSO on the transfectants. Cellular and vesicular transport assays showed that the transport of radiolabeled glutathione in 293/B5-11 cells was similar to that in 293/mock cells. The mRNA expression of genes

ENDURANCE TRAINING AND GLUTATHIONE-DEPENDENT ANTIOXIDANT DEFENSE MECHANISM IN HEART OF THE DIABETIC RATS

Directory of Open Access Journals (Sweden)

Mustafa Atalay

2003-06-01

Full Text Available Regular physical exercise beneficially influences cardiac antioxidant defenses in normal rats. The aim of this study was to test whether endurance training can strengthen glutathione-dependent antioxidant defense mechanism and decrease lipid peroxidation in heart of the streptozotocin-induced diabetic rats. Redox status of glutathione in blood of diabetic rats in response to training and acute exercise was also examined. Eight weeks of treadmill training increased the endurance in streptozotocin-induced diabetic rats. It did not affect glutathione level in heart tissue at rest and also after exercise. On the other hand, endurance training decreased glutathione peroxidase activity in heart, while glutathione reductase and glutathione S-transferase activities were not affected either by acute exhaustive exercise or endurance training. Reduced and oxidized glutathione levels in blood were not affected by either training or acute exercise. Conjugated dienes levels in heart tissue were increased by acute exhaustive exercise and also 8 weeks treadmill training. Longer duration of exhaustion in trained group may have contributed to the increased conjugated dienes levels in heart after acute exercise. Our results suggest that endurance type exercise may make heart more susceptible to oxidative stress. Therefore it may be wise to combine aerobic exercise with insulin treatment to prevent its adverse effects on antioxidant defense in heart in patients with diabetes mellitus

Comparison of preconditioned generalized conjugate gradient methods to two-dimensional neutron and photon transport equation

International Nuclear Information System (INIS)

Chen, G.S.; Yang, D.Y.

1998-01-01

We apply and compare the preconditioned generalized conjugate gradient methods to solve the linear system equation that arises in the two-dimensional neutron and photon transport equation in this paper. Several subroutines are developed on the basis of preconditioned generalized conjugate gradient methods for time-independent, two-dimensional neutron and photon transport equation in the transport theory. These generalized conjugate gradient methods are used: TFQMR (transpose free quasi-minimal residual algorithm) CGS (conjugate gradient square algorithm), Bi-CGSTAB (bi-conjugate gradient stabilized algorithm) and QMRCGSTAB (quasi-minimal residual variant of bi-conjugate gradient stabilized algorithm). These subroutines are connected to computer program DORT. Several problems are tested on a personal computer with Intel Pentium CPU. The reasons to choose the generalized conjugate gradient methods are that the methods have better residual (equivalent to error) control procedures in the computation and have better convergent rate. The pointwise incomplete LU factorization ILU, modified pointwise incomplete LU factorization MILU, block incomplete factorization BILU and modified blockwise incomplete LU factorization MBILU are the preconditioning techniques used in the several testing problems. In Bi-CGSTAB, CGS, TFQMR and QMRCGSTAB method, we find that either CGS or Bi-CGSTAB method combined with preconditioner MBILU is the most efficient algorithm in these methods in the several testing problems. The numerical solution of flux by preconditioned CGS and Bi-CGSTAB methods has the same result as those from Cray computer, obtained by either the point successive relaxation method or the line successive relaxation method combined with Gaussian elimination

Cellular glutathione prevents cytolethality of monomethylarsonic acid

International Nuclear Information System (INIS)

Sakurai, Teruaki; Kojima, Chikara; Ochiai, Masayuki; Ohta, Takami; Sakurai, Masumi H.; Waalkes, Michael P.; Fujiwara, Kitao

2004-01-01

Inorganic arsenicals are clearly toxicants and carcinogens in humans. In mammals, including humans, inorganic arsenic often undergoes methylation, forming compounds such as monomethylarsonic acid (MMAs V ) and dimethylarsinic acid (DMAs V ). However, much less information is available on the in vitro toxic potential or mechanisms of these methylated arsenicals, especially MMAs V . We studied the molecular mechanisms of in vitro cytolethality of MMAs V using a rat liver epithelial cell line (TRL 1215). MMAs V was not cytotoxic in TRL 1215 cells even at concentrations exceeding 10 mM, but it became weakly cytotoxic and induced both necrotic and apoptotic cell death when cellular reduced glutathione (GSH) was depleted with the glutathione synthase inhibitor, L-buthionine-[S,R]-sulfoximine (BSO), or the glutathione reductase inhibitor, carmustine. Similar results were observed in the other mammalian cells, such as human skin TIG-112 cells, chimpanzee skin CRT-1609 cells, and mouse metallothionein (MT) positive and MT negative embryonic cells. Ethacrynic acid (EA), an inhibitor of glutathione S-transferase (GST) that catalyses GSH-substrate conjugation, also enhanced the cytolethality of MMAs V , but aminooxyacetic acid (AOAA), an inhibitor of β-lyase that catalyses the final breakdown of GSH-substrate conjugates, had no effect. Both the cellular GSH levels and the cellular GST activity were increased by the exposure to MMAs V in TRL 1215 cells. On the other hand, the addition of exogenous extracellular GSH enhanced the cytolethality of MMAs V , although cellular GSH levels actually prevented the cytolethality of combined MMAs V and exogenous GSH. These findings indicate that human arsenic metabolite MMAs V is not a highly toxic compound in mammalian cells, and the level of cellular GSH is critical to its eventual toxic effects

Regulation of epigenetic traits of the glutathione S-transferase P1 gene:From detoxification towards cancer prevention and diagnosis

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Marc eDiederich

2014-07-01

Full Text Available Glutathione S-transferases (GSTs are phase II drug detoxifying enzymes that play an essential role in maintenance of cell integrity and protection against DNA damage by catalyzing the conjugation of glutathione to a wide variety of exo- and endogenous electrophilic substrates. GSTP1, the gene encoding the pi­class GST is frequently inactivated by acquired somatic CpG island promoter hypermethylation in multiple cancer subtypes including prostate, breast, liver and blood cancers. Epigenetically mediated GSTP1 silencing is associated with enhanced cancer susceptibility by decreasing its caretaker gene function, which tends to promote neoplastic transformation allowing the cell to acquire additional alterations. Thus, this epigenetic alteration is now considered as a cancer biomarker but could as well play a driving role in multistep cancer development especially well documented in prostate cancer development.The present review discusses application of epigenetic alterations affecting GSTP1 in cancer medicine used alone or in combination with other biomarkers for cancer detection and diagnosis as well as for future targeted preventive and therapeutic interventions including by dietary agents.

A general relationship between disorder, aggregation and charge transport in conjugated polymers

KAUST Repository

Noriega, Rodrigo; Rivnay, Jonathan; Vandewal, Koen; Koch, Felix P. V.; Stingelin, Natalie; Smith, Paul; Toney, Michael F.; Salleo, Alberto

2013-01-01

Conjugated polymer chains have many degrees of conformational freedom and interact weakly with each other, resulting in complex microstructures in the solid state. Understanding charge transport in such systems, which have amorphous and ordered phases exhibiting varying degrees of order, has proved difficult owing to the contribution of electronic processes at various length scales. The growing technological appeal of these semiconductors makes such fundamental knowledge extremely important for materials and process design. We propose a unified model of how charge carriers travel in conjugated polymer films. We show that in high-molecular-weight semiconducting polymers the limiting charge transport step is trapping caused by lattice disorder, and that short-range intermolecular aggregation is sufficient for efficient long-range charge transport. This generalization explains the seemingly contradicting high performance of recently reported, poorly ordered polymers and suggests molecular design strategies to further improve the performance of future generations of organic electronic materials. © 2013 Macmillan Publishers Limited. All rights reserved.

A general relationship between disorder, aggregation and charge transport in conjugated polymers

KAUST Repository

Noriega, Rodrigo

2013-08-04

Conjugated polymer chains have many degrees of conformational freedom and interact weakly with each other, resulting in complex microstructures in the solid state. Understanding charge transport in such systems, which have amorphous and ordered phases exhibiting varying degrees of order, has proved difficult owing to the contribution of electronic processes at various length scales. The growing technological appeal of these semiconductors makes such fundamental knowledge extremely important for materials and process design. We propose a unified model of how charge carriers travel in conjugated polymer films. We show that in high-molecular-weight semiconducting polymers the limiting charge transport step is trapping caused by lattice disorder, and that short-range intermolecular aggregation is sufficient for efficient long-range charge transport. This generalization explains the seemingly contradicting high performance of recently reported, poorly ordered polymers and suggests molecular design strategies to further improve the performance of future generations of organic electronic materials. © 2013 Macmillan Publishers Limited. All rights reserved.

Role of SbmA in the Uptake of Peptide Nucleic Acid (PNA)-Peptide Conjugates in E. coli

DEFF Research Database (Denmark)

Ghosal, Anubrata; Vitali, Ally; Stach, James E M

2013-01-01

Antisense PNA oligomers targeting essential genes (acpP or ftsZ) and conjugated to the delivery peptide L((KFF)(3)K) show complete growth inhibition of wild type E. coli strain (MG1655) with submicromolar MIC. In this study we show that resistant mutants generated against such PNA......-peptide conjugates had disruptions in the region of sbmA, a gene encoding an inner membrane peptide transporter. The wild type sensitivity to the PNA conjugates was re-established in the resistance mutants by complementation with sbmA. Furthermore, deletion of sbmA in E. coli AS19, a strain that is sensitive...

Charge transport in conjugated polymers: a multiscale picture

Science.gov (United States)

Ruehle, Victor; Kirkpatrick, James; Kremer, Kurt; Andrienko, Denis

2009-03-01

A framework to study charge transport in conjugated polymers using realistic morphologies is developed. First, the atomistic force field is refined using first-principles calculations. Systematic coarse graining is then performed to extend simulation times and system sizes accessible to molecular dynamics simulations. Material morphologies are generated using the coarse grained and atomistic models. Finally, the charge mobility is obtained using temperature activated hopping picture for charge transport [1]. The framework is tested on neutral and oxidized polypyrrole with different structural ordering [2]. [4pt] [1] J. Kirkpatrick, V. Marcon, J. Nelson, K. Kremer, D. Andrienko, Phys. Rev. Lett. 98, 227402 (2007)[0pt] [2] V. Ruehle, J. Kirkpatrick, K. Kremer, D. Andrienko, Phys. Stat. Solidi B, 245, 844 (2008)

A conserved helicase processivity factor is needed for conjugation and replication of an integrative and conjugative element.

Directory of Open Access Journals (Sweden)

Jacob Thomas

Full Text Available Integrative and conjugative elements (ICEs are agents of horizontal gene transfer and have major roles in evolution and acquisition of new traits, including antibiotic resistances. ICEs are found integrated in a host chromosome and can excise and transfer to recipient bacteria via conjugation. Conjugation involves nicking of the ICE origin of transfer (oriT by the ICE-encoded relaxase and transfer of the nicked single strand of ICE DNA. For ICEBs1 of Bacillus subtilis, nicking of oriT by the ICEBs1 relaxase NicK also initiates rolling circle replication. This autonomous replication of ICEBs1 is critical for stability of the excised element in growing cells. We found a conserved and previously uncharacterized ICE gene that is required for conjugation and replication of ICEBs1. Our results indicate that this gene, helP (formerly ydcP, encodes a helicase processivity factor that enables the host-encoded helicase PcrA to unwind the double-stranded ICEBs1 DNA. HelP was required for both conjugation and replication of ICEBs1, and HelP and NicK were the only ICEBs1 proteins needed for replication from ICEBs1 oriT. Using chromatin immunoprecipitation, we measured association of HelP, NicK, PcrA, and the host-encoded single-strand DNA binding protein Ssb with ICEBs1. We found that NicK was required for association of HelP and PcrA with ICEBs1 DNA. HelP was required for association of PcrA and Ssb with ICEBs1 regions distal, but not proximal, to oriT, indicating that PcrA needs HelP to progress beyond nicked oriT and unwind ICEBs1. In vitro, HelP directly stimulated the helicase activity of the PcrA homologue UvrD. Our findings demonstrate that HelP is a helicase processivity factor needed for efficient unwinding of ICEBs1 for conjugation and replication. Homologues of HelP and PcrA-type helicases are encoded on many known and putative ICEs. We propose that these factors are essential for ICE conjugation, replication, and genetic stability.

Glutathione in plants: an integrated overview.

Science.gov (United States)

Noctor, Graham; Mhamdi, Amna; Chaouch, Sejir; Han, Yi; Neukermans, Jenny; Marquez-Garcia, Belen; Queval, Guillaume; Foyer, Christine H

2012-02-01

Plants cannot survive without glutathione (γ-glutamylcysteinylglycine) or γ-glutamylcysteine-containing homologues. The reasons why this small molecule is indispensable are not fully understood, but it can be inferred that glutathione has functions in plant development that cannot be performed by other thiols or antioxidants. The known functions of glutathione include roles in biosynthetic pathways, detoxification, antioxidant biochemistry and redox homeostasis. Glutathione can interact in multiple ways with proteins through thiol-disulphide exchange and related processes. Its strategic position between oxidants such as reactive oxygen species and cellular reductants makes the glutathione system perfectly configured for signalling functions. Recent years have witnessed considerable progress in understanding glutathione synthesis, degradation and transport, particularly in relation to cellular redox homeostasis and related signalling under optimal and stress conditions. Here we outline the key recent advances and discuss how alterations in glutathione status, such as those observed during stress, may participate in signal transduction cascades. The discussion highlights some of the issues surrounding the regulation of glutathione contents, the control of glutathione redox potential, and how the functions of glutathione and other thiols are integrated to fine-tune photorespiratory and respiratory metabolism and to modulate phytohormone signalling pathways through appropriate modification of sensitive protein cysteine residues. © 2011 Blackwell Publishing Ltd.

Isolation and characterization of a cDNA clone coding for a glutathione S-transferase class delta enzyme from the biting midge Culicoides variipennis sonorensis Wirth and Jones.

Science.gov (United States)

Abdallah, M A; Pollenz, R S; Droog, F N; Nunamaker, R A; Tabachnick, W J; Murphy, K E

2000-12-01

Culicoides variipennis sonorensis is the primary vector of bluetongue viruses in North America. Glutathione S-transferases (GSTs) are enzymes that catalyze nucleophilic substitutions, converting reactive lipophilic molecules into soluble conjugates. Increased GST activity is associated with development of insecticide resistance. Described here is the isolation of the first cDNA encoding a C. variipennis GST. The clone consists of 720 translated bases encoding a protein with a M(r) of approximately 24,800 composed of 219 amino acids. The deduced amino acid sequence is similar (64%-74%) to class Delta (previously named Theta) GSTs from the dipteran genera Musca, Drosophila, Lucilia and Anopheles. The cDNA was subcloned into pET-11b, expressed in Epicurian coli BL21 (DE3) and has a specific activity of approximately 28,000 units/mg for the substrate 1-chloro-2,4-dinitrobenzene.

Quantum Mechanical/Molecular Mechanical Free Energy Simulations of the Glutathione S-Transferase (M1-1) Reaction with Phenanthrene 9,10-Oxide

NARCIS (Netherlands)

Ridder, L.; Rietjens, I.M.C.M.; Vervoort, J.J.M.; Mulholland, A.J.

2002-01-01

Glutathione S-transferases (GSTs) play an important role in the detoxification of xenobiotics in mammals. They catalyze the conjugation of glutathione to a wide range of electrophilic compounds. Phenanthrene 9,10-oxide is a model substrate for GSTs, representing an important group of epoxide

Preparation and Comparison of Chitosan Nanoparticles with Different Degrees of Glutathione Thiolation

Directory of Open Access Journals (Sweden)

R Dinarvand

2011-12-01

Full Text Available Background: Chitosan has gained considerable attentions as a biocompatible carrier to improve delivery of active agents. Application of this vehicle in the form of nanoparticle could profit advantages of nanotechnology to increase efficacy of active agents. The purpose of this study was to provide detailed information about chitosan-glutathione (Cht-GSHnanoparticles which are gaining popularity because of their high mucoadhesive and extended drug release properties. Methods: Depolymerization of chitosan was carried out using sodium nitrite method.Glutathione was covalently attached to chitosan and the solubility of the resulting conjugates was evaluated. Nanoparticles were prepared by ionic gelation method and then the effect of glutathione immobilization on properties of nanoparticles was investigated. Results: Thiolation efficiency was higher in lower molecular weight chitosan polymers compared to unmodified chitosan nanoparticles. Cht-GSH conjugates of the same molecular weight but with different degrees of thiolation had the same hydrodynamic diameter (995± nm and surface charge (102± mV as unmodified chitosan, but comprised of a denser network structure and lower concentration. Cht-GSH nanoparticles also exhibited greater mucoadhesive strength which was less affected by ionic strength and pH of the environment. Conclusion:Thiolation improves the solubility of chitosan without any significant changes in size and charge of nanoparticles, but affects the nanogel structure.

Carrier Transport Enhancement in Conjugated Polymers through Interfacial Self-Assembly of Solution-State Aggregates

KAUST Repository

Zhao, Kui; Khan, Hadayat Ullah; Li, Ruipeng; Hu, Hanlin; Amassian, Aram

2016-01-01

and extension of the conjugated backbone of the polymer which clearly translate to significant improvements of carrier transport at the semiconductor-dielectric interface in organic thin film transistors. This study points to opportunities in combining

Glutathione peroxidases of the potato cyst nematode Globodera Rostochiensis

NARCIS (Netherlands)

Jones, J.T.; Reavy, B.; Smant, G.; Prior, A.E.

2004-01-01

We report the cloning and characterisation of full-length DNAs complementary to RNA (cDNAs) encoding two glutathione peroxidases (GpXs) from a plant parasitic nematode, the potato cyst nematode (PCN) Globodera rostochiensis. One protein has a functional signal peptide that targets the protein for

Antioxidant role of glutathione S-transferases: 4-Hydroxynonenal, a key molecule in stress-mediated signaling.

Science.gov (United States)

Singhal, Sharad S; Singh, Sharda P; Singhal, Preeti; Horne, David; Singhal, Jyotsana; Awasthi, Sanjay

2015-12-15

4-Hydroxy-2-trans-nonenal (4HNE), one of the major end products of lipid peroxidation (LPO), has been shown to induce apoptosis in a variety of cell lines. It appears to modulate signaling processes in more than one way because it has been suggested to have a role in signaling for differentiation and proliferation. It has been known that glutathione S-transferases (GSTs) can reduce lipid hydroperoxides through their Se-independent glutathione-peroxidase activity and that these enzymes can also detoxify LPO end-products such as 4HNE. Available evidence from earlier studies together with results of recent studies in our laboratories strongly suggests that LPO products, particularly hydroperoxides and 4HNE, are involved in the mechanisms of stress-mediated signaling and that it can be modulated by the alpha-class GSTs through the regulation of the intracellular concentrations of 4HNE. We demonstrate that 4HNE induced apoptosis in various cell lines is accompanied with c-Jun-N-terminal kinase (JNK) and caspase-3 activation. Cells exposed to mild, transient heat or oxidative stress acquire the capacity to exclude intracellular 4HNE at a faster rate by inducing GSTA4-4 which conjugates 4HNE to glutathione (GSH), and RLIP76 which mediates the ATP-dependent transport of the GSH-conjugate of 4HNE (GS-HNE). The balance between formation and exclusion promotes different cellular processes - higher concentrations of 4HNE promote apoptosis; whereas, lower concentrations promote proliferation. In this article, we provide a brief summary of the cellular effects of 4HNE, followed by a review of its GST-catalyzed detoxification, with an emphasis on the structural attributes that play an important role in the interactions with alpha-class GSTA4-4. Taken together, 4HNE is a key signaling molecule and that GSTs being determinants of its intracellular concentrations, can regulate stress-mediated signaling, are reviewed in this article. Copyright © 2015 Elsevier Inc. All rights

The Incomplete Glutathione Puzzle: Just Guessing at Numbers and Figures?

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Deponte, Marcel

2017-11-20

Glutathione metabolism is comparable to a jigsaw puzzle with too many pieces. It is supposed to comprise (i) the reduction of disulfides, hydroperoxides, sulfenic acids, and nitrosothiols, (ii) the detoxification of aldehydes, xenobiotics, and heavy metals, and (iii) the synthesis of eicosanoids, steroids, and iron-sulfur clusters. In addition, glutathione affects oxidative protein folding and redox signaling. Here, I try to provide an overview on the relevance of glutathione-dependent pathways with an emphasis on quantitative data. Recent Advances: Intracellular redox measurements reveal that the cytosol, the nucleus, and mitochondria contain very little glutathione disulfide and that oxidative challenges are rapidly counterbalanced. Genetic approaches suggest that iron metabolism is the centerpiece of the glutathione puzzle in yeast. Furthermore, recent biochemical studies provide novel insights on glutathione transport processes and uncoupling mechanisms. Which parts of the glutathione puzzle are most relevant? Does this explain the high intracellular concentrations of reduced glutathione? How can iron-sulfur cluster biogenesis, oxidative protein folding, or redox signaling occur at high glutathione concentrations? Answers to these questions not only seem to depend on the organism, cell type, and subcellular compartment but also on different ideologies among researchers. A rational approach to compare the relevance of glutathione-dependent pathways is to combine genetic and quantitative kinetic data. However, there are still many missing pieces and too little is known about the compartment-specific repertoire and concentration of numerous metabolites, substrates, enzymes, and transporters as well as rate constants and enzyme kinetic patterns. Gathering this information might require the development of novel tools but is crucial to address potential kinetic competitions and to decipher uncoupling mechanisms to solve the glutathione puzzle. Antioxid. Redox Signal

Cysteine- and glutathione-mediated uptake of lead and cadmium into Zea mays and Brassica napus roots

International Nuclear Information System (INIS)

Vadas, Timothy M.; Ahner, Beth A.

2009-01-01

This study examines a new mechanism for the uptake of Pb and Cd into Brassica napus and Zea mays roots. During hydroponic experiments, the uptake of Pb and Cd was enhanced in the presence of cysteine and glutathione, whereas no or very low uptake was observed in EDTA and penicillamine controls. Uptake rates were also enhanced after pre-exposure to cysteine or glutathione and inhibited in the presence of vanadate, suggesting a biological mechanism of uptake. Increasing concentrations of glutathione in solution resulted in decreasing Pb uptake rates, indicating competition for transport between free-glutathione and Pb-glutathione species. Pb uptake in the presence of increasing cysteine concentrations resulted in decreased uptake initially but linearly increasing uptake at higher concentrations. Experimentation showed concentration dependent Pb uptake rates. We speculate that there are specific transporters for these thiol ligands and describe what barriers remain for application of this novel transport mechanism in chelator-assisted phytoremediation. - Cysteine and glutathione mediate the transport of lead and cadmium into plant roots.

Cysteine- and glutathione-mediated uptake of lead and cadmium into Zea mays and Brassica napus roots

Energy Technology Data Exchange (ETDEWEB)

Vadas, Timothy M., E-mail: tvadas@umbc.ed [Department of Biological and Environmental Engineering, Cornell University, 320 Riley-Robb Hall, Ithaca, NY 14853 (United States); Ahner, Beth A., E-mail: baa7@cornell.ed [Department of Biological and Environmental Engineering, Cornell University, 320 Riley-Robb Hall, Ithaca, NY 14853 (United States)

2009-08-15

This study examines a new mechanism for the uptake of Pb and Cd into Brassica napus and Zea mays roots. During hydroponic experiments, the uptake of Pb and Cd was enhanced in the presence of cysteine and glutathione, whereas no or very low uptake was observed in EDTA and penicillamine controls. Uptake rates were also enhanced after pre-exposure to cysteine or glutathione and inhibited in the presence of vanadate, suggesting a biological mechanism of uptake. Increasing concentrations of glutathione in solution resulted in decreasing Pb uptake rates, indicating competition for transport between free-glutathione and Pb-glutathione species. Pb uptake in the presence of increasing cysteine concentrations resulted in decreased uptake initially but linearly increasing uptake at higher concentrations. Experimentation showed concentration dependent Pb uptake rates. We speculate that there are specific transporters for these thiol ligands and describe what barriers remain for application of this novel transport mechanism in chelator-assisted phytoremediation. - Cysteine and glutathione mediate the transport of lead and cadmium into plant roots.

Escherichia coli yjjPB genes encode a succinate transporter important for succinate production.

Science.gov (United States)

Fukui, Keita; Nanatani, Kei; Hara, Yoshihiko; Yamakami, Suguru; Yahagi, Daiki; Chinen, Akito; Tokura, Mitsunori; Abe, Keietsu

2017-09-01

Under anaerobic conditions, Escherichia coli produces succinate from glucose via the reductive tricarboxylic acid cycle. To date, however, no genes encoding succinate exporters have been established in E. coli. Therefore, we attempted to identify genes encoding succinate exporters by screening an E. coli MG1655 genome library. We identified the yjjPB genes as candidates encoding a succinate transporter, which enhanced succinate production in Pantoea ananatis under aerobic conditions. A complementation assay conducted in Corynebacterium glutamicum strain AJ110655ΔsucE1 demonstrated that both YjjP and YjjB are required for the restoration of succinate production. Furthermore, deletion of yjjPB decreased succinate production in E. coli by 70% under anaerobic conditions. Taken together, these results suggest that YjjPB constitutes a succinate transporter in E. coli and that the products of both genes are required for succinate export.

Characterization of Affinity-Purified Isoforms of Acinetobacter calcoaceticus Y1 Glutathione Transferases

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Chin-Soon Chee

2014-01-01

Full Text Available Glutathione transferases (GST were purified from locally isolated bacteria, Acinetobacter calcoaceticus Y1, by glutathione-affinity chromatography and anion exchange, and their substrate specificities were investigated. SDS-polyacrylamide gel electrophoresis revealed that the purified GST resolved into a single band with a molecular weight (MW of 23 kDa. 2-dimensional (2-D gel electrophoresis showed the presence of two isoforms, GST1 (pI 4.5 and GST2 (pI 6.2 with identical MW. GST1 was reactive towards ethacrynic acid, hydrogen peroxide, 1-chloro-2,4-dinitrobenzene, and trans,trans-hepta-2,4-dienal while GST2 was active towards all substrates except hydrogen peroxide. This demonstrated that GST1 possessed peroxidase activity which was absent in GST2. This study also showed that only GST2 was able to conjugate GSH to isoproturon, a herbicide. GST1 and GST2 were suggested to be similar to F0KLY9 (putative glutathione S-transferase and F0KKB0 (glutathione S-transferase III of Acinetobacter calcoaceticus strain PHEA-2, respectively.

Characterization of Affinity-Purified Isoforms of Acinetobacter calcoaceticus Y1 Glutathione Transferases

Science.gov (United States)

Chee, Chin-Soon; Tan, Irene Kit-Ping; Alias, Zazali

2014-01-01

Glutathione transferases (GST) were purified from locally isolated bacteria, Acinetobacter calcoaceticus Y1, by glutathione-affinity chromatography and anion exchange, and their substrate specificities were investigated. SDS-polyacrylamide gel electrophoresis revealed that the purified GST resolved into a single band with a molecular weight (MW) of 23 kDa. 2-dimensional (2-D) gel electrophoresis showed the presence of two isoforms, GST1 (pI 4.5) and GST2 (pI 6.2) with identical MW. GST1 was reactive towards ethacrynic acid, hydrogen peroxide, 1-chloro-2,4-dinitrobenzene, and trans,trans-hepta-2,4-dienal while GST2 was active towards all substrates except hydrogen peroxide. This demonstrated that GST1 possessed peroxidase activity which was absent in GST2. This study also showed that only GST2 was able to conjugate GSH to isoproturon, a herbicide. GST1 and GST2 were suggested to be similar to F0KLY9 (putative glutathione S-transferase) and F0KKB0 (glutathione S-transferase III) of Acinetobacter calcoaceticus strain PHEA-2, respectively. PMID:24892084

Charge transport through molecular rods with reduced pi-conjugation.

Science.gov (United States)

Lörtscher, Emanuel; Elbing, Mark; Tschudy, Meinrad; von Hänisch, Carsten; Weber, Heiko B; Mayor, Marcel; Riel, Heike

2008-10-24

A series of oligophenylene rods of increasing lengths is synthesized to investigate the charge-transport mechanisms. Methyl groups are attached to the phenyl rings to weaken the electronic overlap of the pi-subsystems along the molecular backbones. Out-of-plane rotation of the phenyl rings is confirmed in the solid state by means of X-ray analysis and in solution by using UV/Vis spectroscopy. The influence of the reduced pi-conjugation on the resonant charge transport is studied at the single-molecule level by using the mechanically controllable break-junction technique. Experiments are performed under ultra-high-vacuum conditions at low temperature (50 K). A linear increase of the conductance gap with increasing number of phenyl rings (from 260 meV for one ring to 580 meV for four rings) is revealed. In addition, the absolute conductance of the first resonant peaks does not depend on the length of the molecular wire. Resonant transport through the first molecular orbital is found to be dominated by charge-carrier injection into the molecule, rather than by the intrinsic resistance of the molecular wire length.

A regulatory review for products containing glutathione

Directory of Open Access Journals (Sweden)

Nur Hidayah Abd Rahim

2016-01-01

Full Text Available Glutathione is a potent antioxidant as well as has important role for DNA synthesis and repair, protein synthesis, amino acid transport, and enzyme activation. Besides this, Glutathione products are now mainly selling as whitening agent which are mainly marketing through social media (Facebook and different websites. Information is not available whether glutathione product are following the regulatory guidelines of National Pharmaceutical Control Bureau of Malaysia (NPCB for selling, advertisement and promotion. This review was carried out by extracting information about glutathione from scientific database using PubMed, Cochrane Library and Embase. Analysis of the available information, case example of glutathione products showed that a brand of glutathione (Glutacaps HQ did not show the product's registration number from NPCB, and also did not show the name, address, contact number of the advertiser, and even not found the name of the manufacture. Without providing the above mentioned information, the product is selling and promoting through social media (fb which is not allowed by the NPCB guidelines part 4.14. So far, only two clinical trials were conducted on glutathione supplementation for 4 weeks duration. There was no serious or systematic adverse effects reported in clinical trials. As the two clinic trials resulted contradictory outcomes, further studies needed for conformation of the clinic benefits of glutathione. Otherwise, random use of glutathione may be risk for the health of the people. Besides, the marketer mainly promoting glutathione as the skin whitening beauty product instead of using as health supplement, it may cause additional and serious risk to the users as the manufacturer not providing sufficient information about the product, its registration number, manufacturing company, etc.

A novel plant glutathione S-transferase/peroxidase suppresses Bax lethality in yeast

DEFF Research Database (Denmark)

Kampranis, S C; Damianova, R; Atallah, M

2000-01-01

The mammalian inducer of apoptosis Bax is lethal when expressed in yeast and plant cells. To identify potential inhibitors of Bax in plants we transformed yeast cells expressing Bax with a tomato cDNA library and we selected for cells surviving after the induction of Bax. This genetic screen allows...... for the identification of plant genes, which inhibit either directly or indirectly the lethal phenotype of Bax. Using this method a number of cDNA clones were isolated, the more potent of which encodes a protein homologous to the class theta glutathione S-transferases. This Bax-inhibiting (BI) protein was expressed...... in Escherichia coli and found to possess glutathione S-transferase (GST) and weak glutathione peroxidase (GPX) activity. Expression of Bax in yeast decreases the intracellular levels of total glutathione, causes a substantial reduction of total cellular phospholipids, diminishes the mitochondrial membrane...

Intestinal glutathione: determinant of mucosal peroxide transport, metabolism, and oxidative susceptibility

International Nuclear Information System (INIS)

Aw, Tak Yee

2005-01-01

The intestine is a primary site of nutrient absorption and a critical defense barrier against dietary-derived mutagens, carcinogens, and oxidants. Accumulation of oxidants like peroxidized lipids in the gut lumen can contribute to impairment of mucosal metabolic pathways, enterocyte dysfunction independent of cell injury, and development of gut pathologies, such as inflammation and cancer. Despite this recognition, we know little of the pathways of intestinal transport, metabolism, and luminal disposition of dietary peroxides in vivo or of the underlying mechanisms of lipid peroxide-induced genesis of intestinal disease processes. This chapter summarizes our current understanding of the determinants of intestinal absorption and metabolism of peroxidized lipids. I will review experimental evidence from our laboratory and others (Table 1) supporting the pivotal role that glutathione (GSH) and reduced nicotinamide adenine dinucleotide phosphate (NADPH) play in mucosal transport and metabolism of lipid hydroperoxides and how reductant availability can be compromised under chronic stress such as hypoxia, and the influence of GSH on oxidative susceptibility, and redox contribution to genesis of gut disorders. The discussion is pertinent to understanding dietary lipid peroxides and GSH redox balance in intestinal physiology and pathophysiology and the significance of luminal GSH in preserving the integrity of the intestinal epithelium

Molecular cloning, biochemical characterization, and expression analysis of two glutathione S-transferase paralogs from the big-belly seahorse (Hippocampus abdominalis).

Science.gov (United States)

Tharuka, M D Neranjan; Bathige, S D N K; Lee, Jehee

2017-12-01

Glutathione S-transferases (GSTs, EC 2.5.1.18) are important Phase II detoxifying enzymes that catalyze hydrophobic, electrophilic xenobiotic substance with the conjugation of reduced glutathione (GSH). In this study, GSTμ and GSTρ paralogs of GST in the big belly seahorse (Hippocampus abdominalis; HaGSTρ, HaGSTμ) were biochemically, molecularly, functionally characterized to determine their detoxification range and protective capacities upon different pathogenic stresses. HaGSTρ and HaGSTμ are composed of coding sequences of 681bp and 654bp, which encode proteins 225 and 217 amino acids, with predicted molecular masses of 26.06kDa and 25.74kDa respectively. Sequence analysis revealed that both HaGSTs comprise the characteristic GSH-binding site in the thioredoxin-like N-terminal domain and substrate binding site in the C-terminal domain. The recombinant HaGSTρ and HaGSTμ proteins catalyzed the model GST substrate 1-chloro-2, 4-dinitrobenzene (CDNB). Enzyme kinetic analysis revealed different K m and V max values for each rHaGST, suggesting that they have different conjugation rates. The optimum conditions (pH, temperature) and inhibitory assays of each protein demonstrated different optimal ranges. However, HaGSTμ was highly expressed in the ovary and gill, whereas HaGSTρ was highly expressed in the gill and pouch. mRNA expression of HaGSTρ and HaGSTμ was significantly elevated upon lipopolysaccharide, Poly (I:C), and Edwardsiella tarda challenges in liver and in blood cells as well as with Streptococcus iniae challenge in blood cells. From these collective experimental results, we propose that HaGSTρ and HaGSTμ are effective in detoxifying xenobiotic toxic agents, and importantly, their mRNA expression could be stimulated by immunological stress signals in the aquatic environment. Copyright © 2017 Elsevier Inc. All rights reserved.

Effect of backbone structure on charge transport along isolated conjugated polymer chains

International Nuclear Information System (INIS)

Siebbeles, Laurens D.A.; Grozema, Ferdinand C.; Haas, Matthijs P. de; Warman, John M.

2005-01-01

Fast charge transport in conjugated polymers is essential for their application in opto-electronic devices. In the present paper, measurements and theoretical modeling of the mobility of excess charges along isolated chains of conjugated polymers in dilute solution are presented. Charge carriers were produced by irradiation of the polymer solution with 3-MeV electrons from a Van de Graaff accelerator. The mobilities of the charges along the polymer chains were obtained from time-resolved microwave conductivity measurements. The mobilities are strongly dependent on the chemical nature of the polymer backbone. Comparison of the experimental data with results from ab initio quantum mechanical calculations shows that the measured mobilities are strongly limited by torsional disorder, chemical defects and chain ends. Improvement of the structure of polymer backbones is therefore expected to significantly enhance the performance of these materials in 'plastic electronics'

Activation of glutathione peroxidase via Nrf1 mediates genistein's protection against oxidative endothelial cell injury

International Nuclear Information System (INIS)

Hernandez-Montes, Eva; Pollard, Susan E.; Vauzour, David; Jofre-Montseny, Laia; Rota, Cristina; Rimbach, Gerald; Weinberg, Peter D.; Spencer, Jeremy P.E.

2006-01-01

Cellular actions of isoflavones may mediate the beneficial health effects associated with high soy consumption. We have investigated protection by genistein and daidzein against oxidative stress-induced endothelial injury. Genistein but not daidzein protected endothelial cells from damage induced by oxidative stress. This protection was accompanied by decreases in intracellular glutathione levels that could be explained by the generation of glutathionyl conjugates of the oxidised genistein metabolite, 5,7,3',4'-tetrahydroxyisoflavone. Both isoflavones evoked increased protein expression of γ-glutamylcysteine synthetase-heavy subunit (γ-GCS-HS) and increased cytosolic accumulation and nuclear translocation of Nrf2. However, only genistein led to increases in the cytosolic accumulation and nuclear translocation of Nrf1 and the increased expression of and activity of glutathione peroxidase. These results suggest that genistein-induced protective effects depend primarily on the activation of glutathione peroxidase mediated by Nrf1 activation, and not on Nrf2 activation or increases in glutathione synthesis

Chemical form of selenium affects its uptake, transport, and glutathione peroxidase activity in the human intestinal Caco-2 cell model.

Science.gov (United States)

Zeng, Huawei; Jackson, Matthew I; Cheng, Wen-Hsing; Combs, Gerald F

2011-11-01

Determining the effect of selenium (Se) chemical form on uptake, transport, and glutathione peroxidase activity in human intestinal cells is critical to assess Se bioavailability at nutritional doses. In this study, we found that two sources of L-selenomethionine (SeMet) and Se-enriched yeast each increased intracellular Se content more effectively than selenite or methylselenocysteine (SeMSC) in the human intestinal Caco-2 cell model. Interestingly, SeMSC, SeMet, and digested Se-enriched yeast were transported at comparable efficacy from the apical to basolateral sides, each being about 3-fold that of selenite. In addition, these forms of Se, whether before or after traversing from apical side to basolateral side, did not change the potential to support glutathione peroxidase (GPx) activity. Although selenoprotein P has been postulated to be a key Se transport protein, its intracellular expression did not differ when selenite, SeMSC, SeMet, or digested Se-enriched yeast was added to serum-contained media. Taken together, our data show, for the first time, that the chemical form of Se at nutritional doses can affect the absorptive (apical to basolateral side) efficacy and retention of Se by intestinal cells; but that, these effects are not directly correlated to the potential to support GPx activity.

Vectorial transport of unconjugated and conjugated bile salts by monolayers of LLC-PK1 cells doubly transfected with human NTCP and BSEP or with rat Ntcp and Bsep.

Science.gov (United States)

Mita, Sachiko; Suzuki, Hiroshi; Akita, Hidetaka; Hayashi, Hisamitsu; Onuki, Reiko; Hofmann, Alan F; Sugiyama, Yuichi

2006-03-01

Na(+)-taurocholate-cotransporting peptide (NTCP)/SLC10A1 and bile salt export pump (BSEP)/ABCB11 synergistically play an important role in the transport of bile salts by the hepatocyte. In this study, we transfected human NTCP and BSEP or rat Ntcp and Bsep into LLC-PK1 cells, a cell line devoid of bile salts transporters. Transport by these cells was characterized with a focus on substrate specificity between rats and humans. The basal to apical flux of taurocholate across NTCP- and BSEP-expressing LLC-PK1 monolayers was 10 times higher than that in the opposite direction, whereas the flux across the monolayer of control and NTCP or BSEP single-expressing cells did not show any vectorial transport. The basal to apical flux of taurocholate was saturated with a K(m) value of 20 microM. Vectorial transcellular transport was also observed for cholate, chenodeoxycholate, ursodeoxycholate, their taurine and glycine conjugates, and taurodeoxycholate and glycodeoxycholate, whereas no transport of lithocholate was detected. To evaluate the respective functions of NTCP and BSEP and to compare them with those of rat Ntcp and Bsep, we calculated the clearance by each transporter in this system. A good correlation in the clearance of the examined bile salts (cholate, chenodeoxycholate, ursodeoxycholate, and their taurine or glycine conjugates) was observed between transport by human and that of rat transporters in terms of their rank order: for NTCP, taurine conjugates > glycine conjugates > unconjugated bile salts, and for BSEP, unconjugated bile salts and glycine conjugates > taurine conjugates. In conclusion, the substrate specificity of human and rat NTCP and BSEP appear to be very similar at least for monovalent bile salts under physiological conditions.

Up-regulation of glutathione-related genes, enzyme activities and transport proteins in human cervical cancer cells treated with doxorubicin.

Science.gov (United States)

Drozd, Ewa; Krzysztoń-Russjan, Jolanta; Marczewska, Jadwiga; Drozd, Janina; Bubko, Irena; Bielak, Magda; Lubelska, Katarzyna; Wiktorska, Katarzyna; Chilmonczyk, Zdzisław; Anuszewska, Elżbieta; Gruber-Bzura, Beata

2016-10-01

Doxorubicin (DOX), one of the most effective anticancer drugs, acts in a variety of ways including DNA damage, enzyme inhibition and generation of reactive oxygen species. Glutathione (GSH) and glutathione-related enzymes including: glutathione peroxidase (GPX), glutathione reductase (GSR) and glutathione S-transferases (GST) may play a role in adaptive detoxification processes in response to the oxidative stress, thus contributing to drug resistance phenotype. In this study, we investigated effects of DOX treatment on expression and activity of GSH-related enzymes and multidrug resistance-associated proteins in cultured human cervical cancer cells displaying different resistance against this drug (HeLa and KB-V1). Determination of expression level of genes encoding GST isoforms and MRP proteins (GCS, GPX, GSR, GSTA1-3, GSTM1, GSTP1, ABCC1-3, MGST1-3) was performed using StellARray™ Technology. Enzymatic activities of GPX and GSR were measured using biochemical methods. Expression of MRP1 was examined by immunofluorescence microscopy. This study showed that native expression levels of GSTM1 and GSTA3 were markedly higher in KB-V1 cells (2000-fold and 200-fold) compared to HeLa cells. Resistant cells have also shown significantly elevated expression of GSTA1 and GSTA2 genes (200-fold and 50-fold) as a result of DOX treatment. In HeLa cells, exposure to DOX increased expression of all genes: GSTM1 (7-fold) and GSTA1-3 (550-fold, 150-fold and 300-fold). Exposure to DOX led to the slight increase of GCS expression as well as GPX activity in KB-V1 cells, while in HeLa cells it did not. Expression of ABCC1 (MRP1) was not increased in any of the tested cell lines. Our results indicate that expression of GSTM1 and GSTA1-3 genes is up-regulated by DOX treatment and suggest that activity of these genes may be associated with drug resistance of the tested cells. At the same time, involvement of MRP1 in DOX resistance in the given experimental conditions is unlikely

Biochemical Characterization and Vaccine Potential of a Heme-Binding Glutathione Transferase from the Adult Hookworm Ancylostoma caninum

Science.gov (United States)

Zhan, Bin; Liu, Sen; Perally, Samirah; Xue, Jian; Fujiwara, Ricardo; Brophy, Peter; Xiao, Shuhua; Liu, Yueyuan; Feng, Jianjun; Williamson, Angela; Wang, Yan; Bueno, Lilian L.; Mendez, Susana; Goud, Gaddam; Bethony, Jeffrey M.; Hawdon, John M.; Loukas, Alex; Jones, Karen; Hotez, Peter J.

2005-01-01

We report the cloning and expression of Ac-GST-1, a novel glutathione S-transferase from the adult hookworm Ancylostoma caninum, and its possible role in parasite blood feeding and as a vaccine target. The predicted Ac-GST-1 open reading frame contains 207 amino acids (mass, 24 kDa) and exhibited up to 65% amino acid identity with other nematode GSTs. mRNA encoding Ac-GST-1 was detected in adults, eggs, and larval stages, but the protein was detected only in adult hookworm somatic extracts and excretory/secretory products. Using antiserum to the recombinant protein, Ac-GST-1 was immunolocalized to the parasite hypodermis and muscle tissue and weakly to the intestine. Recombinant Ac-GST-1 was enzymatically active, as determined by conjugation of glutathione to a model substrate, and exhibited a novel high-affinity binding site for hematin. The possible role of Ac-GST-1 in parasite heme detoxification during hemoglobin digestion or heme uptake prompted interest in evaluating it as a potential vaccine antigen. Vaccination of dogs with Ac-GST-1 resulted in a 39.4% reduction in the mean worm burden and 32.3% reduction in egg counts compared to control dogs following larval challenge, although the reductions were not statistically significant. However, hamsters vaccinated with Ac-GST-1 exhibited statistically significant worm reduction (53.7%) following challenge with heterologous Necator americanus larvae. These studies suggest that Ac-GST-1 is a possible drug and vaccine target for hookworm infection. PMID:16177370

A novel method for screening the glutathione transferase inhibitors

Directory of Open Access Journals (Sweden)

Węgrzyn Grzegorz

2009-03-01

Full Text Available Abstract Background Glutathione transferases (GSTs belong to the family of Phase II detoxification enzymes. GSTs catalyze the conjugation of glutathione to different endogenous and exogenous electrophilic compounds. Over-expression of GSTs was demonstrated in a number of different human cancer cells. It has been found that the resistance to many anticancer chemotherapeutics is directly correlated with the over-expression of GSTs. Therefore, it appears to be important to find new GST inhibitors to prevent the resistance of cells to anticancer drugs. In order to search for glutathione transferase (GST inhibitors, a novel method was designed. Results Our results showed that two fragments of GST, named F1 peptide (GYWKIKGLV and F2 peptide (KWRNKKFELGLEFPNL, can significantly inhibit the GST activity. When these two fragments were compared with several known potent GST inhibitors, the order of inhibition efficiency (measured in reactions with 2,4-dinitrochlorobenzene (CDNB and glutathione as substrates was determined as follows: tannic acid > cibacron blue > F2 peptide > hematin > F1 peptide > ethacrynic acid. Moreover, the F1 peptide appeared to be a noncompetitive inhibitor of the GST-catalyzed reaction, while the F2 peptide was determined as a competitive inhibitor of this reaction. Conclusion It appears that the F2 peptide can be used as a new potent specific GST inhibitor. It is proposed that the novel method, described in this report, might be useful for screening the inhibitors of not only GST but also other enzymes.

Rifampin modulation of xeno- and endobiotic conjugating enzyme mRNA expression and associated microRNAs in human hepatocytes.

Science.gov (United States)

Gufford, Brandon T; Robarge, Jason D; Eadon, Michael T; Gao, Hongyu; Lin, Hai; Liu, Yunlong; Desta, Zeruesenay; Skaar, Todd C

2018-04-01

Rifampin is a pleiotropic inducer of multiple drug metabolizing enzymes and transporters. This work utilized a global approach to evaluate rifampin effects on conjugating enzyme gene expression with relevance to human xeno- and endo-biotic metabolism. Primary human hepatocytes from 7 subjects were treated with rifampin (10 μmol/L, 24 hours). Standard methods for RNA-seq library construction, EZBead preparation, and NextGen sequencing were used to measure UDP-glucuronosyl transferase UGT, sulfonyltransferase SULT, N acetyltransferase NAT, and glutathione-S-transferase GST mRNA expression compared to vehicle control (0.01% MeOH). Rifampin-induced (>1.25-fold) mRNA expression of 13 clinically important phase II drug metabolizing genes and repressed (>1.25-fold) the expression of 3 genes ( P  accounting for simultaneous induction of both CYP3A4 and UGT1A4 predicted a ~10-fold decrease in parent midazolam exposure with only a ~2-fold decrease in midazolam N-glucuronide metabolite exposure. These data reveal differential effects of rifampin on the human conjugating enzyme transcriptome and potential associations with miRNAs that form the basis for future mechanistic studies to elucidate the interplay of conjugating enzyme regulatory elements.

Modelling of charge carrier transport in conjugated polymers doped by polar additives

Czech Academy of Sciences Publication Activity Database

Toman, Petr; Nešpůrek, Stanislav; Bartkowiak, W.

2009-01-01

Roč. 27, č. 3 (2009), s. 797-812 ISSN 0137-1339. [International Conference on Electrical and Related Properties of Organic Solids /11./. Piechowice, 13.07.2008-17.07.2008] R&D Projects: GA ČR GA203/06/0285; GA AV ČR KAN400720701; GA MŠk MEB050815 Institutional research plan: CEZ:AV0Z40500505 Keywords : conjugated polymers * charge carrier transport * molecular electronics Subject RIV: CD - Macromolecular Chemistry Impact factor: 0.384, year: 2009

The promising anticancer drug 3-bromopyruvate is metabolized through glutathione conjugation which affects chemoresistance and clinical practice: An evidence-based view.

Science.gov (United States)

El Sayed, Salah Mohamed; Baghdadi, Hussam; Zolaly, Mohammed; Almaramhy, Hamdi H; Ayat, Mongi; Donki, Jagadish G

2017-03-01

3-Bromopyruvate (3BP) is a promising effective anticancer drug against many different tumors in children and adults. 3BP exhibited strong anticancer effects in both preclinical and human studies e.g. energy depletion, oxidative stress, anti-angiogenesis, anti-metastatic effects, targeting cancer stem cells and antagonizing the Warburg effect. There is no report about 3BP metabolism to guide researchers and oncologists to improve clinical practice and prevent drug resistance. In this article, we provide evidences that 3BP is metabolized through glutathione (GSH) conjugation as a novel report where 3BP was confirmed to be attached to GSH followed by permanent loss of pharmacological effects in a picture similar to cisplatin. Both cisplatin and 3BP are alkylating agents. Reported decrease in endogenous cellular GSH content upon 3BP treatment was confirmed to be due to the formation of 3BP-GSH complex i.e. GSH consumption for conjugation with 3BP. Cancer cells having high endogenous GSH exhibit resistance to 3BP while 3BP sensitive cells acquire resistance upon adding exogenous GSH. Being a thiol blocker, 3BP may attack thiol groups in tissues and serum proteins e.g. albumin and GSH. That may decrease 3BP-induced anticancer effects and the functions of those proteins. We proved here that 3BP metabolism is different from metabolism of hydroxypyruvate that results from metabolism of D-serine using D-amino acid oxidase. Clinically, 3BP administration should be monitored during albumin infusion and protein therapy where GSH should be added to emergency medications. GSH exerts many physiological effects and is safe for human administration both orally and intravenously. Based on that, reported GSH-induced inhibition of 3BP effects makes 3BP effects reversible, easily monitored and easily controlled. This confers a superiority of 3BP over many anticancer agents. Copyright © 2017 Elsevier Ltd. All rights reserved.

Glutathione Preservation during Storage of Rat Lenses in Optisol-GS and Castor Oil

DEFF Research Database (Denmark)

Holm, Thomas; Brøgger-Jensen, Martin Rocho; Johnson, Leif

2013-01-01

Glutathione concentration in the lens decreases in aging and cataractous lenses, providing a marker for tissue condition. Experimental procedures requiring unfrozen lenses from donor banks rely on transportation in storage medium, affecting lens homeostasis and alterations in glutathione levels. ....... The aim of the study was to examine the effects of Optisol-GS and castor oil on lens condition, determined from their ability to maintain glutathione concentrations....

The fate and transport of reproductive hormones and their conjugates in the environment (Invited)

Science.gov (United States)

Casey, F. X.; Shrestha, S. L.; Hakk, H.; Smith, D. J.; Larsen, G. L.; Padmanabhan, G.

2009-12-01

Reproductive steroid hormones can disrupt the endocrine system of some species at ng/L concentrations. Sources of steroid hormones to the environment include human waste water effluents or manure produced at animal feeding operations (AFOs). Steroid hormones, such as 17β-estradiol (E2) and estrone (E1), undergo various fate and transport processes, and laboratory studies have shown that they do not persist long (hours to few days), and have very little if any mobility in soil. Nonetheless, steroid hormones are detected at frequencies and concentrations of concern in the natural environment that would suggest their moderate persistence and mobility. One theory that may partially explain the disparity between field and laboratory studies is that conjugated forms of hormones are more mobile than their deconjugated counterparts. Glucuronide and sulfate conjugates are found in abundance in animal waste and are more soluble than their deconjugated forms. Laboratory studies were conducted to study the fate of a major urinary E2 conjugate, 17β-estradiol glucuronide (E2G), in a Hamar soil (Sandy, mixed, frigid typic Endoaquolls) from the surface and subsurface horizons. Speciation studies using batch sorption indicated that E2G degraded to E2 and E1 within 24 hours in the upper horizon soil with organic carbon content (OC) of 1.35%; whereas it persisted more in the lower horizon soil containing 0.32% OC. For initial concentrations of 2.8-28 mg/L, more than 15% of the applied dose concentration was still intact in the conjugate form in the aqueous phase for 3 - 14 days, in the lower horizon soil. The decline of E2G in the aqueous phase in the upper horizon soil was approximated with a first-order rate constant (k), which ranged from -0.208 to -0.279/h. The k values ranged from -0.006 to -0.016/h for the lower soil horizon. The differences in k values between the two horizons could be attributed to differences in bacterial activity and/or differences in sorption capacities

The poplar phi class glutathione transferase: expression, activity and structure of GSTF1

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Henri ePégeot

2014-12-01

Full Text Available Glutathione transferases (GSTs constitute a superfamily of enzymes with essential roles in cellular detoxification and secondary metabolism in plants as in other organisms. Several plant GSTs, including those of the Phi class (GSTFs, require a conserved catalytic serine residue to perform glutathione (GSH-conjugation reactions. Genomic analyses revealed that terrestrial plants have around 10 GSTFs, 8 in the Populus trichocarpa genome, but their physiological functions and substrates are mostly unknown. Transcript expression analyses showed a predominant expression of all genes both in reproductive (female flowers, fruits, floral buds and vegetative organs (leaves, petioles. Here, we show that the recombinant poplar GSTF1 (PttGSTF1 possesses peroxidase activity towards cumene hydroperoxide and GSH-conjugation activity towards model substrates such as 2,4-dinitrochlorobenzene, benzyl and phenetyl isothiocyanate, 4-nitrophenyl butyrate and 4-hydroxy-2-nonenal but interestingly not on previously identified GSTF-class substrates. In accordance to analytical gel filtration data, crystal structure of PttGSTF1 showed a canonical dimeric organization with bound GSH or MES molecules. The structure of these protein-substrate complexes allowed delineating the residues contributing to both the G and H sites that form the active site cavity. In sum, the presence of GSTF1 transcripts and proteins in most poplar organs especially those rich in secondary metabolites such as flowers and fruits, together with its GSH-conjugation activity and its documented stress-responsive expression suggest that its function is associated with the catalytic transformation of metabolites and/or peroxide removal rather than with ligandin properties as previously reported for other GSTFs.

Multigene families encode the major enzymes of antioxidant metabolism in Eucalyptus grandis L

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Felipe Karam Teixeira

2005-01-01

Full Text Available Antioxidant metabolism protects cells from oxidative damage caused by reactive oxygen species (ROS. In plants, several enzymes act jointly to maintain redox homeostasis. Moreover, isoform diversity contributes to the fine tuning necessary for plant responses to both exogenous and endogenous signals influencing antioxidant metabolism. This study aimed to provide a comprehensive view of the major classes of antioxidant enzymes in the woody species Eucalyptus grandis. A careful survey of the FORESTs data bank revealed 36 clusters as encoding antioxidant enzymes: six clusters encoding ascorbate peroxidase (APx isozymes, three catalase (CAT proteins, three dehydroascorbate reductase (DHAR, two glutathione reductase (GR isozymes, four monodehydroascorbate reductase (MDHAR, six phospholipid hydroperoxide glutathione peroxidases (PhGPx, and 12 encoding superoxide dismutases (SOD isozymes. Phylogenetic analysis demonstrated that all clusters (identified herein grouped with previously characterized antioxidant enzymes, corroborating the analysis performed. With respect to enzymes involved in the ascorbate-glutathione cycle, both cytosolic and chloroplastic isoforms were putatively identified. These sequences were widely distributed among the different ESTs libraries indicating a broad gene expression pattern. Overall, the data indicate the importance of antioxidant metabolism in eucalyptus.

Crystallization and preliminary X-ray diffraction analysis of a glutathione S-transferase from Xylella fastidiosa

International Nuclear Information System (INIS)

Garcia, Wanius; Travensolo, Regiane F.; Rodrigues, Nathalia C.; Muniz, João R. C.; Caruso, Célia S.; Lemos, Eliana G. M.; Araujo, Ana Paula U.; Carrilho, Emanuel

2008-01-01

Glutathione S-transferase from X. fastidiosa (xfGST) has been overexpressed in E. coli, purified and crystallized. Diffraction data were collected to 2.23 Å. Glutathione S-transferases (GSTs) form a group of multifunctional isoenzymes that catalyze the glutathione-dependent conjugation and reduction reactions involved in the cellular detoxification of xenobiotic and endobiotic compounds. GST from Xylella fastidiosa (xfGST) was overexpressed in Escherichia coli and purified by conventional affinity chromatography. In this study, the crystallization and preliminary X-ray analysis of xfGST is described. The purified protein was crystallized by the vapour-diffusion method, producing crystals that belonged to the triclinic space group P1. The unit-cell parameters were a = 47.73, b = 87.73, c = 90.74 Å, α = 63.45, β = 80.66, γ = 94.55°. xfGST crystals diffracted to 2.23 Å resolution on a rotating-anode X-ray source

Crystallization and preliminary X-ray diffraction analysis of a glutathione S-transferase from Xylella fastidiosa

Energy Technology Data Exchange (ETDEWEB)

Garcia, Wanius, E-mail: wanius@if.sc.usp.br [Laboratório de Biofísica Molecular ‘Sérgio Mascarenhas’, Instituto de Física de São Carlos, Universidade de São Paulo (USP), São Carlos (Brazil); Travensolo, Regiane F. [Grupo de Bioanalítica, Microfabricação e Separações, Instituto de Química de São Carlos, Universidade de São Paulo (USP), São Carlos (Brazil); Rodrigues, Nathalia C.; Muniz, João R. C. [Laboratório de Biofísica Molecular ‘Sérgio Mascarenhas’, Instituto de Física de São Carlos, Universidade de São Paulo (USP), São Carlos (Brazil); Caruso, Célia S. [Grupo de Bioanalítica, Microfabricação e Separações, Instituto de Química de São Carlos, Universidade de São Paulo (USP), São Carlos (Brazil); Lemos, Eliana G. M. [Laboratório de Bioquímica de Microrganismos e de Plantas, Departamento de Tecnologia, UNESP, Jaboticabal (Brazil); Araujo, Ana Paula U. [Laboratório de Biofísica Molecular ‘Sérgio Mascarenhas’, Instituto de Física de São Carlos, Universidade de São Paulo (USP), São Carlos (Brazil); Carrilho, Emanuel, E-mail: wanius@if.sc.usp.br [Grupo de Bioanalítica, Microfabricação e Separações, Instituto de Química de São Carlos, Universidade de São Paulo (USP), São Carlos (Brazil); Laboratório de Biofísica Molecular ‘Sérgio Mascarenhas’, Instituto de Física de São Carlos, Universidade de São Paulo (USP), São Carlos (Brazil)

2008-02-01

Glutathione S-transferase from X. fastidiosa (xfGST) has been overexpressed in E. coli, purified and crystallized. Diffraction data were collected to 2.23 Å. Glutathione S-transferases (GSTs) form a group of multifunctional isoenzymes that catalyze the glutathione-dependent conjugation and reduction reactions involved in the cellular detoxification of xenobiotic and endobiotic compounds. GST from Xylella fastidiosa (xfGST) was overexpressed in Escherichia coli and purified by conventional affinity chromatography. In this study, the crystallization and preliminary X-ray analysis of xfGST is described. The purified protein was crystallized by the vapour-diffusion method, producing crystals that belonged to the triclinic space group P1. The unit-cell parameters were a = 47.73, b = 87.73, c = 90.74 Å, α = 63.45, β = 80.66, γ = 94.55°. xfGST crystals diffracted to 2.23 Å resolution on a rotating-anode X-ray source.

Delineation of the Pasteurellaceae-specific GbpA-family of glutathione-binding proteins

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Vergauwen Bjorn

2011-11-01

Full Text Available Abstract Background The Gram-negative bacterium Haemophilus influenzae is a glutathione auxotroph and acquires the redox-active tripeptide by import. The dedicated glutathione transporter belongs to the ATP-binding cassette (ABC-transporter superfamily and displays more than 60% overall sequence identity with the well-studied dipeptide (Dpp permease of Escherichia coli. The solute binding protein (SBP that mediates glutathione transport in H. influenzae is a lipoprotein termed GbpA and is 54% identical to E. coli DppA, a well-studied member of family 5 SBP's. The discovery linking GbpA to glutathione import came rather unexpectedly as this import-priming SBP was previously annotated as a heme-binding protein (HbpA, and was thought to mediate heme acquisition. Nonetheless, although many SBP's have been implicated in more than one function, a prominent physiological role for GbpA and its partner permease in heme acquisition appears to be very unlikely. Here, we sought to characterize five representative GbpA homologs in an effort to delineate the novel GbpA-family of glutathione-specific family 5 SBPs and to further clarify their functional role in terms of ligand preferences. Results Lipoprotein and non-lipoprotein GbpA homologs were expressed in soluble form and substrate specificity was evaluated via a number of ligand binding assays. A physiologically insignificant affinity for hemin was observed for all five GbpA homologous test proteins. Three out of five test proteins were found to bind glutathione and some of its physiologically relevant derivatives with low- or submicromolar affinity. None of the tested SBP family 5 allocrites interacted with the remaining two GbpA test proteins. Structure-based sequence alignments and phylogenetic analysis show that the two binding-inert GbpA homologs clearly form a separate phylogenetic cluster. To elucidate a structure-function rationale for this phylogenetic differentiation, we determined the crystal

ORF Alignment: NC_001144 [GENIUS II[Archive

Lifescience Database Archive (English)

Full Text Available ... unconjugated bilirubin and in heavy metal detoxification ... via glutathione conjugates, al...mbrane transporter of MRP/CFTR family, found in ... vacuolar membrane, involved in the transport of ...

Metabolism of tert-butylhydroquinone to S-substituted conjugates in the male fischer 344 rat

NARCIS (Netherlands)

Peters, M.M.C.G.; Lau, S.S.; Dulik, D.; Murphy, D.; Ommen, B. van; Bladeren, P.J. van; Monks, T.J.

1996-01-01

tert-Butyl-4-hydroxyanisole (BHA) and its demethylated analog, tert- butyl-hydroquinone (TBHQ), are antioxidants used in food. Both BHA and TBHQ have been shown to promote kidney and bladder carcinogenesis in the rat. We have previously demonstrated that glutathione (GSH) conjugates of a variety of

Impact of the Crystallite Orientation Distribution on Exciton Transport in Donor–Acceptor Conjugated Polymers

KAUST Repository

Ayzner, Alexander L.; Mei, Jianguo; Appleton, Anthony; DeLongchamp, Dean; Nardes, Alexandre; Benight, Stephanie; Kopidakis, Nikos; Toney, Michael F.; Bao, Zhenan

2015-01-01

© 2015 American Chemical Society. Conjugated polymers are widely used materials in organic photovoltaic devices. Owing to their extended electronic wave functions, they often form semicrystalline thin films. In this work, we aim to understand whether distribution of crystallographic orientations affects exciton diffusion using a low-band-gap polymer backbone motif that is representative of the donor/acceptor copolymer class. Using the fact that the polymer side chain can tune the dominant crystallographic orientation in the thin film, we have measured the quenching of polymer photoluminescence, and thus the extent of exciton dissociation, as a function of crystal orientation with respect to a quenching substrate. We find that the crystallite orientation distribution has little effect on the average exciton diffusion length. We suggest several possibilities for the lack of correlation between crystallographic texture and exciton transport in semicrystalline conjugated polymer films.

Impact of the Crystallite Orientation Distribution on Exciton Transport in Donor–Acceptor Conjugated Polymers

KAUST Repository

Ayzner, Alexander L.

2015-12-30

© 2015 American Chemical Society. Conjugated polymers are widely used materials in organic photovoltaic devices. Owing to their extended electronic wave functions, they often form semicrystalline thin films. In this work, we aim to understand whether distribution of crystallographic orientations affects exciton diffusion using a low-band-gap polymer backbone motif that is representative of the donor/acceptor copolymer class. Using the fact that the polymer side chain can tune the dominant crystallographic orientation in the thin film, we have measured the quenching of polymer photoluminescence, and thus the extent of exciton dissociation, as a function of crystal orientation with respect to a quenching substrate. We find that the crystallite orientation distribution has little effect on the average exciton diffusion length. We suggest several possibilities for the lack of correlation between crystallographic texture and exciton transport in semicrystalline conjugated polymer films.

Activity-Based Probes for Isoenzyme- and Site-Specific Functional Characterization of Glutathione S -Transferases

Energy Technology Data Exchange (ETDEWEB)

Stoddard, Ethan G. [Chemical Biology and Exposure; Killinger, Bryan J. [Chemical Biology and Exposure; Nair, Reji N. [Chemical Biology and Exposure; Sadler, Natalie C. [Chemical Biology and Exposure; Volk, Regan F. [Chemical Biology and Exposure; Purvine, Samuel O. [Chemical Biology and Exposure; Shukla, Anil K. [Chemical Biology and Exposure; Smith, Jordan N. [Chemical Biology and Exposure; Wright, Aaron T. [Chemical Biology and Exposure

2017-11-01

Glutathione S-transferases (GSTs) comprise a highly diverse family of phase II drug metabolizing enzymes whose shared function is the conjugation of reduced glutathione to various endo- and xenobiotics. Although the conglomerate activity of these enzymes can be measured by colorimetric assays, measurement of the individual contribution from specific isoforms and their contribution to the detoxification of xenobiotics in complex biological samples has not been possible. For this reason, we have developed two activity-based probes that characterize active glutathione transferases in mammalian tissues. The GST active site is comprised of a glutathione binding “G site” and a distinct substrate binding “H site”. Therefore, we developed (1) a glutathione-based photoaffinity probe (GSH-ABP) to target the “G site”, and (2) a probe designed to mimic a substrate molecule and show “H site” activity (GST-ABP). The GSH-ABP features a photoreactive moiety for UV-induced covalent binding to GSTs and glutathione-binding enzymes. The GST-ABP is a derivative of a known mechanism-based GST inhibitor that binds within the active site and inhibits GST activity. Validation of probe targets and “G” and “H” site specificity was carried out using a series of competitors in liver homogenates. Herein, we present robust tools for the novel characterization of enzyme- and active site-specific GST activity in mammalian model systems.

Transferrin-conjugated magnetic dextran-spermine nanoparticles for targeted drug transport across blood-brain barrier.

Science.gov (United States)

Ghadiri, Maryam; Vasheghani-Farahani, Ebrahim; Atyabi, Fatemeh; Kobarfard, Farzad; Mohamadyar-Toupkanlou, Farzaneh; Hosseinkhani, Hossein

2017-10-01

Application of many vital hydrophilic medicines have been restricted by blood-brain barrier (BBB) for treatment of brain diseases. In this study, a targeted drug delivery system based on dextran-spermine biopolymer was developed for drug transport across BBB. Drug loaded magnetic dextran-spermine nanoparticles (DS-NPs) were prepared via ionic gelation followed by transferrin (Tf) conjugation as targeting moiety. The characteristics of Tf conjugated nanoparticles (TDS-NPs) were analyzed by different methods and their cytotoxicity effects on U87MG cells were tested. The superparamagnetic characteristic of TDS-NPs was verified by vibration simple magnetometer. Capecitabine loaded TDS-NPs exhibited pH-sensitive release behavior with enhanced cytotoxicity against U87MG cells, compared to DS-NPs and free capecitabine. Prussian-blue staining and TEM-imaging showed the significant cellular uptake of TDS-NPs. Furthermore, a remarkable increase of Fe concentrations in brain was observed following their biodistribution and histological studies in vivo, after 1 and 7 days of post-injection. Enhanced drug transport across BBB and pH-triggered cellular uptake of TDS-NPs indicated that these theranostic nanocarriers are promising candidate for the brain malignance treatment. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 2851-2864, 2017. © 2017 Wiley Periodicals, Inc.

Carrier Transport Enhancement in Conjugated Polymers through Interfacial Self-Assembly of Solution-State Aggregates

KAUST Repository

Zhao, Kui

2016-07-13

We demonstrate that local and long range orders of poly(3-hexylthiophene) (P3HT) semicrystalline films can be synergistically improved by combining chemical functionalization of the dielectric surface with solution-state disentanglement and pre-aggregation of P3HT in a theta solvent, leading to a very significant enhancement of the field effect carrier mobility. The pre-aggregation and surface functionalization effects combine to enhance the carrier mobility nearly 100-fold as compared with standard film preparation by spin-coating, and nearly 10-fold increase over the benefits of pre-aggregation alone. In situ quartz crystal microbalance with dissipation (QCM-D) experiments reveal enhanced deposition of pre-aggregates on surfaces modified with an alkyl-terminated self-assembled monolayer (SAM) in comparison to un-aggregated polymer chains. Additional investigations reveal the combined pre-aggregation and surface functionalization significantly enhances local order of the conjugated polymer through planarization and extension of the conjugated backbone of the polymer which clearly translate to significant improvements of carrier transport at the semiconductor-dielectric interface in organic thin film transistors. This study points to opportunities in combining complementary routes, such as well-known pre-aggregation with substrate chemical functionalization, to enhance the polymer self-assembly and improve its interfacial order with benefits for transport properties.

Treatment with Cefotaxime Affects Expression of Conjugation Associated Proteins and Conjugation Transfer Frequency of an IncI1 Plasmid in Escherichia coli

DEFF Research Database (Denmark)

Møller, Thea S B; Liu, Gang; Boysen, Anders

2017-01-01

research suggests that the effect of antibiotic treatment on plasmid conjugation frequencies, and hence the spread of resistance plasmids, may have been overestimated. We addressed the question by quantifying transfer proteins and conjugation frequencies of a blaCTX-M-1 encoding IncI1 resistance plasmid....... The frequency of plasmid conjugation, measured in an antibiotic free environment, increased significantly when the donor was pre-grown in broth containing CTX compared to growth without this drug, regardless of whether blaCTX-M-1 was located on the plasmid or in trans on the chromosome. The results shows...

Type 3 Fimbriae Encoded on Plasmids Are Expressed from a Unique Promoter without Affecting Host Motility, Facilitating an Exceptional Phenotype That Enhances Conjugal Plasmid Transfer

DEFF Research Database (Denmark)

Madsen, Jonas Stenlokke; Riber, Leise; Kot, Witold

2016-01-01

Horizontal gene transfer (HGT), the transmission of genetic material to a recipient that is not the progeny of the donor, is fundamental in bacterial evolution. HGT is often mediated by mobile genetic elements such as conjugative plasmids, which may be in conflict with the chromosomal elements...... of the genome because they are independent replicons that may petition their own evolutionary strategy. Here we study differences between type 3 fimbriae encoded on wild type plasmids and in chromosomes. Using known and newly characterized plasmids we show that the expression of type 3 fimbriae encoded...... on plasmids is systematically different, as MrkH, a c-di-GMP dependent transcriptional activator is not needed for strong expression of the fimbriae. MrkH is required for expression of type 3 fimbriae of the Klebsiella pneumoniae chromosome, wherefrom the fimbriae operon (mrkABCDF) of plasmids is believed...

Hijacking membrane transporters for arsenic phytoextraction

Science.gov (United States)

LeBlanc, Melissa S.; McKinney, Elizabeth C.; Meagher, Richard B.; Smith, Aaron P.

2012-01-01

Arsenic is a toxic metalloid and recognized carcinogen. Arsenate and arsenite are the most common arsenic species available for uptake by plants. As an inorganic phosphate (Pi) analog, arsenate is acquired by plant roots through endogenous Pi transport systems. Inside the cell, arsenate is reduced to the thiol-reactive form arsenite. Glutathione (GSH)-conjugates of arsenite may be extruded from the cell or sequestered in vacuoles by members of the ATP-binding cassette (ABC) family of transporters. In the present study we sought to enhance both plant arsenic uptake through Pi transporter overexpression, and plant arsenic tolerance through ABC transporter overexpression. We demonstrate that Arabidopsis thaliana plants overexpressing the high-affinity Pi transporter family members, AtPht1;1 or AtPht1;7, are hypersensitive to arsenate due to increased arsenate uptake. These plants do not exhibit increased sensitivity to arsenite. Co-overexpression of the yeast ABC transporter YCF1 in combination with AtPht1;1 or AtPht1;7 suppresses the arsenate-sensitive phenotype while further enhancing arsenic uptake. Taken together, our results support an arsenic transport mechanism in which arsenate uptake is increased through Pi transporter overexpression, and arsenic tolerance is enhanced through YCF1-mediated vacuolar sequestration. This work substantiates the viability of coupling enhanced uptake and vacuolar sequestration as a means for developing a prototypical engineered arsenic hyperaccumulator. PMID:23108027

Impact of haloperidol and quetiapine on the expression of genes encoding antioxidant enzymes in human neuroblastoma SH-SY5Y cells.

Science.gov (United States)

Schmidt, Andreas Johannes; Hemmeter, Ulrich Michael; Krieg, Jürgen-Christian; Vedder, Helmut; Heiser, Philip

2009-05-01

Antipsychotics are known to alter antioxidant activities in vivo. Therefore, the aim of the present study was to examine in the human neuroblastoma SH-SY5Y cell line the impact of a typical (haloperidol) and an atypical (quetiapine) antipsychotic on the expression of genes encoding the key enzymes of the antioxidant metabolism (Cu, Zn superoxide dismutase; Mn superoxide dismutase; glutathione peroxidase; catalase) and enzymes of the glutathione metabolism (gamma-glutamyl cysteine synthetase, glutathione-S-transferase, gamma-glutamyltranspeptidase, glutathione reductase). The cells were incubated for 24h with 0.3, 3, 30 and 300microM haloperidol and quetiapine, respectively; mRNA levels were measured by polymerase chain reaction. In the present study, we observed mostly significant decreases of mRNA contents. With respect to the key pathways, we detected mainly effects on the mRNA levels of the hydrogen peroxide detoxifying enzymes. Among the enzymes of the glutathione metabolism, glutathione-S-transferase- and gamma-glutamyltranspeptidase-mRNA levels showed the most prominent effects. Taken together, our results demonstrate a significantly reduced expression of genes encoding for antioxidant enzymes after treatment with the antipsychotics, haloperidol and quetiapine.

CONJUGATED POLYMERS AND POLYELECTROLYTES IN SOLAR PHOTOCONVERSION, Final Technical Report

Energy Technology Data Exchange (ETDEWEB)

Schanze, Kirk S [University of Florida

2014-08-05

This DOE-supported program investigated the fundamental properties of conjugated polyelectrolytes, with emphasis placed on studies of excited state energy transport, self-assembly into conjugated polyelectroyte (CPE) based films and colloids, and exciton transport and charge injection in CPE films constructed atop wide bandgap semiconductors. In the most recent grant period we have also extended efforts to examine the properties of low-bandgap donor-acceptor conjugated polyelectrolytes that feature strong visible light absorption and the ability to adsorb to metal-oxide interfaces.

Molecular docking simulations provide insights in the substrate binding sites and possible substrates of the ABCC6 transporter.

Directory of Open Access Journals (Sweden)

Mohammad Jakir Hosen

Full Text Available The human ATP-binding cassette family C member 6 (ABCC6 gene encodes an ABC transporter protein (ABCC6, primarily expressed in liver and kidney. Mutations in the ABCC6 gene cause pseudoxanthoma elasticum (PXE, an autosomal recessive connective tissue disease characterized by ectopic mineralization of the elastic fibers. The pathophysiology underlying PXE is incompletely understood, which can at least partly be explained by the undetermined nature of the ABCC6 substrates as well as the unknown substrate recognition and binding sites. Several compounds, including anionic glutathione conjugates (N-ethylmaleimide; NEM-GS and leukotriene C4 (LTC4 were shown to be modestly transported in vitro; conversely, vitamin K3 (VK3 was demonstrated not to be transported by ABCC6. To predict the possible substrate binding pockets of the ABCC6 transporter, we generated a 3D homology model of ABCC6 in both open and closed conformation, qualified for molecular docking and virtual screening approaches. By docking 10 reported in vitro substrates in our ABCC6 3D homology models, we were able to predict the substrate binding residues of ABCC6. Further, virtual screening of 4651 metabolites from the Human Serum Metabolome Database against our open conformation model disclosed possible substrates for ABCC6, which are mostly lipid and biliary secretion compounds, some of which are found to be involved in mineralization. Docking of these possible substrates in the closed conformation model also showed high affinity. Virtual screening expands this possibility to explore more compounds that can interact with ABCC6, and may aid in understanding the mechanisms leading to PXE.

Epoxidation of the methamphetamine pyrolysis product, trans-phenylpropene, to trans-phenylpropylene oxide by CYP enzymes and stereoselective glutathione adduct formation

International Nuclear Information System (INIS)

Sanga, Madhu; Younis, Islam R.; Tirumalai, Padma S.; Bland, Tina M.; Banaszewska, Monica; Konat, Gregory W.; Tracy, Timothy S.; Gannett, Peter M.; Callery, Patrick S.

2006-01-01

Pyrolytic products of smoked methamphetamine hydrochloride are well established. Among the various degradation products formed, trans-phenylpropene (trans-β-methylstyrene) is structurally similar to styrene analogues known to be bioactivated by CYP enzymes. In human liver microsomes, trans-phenylpropene was converted to the epoxide trans-phenylpropylene oxide (trans-2-methyl-3-phenyloxirane) and cinnamyl alcohol. Incubation of trans-phenylpropene with microsomes in the presence of enzyme-specific P450 enzyme inhibitors indicated the involvement of CYP2E1, CYP1A2, and CYP3A4 enzymes. Both (R,R)-phenylpropylene oxide and (S,S)-phenylpropylene oxide were formed in human liver microsomal preparations. Enantiomers of trans-phenylpropylene oxide were stereoselectively and regioselectively conjugated in a Phase II drug metabolism reaction catalyzed by human liver cytosolic enzymes consisting of conjugation with glutathione. The structure of the phenylpropylene oxide-glutathione adduct is consistent with nucleophilic ring-opening by attack at the benzylic carbon. Exposure of cultured C6 glial cells to (S,S)-phenylpropylene oxide produced a cytotoxic response in a concentration-dependent manner based on cell degeneration and death

Effect of a Fusion Peptide by Covalent Conjugation of a Mitochondrial Cell-Penetrating Peptide and a Glutathione Analog Peptide

Directory of Open Access Journals (Sweden)

Carmine Pasquale Cerrato

2017-06-01

Full Text Available Previously, we designed and synthesized a library of mitochondrial antioxidative cell-penetrating peptides (mtCPPs superior to the parent peptide, SS31, to protect mitochondria from oxidative damage. A library of antioxidative glutathione analogs called glutathione peptides (UPFs, exceptional in hydroxyl radical elimination compared with glutathione, were also designed and synthesized. Here, a follow-up study is described, investigating the effects of the most promising members from both libraries on reactive oxidative species scavenging ability. None of the peptides influenced cell viability at the concentrations used. Fluorescence microscopy studies showed that the fluorescein-mtCPP1-UPF25 (mtgCPP internalized into cells, and spectrofluorometric analysis determined the presence and extent of peptide into different cell compartments. mtgCPP has superior antioxidative activity compared with mtCPP1 and UPF25 against H2O2 insult, preventing ROS formation by 2- and 3-fold, respectively. Moreover, we neither observed effects on mitochondrial membrane potential nor production of ATP. These data indicate that mtgCPP is targeting mitochondria, protecting them from oxidative damage, while also being present in the cytosol. Our hypothesis is based on a synergistic effect resulting from the fused peptide. The mitochondrial peptide segment is targeting mitochondria, whereas the glutathione analog peptide segment is active in the cytosol, resulting in increased scavenging ability.

The diverse roles of glutathione-associated cell resistance against hypericin photodynamic therapy

Directory of Open Access Journals (Sweden)

Theodossis A. Theodossiou

2017-08-01

Full Text Available The diverse responses of different cancers to treatments such as photodynamic therapy of cancer (PDT have fueled a growing need for reliable predictive markers for treatment outcome. In the present work we have studied the differential response of two phenotypically and genotypically different breast adenocarcinoma cell lines, MCF7 and MDA-MB-231, to hypericin PDT (HYP-PDT. MDA-MB-231 cells were 70% more sensitive to HYP PDT than MCF7 cells at LD50. MCF7 were found to express a substantially higher level of glutathione peroxidase (GPX4 than MDA-MB-231, while MDA-MB-231 differentially expressed glutathione-S-transferase (GSTP1, mainly used for xenobiotic detoxification. Eighty % reduction of intracellular glutathione (GSH by buthionine sulfoximine (BSO, largely enhanced the sensitivity of the GSTP1 expressing MDA-MB-231 cells to HYP-PDT, but not in MCF7 cells. Further inhibition of the GSH reduction however by carmustine (BCNU resulted in an enhanced sensitivity of MCF7 to HYP-PDT. HYP loading studies suggested that HYP can be a substrate of GSTP for GSH conjugation as BSO enhanced the cellular HYP accumulation by 20% in MDA-MB-231 cells, but not in MCF7 cells. Studies in solutions showed that L-cysteine can bind the GSTP substrate CDNB in the absence of GSTP. This means that the GSTP-lacking MCF7 may use L-cysteine for xenobiotic detoxification, especially during GSH synthesis inhibition, which leads to L-cysteine build-up. This was confirmed by the lowered accumulation of HYP in both cell lines in the presence of BSO and the L-cysteine source NAC. NAC reduced the sensitivity of MCF7, but not MDA-MB-231, cells to HYP PDT which is in accordance with the antioxidant effects of L-cysteine and its potential as a GSTP substrate. As a conclusion we have herein shown that the different GSH based cell defense mechanisms can be utilized as predictive markers for the outcome of PDT and as a guide for selecting optimal combination strategies. Keywords

Cross-conjugation and quantum interference: a general correlation?

DEFF Research Database (Denmark)

Valkenier, Hennie; Guedon, Constant M.; Markussen, Troels

2014-01-01

We discuss the relationship between the pi-conjugation pattern, molecular length, and charge transport properties of molecular wires, both from an experimental and a theoretical viewpoint. Specifically, we focus on the role of quantum interference in the conductance properties of cross-conjugated...

The Mimivirus Genome Encodes a Mitochondrial Carrier That Transports dATP and dTTP▿

Science.gov (United States)

Monné, Magnus; Robinson, Alan J.; Boes, Christoph; Harbour, Michael E.; Fearnley, Ian M.; Kunji, Edmund R. S.

2007-01-01

Members of the mitochondrial carrier family have been reported in eukaryotes only, where they transport metabolites and cofactors across the mitochondrial inner membrane to link the metabolic pathways of the cytosol and the matrix. The genome of the giant virus Mimiviridae mimivirus encodes a member of the mitochondrial carrier family of transport proteins. This viral protein has been expressed in Lactococcus lactis and is shown to transport dATP and dTTP. As the 1.2-Mb double-stranded DNA mimivirus genome is rich in A and T residues, we speculate that the virus is using this protein to target the host mitochondria as a source of deoxynucleotides for its replication. PMID:17229695

Characterization of biliary conjugates of 4,4'-methylenedianiline in male versus female rats

International Nuclear Information System (INIS)

Chen, Kan; Cole, Richard B.; Santa Cruz, Vicente; Blakeney, Ernest W.; Kanz, Mary F.; Dugas, Tammy R.

2008-01-01

4,4'-Methylenedianiline (4,4'-diaminodiphenylmethane; DAPM) is an aromatic diamine used in the production of numerous polyurethane foams and epoxy resins. Previous studies in rats revealed that DAPM initially injures biliary epithelial cells of the liver, that the toxicity is greater in female than in male rats, and that the toxic metabolites of DAPM are excreted into bile. Since male and female rats exhibit differences in the expression of both phase I and phase II enzymes, our hypothesis was that female rats either metabolize DAPM to more toxic metabolites or have a decreased capacity to conjugate metabolites to less toxic intermediates. Our objective was thus to isolate, characterize, and quantify DAPM metabolites excreted into bile in both male and female bile duct-cannulated Sprague Dawley rats. The rats were gavaged with [ 14 C]-DAPM, and the collected bile was subjected to reversed-phase HPLC with radioisotope detection. Peaks eluting from HPLC were collected and analyzed using electrospray MS and NMR spectroscopy. HPLC analysis indicated numerous metabolites in both sexes, but male rats excreted greater amounts of glutathione and glucuronide conjugates than females. Electrospray MS and NMR spectra of HPLC fractions revealed that the most prominent metabolite found in bile of both sexes was a glutathione conjugate of an imine metabolite of a 4'-nitroso-DAPM. Seven other metabolites were identified, including acetylated, cysteinyl-glycine, glutamyl-cysteine, glycine, and glucuronide conjugates. While our prior studies demonstrated increased covalent binding of DAPM in the liver and bile of female compared to male rats, in these studies, SDS-PAGE with autoradiography revealed 4-5 radiolabeled protein bands in the bile of rats treated with [ 14 C]-DAPM. In addition, these bands were much more prominent in female than in male rats. These studies thus suggest that a plausible mechanism for the increased sensitivity of female rats to DAPM toxicity may be decreased

Glutathione S-transferase M1 and T1, CYP1A2-2467T/delT ...

African Journals Online (AJOL)

The present study investigated the impact of metabolic gene polymorphisms in modulating lung cancer risk susceptibility. Gene polymorphisms encoding Cytochrome 1A2 (CYP1A2) and Glutathione-S-transferases (GSTT1 and GSTM1) are involved in the bioactivation and detoxification of tobacco carcinogens and may ...

Requirement for Vibrio cholerae integration host factor in conjugative DNA transfer.

Science.gov (United States)

McLeod, Sarah M; Burrus, Vincent; Waldor, Matthew K

2006-08-01

The requirement for host factors in the transmission of integrative and conjugative elements (ICEs) has not been extensively explored. Here we tested whether integration host factor (IHF) or Fis, two host-encoded nucleoid proteins, are required for transfer of SXT, a Vibrio cholerae-derived ICE that can be transmitted to many gram-negative species. Fis did not influence the transfer of SXT to or from V. cholerae. In contrast, IHF proved to be required for V. cholerae to act as an SXT donor. In the absence of IHF, V. cholerae displayed a modest defect for serving as an SXT recipient. Surprisingly, SXT integration into or excision from the V. cholerae chromosome, which requires an SXT-encoded integrase related to lambda integrase, did not require IHF. Therefore, the defect in SXT transmission in the V. cholerae IHF mutant is probably not related to IHF's ability to promote DNA recombination. The V. cholerae IHF mutant was also highly impaired as a donor of RP4, a broad-host-range conjugative plasmid. Thus, the V. cholerae IHF mutant appears to have a general defect in conjugation. Escherichia coli IHF mutants were not impaired as donors or recipients of SXT or RP4, indicating that IHF is a V. cholerae-specific conjugation factor.

Genes encoding novel lipid transporters and their use to increase oil production in vegetative tissues of plants

Science.gov (United States)

Xu, Changcheng; Fan, Jilian; Yan, Chengshi; Shanklin, John

2017-12-26

The present invention discloses a novel gene encoding a transporter protein trigalactosyldiacylglycerol-5 (TGD5), mutations thereof and their use to enhance TAG production and retention in plant vegetative tissue.

Glutathione in the human brain: Review of its roles and measurement by magnetic resonance spectroscopy.

Science.gov (United States)

Rae, Caroline D; Williams, Stephen R

2017-07-15

We review the transport, synthesis and catabolism of glutathione in the brain as well as its compartmentation and biochemistry in different brain cells. The major reactions involving glutathione are reviewed and the factors limiting its availability in brain cells are discussed. We also describe and critique current methods for measuring glutathione in the human brain using magnetic resonance spectroscopy, and review the literature on glutathione measurements in healthy brains and in neurological, psychiatric, neurodegenerative and neurodevelopmental conditions In summary: Healthy human brain glutathione concentration is ∼1-2 mM, but it varies by brain region, with evidence of gender differences and age effects; in neurological disease glutathione appears reduced in multiple sclerosis, motor neurone disease and epilepsy, while being increased in meningiomas; in psychiatric disease the picture is complex and confounded by methodological differences, regional effects, length of disease and drug-treatment. Both increases and decreases in glutathione have been reported in depression and schizophrenia. In Alzheimer's disease and mild cognitive impairment there is evidence for a decrease in glutathione compared to age-matched healthy controls. Improved methods to measure glutathione in vivo will provide better precision in glutathione determination and help resolve the complex biochemistry of this molecule in health and disease. Copyright © 2017 Elsevier Inc. All rights reserved.

Interaction of gamma-glutamyltranspeptidase with clofibryl-S-acyl-glutathione in vitro and in vivo in rat.

Science.gov (United States)

Grillo, M P; Benet, L Z

2001-08-01

Clofibric acid (CA) is metabolized to chemically reactive acylating products that can transacylate glutathione to form clofibryl-S-acyl-glutathione (CA-SG) in vitro and in vivo. We investigated the first step in the degradation of CA-SG to the mercapturic acid conjugate, clofibryl-S-acyl-N-acetylcysteine (CA-SNAC), which is catalyzed by gamma-glutamyltranspeptidase (gamma-GT). After gamma-GT mediated cleavage of glutamate from CA-SG, the product clofibryl-S-acyl-cysteinylglycine (CA-S-CG) should undergo an intramolecular rearrangement reaction [Tate, S. S. (1975) FEBS Lett. 54, 319-322] to form clofibryl-N-acyl-cysteinylglycine (CA-N-CG). We performed in vitro studies incubating CA-SG with gamma-GT to determine the products formed, and in vivo studies examining the products excreted in urine after dosing rats with CA-SG or CA. Thus, CA-SG (0.1 mM) was incubated with gamma-GT (0.1 unit/mL) in buffer (pH 7.4, 25 degrees C) and analyzed for products formed by reversed-phase HPLC and electrospray mass spectrometry (ESI/MS). Results showed that CA-SG is degraded completely after 6 h of incubation leading to the formation of two products, CA-N-CG and its disulfide, with no detection of CA-S-CG thioester. After 36 h of incubation, only the disulfide remained in the incubation. Treatment of the disulfide with dithiothreitol led to the reappearance of CA-N-CG. ESI/LC/MS analysis of urine (16 h) extracts of CA-SG-dosed rats (200 mg/kg, iv) showed that CA-SG is degraded to CA-N-CG, CA-N-acyl-cysteine (CA-N-C) and their respective S-methylated products. The mercapturic acid conjugate (CA-SNAC) was found as a minor product. Analysis of urine extracts from CA-dosed rats (200 mg/kg, ip) resulted in the detection of clofibryl-N-acyl-cysteine (CA-N-C), but no evidence for the formation of CA-SNAC was obtained. These in vitro and in vivo experiments indicate that gamma-GT mediated degradation of clofibryl-S-acyl-glutathione leads primarily to the formation and excretion of clofibryl

Brain-targeted delivery of trans-activating transcriptor-conjugated magnetic PLGA/lipid nanoparticles.

Directory of Open Access Journals (Sweden)

Xiangru Wen

Full Text Available Magnetic poly (D,L-lactide-co-glycolide (PLGA/lipid nanoparticles (MPLs were fabricated from PLGA, L-α-phosphatidylethanolamine (DOPE, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-amino (polyethylene glycol (DSPE-PEG-NH2, and magnetic nanoparticles (NPs, and then conjugated to trans-activating transcriptor (TAT peptide. The TAT-MPLs were designed to target the brain by magnetic guidance and TAT conjugation. The drugs hesperidin (HES, naringin (NAR, and glutathione (GSH were encapsulated in MPLs with drug loading capacity (>10% and drug encapsulation efficiency (>90%. The therapeutic efficacy of the drug-loaded TAT-MPLs in bEnd.3 cells was compared with that of drug-loaded MPLs. The cells accumulated higher levels of TAT-MPLs than MPLs. In addition, the accumulation of QD-loaded fluorescein isothiocyanate (FITC-labeled TAT-MPLs in bEnd.3 cells was dose and time dependent. Our results show that TAT-conjugated MPLs may function as an effective drug delivery system that crosses the blood brain barrier to the brain.

Enhancement of anti-tumor activity of hybrid peptide in conjugation with carboxymethyl dextran via disulfide linkers.

Science.gov (United States)

Gaowa, Arong; Horibe, Tomohisa; Kohno, Masayuki; Tabata, Yasuhiko; Harada, Hiroshi; Hiraoka, Masahiro; Kawakami, Koji

2015-05-01

To improve the anti-tumor activity of EGFR2R-lytic hybrid peptide, we prepared peptide-modified dextran conjugates with the disulfide bonds between thiolated carboxymethyl dextran (CMD-Cys) and cysteine-conjugated peptide (EGFR2R-lytic-Cys). In vitro release studies showed that the peptide was released from the CMD-s-s-peptide conjugate in a concentration-dependent manner in the presence of glutathione (GSH, 2μM-2mM). The CMD-s-s-peptide conjugate exhibited a similar cytotoxic activity with free peptide alone against human pancreatic cancer BxPC-3 cells in vitro. Furthermore, it was shown that the CMD-s-s-peptide conjugates were highly accumulated in tumor tissue in a mouse xenograft model using BxPC-3 cells, and the anti-tumor activity of the conjugate was more effective than that of the free peptide. In addition, the plasma concentrations of peptide were moderately increased and the elimination half-life of the peptide was prolonged after intravenous injection of CMD-s-s-peptide conjugates. These results demonstrated that the conjugate based on thiolated CMD polymer would be potentially useful carriers for the sustained release of the hybrid peptide in vivo. Copyright © 2015 Elsevier B.V. All rights reserved.

Novel interaction of diethyldithiocarbamate with the glutathione/glutathione peroxidase system

International Nuclear Information System (INIS)

Kumar, K.S.; Sancho, A.M.; Weiss, J.F.

1986-01-01

Diethyldithiocarbamate (DDC) exhibits a variety of pharmacologic activities, including both radioprotective and sensitizing properties. Since the glutathione/glutathione peroxidase system may be a significant factor in determining radiation sensitivity, the potential mechanisms of action of DDC in relation to this system were examined in vitro. The interaction of DDC with reduced glutathione (GSH) was tested using a simple system based on the reduction of cytochrome c. When DDC (0.005 mM) was incubated with GSH (0.5 mM), the reduction of cytochrome c was eightfold greater than that expected from an additive effect of DDC and GSH. GSH could be replaced by oxidized glutathione and glutathione reductase. Cytochrome c reduced by DDC was oxidized by mitochondria. The interaction of DDC with both the hexosemonophosphate shunt pathway and the mitochondrial respiratory chain suggests the possibility of linking these two pathways through DDC. Oxidation of DDC by peroxide and reversal by GSH indicated that the drug can engage in a cyclic reaction with peroxide and GSH. This was confirmed when DDC was used in the assay system for glutathione peroxidase (GSHPx) without GSHPx. DDC at a concentration of 0.25 mM was more active than 0.01 unit of pure GSHPx in eliminating peroxide, and much more active than the other sulfhydryl compounds tested. These studies indicate that DDC can supplement GSHPx activity or substitute for it in detoxifying peroxides, and suggests a unique role in the chemical modification of radiation sensitivity

Interaction of uranium(VI) towards glutathione. An example to study different functional groups in one molecule

International Nuclear Information System (INIS)

Frost, L.; Geipel, G.; Viehweger, K.; Bernhard, G.

2011-01-01

Glutathione, the most abundant thiol compound of the cell, has a great binding potential towards heavy metal ions. Hence it might influence the distribution of actinides on a cellular level. The unknown strength of the interaction of uranium(VI) with glutathione at physiologically relevant pH is subject of this paper and was studied with UV-vis spectroscopy and time-resolved laserinduced fluorescence spectroscopy (TRLFS). The complex stability constant of UO 2 H 2 GS + , logβ 121 0 , was calculated to be 39.09±0.15 and 39.04±0.02 in case of UV-vis spectroscopy and TRLFS respectively. Therefore the average formation constant for UO 2 2+ +H 2 GS - = UO 2 H 2 GS + can be assigned to be log K 0 11 = 19.83±0.15. Furthermore it was demonstrated that derivatization of the ligand associated with an enhancement of the ligand's spectroscopic properties can be used for the determination of complex stability constants and to assess the coordination chemistry in more detail. Using UV-vis spectroscopy, the stability constant of the complex between UO 2 2+ and glutathione pyruvate S-conjugate, a well absorbing ligand in contrast to glutathione, was calculated to be > 39.24±0.08. Furthermore the interaction of UO 2 2+ with glutathione derivatized with the fluorescent label monobromobimane was examined with femtosecond laser fluorescence spectroscopy. Thereby the stability constant of the 1: 1 complex was determined to be > 39.35±0.02. Although the thiol group of glutathione was blocked a strong coordination was found. Thus a significant involvement of the thiol group in the coordination of U(VI) can be excluded. (orig.)

Plasmid Conjugation in E. coli and Drug Resistance | Igwe ...

African Journals Online (AJOL)

This study aimed at determining the antibiotics susceptibility pattern of E. coli isolates claimed to be multidrug resistance using disc diffusion method. It also determined the presence of transferable resistance plasmids through conjugation and evaluated the medical significance of plasmid encoding E. coli and drug ...

Correction of glutathione metabolism in the liver of albino rats affected by low radiation doses

International Nuclear Information System (INIS)

Moiseenok, A.G.; Slyshenkov, V.S.; Khomich, T.I.; Zimatkina, T.I.; Kanunnikova, N.P.

1997-01-01

The levels of total glutathione GSH, GSSG and the activities of glutathione reductase and glutathione peroxidase were studied in the liver of adult albino rats subjected to 3-fold external γ-irradiation throughout 2 weeks at the overall dose of 0.75 Gy after 15 h, 2 and 5 days from the last irradiation. Some animals were injected intraperitoneally with the pantothenate containing complex > 3 times on days 1-3 before the irradiation. The radiation related decrease of GSH, GSH/GSSG and the total glutathione level was prevented by the prophylactic administration of the complex and probably at the expense of the activation of the G-SH biosynthesis and/or transport in the liver by the CoA biosynthetic precursor. (author)

Self-assembly of pi-conjugated peptides in aqueous environments leading to energy-transporting bioelectronic nanostructures

Energy Technology Data Exchange (ETDEWEB)

Tavor, John [Johns Hopkins Univ., Baltimore, MD (United States)

2016-12-06

The realization of new supramolecular pi-conjugated organic structures inspired and driven by peptide-based self-assembly will offer a new approach to interface with the biotic environment in a way that will help to meet many DOE-recognized grand challenges. Previously, we developed pi-conjugated peptides that undergo supramolecular self-assembly into one-dimensional (1-D) organic electronic nanomaterials under benign aqueous conditions. The intermolecular interactions among the pi-conjugated organic segments within these nanomaterials lead to defined perturbations of their optoelectronic properties and yield nanoscale conduits that support energy transport within individual nanostructures and throughout bulk macroscopic collections of nanomaterials. Our objectives for future research are to construct and study biomimetic electronic materials for energy-related technology optimized for harsher non-biological environments where peptide-driven self-assembly enhances pi-stacking within nanostructured biomaterials, as detailed in the following specific tasks: (1) synthesis and detailed optoelectronic characterization of new pi-electron units to embed within homogeneous self assembling peptides, (2) molecular and data-driven modeling of the nanomaterial aggregates and their higher-order assemblies, and (3) development of new hierarchical assembly paradigms to organize multiple electronic subunits within the nanomaterials leading to heterogeneous electronic properties (i.e. gradients and localized electric fields). These intertwined research tasks will lead to the continued development and fundamental mechanistic understanding of a powerful bioinspired materials set capable of making connections between nanoscale electronic materials and macroscopic bulk interfaces, be they those of a cell, a protein or a device.

Intestinal transport and metabolism of acrylamide

International Nuclear Information System (INIS)

Zoedl, Bettina; Schmid, Diethart; Wassler, Georg; Gundacker, Claudia; Leibetseder, Valentin; Thalhammer, Theresia; Ekmekcioglu, Cem

2007-01-01

There has been an intensive debate whether dietary exposure to acrylamide could increase the risk of human cancer since the first description of the presence of acrylamide in food in 2002. As the intestinal mechanisms of acrylamide absorption are poorly investigated we studied the transport of acrylamide in differentiated Caco-2 cells and its effects on biotransformation enzymes (CYP2E1 and glutathione S-transferase) and glutathione levels. We found that the apparent permeability of [1- 14 C] acrylamide from the basal to the apical compartment was approximately 20% higher compared to that in the opposite direction. No differences were detected for apical-basal transport against a basal gradient. Transport rates from the apical to the basal chamber at 4 deg. C were about 50% lower than at 37 deg. C. Concentration dependent transport from apical to basal was linear. Predominantly, basal to apical transport was decreased when energy metabolism of the cells was inhibited by application of sodium azide and 2-deoxy-D-glucose. Finally, more acrylamide was transported at luminal pH 6 compared to pH 7.4 from basal to the apical direction. Increasing levels of acrylamide showed no effects on the activity of glutathione S-transferase but resulted in a depletion of total glutathione concentrations. In conclusion transport of acrylamide in the intestine is mediated primarily by passive processes possibly combined with a modest energy- and pH-dependent active secretory component. Depletion of cellular glutathione levels may be one potential mechanism for acrylamide (geno)toxicity

Catalysis of Silver catfish Major Hepatic Glutathione Transferase proceeds via rapid equilibrium sequential random Mechanism

Directory of Open Access Journals (Sweden)

Ayodele O. Kolawole

Full Text Available Fish hepatic glutathione transferases are connected with the elimination of intracellular pollutants and detoxification of organic micro-pollutants in their aquatic ecosystem. The two-substrate steady state kinetic mechanism of Silver catfish (Synodontis eupterus major hepatic glutathione transferases purified to apparent homogeneity was explored. The enzyme was dimeric enzyme with a monomeric size of 25.6 kDa. Initial-velocity studies and Product inhibition patterns by methyl glutathione and chloride with respect to GSH-CDNB; GSH-ρ-nitrophenylacetate; and GSH-Ethacrynic acid all conforms to a rapid equilibrium sequential random Bi Bi kinetic mechanism rather than steady state sequential random Bi Bi kinetic. α was 2.96 ± 0.35 for the model. The pH profile of Vmax/KM (with saturating 1-chloro-2,4-dinitrobenzene and variable GSH concentrations showed apparent pKa value of 6.88 and 9.86. Inhibition studies as a function of inhibitor concentration show that the enzyme is a homodimer and near neutral GST. The enzyme poorly conjugates 4-hydroxylnonenal and cumene hydroperoxide and may not be involved in oxidative stress protection. The seGST is unique and overwhelmingly shows characteristics similar to those of homodimeric class Pi GSTs, as was indicated by its kinetic mechanism, substrate specificity and inhibition studies. The rate- limiting step, probably the product release, of the reaction is viscosity-dependent and is consequential if macro-viscosogen or micro-viscosogen. Keywords: Silver catfish, Glutathione transferase, Steady-state, Kinetic mechanism, Inhibition

Entanglement of conjugated polymer chains influences molecular self-assembly and carrier transport

KAUST Repository

Zhao, Kui; Khan, Hadayat Ullah; Li, Ruipeng; Su, Yisong; Amassian, Aram

2013-01-01

The influence of polymer entanglement on the self-assembly, molecular packing structure, and microstructure of low-Mw (lightly entangled) and high-Mw (highly entangled) poly (3-hexylthiophene) (P3HT), and the carrier transport in thin-film transistors, are investigated. The polymer chains are gradually disentangled in a marginal solvent via ultrasonication of the polymer solution, and demonstrate improved diffusivity of precursor species (coils, aggregates, and microcrystallites), enhanced nucleation and crystallization of P3HT in solution, and self-assembly of well-ordered and highly textured fibrils at the solid-liquid interface. In low-Mw P3HT, reducing chain entanglement enhances interchain and intrachain ordering, but reduces the interconnectivity of ordered domains (tie molecules) due to the presence of short chains, thus deteriorating carrier transport even in the face of improving crystallinity. Reducing chain entanglement in high-Mw P3HT solutions increases carrier mobility up to ≈20-fold, by enhancing interchain and intrachain ordering while maintaining a sufficiently large number of tie molecules between ordered domains. These results indicate that charge carrier mobility is strongly governed by the balancing of intrachain and interchain ordering, on the one hand, and interconnectivity of ordered domains, on the other hand. In high-Mw P3HT, intrachain and interchain ordering appear to be the key bottlenecks to charge transport, whereas in low-Mw P3HT, the limited interconnectivity of the ordered domains acts as the primary bottleneck to charge transport. Conjugated polymer chains of poly(3-hexylthiophene) (P3HT) are gradually disentangled in solution and trends in carrier transport mechanisms in organic thin film transistors for low- and high-molecular weight P3HT are investigated. While intrachain and interchain ordering within ordered domains are the key bottlenecks to charge transport in high-Mw P3HT films, the limited interconnectivity of ordered

Entanglement of conjugated polymer chains influences molecular self-assembly and carrier transport

KAUST Repository

Zhao, Kui

2013-06-26

The influence of polymer entanglement on the self-assembly, molecular packing structure, and microstructure of low-Mw (lightly entangled) and high-Mw (highly entangled) poly (3-hexylthiophene) (P3HT), and the carrier transport in thin-film transistors, are investigated. The polymer chains are gradually disentangled in a marginal solvent via ultrasonication of the polymer solution, and demonstrate improved diffusivity of precursor species (coils, aggregates, and microcrystallites), enhanced nucleation and crystallization of P3HT in solution, and self-assembly of well-ordered and highly textured fibrils at the solid-liquid interface. In low-Mw P3HT, reducing chain entanglement enhances interchain and intrachain ordering, but reduces the interconnectivity of ordered domains (tie molecules) due to the presence of short chains, thus deteriorating carrier transport even in the face of improving crystallinity. Reducing chain entanglement in high-Mw P3HT solutions increases carrier mobility up to ≈20-fold, by enhancing interchain and intrachain ordering while maintaining a sufficiently large number of tie molecules between ordered domains. These results indicate that charge carrier mobility is strongly governed by the balancing of intrachain and interchain ordering, on the one hand, and interconnectivity of ordered domains, on the other hand. In high-Mw P3HT, intrachain and interchain ordering appear to be the key bottlenecks to charge transport, whereas in low-Mw P3HT, the limited interconnectivity of the ordered domains acts as the primary bottleneck to charge transport. Conjugated polymer chains of poly(3-hexylthiophene) (P3HT) are gradually disentangled in solution and trends in carrier transport mechanisms in organic thin film transistors for low- and high-molecular weight P3HT are investigated. While intrachain and interchain ordering within ordered domains are the key bottlenecks to charge transport in high-Mw P3HT films, the limited interconnectivity of ordered

Multidrug-resistance-associated protein plays a protective role in menadione-induced oxidative stress in endothelial cells.

Science.gov (United States)

Takahashi, Kyohei; Shibata, Tomohito; Oba, Tatsuya; Ishikawa, Tetsuya; Yoshikawa, Masahito; Tatsunami, Ryosuke; Takahashi, Kazuhiko; Tampo, Yoshiko

2009-02-13

Menadione, a redox-cycling quinone known to cause oxidative stress, binds to reduced glutathione (GSH) to form glutathione S-conjugate. Glutathione S-conjugates efflux is often mediated by multidrug-resistance-associated protein (MRP). We investigated the effect of a transporter inhibitor, MK571 (3-[[3-[2-(7-chloroquinolin-2-yl)vinyl]phenyl]-(2-dimethylcarbamoylethylsulfanyl)methylsulfanyl] propionic acid), on menadione-induced oxidative stress in bovine aortic endothelial cells (BAECs). BAECs were treated with menadione and MK571, and cell viability was measured. Modulation of intracellular GSH levels was performed with buthionine sulfoximine and GSH ethyl ester treatments. Intracellular superoxide was estimated by dihydroethidium oxidation using fluorescence microscopy or flow cytometry. Expression of MRP was determined by flow cytometry using phycoerythrin-conjugated anti-MRP monoclonal antibody. Intracellular GSH depletion by buthionine sulfoximine promoted the loss of viability of BAECs exposed to menadione. Exogenous GSH, which does not permeate the cell membrane, or GSH ethyl ester protected BAECs against the loss of viability induced by menadione. The results suggest that GSH binds to menadione outside the cells as well as inside. Pretreatment of BAECs with MK571 dramatically increased intracellular levels of superoxide generated from menadione, indicating that menadione may accumulate in the intracellular milieu. Finally, we found that MK571 aggravated menadione-induced toxicity in BAECs and that MRP levels were increased in menadione-treated cells. We conclude that MRP plays a vital role in protecting BAECs against menadione-induced oxidative stress, presumably due to its ability to transport glutathione S-conjugate.

Development and Synthesis of DNA-Encoded Benzimidazole Library.

Science.gov (United States)

Ding, Yun; Chai, Jing; Centrella, Paolo A; Gondo, Chenaimwoyo; DeLorey, Jennifer L; Clark, Matthew A

2018-04-25

Encoded library technology (ELT) is an effective approach to the discovery of novel small-molecule ligands for biological targets. A key factor for the success of the technology is the chemical diversity of the libraries. Here we report the development of DNA-conjugated benzimidazoles. Using 4-fluoro-3-nitrobenzoic acid as a key synthon, we synthesized a 320 million-member DNA-encoded benzimidazole library using Fmoc-protected amino acids, amines and aldehydes as diversity elements. Affinity selection of the library led to the discovery of a novel, potent and specific antagonist of the NK3 receptor.

Glutathione preservation during storage of rat lenses in optisol-GS and castor oil.

Science.gov (United States)

Holm, Thomas; Brøgger-Jensen, Martin Rocho; Johnson, Leif; Kessel, Line

2013-01-01

Glutathione concentration in the lens decreases in aging and cataractous lenses, providing a marker for tissue condition. Experimental procedures requiring unfrozen lenses from donor banks rely on transportation in storage medium, affecting lens homeostasis and alterations in glutathione levels. The aim of the study was to examine the effects of Optisol-GS and castor oil on lens condition, determined from their ability to maintain glutathione concentrations. Rat lenses were stored in the two types of storage media at varying time intervals up to 3 days. Glutathione concentration was afterwards determined in an enzymatic detection assay, specific for both reduced and oxidized forms. Lenses removed immediately after death exhibited a glutathione concentration of 4.70±0.29 mM. In vitro stored lenses in Optisol-GS lost glutathione quickly, ending with a concentration of 0.60±0.34 mM after 3 days while castor oil stored lenses exhibited a slower decline and ended at 3 times the concentration. A group of lenses were additionally stored under post mortem conditions within the host for 6 hours before its removal. Total glutathione after 6 hours was similar to that of lenses removed immediately after death, but with altered GSH and GSSG concentrations. Subsequent storage of these lenses in media showed changes similar to those in the first series of experiments, albeit to a lesser degree. It was determined that storage in Optisol-GS resulted in a higher loss of glutathione than lenses stored in castor oil. Storage for more than 12 hours reduced glutathione to half its original concentration, and was considered unusable after 24 hours.

Glutathione preservation during storage of rat lenses in optisol-GS and castor oil.

Directory of Open Access Journals (Sweden)

Thomas Holm

Full Text Available BACKGROUND: Glutathione concentration in the lens decreases in aging and cataractous lenses, providing a marker for tissue condition. Experimental procedures requiring unfrozen lenses from donor banks rely on transportation in storage medium, affecting lens homeostasis and alterations in glutathione levels. The aim of the study was to examine the effects of Optisol-GS and castor oil on lens condition, determined from their ability to maintain glutathione concentrations. METHODOLOGY/PRINCIPAL FINDINGS: Rat lenses were stored in the two types of storage media at varying time intervals up to 3 days. Glutathione concentration was afterwards determined in an enzymatic detection assay, specific for both reduced and oxidized forms. Lenses removed immediately after death exhibited a glutathione concentration of 4.70±0.29 mM. In vitro stored lenses in Optisol-GS lost glutathione quickly, ending with a concentration of 0.60±0.34 mM after 3 days while castor oil stored lenses exhibited a slower decline and ended at 3 times the concentration. A group of lenses were additionally stored under post mortem conditions within the host for 6 hours before its removal. Total glutathione after 6 hours was similar to that of lenses removed immediately after death, but with altered GSH and GSSG concentrations. Subsequent storage of these lenses in media showed changes similar to those in the first series of experiments, albeit to a lesser degree. CONCLUSIONS/SIGNIFICANCE: It was determined that storage in Optisol-GS resulted in a higher loss of glutathione than lenses stored in castor oil. Storage for more than 12 hours reduced glutathione to half its original concentration, and was considered unusable after 24 hours.

Hepatocyte Hyperproliferation upon Liver-Specific Co-disruption of Thioredoxin-1, Thioredoxin Reductase-1, and Glutathione Reductase

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Justin R. Prigge

2017-06-01

Full Text Available Energetic nutrients are oxidized to sustain high intracellular NADPH/NADP+ ratios. NADPH-dependent reduction of thioredoxin-1 (Trx1 disulfide and glutathione disulfide by thioredoxin reductase-1 (TrxR1 and glutathione reductase (Gsr, respectively, fuels antioxidant systems and deoxyribonucleotide synthesis. Mouse livers lacking both TrxR1 and Gsr sustain these essential activities using an NADPH-independent methionine-consuming pathway; however, it remains unclear how this reducing power is distributed. Here, we show that liver-specific co-disruption of the genes encoding Trx1, TrxR1, and Gsr (triple-null causes dramatic hepatocyte hyperproliferation. Thus, even in the absence of Trx1, methionine-fueled glutathione production supports hepatocyte S phase deoxyribonucleotide production. Also, Trx1 in the absence of TrxR1 provides a survival advantage to cells under hyperglycemic stress, suggesting that glutathione, likely via glutaredoxins, can reduce Trx1 disulfide in vivo. In triple-null livers like in many cancers, deoxyribonucleotide synthesis places a critical yet relatively low-volume demand on these reductase systems, thereby favoring high hepatocyte turnover over sustained hepatocyte integrity.

One-electron oxidation of diclofenac by human cytochrome P450s as a potential bioactivation mechanism for formation of 2'-(glutathion-S-yl)-deschloro-diclofenac.

Science.gov (United States)

Boerma, Jan Simon; Vermeulen, Nico P E; Commandeur, Jan N M

2014-01-25

Reactive metabolites have been suggested to play a role in the idiosyncratic hepatotoxicity observed with diclofenac (DF). By structural identification of the GSH conjugates formed after P450-catalyzed bioactivation of DF, it was shown that three types of reactive intermediates were formed: p-benzoquinone imines, o-imine methide and arene-oxide. Recently, detection of 2'-(glutathion-S-yl)-deschloro-diclofenac (DDF-SG), resulting from chlorine substitution, suggested the existence of a fourth type of P450-dependent reactive intermediate whose inactivation by GSH is completely dependent on presence of glutathione S-transferase. In this study, fourteen recombinant cytochrome P450s and three flavin-containing monooxygenases were tested for their ability to produce oxidative DF metabolites and their corresponding GSH conjugates. Concerning the hydroxymetabolites and their GSH conjugates, results were consistent with previous studies. Unexpectedly, all tested recombinant P450s were able to form DDF-SG to almost similar extent. DDF-SG formation was found to be partially independent of NADPH and even occurred by heat-inactivated P450. However, product formation was fully dependent on both GSH and glutathione-S-transferase P1-1. DDF-SG formation was also observed in reactions with horseradish peroxidase in absence of hydrogen peroxide. Because DDF-SG was not formed by free iron, it appears that DF can be bioactivated by iron in hemeproteins. This was confirmed by DDF-SG formation by other hemeproteins such as hemoglobin. As a mechanism, we propose that DF is subject to heme-dependent one-electron oxidation. The resulting nitrogen radical cation, which might activate the chlorines of DF, then undergoes a GST-catalyzed nucleophilic aromatic substitution reaction in which the chlorine atom of the DF moiety is replaced by GSH. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Correction of glutathione deficiency in the lower respiratory tract of HIV seropositive individuals by glutathione aerosol treatment.

Science.gov (United States)

Holroyd, K J; Buhl, R; Borok, Z; Roum, J H; Bokser, A D; Grimes, G J; Czerski, D; Cantin, A M; Crystal, R G

1993-10-01

Concentrations of glutathione, a ubiquitous tripeptide with immune enhancing and antioxidant properties, are decreased in the blood and lung epithelial lining fluid of human immunodeficiency virus (HIV) seropositive individuals. Since the lung is the most common site of infection in those who progress to AIDS it is rational to consider whether it is possible to safely augment glutathione levels in the epithelial lining fluid of HIV seropositive individuals, thus potentially improving local host defence. Purified reduced glutathione was delivered by aerosol to HIV seropositive individuals (n = 14) and the glutathione levels in lung epithelial lining fluid were compared before and at one, two, and three hours after aerosol administration. Before treatment total glutathione concentrations in the epithelial lining fluid were approximately 60% of controls. After three days of twice daily doses each of 600 mg reduced glutathione, total glutathione levels in the epithelial lining fluid increased and remained in the normal range for at least three hours after treatment. Strikingly, even though > 95% of the glutathione in the aerosol was in its reduced form, the percentage of oxidised glutathione in epithelial lining fluid increased from 5% before treatment to about 40% three hours after treatment, probably reflecting the use of glutathione as an antioxidant in vivo. No adverse effects were observed. It is feasible and safe to use aerosolised reduced glutathione to augment the deficient glutathione levels of the lower respiratory tract of HIV seropositive individuals. It is rational to evaluate further the efficacy of this tripeptide in improving host defence in HIV seropositive individuals.

The extended regulatory networks of SXT/R391 integrative and conjugative elements and IncA/C conjugative plasmids.

Science.gov (United States)

Poulin-Laprade, Dominic; Carraro, Nicolas; Burrus, Vincent

2015-01-01

Nowadays, healthcare systems are challenged by a major worldwide drug resistance crisis caused by the massive and rapid dissemination of antibiotic resistance genes and associated emergence of multidrug resistant pathogenic bacteria, in both clinical and environmental settings. Conjugation is the main driving force of gene transfer among microorganisms. This mechanism of horizontal gene transfer mediates the translocation of large DNA fragments between two bacterial cells in direct contact. Integrative and conjugative elements (ICEs) of the SXT/R391 family (SRIs) and IncA/C conjugative plasmids (ACPs) are responsible for the dissemination of a broad spectrum of antibiotic resistance genes among diverse species of Enterobacteriaceae and Vibrionaceae. The biology, diversity, prevalence and distribution of these two families of conjugative elements have been the subject of extensive studies for the past 15 years. Recently, the transcriptional regulators that govern their dissemination through the expression of ICE- or plasmid-encoded transfer genes have been described. Unrelated repressors control the activation of conjugation by preventing the expression of two related master activator complexes in both types of elements, i.e., SetCD in SXT/R391 ICEs and AcaCD in IncA/C plasmids. Finally, in addition to activating ICE- or plasmid-borne genes, these master activators have been shown to specifically activate phylogenetically unrelated mobilizable genomic islands (MGIs) that also disseminate antibiotic resistance genes and other adaptive traits among a plethora of pathogens such as Vibrio cholerae and Salmonella enterica.

The extended regulatory networks of SXT/R391 integrative and conjugative elements and IncA/C conjugative plasmids.

Directory of Open Access Journals (Sweden)

Dominic ePoulin-Laprade

2015-08-01

Full Text Available Nowadays, healthcare systems are challenged by a major worldwide drug resistance crisis caused by the massive and rapid dissemination of antibiotic resistance genes and associated emergence of multidrug resistant pathogenic bacteria, in both clinical and environmental settings. Conjugation is the main driving force of gene transfer among microorganisms. This mechanism of horizontal gene transfer mediates the translocation of large DNA fragments between two bacterial cells in direct contact. Integrative and conjugative elements (ICEs of the SXT/R391 family (SRIs and IncA/C conjugative plasmids (ACPs are responsible for the dissemination of a broad spectrum of antibiotic resistance genes among diverse species of Enterobacteriaceae and Vibrionaceae. The biology, diversity, prevalence and distribution of these two families of conjugative elements have been the subject of extensive studies for the past 15 years. Recently, the transcriptional regulators that govern their dissemination through the expression of ICE- or plasmid-encoded transfer genes have been described. Unrelated repressors control the activation of conjugation by preventing the expression of two related master activator complexes in both types of elements, i.e. SetCD in SXT/R391 ICEs and AcaCD in IncA/C plasmids. Finally, in addition to activating ICE- or plasmid-borne genes, these master activators have been shown to specifically activate phylogenetically unrelated mobilizable genomic islands (MGIs that also disseminate antibiotic resistance genes and other adaptive traits among a plethora of pathogens such as Vibrio cholerae and Salmonella enterica.

Reduction of antinutritional glucosinolates in Brassica oilseeds by mutation of genes encoding transporters

DEFF Research Database (Denmark)

Nour-Eldin, Hussam Hassan; Madsen, Svend Roesen; Engelen, Steven

2017-01-01

The nutritional value of Brassica seed meals is reduced by the presence of glucosinolates, which are toxic compounds involved in plant defense. Mutation of the genes encoding two glucosinolate transporters (GTRs) eliminated glucosinolates from Arabidopsis thaliana seeds, but translation of loss......-of-function phenotypes into Brassica crops is challenging because Brassica is polyploid. We mutated one of seven and four of 12 GTR orthologs and reduced glucosinolate levels in seeds by 60-70% in two different Brassica species (Brassica rapa and Brassica juncea). Reduction in seed glucosinolates was stably inherited...... over multiple generations and maintained in field trials of two mutant populations at three locations. Successful translation of the gtr loss-of-function phenotype from model plant to two Brassica crops suggests that our transport engineering approach could be broadly applied to reduce seed...

Glutathione peroxidases of the potato cyst nematode Globodera Rostochiensis.

Science.gov (United States)

Jones, J T; Reavy, B; Smant, G; Prior, A E

2004-01-07

We report the cloning and characterisation of full-length DNAs complementary to RNA (cDNAs) encoding two glutathione peroxidases (GpXs) from a plant parasitic nematode, the potato cyst nematode (PCN) Globodera rostochiensis. One protein has a functional signal peptide that targets the protein for secretion from animal cells while the other is predicted to be intracellular. Both genes are expressed in all parasite stages tested. The mRNA encoding the intracellular GpX is present throughout the nematode second stage juvenile and is particularly abundant in metabolically active tissues including the genital primordia. The mRNA encoding the secreted GpX is restricted to the hypodermis, the outermost cellular layer of the nematode, a location from which it is likely to be secreted to the parasite surface. Biochemical studies confirmed the secreted protein as a functional GpX and showed that, like secreted GpXs of other parasitic nematodes, it does not metabolise hydrogen peroxide but has a preference for larger hydroperoxide substrates. The intracellular protein is likely to have a role in metabolism of active oxygen species derived from internal body metabolism while the secreted protein may protect the parasite from host defences. Other functional roles for this protein are discussed.

Excitons in conjugated polymers: Do we need a paradigma change?

Energy Technology Data Exchange (ETDEWEB)

Beenken, Wichard J.D. [Department of Theoretical Physics I, Ilmenau University of Thechnology (Germany)

2009-12-15

We have previously shown that both, polymer conformation and dynamics are crucial for the exciton transport in conjugated polymers. Thereby we found that the usual Foerster-type hopping transfer model - even if one applies the line-dipole approximation - falls short in one crucial aspect: the nature of the sites the excitons are transferred between is still unclear. We found that the simple model of spectroscopic units defined as segments of the polymer chains separated by structural defects breaking the {pi}-conjugation is only justified for chemical defects like hydrogenated double bonds, or extreme gauche (90 ) torsions between the monomers. Both defects are far too rare in a well-prepared conjugated polymer to explain the mean spectroscopic-unit length of typically 6-7 monomers. Meanwhile, also the concept of dynamical formation of the spectroscopic units, we had previously suggested, has also failed. Thus the question of a paradigma change concerning the exciton transport in conjugated polymers appears on the agenda. (Abstract Copyright [2009], Wiley Periodicals, Inc.)

Human Intestinal Cells Modulate Conjugational Transfer of Multidrug Resistance Plasmids between Clinical Escherichia coli Isolates

DEFF Research Database (Denmark)

Machado, Ana Manuel; Sommer, Morten

2014-01-01

Bacterial conjugation in the human gut microbiota is believed to play a major role in the dissemination of antibiotic resistance genes and virulence plasmids. However, the modulation of bacterial conjugation by the human host remains poorly understood and there is a need for controlled systems...... to study this process. We established an in vitro co-culture system to study the interaction between human intestinal cells and bacteria. We show that the conjugation efficiency of a plasmid encoding an extended spectrum beta-lactamase is reduced when clinical isolates of Escherichia coli are co...... of the intestinal cells exposed to bacteria leading to a two-fold reduction in conjugation efficiency. These results show that human gut epithelial cells can modulate bacterial conjugation and may have relevance to gene exchange in the gut....

Hepatic uptake of conjugated bile acids is mediated by both sodium taurocholate cotransporting polypeptide and organic anion transporting polypeptides and modulated by intestinal sensing of plasma bile acid levels in mice.

Science.gov (United States)

Slijepcevic, Davor; Roscam Abbing, Reinout L P; Katafuchi, Takeshi; Blank, Antje; Donkers, Joanne M; van Hoppe, Stéphanie; de Waart, Dirk R; Tolenaars, Dagmar; van der Meer, Jonathan H M; Wildenberg, Manon; Beuers, Ulrich; Oude Elferink, Ronald P J; Schinkel, Alfred H; van de Graaf, Stan F J

2017-11-01

The Na + -taurocholate cotransporting polypeptide (NTCP/SLC10A1) is believed to be pivotal for hepatic uptake of conjugated bile acids. However, plasma bile acid levels are normal in a subset of NTCP knockout mice and in mice treated with myrcludex B, a specific NTCP inhibitor. Here, we elucidated which transport proteins mediate the hepatic uptake of conjugated bile acids and demonstrated intestinal sensing of elevated bile acid levels in plasma in mice. Mice or healthy volunteers were treated with myrcludex B. Hepatic bile acid uptake kinetics were determined in wild-type (WT), organic anion transporting polypeptide (OATP) knockout mice (lacking Slco1a/1b isoforms), and human OATP1B1-transgenic mice. Effects of fibroblast growth factor 19 (FGF19) on hepatic transporter mRNA levels were assessed in rat hepatoma cells and in mice by peptide injection or adeno-associated virus-mediated overexpression. NTCP inhibition using myrcludex B had only moderate effects on bile acid kinetics in WT mice, but completely inhibited active transport of conjugated bile acid species in OATP knockout mice. Cholesterol 7α-hydroxylase Cyp7a1 expression was strongly down-regulated upon prolonged inhibition of hepatic uptake of conjugated bile acids. Fgf15 (mouse counterpart of FGF19) expression was induced in hypercholanemic OATP and NTCP knockout mice, as well as in myrcludex B-treated cholestatic mice, whereas plasma FGF19 was not induced in humans treated with myrcludex B. Fgf15/FGF19 expression was induced in polarized human enterocyte-models and mouse organoids by basolateral incubation with a high concentration (1 mM) of conjugated bile acids. NTCP and OATPs contribute to hepatic uptake of conjugated bile acids in mice, whereas the predominant uptake in humans is NTCP mediated. Enterocytes sense highly elevated levels of (conjugated) bile acids in the systemic circulation to induce FGF15/19, which modulates hepatic bile acid synthesis and uptake. (Hepatology 2017;66:1631-1643). �

Conjugative Plasmid Transfer in Xylella fastidiosa Is Dependent on tra and trb Operon Functions

OpenAIRE

Burbank, Lindsey P.; Van Horn, Christopher R.

2017-01-01

The insect-transmitted plant pathogen Xylella fastidiosa is capable of efficient horizontal gene transfer (HGT) and recombination. Natural transformation occurs at high rates in X. fastidiosa, but there also is evidence that certain strains of X. fastidiosa carry native plasmids equipped with transfer and mobilization genes, suggesting conjugation as an additional mechanism of HGT in some instances. Two operons, tra and trb, putatively encoding a conjugative type IV secretion system, are foun...

Molecular mechanisms of reduced glutathione transport: role of the MRP/CFTR/ABCC and OATP/SLC21A families of membrane proteins

International Nuclear Information System (INIS)

Ballatori, Nazzareno; Hammond, Christine L.; Cunningham, Jennifer B.; Krance, Suzanne M.; Marchan, Rosemarie

2005-01-01

The initial step in reduced glutathione (GSH) turnover in all mammalian cells is its transport across the plasma membrane into the extracellular space; however, the mechanisms of GSH transport are not clearly defined. GSH export is required for the delivery of its constituent amino acids to other tissues, detoxification of drugs, metals, and other reactive compounds of both endogenous and exogenous origin, protection against oxidant stress, and secretion of hepatic bile. Recent studies indicate that some members of the multidrug resistance-associated protein (MRP/CFTR or ABCC) family of ATP-binding cassette (ABC) proteins, as well as some members of the organic anion transporting polypeptide (OATP or SLC21A) family of transporters contribute to this process. In particular, five of the 12 members of the MRP/CFTR family appear to mediate GSH export from cells namely, MRP1, MRP2, MRP4, MRP5, and CFTR. Additionally, two members of the OATP family, rat Oatp1 and Oatp2, have been identified as GSH transporters. For the Oatp1 transporter, efflux of GSH may provide the driving force for the uptake of extracellular substrates. In humans, OATP-B and OATP8 do not appear to transport GSH; however, other members of this family have yet to be characterized in regards to GSH transport. In yeast, the ABC proteins Ycf1p and Bpt1p transport GSH from the cytosol into the vacuole, whereas Hgt1p mediates GSH uptake across the plasma membrane. Because transport is a key step in GSH homeostasis and is intimately linked to its biological functions, GSH export proteins are likely to modulate essential cellular functions

Corneal endothelial glutathione after photodynamic change

International Nuclear Information System (INIS)

Hull, D.S.; Riley, M.V.; Csukas, S.; Green, K.

1982-01-01

Rabbit corneal endothelial cells perfused with 5 X 10(-6)M rose bengal and exposed to incandescent light demonstrated no alteration of either total of or percent oxidized glutathione after 1 hr. Addition of 5400 U/ml catalase to the perfusing solution had no effect on total glutathione levels but caused a marked reduction in percent oxidized glutathione in corneas exposed to light as well as in those not exposed to light. Substitution of sucrose for glucose in the perfusing solution had no effect on total or percent oxidized glutathione. Perfusion of rabbit corneal endothelium with 0.5 mM chlorpromazine and exposure to ultraviolet (UV) light resulted in no change in total glutathione content. A marked reduction in percent oxidized glutathione occurred, however, in corneas perfused with 0.5 mM chlorpromazine both in the presence and absence of UV light. It is concluded that photodynamically induced swelling of corneas is not the result of a failure of the glutathione redox system

Optical pulse coupling in a photorefractive crystal, propagation of encoded pulses in an optical fiber, and phase conjugate optical interconnections

Energy Technology Data Exchange (ETDEWEB)

Yao, X.S.

1992-01-01

In Part I, the author presents a theory to describe the interaction between short optical pulses in a photorefractive crystal. This theory provides an analytical framework for pulse coherence length measurements using a photorefractive crystal. The theory also predicts how a pulse changes its temporal shape due to its coupling with another pulse in a photorefractive crystal. The author describes experiments to demonstrate how photorefractive coupling alters the temporal shape and the frequency spectrum of an optical pulse. The author describes a compact optical field correlator. Using this correlator, the author measured the field cross-correlation function of optical pulses using a photorefractive crystal. The author presents a more sophisticated theory to describe the photorefractive coupling of optical pulses that are too short for the previous theory to be valid. In Part II of this dissertation, the author analyzes how the group-velocity dispersion and the optical nonlinearity of an optical fiber ruin an fiberoptic code-division multiple-access (CDMA) communication system. The author treats the optical fiber's nonlinear response with a novel approach and derives the pulse propagation equation. Through analysis and numerically simulations, the author obtains the maximum and the maximum allowed peak pulse power, as well as the minimum and the maximum allowed pulse width for the communication system to function properly. The author simulates how the relative misalignment between the encoding and the decoding masks affects the system's performance. In Part III the author demonstrates a novel optical interconnection device based on a mutually pumped phase conjugator. This device automatically routes light from selected information-sending channels to selected information-receiving channels, and vice versa. The phase conjugator eliminates the need for critical alignment. It is shown that a large number of optical channels can be interconnected using this

Menadione stress in Saccharomyces cerevisiae strains deficient in the glutathione transferases.

Science.gov (United States)

Castro, F A V; Herdeiro, R S; Panek, A D; Eleutherio, E C A; Pereira, M D

2007-02-01

Using S. cerevisiae as a eukaryotic cell model we have analyzed the involvement of both glutathione transferase isoforms, Gtt1 and Gtt2, in constitutive resistance and adaptive response to menadione, a quinone which can exert its toxicity as redox cycling and/or electrophiles. The detoxification properties, of these enzymes, have also been analyzed by the appearance of S-conjugates in the media. Direct exposure to menadione (20 mM/60 min) showed to be lethal for cells deficient on both Gtt1 and Gtt2 isoforms. However, after pre-treatment with a low menadione concentration, cells deficient in Gtt2 displayed reduced ability to acquire tolerance when compared with the control and the Gtt1 deficient strains. Analyzing the toxic effects of menadione we observed that the gtt2 mutant showed no reduction in lipid peroxidation levels. Moreover, measuring the levels of intracellular oxidation during menadione stress we have shown that the increase of this oxidative stress parameter was due to the capacity menadione possesses in generating reactive oxygen species (ROS) and that both GSH and Gtt2 isoform were required to enhance ROS production. Furthermore, the efflux of the menadione-GSH conjugate, which is related with detoxification of xenobiotic pathways, was not detected in the gtt2 mutant. Taken together, these results suggest that acquisition of tolerance against stress generated by menadione and the process of detoxification through S-conjugates are dependent upon Gtt2 activity. This assessment was corroborated by the increase of GTT2 expression, and not of GTT1, after menadione treatment.

The role of glutathione transferases in renal cell carcinoma

Directory of Open Access Journals (Sweden)

Ćorić Vesna

2016-01-01

Full Text Available Mounting evidence suggest that members of the subfamily of cytosolic glutathione S-transferases (GSTs possess roles far beyond the classical glutathione-dependent enzymatic conjugation of electrophilic metabolites and xenobiotics. Namely, monomeric forms of certain GSTs are capable of forming protein: protein interactions with protein kinases and regulate cell apoptotic pathways. Due to this dual functionality of cytosolic GSTs, they might be implicated in both the development and the progression of renal cell carcinoma (RCC. Prominent genetic heterogeneity, resulting from the gene deletions, as well as from SNPs in the coding and non-coding regions of GST genes, might affect GST isoenzyme profiles in renal parenchyma and therefore serve as a valuable indicator for predicting the risk of cancer development. Namely, GSTs are involved in the biotransformation of several compounds recognized as risk factors for RCC. The most potent carcinogen of polycyclic aromatic hydrocarbon diol epoxides, present in cigarette smoke, is of benzo(apyrene (BPDE, detoxified by GSTs. So far, the relationship between GST genotype and BPDE-DNA adduct formation, in determining the risk for RCC, has not been evaluated in patients with RCC. Although the association between certain individual and combined GST genotypes and RCC risk has been debated in a the literature, the data on the prognostic value of GST polymorphism in patients with RCC are scarce, probably due to the fact that the molecular mechanism supporting the role of GSTs in RCC progression has not been clarified as yet.

Reactivity of Biliatresone, a Natural Biliary Toxin, with Glutathione, Histamine, and Amino Acids.

Science.gov (United States)

Koo, Kyung A; Waisbourd-Zinman, Orith; Wells, Rebecca G; Pack, Michael; Porter, John R

2016-02-15

In our previous work, we identified a natural toxin, biliatresone, from Dysphania glomulifera and D. littoralis, endemic plants associated with outbreaks of biliary atresia in Australian neonatal livestock. Biliatresone is a very rare isoflavonoid with an α-methylene ketone between two phenyls, 1,2-diaryl-2-propenone, along with methylenedioxy, dimethoxyl, and hydroxyl functional groups, that causes extrahepatic biliary toxicity in zebrafish. The toxic core of biliatresone is a methylene in the α-position relative to the ketone of 1,2-diaryl-2-propenone that serves as an electrophilic Michael acceptor. The α-methylene of biliatresone spontaneously conjugated with water and methanol (MeOH), respectively, via Michael addition in a reverse phase high-performance liquid chromatography (RP-HPLC) analysis. We here report the reactivity of biliatresone toward glutathione (GSH), several amino acids, and other thiol- or imidazole-containing biomolecules. LC-MS and HPLC analysis of the conjugation reaction showed the reactivity of biliatresone to be in the order histidine > N-acetyl-d-cysteine (D-NAC) = N-acetyl-l-cysteine (L-NAC) > histamine > glutathione ≥ cysteine ≫ glycine > glutamate > phenylalanine, while serine and adenine had no reactivity due to intramolecular hydrogen bonding in the protic solvents. The reactivity of ethyl vinyl ketone (EVK, 1-penten-3-one), an example of a highly reactive α,ß-unsaturated ketone, toward GSH gave a 6.7-fold lower reaction rate constant than that of biliatresone. The reaction rate constant of synthetic 1,2-diaryl-2-propen-1-one (DP), a core structure of the toxic molecule, was 10-fold and 1.5-fold weaker in potency compared to the reaction rate constants of biliatresone and EVK, respectively. These results demostrated that the methylenedioxy, dimethoxyl, and hydroxyl functional groups of biliatresone contribute to the stronger reactivity of the Michael acceptor α-methylene ketone toward nucleophiles compared to that of DP

A maize gene encoding an NADPH binding enzyme highly homologous to isoflavone reductases is activated in response to sulfur starvation.

Science.gov (United States)

Petrucco, S; Bolchi, A; Foroni, C; Percudani, R; Rossi, G L; Ottonello, S

1996-01-01

we isolated a novel gene that is selectively induced both in roots and shoots in response to sulfur starvation. This gene encodes a cytosolic, monomeric protein of 33 kD that selectively binds NADPH. The predicted polypeptide is highly homologous ( > 70%) to leguminous isoflavone reductases (IFRs), but the maize protein (IRL for isoflavone reductase-like) belongs to a novel family of proteins present in a variety of plants. Anti-IRL antibodies specifically recognize IFR polypeptides, yet the maize protein is unable to use various isoflavonoids as substrates. IRL expression is correlated closely to glutathione availability: it is persistently induced in seedlings whose glutathione content is about fourfold lower than controls, and it is down-regulated rapidly when control levels of glutathione are restored. This glutathione-dependent regulation indicates that maize IRL may play a crucial role in the establishment of a thiol-independent response to oxidative stress under glutathione shortage conditions.

gamma-Glutamyl amino acids. Transport and conversion to 5-oxoproline in the kidney

International Nuclear Information System (INIS)

Bridges, R.J.; Meister, A.

1985-01-01

Transport of gamma-glutamyl amino acids, a step in the proposed glutathione-gamma-glutamyl transpeptidase-mediated amino acid transport pathway, was examined in mouse kidney. The transport of gamma-glutamyl amino acids was demonstrated in vitro in studies on kidney slices. Transport was followed by measuring uptake of 35 S after incubation of the slices in media containing gamma-glutamyl methionine [ 35 S]sulfone. The experimental complication associated with extracellular conversion of the gamma-glutamyl amino acid to amino acid and uptake of the latter by slices was overcome by using 5-oxoproline formation (catalyzed by intracellular gamma-glutamyl-cyclotransferase) as an indicator of gamma-glutamyl amino acid transport. This method was also successfully applied to studies on transport of gamma-glutamyl amino acids in vivo. Transport of gamma-glutamyl amino acids in vitro and in vivo is inhibited by several inhibitors of gamma-glutamyl transpeptidase and also by high extracellular levels of glutathione. This seems to explain urinary excretion of gamma-glutamylcystine by humans with gamma-glutamyl transpeptidase deficiency and by mice treated with inhibitors of this enzyme. Mice depleted of glutathione by treatment with buthionine sulfoximine (which inhibits glutathione synthesis) or by treatment with 2,6-dimethyl-2,5-heptadiene-4-one (which effectively interacts with tissue glutathione) exhibited significantly less transport of gamma-glutamyl amino acids than did untreated controls. The findings suggest that intracellular glutathione functions in transport of gamma-glutamyl amino acids. Evidence was also obtained for transport of gamma-glutamyl gamma-glutamylphenylalanine into kidney slices

The cell-based L-glutathione protection assays to study endocytosis and recycling of plasma membrane proteins.

Science.gov (United States)

Cihil, Kristine M; Swiatecka-Urban, Agnieszka

2013-12-13

Membrane trafficking involves transport of proteins from the plasma membrane to the cell interior (i.e. endocytosis) followed by trafficking to lysosomes for degradation or to the plasma membrane for recycling. The cell based L-glutathione protection assays can be used to study endocytosis and recycling of protein receptors, channels, transporters, and adhesion molecules localized at the cell surface. The endocytic assay requires labeling of cell surface proteins with a cell membrane impermeable biotin containing a disulfide bond and the N-hydroxysuccinimide (NHS) ester at 4 ºC - a temperature at which membrane trafficking does not occur. Endocytosis of biotinylated plasma membrane proteins is induced by incubation at 37 ºC. Next, the temperature is decreased again to 4 ºC to stop endocytic trafficking and the disulfide bond in biotin covalently attached to proteins that have remained at the plasma membrane is reduced with L-glutathione. At this point, only proteins that were endocytosed remain protected from L-glutathione and thus remain biotinylated. After cell lysis, biotinylated proteins are isolated with streptavidin agarose, eluted from agarose, and the biotinylated protein of interest is detected by western blotting. During the recycling assay, after biotinylation cells are incubated at 37 °C to load endocytic vesicles with biotinylated proteins and the disulfide bond in biotin covalently attached to proteins remaining at the plasma membrane is reduced with L-glutathione at 4 ºC as in the endocytic assay. Next, cells are incubated again at 37 °C to allow biotinylated proteins from endocytic vesicles to recycle to the plasma membrane. Cells are then incubated at 4 ºC, and the disulfide bond in biotin attached to proteins that recycled to the plasma membranes is reduced with L-glutathione. The biotinylated proteins protected from L-glutathione are those that did not recycle to the plasma membrane.

Organometallic DNA-B12 Conjugates as Potential Oligonucleotide Vectors: Synthesis and Structural and Binding Studies with Human Cobalamin-Transport Proteins.

Science.gov (United States)

Mutti, Elena; Hunger, Miriam; Fedosov, Sergey; Nexo, Ebba; Kräutler, Bernhard

2017-11-16

The synthesis and structural characterization of Co-(dN) 25 -Cbl (Cbl: cobalamin; dN: deoxynucleotide) and Co-(dN) 39 -Cbl, which are organometallic DNA-B 12 conjugates with single DNA strands consisting of 25 and 39 deoxynucleotides, respectively, and binding studies of these two DNA-Cbl conjugates to three homologous human Cbl transporting proteins, transcobalamin (TC), intrinsic factor (IF), and haptocorrin (HC), are reported. This investigation tests the suitability of such DNA-Cbls for the task of eventual in vivo oligonucleotide delivery. The binding of DNA-Cbl to TC, IF, and HC was investigated in competition with either a fluorescent Cbl derivative and Co-(dN) 25 -Cbl, or radiolabeled vitamin B 12 ( 57 Co-CNCbl) and Co-(dN) 25 -Cbl or Co-(dN) 39 -Cbl. Binding of the new DNA-Cbl conjugates was fast and tight with TC, but poorer with HC and IF, which extends a similar original finding with the simpler DNA-Cbl, Co-(dN) 18 -Cbl. The contrasting affinities of TC versus IF and HC for the DNA-Cbl conjugates are rationalized herein by a stepwise mechanism of Cbl binding. Critical contributions to overall affinity result from gradual conformational adaptations of the Cbl-binding proteins to the DNA-Cbl, which is first bound to the respective β domains. This transition is fast with TC, but slow with IF and HC, with which weaker binding results. The invariably tight interaction of the DNA-Cbl conjugates with TC makes the Cbl moiety a potential natural vector for the specific delivery of oligonucleotide loads from the blood into cells. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Changes of reduced glutathion, glutathion reductase, and glutathione peroxidase after radiation in guinea pigs

International Nuclear Information System (INIS)

Erden, M.; Bor, N.M.

1984-01-01

In this series of experiments the protective action of reduced glutathion due to ionizing radiation has been studied. In the experimental group 18 guinea pigs were exposed to successive radiations of 150 rad 3 or 4 days apart. Total dose given amounted to 750 rad which is the LD50 for guinea pigs. Blood samples were taken 30 min after each exposure. The control series were sham radiated but otherwise treated identically. The cells of the removed blood samples were separated by centrifugation and were subjected to the reduced glutathion stability test. GSSGR, GPer, and LDH enzyme activities were also measured of which the latter served as a marked enzyme. It was found that LDH did not show any alteration after radiation. The reduced glutathion stability test showed a consistent but minor reduction (P greater than 0.05), in the experimental group. GSSGR enzyme activity on the other hand was reduced significantly (from 176.48 +/- 11.32 to 41.34 +/- 1.17 IU/ml of packed erythrocytes, P less than 0.001) in the same group. GPer activity showed a consistent but minor elevation during the early phase of the experimental group. It was later increased significantly beginning after 600 rad total radiation on the fourth session (P less than 0.050)

Modelling the effect of nonplanarity on charge transport along conjugated polymer chains

International Nuclear Information System (INIS)

Correia, Helena M.G.; Ramos, Marta M.D.

2007-01-01

Conjugated polymers show interesting properties that make them appropriated for nanoelectronics. Several studies of poly(p-phenylene vinylene) (PPV) have suggested that each polymer chain consists of several planar segments, with conjugation length of nanoscale dimension, linked by twists or kinks. A pronounced twist between two planar segments in a PPV chain not only causes loss of main-chain conjugation but it may also alter electron and hole mobility along the chain, which has further implications for the percolation of charge through the polymer film. We used self-consistent quantum molecular dynamics calculations to provide information on the electric field needed to move the injected charges (either electrons or holes) along the planar segments of PPV and to cross the twist between two planar segments perpendicular to each other. Field-dependent charge mobility was also estimated for conjugated segments of various lengths. Our results suggest that electrons can cross the twist between adjacent planar segments for lower applied electric fields than holes if there is no more than one electronic charge (electron or hole) on the PPV chain, otherwise similar fields are needed

Structural Understanding of the Glutathione-dependent Reduction Mechanism of Glutathionyl-Hydroquinone Reductases*

Science.gov (United States)

Green, Abigail R.; Hayes, Robert P.; Xun, Luying; Kang, ChulHee

2012-01-01

Glutathionyl-hydroquinone reductases (GS- HQRs) are a newly identified group of glutathione transferases, and they are widely distributed in bacteria, halobacteria, fungi, and plants. GS-HQRs catalyze glutathione (GSH)-dependent reduction of glutathionyl-hydroquinones (GS-hydroquinones) to hydroquinones. GS-hydroquinones can be spontaneously formed from benzoquinones reacting with reduced GSH via Michael addition, and GS-HQRs convert the conjugates to hydroquinones. In this report we have determined the structures of two bacterial GS-HQRs, PcpF of Sphingobium chlorophenolicum and YqjG of Escherichia coli. The two structures and the previously reported structure of a fungal GS-HQR shared many features and displayed complete conservation for all the critical residues. Furthermore, we obtained the binary complex structures with GS-menadione, which in its reduced form, GS-menadiol, is a substrate. The structure revealed a large H-site that could accommodate various substituted hydroquinones and a hydrogen network of three Tyr residues that could provide the proton for reductive deglutathionylation. Mutation of the Tyr residues and the position of two GSH molecules confirmed the proposed mechanism of GS-HQRs. The conservation of GS-HQRs across bacteria, halobacteria, fungi, and plants potentiates the physiological role of these enzymes in quinone metabolism. PMID:22955277

Glutathione-mediated detoxification of halobenzoquinone drinking water disinfection byproducts in T24 cells.

Science.gov (United States)

Li, Jinhua; Wang, Wei; Zhang, Hongquan; Le, X Chris; Li, Xing-Fang

2014-10-01

Halobenzoquinones (HBQs) are a new class of drinking water disinfection byproducts (DBPs) and are capable of producing reactive oxygen species and causing oxidative damage to proteins and DNA in T24 human bladder carcinoma cells. However, the exact mechanism of the cytotoxicity of HBQs is unknown. Here, we investigated the role of glutathione (GSH) and GSH-related enzymes including glutathione S-transferase (GST) and glutathione peroxidase (GPx) in defense against HBQ-induced cytotoxicity in T24 cells. The HBQs are 2,6-dichloro-1,4-benzoquinone (DCBQ), 2,6-dichloro-3-methyl-1,4-benzoquinone (DCMBQ), 2,3,6-trichloro-1,4-benzoquinone (TriCBQ), and 2,6-dibromobenzoquinone (DBBQ). We found that depletion of cellular GSH could sensitize cells to HBQs and extracellular GSH supplementation could attenuate HBQ-induced cytotoxicity. HBQs caused significant cellular GSH depletion and increased cellular GST activities in a concentration-dependent manner. Our mass spectrometry study confirms that HBQs can conjugate with GSH, explaining in part the mechanism of GSH depletion by HBQs. The effects of HBQs on GPx activity are compound dependent; DCMBQ and DBBQ decrease cellular GPx activities, whereas DCBQ and TriCBQ have no significant effects. Pearson correlation analysis shows that the cellular GSH level is inversely correlated with ROS production and cellular GST activity in HBQ-treated cells. These results support a GSH and GSH-related enzyme-mediated detoxification mechanism of HBQs in T24 cells. © The Author 2014. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Contribution of Arginine 13 to the Catalytic Activity of Human Class Pi Glutathione Transferase P1-1

International Nuclear Information System (INIS)

Kong, Ji Na; Jo, Dong Hyeon; Do, Hyun Dong; Lee, Jin Ju; Kong, Kwang Hoon

2010-01-01

Arg13 is a conserved active-site residue in all known Pi class glutathione S-transferases (GSTs) and in most Alpha class GSTs. To evaluate its contribution to substrate binding and catalysis of this residue, three mutants (R13A, R13K, and R13L) were expressed in Escherichia coli and purified by GSH affinity chromatography. The substitutions of Arg13 significantly affected GSH-conjugation activity, while scarcely affecting glutathione peroxidase or steroid isomerase activities. Mutation of Arg13 into Ala largely reduced the GSH-conjugation activity by approximately 85 - 95%, whereas substitutions by Lys and Leu barely affected activity. These results suggest that, in the GSH-conjugation activity of hGST P1-1, the contribution of Arg13 toward catalytic activity is highly dependent on substrate specificities and the size of the side chain at position 13. From the kinetic parameters, introduction of larger side chains at position 13 results in stronger affinity (Leu > Lys, Arg > Ala) towards GSH. The substitutions of Arg13 with alanine and leucine significantly affected k cat , whereas substitution with Lys was similar to that of the wild type, indicating the significance of a positively charged residue at position 13. From the plots of log (k cat /K m CDNB ) against pH, the pK a values of the thiol group of GSH bound in R13A, R13K, and R13L were estimated to be 1.8, 1.4, and 1.8 pK units higher than the pK a value of the wildtype enzyme, demonstrating the contribution of the Arg13 guanidinium group to the electrostatic field in the active site. From these results, we suggest that contribution of Arg13 in substrate binding is highly dependent on the nature of the electrophilic substrates, while in the catalytic mechanism, it stabilizes the GSH thiolate through hydrogen bonding

Isolation, nucleotide sequence and expression of a cDNA encoding feline granulocyte colony-stimulating factor.

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Dunham, S P; Onions, D E

2001-06-21

A cDNA encoding feline granulocyte colony stimulating factor (fG-CSF) was cloned from alveolar macrophages using the reverse transcriptase-polymerase chain reaction. The cDNA is 949 bp in length and encodes a predicted mature protein of 174 amino acids. Recombinant fG-CSF was expressed as a glutathione S-transferase fusion and purified by affinity chromatography. Biological activity of the recombinant protein was demonstrated using the murine myeloblastic cell line GNFS-60, which showed an ED50 for fG-CSF of approximately 2 ng/ml. Copyright 2001 Academic Press.

A conjugative 38 kB plasmid is present in multiple subspecies of Xylella fastidiosa.

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Rogers, Elizabeth E; Stenger, Drake C

2012-01-01

A ≈ 38kB plasmid (pXF-RIV5) was present in the Riv5 strain of Xylella fastidiosa subsp. multiplex isolated from ornamental plum in southern California. The complete nucleotide sequence of pXF-RIV5 is almost identical to that of pXFAS01 from X. fastidiosa subsp. fastidiosa strain M23; the two plasmids vary at only 6 nucleotide positions. BLAST searches and phylogenetic analyses indicate pXF-RIV5 and pXFAS01 share some similarity to chromosomal and plasmid (pXF51) sequences of X. fastidiosa subsp. pauca strain 9a5c and more distant similarity to plasmids from a wide variety of bacteria. Both pXF-RIV5 and pXFAS01 encode homologues of a complete Type IV secretion system involved in conjugation and DNA transfer among bacteria. Mating pair formation proteins (Trb) from Yersinia pseudotuberculosis IP31758 are the mostly closely related non-X. fastidiosa proteins to most of the Trb proteins encoded by pXF-RIV5 and pXFAS01. Unlike many bacterial conjugative plasmids, pXF-RIV5 and pXFAS01 do not carry homologues of known accessory modules that confer selective advantage on host bacteria. However, both plasmids encode seven hypothetical proteins of unknown function and possess a small transposon-associated region encoding a putative transposase and associated factor. Vegetative replication of pXF-RIV5 and pXFAS01 appears to be under control of RepA protein and both plasmids have an origin of DNA replication (oriV) similar to that of pRP4 and pR751 from Escherichia coli. In contrast, conjugative plasmids commonly encode TrfA and have an oriV similar to those found in IncP-1 incompatibility group plasmids. The presence of nearly identical plasmids in single strains from two distinct subspecies of X. fastidiosa is indicative of recent horizontal transfer, probably subsequent to the introduction of subspecies fastidiosa to the United States in the late 19(th) century.

A conjugative 38 kB plasmid is present in multiple subspecies of Xylella fastidiosa.

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Elizabeth E Rogers

Full Text Available A ≈ 38kB plasmid (pXF-RIV5 was present in the Riv5 strain of Xylella fastidiosa subsp. multiplex isolated from ornamental plum in southern California. The complete nucleotide sequence of pXF-RIV5 is almost identical to that of pXFAS01 from X. fastidiosa subsp. fastidiosa strain M23; the two plasmids vary at only 6 nucleotide positions. BLAST searches and phylogenetic analyses indicate pXF-RIV5 and pXFAS01 share some similarity to chromosomal and plasmid (pXF51 sequences of X. fastidiosa subsp. pauca strain 9a5c and more distant similarity to plasmids from a wide variety of bacteria. Both pXF-RIV5 and pXFAS01 encode homologues of a complete Type IV secretion system involved in conjugation and DNA transfer among bacteria. Mating pair formation proteins (Trb from Yersinia pseudotuberculosis IP31758 are the mostly closely related non-X. fastidiosa proteins to most of the Trb proteins encoded by pXF-RIV5 and pXFAS01. Unlike many bacterial conjugative plasmids, pXF-RIV5 and pXFAS01 do not carry homologues of known accessory modules that confer selective advantage on host bacteria. However, both plasmids encode seven hypothetical proteins of unknown function and possess a small transposon-associated region encoding a putative transposase and associated factor. Vegetative replication of pXF-RIV5 and pXFAS01 appears to be under control of RepA protein and both plasmids have an origin of DNA replication (oriV similar to that of pRP4 and pR751 from Escherichia coli. In contrast, conjugative plasmids commonly encode TrfA and have an oriV similar to those found in IncP-1 incompatibility group plasmids. The presence of nearly identical plasmids in single strains from two distinct subspecies of X. fastidiosa is indicative of recent horizontal transfer, probably subsequent to the introduction of subspecies fastidiosa to the United States in the late 19(th century.

Adsorption mechanisms of L-Glutathione on Au and controlled nano-patterning through Dip Pen Nanolithography

Energy Technology Data Exchange (ETDEWEB)

Calborean, A., E-mail: adrian.calborean@gmail.com [National Institute for Research and Development of Isotopic and Molecular Technologies, Donat 67-103, 400293 Cluj-Napoca (Romania); Martin, F.; Marconi, D.; Turcu, R.; Kacso, I.E.; Buimaga-Iarinca, L. [National Institute for Research and Development of Isotopic and Molecular Technologies, Donat 67-103, 400293 Cluj-Napoca (Romania); Graur, F. [Iuliu Haţieganu University of Medicine and Pharmacy Cluj-Napoca, Babeş 8, 400012 Cluj-Napoca (Romania); Turcu, I. [National Institute for Research and Development of Isotopic and Molecular Technologies, Donat 67-103, 400293 Cluj-Napoca (Romania)

2015-12-01

Dip Pen Nanolithography technique has been employed for patterning L-Glutathione tripeptide (L-y-glutamyl-L-cysteinyl-glycine) nanostructures at specific locations on metallic Au(111) substrate. The formed supramolecular architectures were designed through straight lines and dots serving as precursors for building blocks assemblies in nano-bio-electronics applications or as template structures for functionalized particles in the form of host–guest networks. Tween 20 polyoxyethylene surfactant concentrations ranging from 0.005 to 0.1% (v/v) into initial L-Glutathione tripeptide (2 mg mL{sup −1}) ink solutions were sequentially tested for the improvement of the ink delivery process and to assure an optimum uniformity and homogeneity over the patterned space. A strong relationship was found between the coated atomic force microscope (AFM) cantilever within the highly effective Tween 20 activator adjuvant and the molecular diffusion along concentration gradients. An increase in the driving force for ink transport from the AFM tip has been demonstrated within the highest 0.1% (v/v) TW 20 surfactant concentration, favoring the patterning of GSH molecules routinely with sub-100 nm resolution. Self-assembled monolayers of GSH were also fabricated and characterized in the light of X-ray photoemission spectroscopy (XPS) and ellipsometric optical measurements. Adsorption from water of L-Glutathione to the gold substrate is proven to be made by the thiol group of cysteine. Theoretical DFT approaches were applied for quantum chemical studies dedicated to electronic processes underneath molecular GSH/Au(111) systems. - Highlights: • Controlled nano-patterning of L-Glutathione was performed on Au by DPN. • Tween 20 was used for increasing driving force of molecular ink transport. • SAM's formation has described the adsorption mechanisms of L-Glutathione on Au. • Electronic properties of hybrid GSH/Au(111) structures were investigated by DFT. • Validation of

Interplay between MRP Inhibition and Metabolism of MRP Inhibitors: The Case of Curcumin

NARCIS (Netherlands)

Wortelboer, H.M.; Usta, M.; Velde, A.E. van der; Boersma, M.G.; Spenkelink, B.; Zanden, J.J. van; Rietjens, I.M.C.M.; Bladeren, P.J. van; Cnubben, N.H.P.

2003-01-01

The multidrug resistance proteins MRP1 and MRP2 are efflux transporters with broad substrate specificity, including glutathione, glucuronide, and sulfate conjugates. In the present study, the interaction of the dietary polyphenol curcumin with MRP1 and MRP2 and the interplay between

Interplay between MRP-inhibition and metabolism of MRP-inhibitors: the case of curcumin

NARCIS (Netherlands)

Wortelboer, H.M.; Usta, M.; Velde, van der A.E.; Boersma, M.G.; Spenkelink, A.; Zanden, van J.J.; Rietjens, I.M.C.M.; Bladeren, van P.J.; Cnubben, N.H.P.

2003-01-01

The multidrug resistance proteins MRP1 and MRP2 are efflux transporters with broad substrate specificity, including glutathione, glucuronide, and sulfate conjugates. In the present study, the interaction of the dietary polyphenol curcumin with MRP1 and MRP2 and the interplay between

Charge Transport in 4 nm Molecular Wires with Interrupted Conjugation: Combined Experimental and Computational Evidence for Thermally Assisted Polaron Tunneling.

Science.gov (United States)

Taherinia, Davood; Smith, Christopher E; Ghosh, Soumen; Odoh, Samuel O; Balhorn, Luke; Gagliardi, Laura; Cramer, Christopher J; Frisbie, C Daniel

2016-04-26

We report the synthesis, transport measurements, and electronic structure of conjugation-broken oligophenyleneimine (CB-OPI 6) molecular wires with lengths of ∼4 nm. The wires were grown from Au surfaces using stepwise aryl imine condensation reactions between 1,4-diaminobenzene and terephthalaldehyde (1,4-benzenedicarbaldehyde). Saturated spacers (conjugation breakers) were introduced into the molecular backbone by replacing the aromatic diamine with trans-1,4-diaminocyclohexane at specific steps during the growth processes. FT-IR and ellipsometry were used to follow the imination reactions on Au surfaces. Surface coverages (∼4 molecules/nm(2)) and electronic structures of the wires were determined by cyclic voltammetry and UV-vis spectroscopy, respectively. The current-voltage (I-V) characteristics of the wires were acquired using conducting probe atomic force microscopy (CP-AFM) in which an Au-coated AFM probe was brought into contact with the wires to form metal-molecule-metal junctions with contact areas of ∼50 nm(2). The low bias resistance increased with the number of saturated spacers, but was not sensitive to the position of the spacer within the wire. Temperature dependent measurements of resistance were consistent with a localized charge (polaron) hopping mechanism in all of the wires. Activation energies were in the range of 0.18-0.26 eV (4.2-6.0 kcal/mol) with the highest belonging to the fully conjugated OPI 6 wire and the lowest to the CB3,5-OPI 6 wire (the wire with two saturated spacers). For the two other wires with a single conjugation breaker, CB3-OPI 6 and CB5-OPI 6, activation energies of 0.20 eV (4.6 kcal/mol) and 0.21 eV (4.8 kcal/mol) were found, respectively. Computational studies using density functional theory confirmed the polaronic nature of charge carriers but predicted that the semiclassical activation energy of hopping should be higher for CB-OPI molecular wires than for the OPI 6 wire. To reconcile the experimental and

Genome-wide identification and expression characterization of ABCC-MRP transporters in hexaploid wheat.

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Bhati, Kaushal K; Sharma, Shivani; Aggarwal, Sipla; Kaur, Mandeep; Shukla, Vishnu; Kaur, Jagdeep; Mantri, Shrikant; Pandey, Ajay K

2015-01-01

The ABCC multidrug resistance associated proteins (ABCC-MRP), a subclass of ABC transporters are involved in multiple physiological processes that include cellular homeostasis, metal detoxification, and transport of glutathione-conjugates. Although they are well-studied in humans, yeast, and Arabidopsis, limited efforts have been made to address their possible role in crop like wheat. In the present work, 18 wheat ABCC-MRP proteins were identified that showed the uniform distribution with sub-families from rice and Arabidopsis. Organ-specific quantitative expression analysis of wheat ABCC genes indicated significantly higher accumulation in roots (TaABCC2, TaABCC3, and TaABCC11 and TaABCC12), stem (TaABCC1), leaves (TaABCC16 and TaABCC17), flag leaf (TaABCC14 and TaABCC15), and seeds (TaABCC6, TaABCC8, TaABCC12, TaABCC13, and TaABCC17) implicating their role in the respective tissues. Differential transcript expression patterns were observed for TaABCC genes during grain maturation speculating their role during seed development. Hormone treatment experiments indicated that some of the ABCC genes could be transcriptionally regulated during seed development. In the presence of Cd or hydrogen peroxide, distinct molecular expression of wheat ABCC genes was observed in the wheat seedlings, suggesting their possible role during heavy metal generated oxidative stress. Functional characterization of the wheat transporter, TaABCC13 a homolog of maize LPA1 confirms its role in glutathione-mediated detoxification pathway and is able to utilize adenine biosynthetic intermediates as a substrate. This is the first comprehensive inventory of wheat ABCC-MRP gene subfamily.

Subcellular distribution of glutathione and cysteine in cyanobacteria.

Science.gov (United States)

Zechmann, Bernd; Tomasić, Ana; Horvat, Lucija; Fulgosi, Hrvoje

2010-10-01

Glutathione plays numerous important functions in eukaryotic and prokaryotic cells. Whereas it can be found in virtually all eukaryotic cells, its production in prokaryotes is restricted to cyanobacteria and proteobacteria and a few strains of gram-positive bacteria. In bacteria, it is involved in the protection against reactive oxygen species (ROS), osmotic shock, acidic conditions, toxic chemicals, and heavy metals. Glutathione synthesis in bacteria takes place in two steps out of cysteine, glutamate, and glycine. Cysteine is the limiting factor for glutathione biosynthesis which can be especially crucial for cyanobacteria, which rely on both the sufficient sulfur supply from the growth media and on the protection of glutathione against ROS that are produced during photosynthesis. In this study, we report a method that allows detection and visualization of the subcellular distribution of glutathione in Synechocystis sp. This method is based on immunogold cytochemistry with glutathione and cysteine antisera and computer-supported transmission electron microscopy. Labeling of glutathione and cysteine was restricted to the cytosol and interthylakoidal spaces. Glutathione and cysteine could not be detected in carboxysomes, cyanophycin granules, cell walls, intrathylakoidal spaces, periplasm, and vacuoles. The accuracy of the glutathione and cysteine labeling is supported by two observations. First, preadsorption of the antiglutathione and anticysteine antisera with glutathione and cysteine, respectively, reduced the density of the gold particles to background levels. Second, labeling of glutathione and cysteine was strongly decreased by 98.5% and 100%, respectively, in Synechocystis sp. cells grown on media without sulfur. This study indicates a strong similarity of the subcellular distribution of glutathione and cysteine in cyanobacteria and plastids of plants and provides a deeper insight into glutathione metabolism in bacteria.

Mercury toxicokinetics of the healthy human term placenta involve amino acid transporters and ABC transporters

International Nuclear Information System (INIS)

Straka, Elisabeth; Ellinger, Isabella; Balthasar, Christina; Scheinast, Matthias; Schatz, Jasmin; Szattler, Tamara; Bleichert, Sonja; Saleh, Leila; Knöfler, Martin; Zeisler, Harald; Hengstschläger, Markus; Rosner, Margit; Salzer, Hans; Gundacker, Claudia

2016-01-01

Highlights: • It is known that MeHg is able to pass the placenta and to affect fetal brain development. • Uptake and efflux transporters were examined in human primary trophoblast cells and BeWo cells. • Involvement in mercury transfer was assessed by measurement of cellular mercury content upon siRNA mediated gene knockdown. • Localization of transporters was determined by immunofluorescence microscopy. • LAT1 and rBAT at the apical membrane of the syncytiotrophoblast (STB) are involved in MeHg uptake. • MRP1 located at basal membrane of STB mediates mercury efflux. - Abstract: Background: The capacity of the human placenta to handle exogenous stressors is poorly understood. The heavy metal mercury is well-known to pass the placenta and to affect brain development. An active transport across the placenta has been assumed. The underlying mechanisms however are virtually unknown. Objectives: Uptake and efflux transporters (17 candidate proteins) assumed to play a key role in placental mercury transfer were examined for expression, localization and function in human primary trophoblast cells and the trophoblast-derived choriocarcinoma cell line BeWo. Methods: To prove involvement of the transporters, we used small interfering RNA (siRNA) and exposed cells to methylmercury (MeHg). Total mercury contents of cells were analyzed by Cold vapor-atomic fluorescence spectrometry (CV-AFS). Localization of the proteins in human term placenta sections was determined via immunofluorescence microscopy. Results: We found the amino acid transporter subunits L-type amino acid transporter (LAT)1 and rBAT (related to b 0,+ type amino acid transporter) as well as the efflux transporter multidrug resistance associated protein (MRP)1 to be involved in mercury kinetics of trophoblast cells (t-test P < 0.05). Conclusion: The amino acid transporters located at the apical side of the syncytiotrophoblast (STB) manage uptake of MeHg. Mercury conjugated to glutathione (GSH) is

Glutathione Transferases Superfamily: Cold-Inducible Expression of Distinct GST Genes in Brassica oleracea

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Harshavardhanan Vijayakumar

2016-07-01

Full Text Available Plants, as sessile organisms, can suffer serious growth and developmental consequences under cold stress conditions. Glutathione transferases (GSTs, EC 2.5.1.18 are ubiquitous and multifunctional conjugating proteins, which play a major role in stress responses by preventing oxidative damage by reactive oxygen species (ROS. Currently, understanding of their function(s during different biochemical and signaling pathways under cold stress condition remain unclear. In this study, using combined computational strategy, we identified 65 Brassica oleracea glutathione transferases (BoGST and characterized them based on evolutionary analysis into 11 classes. Inter-species and intra-species duplication was evident between BoGSTs and Arabidopsis GSTs. Based on localization analyses, we propose possible pathways in which GST genes are involved during cold stress. Further, expression analysis of the predicted putative functions for GST genes were investigated in two cold contrasting genotypes (cold tolerance and susceptible under cold condition, most of these genes were highly expressed at 6 h and 1 h in the cold tolerant (CT and cold susceptible (CS lines, respectively. Overall, BoGSTU19, BoGSTU24, BoGSTF10 are candidate genes highly expressed in B. oleracea. Further investigation of GST superfamily in B. oleracea will aid in understanding complex mechanism underlying cold tolerance in plants.

Expression Patterns of Genes Involved in Ascorbate-Glutathione Cycle in Aphid-Infested Maize (Zea mays L.) Seedlings.

Science.gov (United States)

Sytykiewicz, Hubert

2016-02-23

Reduced forms of ascorbate (AsA) and glutathione (GSH) are among the most important non-enzymatic foliar antioxidants in maize (Zea mays L.). The survey was aimed to evaluate impact of bird cherry-oat aphid (Rhopalosiphum padi L.) or grain aphid (Sitobion avenae F.) herbivory on expression of genes related to ascorbate-glutathione (AsA-GSH) cycle in seedlings of six maize varieties (Ambrozja, Nana, Tasty Sweet, Touran, Waza, Złota Karłowa), differing in resistance to the cereal aphids. Relative expression of sixteen maize genes encoding isoenzymes of ascorbate peroxidase (APX1, APX2, APX3, APX4, APX5, APX6, APX7), monodehydroascorbate reductase (MDHAR1, MDHAR2, MDHAR3, MDHAR4), dehydroascorbate reductase (DHAR1, DHAR2, DHAR3) and glutathione reductase (GR1, GR2) was quantified. Furthermore, effect of hemipterans' attack on activity of APX, MDHAR, DHAR and GR enzymes, and the content of reduced and oxidized ascorbate and glutathione in maize plants were assessed. Seedling leaves of more resistant Z. mays varieties responded higher elevations in abundance of target transcripts. In addition, earlier and stronger aphid-triggered changes in activity of APX, MDHAR, DHAR and GR enzymes, and greater modulations in amount of the analyzed antioxidative metabolites were detected in foliar tissues of highly resistant Ambrozja genotype in relation to susceptible Tasty Sweet plants.

HARD X-RAY ASYMMETRY LIMITS IN SOLAR FLARE CONJUGATE FOOTPOINTS

Energy Technology Data Exchange (ETDEWEB)

Daou, Antoun G.; Alexander, David, E-mail: agdaou@rice.edu, E-mail: dalex@rice.edu [Department of Physics and Astronomy, Rice University, 6100 Main Street, MS 108, Houston, TX, 77005 (United States)

2016-11-20

The transport of energetic electrons in a solar flare is modeled using a time-dependent one-dimensional Fokker–Planck code that incorporates asymmetric magnetic convergence. We derive the temporal and spectral evolution of the resulting hard X-ray (HXR) emission in the conjugate chromospheric footpoints, assuming thick target photon production, and characterize the time evolution of the numerically simulated footpoint asymmetry and its relationship to the photospheric magnetic configuration. The thick target HXR asymmetry in the conjugate footpoints is found to increase with magnetic field ratio as expected. However, we find that the footpoint HXR asymmetry saturates for conjugate footpoint magnetic field ratios ≥4. This result is borne out in a direct comparison with observations of 44 double-footpoint flares. The presence of such a limit has not been reported before, and may serve as both a theoretical and observational benchmark for testing a range of particle transport and flare morphology constraints, particularly as a means to differentiate between isotropic and anisotropic particle injection.

Side Chain Engineering in Solution-Processable Conjugated Polymers

KAUST Repository

Mei, Jianguo

2014-01-14

Side chains in conjugated polymers have been primarily utilized as solubilizing groups. However, these side chains have roles that are far beyond. We advocate using side chain engineering to tune a polymer\\'s physical properties, including absorption, emission, energy level, molecular packing, and charge transport. To date, numerous flexible substituents suitable for constructing side chains have been reported. In this Perspective article, we advocate that the side chain engineering approach can advance better designs for next-generation conjugated polymers. © 2013 American Chemical Society.

Scanning-tunneling spectroscopy on conjugated polymer films

NARCIS (Netherlands)

Kemerink, M.; Alvarado, S.F.; Koenraad, P.M.; Janssen, R.A.J.; Salemink, H.W.M.; Wolter, J.H.; Blom, P.W.M.

2003-01-01

Scanning-tunneling spectroscopy experiments have been performed on conjugated polymer films and have been compared to a three-dimensional numerical model for charge injection and transport. It is found that field enhancement near the tip apex leads to significant changes in the injected current,

Effects of Combined Low Glutathione with Mild Oxidative and Low Phosphorus Stress on the Metabolism of Arabidopsis thaliana

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Atsushi Fukushima

2017-08-01

Full Text Available Plants possess highly sensitive mechanisms that monitor environmental stress levels for a dose-dependent fine-tuning of their growth and development. Differences in plant responses to severe and mild abiotic stresses have been recognized. Although many studies have revealed that glutathione can contribute to plant tolerance to various environmental stresses, little is known about the relationship between glutathione and mild abiotic stress, especially the effect of stress-induced altered glutathione levels on the metabolism. Here, we applied a systems biology approach to identify key pathways involved in the gene-to-metabolite networks perturbed by low glutathione content under mild abiotic stress in Arabidopsis thaliana. We used glutathione synthesis mutants (cad2-1 and pad2-1 and plants overexpressing the gene encoding γ-glutamylcysteine synthetase, the first enzyme of the glutathione biosynthetic pathway. The plants were exposed to two mild stress conditions—oxidative stress elicited by methyl viologen and stress induced by the limited availability of phosphate. We observed that the mutants and transgenic plants showed similar shoot growth as that of the wild-type plants under mild abiotic stress. We then selected the synthesis mutants and performed multi-platform metabolomics and microarray experiments to evaluate the possible effects on the overall metabolome and the transcriptome. As a common oxidative stress response, several flavonoids that we assessed showed overaccumulation, whereas the mild phosphate stress resulted in increased levels of specific kaempferol- and quercetin-glycosides. Remarkably, in addition to a significant increased level of sugar, osmolytes, and lipids as mild oxidative stress-responsive metabolites, short-chain aliphatic glucosinolates over-accumulated in the mutants, whereas the level of long-chain aliphatic glucosinolates and specific lipids decreased. Coordinated gene expressions related to glucosinolate and

Evaluation of the impact of genetic polymorphisms in glutathione-related genes on the association between methylmercury or n-3 polyunsaturated long chain fatty acids and risk of myocardial infarction: a case-control study

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Norberg Margareta

2011-04-01

Full Text Available Abstract Background The n-3 polyunsaturated fatty acids eicosapentaenoic acid and docosahexaenoic acid, which are present in fish, are protective against myocardial infarction. However, fish also contains methylmercury, which influences the risk of myocardial infarction, possibly by generating oxidative stress. Methylmercury is metabolized by conjugation to glutathione, which facilitates elimination. Glutathione is also an antioxidant. Individuals with certain polymorphisms in glutathione-related genes may tolerate higher exposures to methylmercury, due to faster metabolism and elimination and/or better glutathione-associated antioxidative capacity. They would thus benefit more from the protective agents in fish, such as eicosapentaenoic+docosahexaenoic acid and selenium. The objective for this study was to elucidate whether genetic polymorphisms in glutathione-related genes modify the association between eicosapentaenoic+docosahexaenoic acid or methylmercury and risk of first ever myocardial infarction. Methods Polymorphisms in glutathione-synthesizing (glutamyl-cysteine ligase catalytic subunit, GCLC and glutamyl-cysteine ligase modifier subunit, GCLM or glutathione-conjugating (glutathione S-transferase P, GSTP1 genes were genotyped in 1027 individuals from northern Sweden (458 cases of first-ever myocardial infarction and 569 matched controls. The impact of these polymorphisms on the association between erythrocyte-mercury (proxy for methylmercury and risk of myocardial infarction, as well as between plasma eicosapentaenoic+docosahexaenoic acid and risk of myocardial infarction, was evaluated by conditional logistic regression. The effect of erythrocyte-selenium on risk of myocardial infarction was also taken into consideration. Results There were no strong genetic modifying effects on the association between plasma eicosapentaenoic+docosahexaenoic acid or erythrocyte-mercury and risk of myocardial infarction risk. When eicosapentaenoic

RLIP76, a glutathione-conjugate transporter, plays a major role in the pathogenesis of metabolic syndrome.

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Jyotsana Singhal

Full Text Available PURPOSE: Characteristic hypoglycemia, hypotriglyceridemia, hypocholesterolemia, lower body mass, and fat as well as pronounced insulin-sensitivity of RLIP76⁻/⁻ mice suggested to us the possibility that elevation of RLIP76 in response to stress could itself elicit metabolic syndrome (MSy. Indeed, if it were required for MSy, drugs used to treat MSy should have no effect on RLIP76⁻/⁻ mice. RESEARCH DESIGN AND METHODS: Blood glucose (BG and lipid measurements were performed in RLIP76⁺/⁺ and RLIP76⁻/⁻ mice, using Ascensia Elite Glucometer® for glucose and ID Labs kits for cholesterol and triglycerides assays. The ultimate effectors of gluconeogenesis are the three enzymes: PEPCK, F-1,6-BPase, and G6Pase, and their expression is regulated by PPARγ and AMPK. The activity of these enzymes was tested by protocols standardized by us. Expressions of RLIP76, PPARα, PPARγ, HMGCR, pJNK, pAkt, and AMPK were performed by Western-blot and tissue staining. RESULTS: The concomitant activation of AMPK and PPARγ by inhibiting transport activity of RLIP76, despite inhibited activity of key glucocorticoid-regulated hepatic gluconeogenic enzymes like PEPCK, G6Pase and F-1,6-BP in RLIP76⁻/⁻ mice, is a salient finding of our studies. The decrease in RLIP76 protein expression by rosiglitazone and metformin is associated with an up-regulation of PPARγ and AMPK. CONCLUSIONS/SIGNIFICANCE: All four drugs, rosiglitazone, metformin, gemfibrozil and atorvastatin failed to affect glucose and lipid metabolism in RLIP76⁻/⁻ mice. Studies confirmed a model in which RLIP76 plays a central role in the pathogenesis of MSy and RLIP76 loss causes profound and global alterations of MSy signaling functions. RLIP76 is a novel target for single-molecule therapeutics for metabolic syndrome.

Determination of glutathione and glutathione disulfide in biological samples: an in-depth review.

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Monostori, Péter; Wittmann, Gyula; Karg, Eszter; Túri, Sándor

2009-10-15

Glutathione (GSH) is a thiol-containing tripeptide, which plays central roles in the defence against oxidative damage and in signaling pathways. Upon oxidation, GSH is transformed to glutathione disulfide (GSSG). The concentrations of GSH and GSSG and their molar ratio are indicators of cell functionality and oxidative stress. Assessment of redox homeostasis in various clinical states and medical applications for restoration of the glutathione status are of growing importance. This review is intended to provide a state-of-the-art overview of issues relating to sample pretreatment and choices for the separation and detection of GSH and GSSG. High-performance liquid chromatography, capillary electrophoresis and gas chromatography (as techniques with a separation step) with photometric, fluorimetric, electrochemical and mass spectrometric detection are discussed, stress being laid on novel approaches.

[Cloning and expression analysis of a zinc-regulated transporters (ZRT), iron-regulated transporter (IRT)-like protein encoding gene in Dendrobium officinale].

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Zhang, Gang; Li, Yi-Min; Li, Biao; Zhang, Da-Wei; Guo, Shun-Xing

2015-01-01

The zinc-regulated transporters (ZRT), iron-regulated transporter (IRT)-like protein (ZIP) plays an important role in the growth and development of plant. In this study, a full length cDNA of ZIP encoding gene, designed as DoZIP1 (GenBank accession KJ946203), was identified from Dendrobium officinale using RT-PCR and RACE. Bioinformatics analysis showed that DoZIP1 consisted of a 1,056 bp open reading frame (ORF) encoded a 351-aa protein with a molecular weight of 37.57 kDa and an isoelectric point (pI) of 6.09. The deduced DoZIP1 protein contained the conserved ZIP domain, and its secondary structure was composed of 50.71% alpha helix, 11.11% extended strand, 36.18% random coil, and beta turn 1.99%. DoZIP1 protein exhibited a signal peptide and eight transmembrane domains, presumably locating in cell membrane. The amino acid sequence had high homology with ZIP proteins from Arabidopsis, alfalfa and rice. A phylogenetic tree analysis demonstrated that DoZIP1 was closely related to AtZIP10 and OsZIP3, and they were clustered into one clade. Real time quantitative PCR analysis demonstrated that the transcription level of DoZIP1 in D. officinale roots was the highest (4.19 fold higher than that of stems), followed by that of leaves (1.12 fold). Molecular characters of DoZIP1 will be useful for further functional determination of the gene involving in the growth and development of D. officinale.

Discovery of a Conjugative Megaplasmid in Bifidobacterium breve

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Bottacini, Francesca; O'Connell Motherway, Mary; Casey, Eoghan; McDonnell, Brian; Mahony, Jennifer; Ventura, Marco

2014-01-01

Bifidobacterium breve is a common and sometimes very abundant inhabitant of the human gut. Genome sequencing of B. breve JCM 7017 revealed the presence of an extrachromosomal element, designated pMP7017 consisting of >190 kb, thus representing the first reported bifidobacterial megaplasmid. In silico characterization of this element revealed several genomic features supporting a stable establishment of the megaplasmid in its host, illustrated by predicted CRISPR-Cas functions that are known to protect the host against intrusion of foreign DNA. Interestingly, pMP7017 is also predicted to encode a conjugative DNA transfer apparatus and, consistent with this notion, we demonstrate here the conjugal transfer of pMP7017 to representative strains of B. breve and B. longum subsp. longum. We also demonstrate the presence of a megaplasmid with homology to pMP7017 in three B. longum subsp. longum strains. PMID:25326305

The Down regulated in Adenoma (dra) gene encodes an intestine-specific membrane sulfate transport protein.

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Silberg, D G; Wang, W; Moseley, R H; Traber, P G

1995-05-19

A gene has been described, Down Regulated in Adenoma (dra), which is expressed in normal colon but is absent in the majority of colon adenomas and adenocarcinomas. However, the function of this protein is unknown. Because of sequence similarity to a recently cloned membrane sulfate transporter in rat liver, the transport function of Dra was examined. We established that dra encodes for a Na(+)-independent transporter for both sulfate and oxalate using microinjected Xenopus oocytes as an assay system. Sulfate transport was sensitive to the anion exchange inhibitor DIDS (4,4'-diisothiocyano-2,2' disulfonic acid stilbene). Using an RNase protection assay, we found that dra mRNA expression is limited to the small intestine and colon in mouse, therefore identifying Dra as an intestine-specific sulfate transporter. dra also had a unique pattern of expression during intestinal development. Northern blot analysis revealed a low level of expression in colon at birth with a marked increase in the first 2 postnatal weeks. In contrast, there was a lower, constant level of expression in small intestine in the postnatal period. Caco-2 cells, a colon carcinoma cell line that differentiates over time in culture, demonstrated a marked induction of dra mRNA as cells progressed from the preconfluent (undifferentiated) to the postconfluent (differentiated) state. These results show that Dra is an intestine-specific Na(+)-independent sulfate transporter that has differential expression during colonic development. This functional characterization provides the foundation for investigation of the role of Dra in intestinal sulfate transport and in the malignant phenotype.

Mobility of the native Bacillus subtilis conjugative plasmid pLS20 is regulated by intercellular signaling.

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Singh, Praveen K; Ramachandran, Gayetri; Ramos-Ruiz, Ricardo; Peiró-Pastor, Ramón; Abia, David; Wu, Ling J; Meijer, Wilfried J J

2013-10-01

Horizontal gene transfer mediated by plasmid conjugation plays a significant role in the evolution of bacterial species, as well as in the dissemination of antibiotic resistance and pathogenicity determinants. Characterization of their regulation is important for gaining insights into these features. Relatively little is known about how conjugation of Gram-positive plasmids is regulated. We have characterized conjugation of the native Bacillus subtilis plasmid pLS20. Contrary to the enterococcal plasmids, conjugation of pLS20 is not activated by recipient-produced pheromones but by pLS20-encoded proteins that regulate expression of the conjugation genes. We show that conjugation is kept in the default "OFF" state and identified the master repressor responsible for this. Activation of the conjugation genes requires relief of repression, which is mediated by an anti-repressor that belongs to the Rap family of proteins. Using both RNA sequencing methodology and genetic approaches, we have determined the regulatory effects of the repressor and anti-repressor on expression of the pLS20 genes. We also show that the activity of the anti-repressor is in turn regulated by an intercellular signaling peptide. Ultimately, this peptide dictates the timing of conjugation. The implications of this regulatory mechanism and comparison with other mobile systems are discussed.

SXT/R391 ICE elements encode a novel ‘trap-door’ strategy for mobile element escape

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Michael P Ryan

2016-05-01

Full Text Available Integrative Conjugative Elements (ICEs are a class of bacterial mobile elements that have the ability to mediate their own integration, excision and transfer from one host genome to another by a mechanism of site-specific recombination, self-circularisation and conjugative transfer. Members of the SXT/R391 ICE family of enterobacterial mobile genetic elements display an unusual UV-inducible sensitisation function which results in stress induced killing of bacterial cells harbouring the ICE. This sensitisation has been shown to be associated with a stress induced overexpression of a mobile element encoded conjugative transfer gene, orf43, a traV homolog. This results in cell lysis and release of a circular form of the ICE. Induction of this novel system may allow transfer of an ICE, enhancing its survival potential under conditions not conducive to conjugative transfer.

Biotin conjugated organic molecules and proteins for cancer therapy: A review.

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Maiti, Santanu; Paira, Priyankar

2018-02-10

The main transporter for biotin is sodium dependent multivitamin transporter (SMVT), which is overexpressed in various aggressive cancer cell lines such as ovarian (OV 2008, ID8), leukemia (L1210FR), mastocytoma (P815), colon (Colo-26), breast (4T1, JC, MMT06056), renal (RENCA, RD0995), and lung (M109) cancer cell lines. Furthermore, its overexpression was found higher to that of folate receptor. Therefore, biotin demand in the rapidly growing tumors is higher than normal tissues. Several biotin conjugated organic molecules has been reported here for selective delivery of the drug in cancer cell. Biotin conjugated molecules are showing higher fold of cytotoxicity in biotin positive cancer cell lines than the normal cell. Nanoparticles and polymer surface modified drugs and biotin mediated cancer theranostic strategy was highlighted in this review. The cytotoxicity and selectivity of the drug in cancer cells has enhanced after biotin conjugation. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Stability of cytochromes P450 and phase II conjugation systems in precision-cut rat lung slices cultured up to 72 h.

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Umachandran, Meera; Ioannides, Costas

2006-07-05

The objective of the present study was to evaluate the stability of cytochrome P450 enzymes and of the conjugation enzyme systems epoxide hydrolase, glucuronosyl transferase, sulphotransferase and glutathione S-transferase in precision-cut rat lung slices incubated in RPMI media for different time periods up to 72 h. Moreover, the effect of culturing of lung slices on total glutathione levels and glutathione reductase was also investigated. Monitoring of cytochrome P450 activity was achieved using established diagnostic probes, but when activity in the lung was low the maintenance of the various enzymes in culture was determined immunologically using Western blotting. The dealkylation of pentoxyresorufin declined markedly during the first 4h of incubation but in the case of ethoxyresorufin loss of activity was more gradual and less severe. Western blot analysis revealed that the rate of decrease in cytochrome P450 apoprotein levels was isoform-specific with CYP2E1 being the most stable and CYP3A the least stable. Generally, phase II activities, especially cytosolic sulphotransferase, were relatively more stable throughout the incubation period compared with cytochromes P450. Finally, glutathione reductase activity and total glutathione levels were maintained throughout the 72 h incubation. The present studies indicate that xenobiotic-metabolising enzymes in precision-cut rat lung slices decline in culture, but the rate of loss differs and depends on the nature of the enzyme.

Variable Levels of Glutathione S-Transferases Are Responsible for the Differential Tolerance to Metolachlor between Maize (Zea mays) Shoots and Roots.

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Li, Dongzhi; Xu, Li; Pang, Sen; Liu, Zhiqian; Wang, Kai; Wang, Chengju

2017-01-11

Glutathione S-transferases (GSTs) play important roles in herbicide tolerance. However, studies on GST function in herbicide tolerance among plant tissues are still lacking. To explore the mechanism of metolachlor tolerance difference between maize shoots and roots, the effects of metolachlor on growth, GST activity, and the expression of the entire GST gene family were investigated. It was found that this differential tolerance to metolachlor was correlated with contrasting GST activity between the two tissues and can be eliminated by a GST inhibitor. An in vitro metolachlor-glutathione conjugation assay confirmed that the transformation of metolachlor is 2-fold faster in roots than in shoots. The expression analysis of the GST gene family revealed that most GST genes are expressed much higher in roots than shoots, both in control and in metolachlor-treated plants. Taken together, higher level expression of most GST genes, leading to higher GST activity and faster herbicide transformation, appears to be responsible for the higher tolerance to metolachlor of maize roots than shoots.

Comparison of plasma malondialdehyde, glutathione, glutathione peroxidase, hydroxyproline and selenium levels in patients with vitiligo and healthy controls

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Ozturk I

2008-01-01

Full Text Available Background: The etiology and pathophysiologic mechanism of vitiligo are still unclear. The relationship between increased oxidative stress due to the accumulation of radicals and reactive oxygen species and the associated changes in blood and epidermal component of vitiliginous skin have been reported many times. We investigated the possible changes of plasma malondialdehyde, glutathione, selenium, hydroxyproline and glutathione peroxidase activity levels in patients with vitiligo in order to evaluate the relationship between oxidative stress and etiopathogenesis of vitiligo. Materials and Methods: Plasma malondialdehyde, glutathione, hydroxyproline and glutathione peroxidase activity levels were measured by spectrophotometric methods, and HPLC was used for measurement of selenium concentrations. Results: Our results showed increased malondialdehyde, hydroxyproline and glutathione peroxidase activity levels in plasma of vitiligo group ( P < 0.05. Conclusion: Support of antioxidant system via nonenzymatic antioxidant compounds and antioxidant enzymes may be useful to prevent of melanocyte degeneration which occur due to oxidative damage in vitiligo.

The multidrug resistance IncA/C transferable plasmid encodes a novel domain-swapped dimeric protein-disulfide isomerase.

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Premkumar, Lakshmanane; Kurth, Fabian; Neyer, Simon; Schembri, Mark A; Martin, Jennifer L

2014-01-31

The multidrug resistance-encoding IncA/C conjugative plasmids disseminate antibiotic resistance genes among clinically relevant enteric bacteria. A plasmid-encoded disulfide isomerase is associated with conjugation. Sequence analysis of several IncA/C plasmids and IncA/C-related integrative and conjugative elements (ICE) from commensal and pathogenic bacteria identified a conserved DsbC/DsbG homolog (DsbP). The crystal structure of DsbP reveals an N-terminal domain, a linker region, and a C-terminal catalytic domain. A DsbP homodimer is formed through domain swapping of two DsbP N-terminal domains. The catalytic domain incorporates a thioredoxin-fold with characteristic CXXC and cis-Pro motifs. Overall, the structure and redox properties of DsbP diverge from the Escherichia coli DsbC and DsbG disulfide isomerases. Specifically, the V-shaped dimer of DsbP is inverted compared with EcDsbC and EcDsbG. In addition, the redox potential of DsbP (-161 mV) is more reducing than EcDsbC (-130 mV) and EcDsbG (-126 mV). Other catalytic properties of DsbP more closely resemble those of EcDsbG than EcDsbC. These catalytic differences are in part a consequence of the unusual active site motif of DsbP (CAVC); substitution to the EcDsbC-like (CGYC) motif converts the catalytic properties to those of EcDsbC. Structural comparison of the 12 independent subunit structures of DsbP that we determined revealed that conformational changes in the linker region contribute to mobility of the catalytic domain, providing mechanistic insight into DsbP function. In summary, our data reveal that the conserved plasmid-encoded DsbP protein is a bona fide disulfide isomerase and suggest that a dedicated oxidative folding enzyme is important for conjugative plasmid transfer.

Peptide π-Electron Conjugates: Organic Electronics for Biology?

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Ardoña, Herdeline Ann M; Tovar, John D

2015-12-16

Highly ordered arrays of π-conjugated molecules are often viewed as a prerequisite for effective charge-transporting materials. Studies involving these materials have traditionally focused on organic electronic devices, with more recent emphasis on biological systems. In order to facilitate the transition to biological environments, biomolecules that can promote hierarchical ordering and water solubility are often covalently appended to the π-electron unit. This review highlights recent work on π-conjugated systems bound to peptide moieties that exhibit self-assembly and aims to provide an overview on the development and emerging applications of peptide-based supramolecular π-electron systems.

Glutathione content in sperm cells of infertile men

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R. V. Fafula

2017-04-01

Full Text Available Hyperproduction of reactive oxygen species can damage sperm cells and is considered to be one of the mechanisms of male infertility. Cell protection from the damaging effects of free radicals and lipid peroxidation products is generally determined by the degree of antioxidant protection. Glutathione is non-enzymatic antioxidant which plays an important protective role against oxidative damages and lipid peroxidation. The aim of the present work is to determine the content of reduced and oxidized glutathione in sperm cells of infertile men. Semen samples from 20 fertile men (normozoospermics and 72 infertile patients (12 oligozoospermics, 17 asthenozoospermics, 10 oligoasthenozoosper­mics and 33 leucocytospermic were used. The total, oxidized (GSSG and reduced (GSH glutathione levels were measured spectrophotometrically. The levels of total glutathione were significantly lower in the spermatozoa of patients with oligozoo-, asthenozoo- and oligoasthenozoospermia than in the control. Infertile groups showed significantly decreased values of reduced glutathione in sperm cells vs. fertile men, indicating an alteration of oxidative status. The oxidized glutathione levels in sperm cells of infertile men did not differ from those of normozoospermic men with proven fertility. The GSH/GSSG ratio was significantly decreased in the oligo-, astheno- and oligoasthenozoospermic groups compared to the normozoospermic group. In patients with leucocytospermia the GSH/GSSG ratio was lower but these changes were not significant. In addition, glutathione peroxidase activity in sperm cells was decreased in patients with oligozoo-, astenozoo-, oligoastenozoospermia and with leucocytospermia. The most significant changes in glutathione peroxidase activity were observed in infertile men with leucocytospermia. Decreased GSH/GSSG ratio indicates a decline in redox-potential of the glutathione system in sperm cells of men with decreased fertilizing potential

Discovery of a conjugative megaplasmid in Bifidobacterium breve.

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Bottacini, Francesca; O'Connell Motherway, Mary; Casey, Eoghan; McDonnell, Brian; Mahony, Jennifer; Ventura, Marco; van Sinderen, Douwe

2015-01-01

Bifidobacterium breve is a common and sometimes very abundant inhabitant of the human gut. Genome sequencing of B. breve JCM 7017 revealed the presence of an extrachromosomal element, designated pMP7017 consisting of >190 kb, thus representing the first reported bifidobacterial megaplasmid. In silico characterization of this element revealed several genomic features supporting a stable establishment of the megaplasmid in its host, illustrated by predicted CRISPR-Cas functions that are known to protect the host against intrusion of foreign DNA. Interestingly, pMP7017 is also predicted to encode a conjugative DNA transfer apparatus and, consistent with this notion, we demonstrate here the conjugal transfer of pMP7017 to representative strains of B. breve and B. longum subsp. longum. We also demonstrate the presence of a megaplasmid with homology to pMP7017 in three B. longum subsp. longum strains. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Novel substrate specificity of glutathione synthesis enzymes from Streptococcus agalactiae and Clostridium acetobutylicum

International Nuclear Information System (INIS)

Kino, Kuniki; Kuratsu, Shoko; Noguchi, Atsushi; Kokubo, Masahiro; Nakazawa, Yuji; Arai, Toshinobu; Yagasaki, Makoto; Kirimura, Kohtaro

2007-01-01

Glutathione (GSH) is synthesized by γ-glutamylcysteine synthetase (γ-GCS) and glutathione synthetase (GS) in living organisms. Recently, bifunctional fusion protein, termed γ-GCS-GS catalyzing both γ-GCS and GS reactions from gram-positive firmicutes Streptococcus agalactiae, has been reported. We revealed that in the γ-GCS activity, S. agalactiae γ-GCS-GS had different substrate specificities from those of Escherichia coli γ-GCS. Furthermore, S. agalactiae γ-GCS-GS synthesized several kinds of γ-glutamyltripeptide, γ-Glu-X aa -Gly, from free three amino acids. In Clostridium acetobutylicum, the genes encoding γ-GCS and putative GS were found to be immediately adjacent by BLAST search, and had amino acid sequence homology with S. agalactiae γ-GCS-GS, respectively. We confirmed that the proteins expressed from each gene showed γ-GCS and GS activity, respectively. C. acetobutylicum GS had broad substrate specificities and synthesized several kinds of γ-glutamyltripeptide, γ-Glu-Cys-X aa . Whereas the substrate specificities of γ-GCS domain protein and GS domain protein of S. agalactiae γ-GCS-GS were the same as those of S. agalactiae γ-GCS-GS

Transport and activation of S-(1,2-dichlorovinyl)-L-cysteine and N-acetyl-S-(1,2-dichlorovinyl)-L-cysteine in rat kidney proximal tubules

International Nuclear Information System (INIS)

Zhang, G.H.; Stevens, J.L.

1989-01-01

An important step in understanding the mechanism underlying the tubular specificity of the nephrotoxicity of toxic cysteine conjugates is to identify the rate-limiting steps in their activation. The rate-limiting steps in the activation of toxic cysteine conjugates were characterized using isolated proximal tubules from the rat and 35S-labeled S-(1,2-dichlorovinyl)-L-cysteine (DCVC) and N-acetyl-S-(1,2-dichlorovinyl)-L-cysteine (NAC-DCVC) as model compounds. The accumulation by tubules of 35S radiolabel from both DCVC and NAC-DCVC was time and temperature dependent and was mediated by both Na+-dependent and independent processes. Kinetic studies with DCVC in the presence of sodium revealed the presence of two components with apparent Km and Vmax values of (1) 46 microM and 0.21 nmol/mg min and (2) 2080 microM and 7.3 nmol/mg.min. NAC-DVVC uptake was via a single system with apparent Km and Vmax values of 157 microM and 0.65 nmol/mg.min, respectively. Probenecid, an inhibitor of the renal organic anion transport system, inhibited accumulation of radiolabel from NAC-DCVC, but not from DCVC. The covalent binding of 35S label to cellular macromolecules was much greater from [35S]DCVC than from NAC-[35S]DCVC. Analysis of metabolites showed that a substantial amount of the cellular NAC-[35S]DCVC was unmetabolized while [35S]DCVC was rapidly metabolized to bound 35S-labeled material and unidentified products. The data suggest that DCVC is rapidly metabolized following transport, but that activation of NAC-DCVC depends on a slower rate of deacetylation. The results are discussed with regard to the segment specificity of cysteine conjugate toxicity and the role of disposition in vivo in the nephrotoxicity of glutathione conjugates

Inhibition of bacterial conjugation by phage M13 and its protein g3p: quantitative analysis and model.

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Abraham Lin

Full Text Available Conjugation is the main mode of horizontal gene transfer that spreads antibiotic resistance among bacteria. Strategies for inhibiting conjugation may be useful for preserving the effectiveness of antibiotics and preventing the emergence of bacterial strains with multiple resistances. Filamentous bacteriophages were first observed to inhibit conjugation several decades ago. Here we investigate the mechanism of inhibition and find that the primary effect on conjugation is occlusion of the conjugative pilus by phage particles. This interaction is mediated primarily by phage coat protein g3p, and exogenous addition of the soluble fragment of g3p inhibited conjugation at low nanomolar concentrations. Our data are quantitatively consistent with a simple model in which association between the pili and phage particles or g3p prevents transmission of an F plasmid encoding tetracycline resistance. We also observe a decrease in the donor ability of infected cells, which is quantitatively consistent with a reduction in pili elaboration. Since many antibiotic-resistance factors confer susceptibility to phage infection through expression of conjugative pili (the receptor for filamentous phage, these results suggest that phage may be a source of soluble proteins that slow the spread of antibiotic resistance genes.

Measurement of glutathione-protein mixed disulfides

International Nuclear Information System (INIS)

Livesey, J.C.; Reed, D.J.

1984-01-01

The development of a sensitive and highly specific assay for the presence of mixed disulfides between protein thiol groups and endogenous thiols has been undertaken. Previous investigations on the concentrations of glutathione (GSH), glutathione disulfide (GSSG) and protein glutathione mixed disulfides (ProSSG) have been of limited usefulness because of the poor specificity of the assays used. Our assay for these forms of glutathione is based on high performance liquid chromatography (HPLC) and is an extension of an earlier method. After perchloric acid precipitation, the protein sample is washed with an organic solvent to fully denature the protein. Up to a 10-fold increase in GSH released from fetal bovine serum (FBS) protein has been found when the protein precipitate is washed with ethanol rather than ether, as earlier suggested. Similar effects have been observed with an as yet unidentified thiol which elutes in the chromatography system with a retention volume similar to cysteine

Active biomonitoring of a subtropical river using glutathione-S ...

African Journals Online (AJOL)

Active biomonitoring of a subtropical river using glutathione-S-transferase (GST) and heat shock proteins (HSP 70) in. Oreochromis niloticusas surrogate biomarkers of metal contamination. Victor Kurauone Muposhi1, Beaven Utete1*, Idah Sithole-Niang2 and Stanley Mukangenyama2. 1Wildlife Ecology and Conservation, ...

Separation of water-soluble metabolites of benzo[a]pyrene formed by cultured human colon

DEFF Research Database (Denmark)

Autrup, Herman

1979-01-01

A method has been developed to separate conjugated metabolites of benzo[a]pyrene into three major fractions: sulfate esters, glucuronides and glutathione conjugates. In cultured human colon, formation of sulfate esters and glutathione conjugates is the major conjugation pathway, while formation......-hydroxybenzo[a]pyrene were the major substrates for sulfotransferase in cultured human colon....

Purification of SUMO conjugating enzymes and kinetic analysis of substrate conjugation

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Yunus, Ali A.; Lima, Christopher D.

2009-01-01

SUMO conjugation to protein substrates requires the concerted action of a dedicated E2 ubiquitin conjugation enzyme (Ubc9) and associated E3 ligases. Although Ubc9 can directly recognize and modify substrate lysine residues that occur within a consensus site for SUMO modification, E3 ligases can redirect specificity and enhance conjugation rates during SUMO conjugation in vitro and in vivo. In this chapter, we will describe methods utilized to purify SUMO conjugating enzymes and model substrates which can be used for analysis of SUMO conjugation in vitro. We will also describe methods to extract kinetic parameters during E3-dependent or E3-independent substrate conjugation. PMID:19107417

Peptide- and saccharide-conjugated dendrimers for targeted drug delivery: a concise review

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Liu, Jie; Gray, Warren D.; Davis, Michael E.; Luo, Ying

2012-01-01

Dendrimers comprise a category of branched materials with diverse functions that can be constructed with defined architectural and chemical structures. When decorated with bioactive ligands made of peptides and saccharides through peripheral chemical groups, dendrimer conjugates are turned into nanomaterials possessing attractive binding properties with the cognate receptors. At the cellular level, bioactive dendrimer conjugates can interact with cells with avidity and selectivity, and this function has particularly stimulated interests in investigating the targeting potential of dendrimer materials for the design of drug delivery systems. In addition, bioactive dendrimer conjugates have so far been studied for their versatile capabilities to enhance stability, solubility and absorption of various types of therapeutics. This review presents a brief discussion on three aspects of the recent studies to use peptide- and saccharide-conjugated dendrimers for drug delivery: (i) synthesis methods, (ii) cell- and tissue-targeting properties and (iii) applications of conjugated dendrimers in drug delivery nanodevices. With more studies to elucidate the structure–function relationship of ligand–dendrimer conjugates in transporting drugs, the conjugated dendrimers hold promise to facilitate targeted delivery and improve drug efficacy for discovery and development of modern pharmaceutics. PMID:23741608

Membrane accessibility of glutathione

DEFF Research Database (Denmark)

Garcia, Almudena; Eljack, N., D.; Sani, ND

2015-01-01

Regulation of the ion pumping activity of the Na(+),K(+)-ATPase is crucial to the survival of animal cells. Recent evidence has suggested that the activity of the enzyme could be controlled by glutathionylation of cysteine residue 45 of the β-subunit. Crystal structures so far available indicate...... that this cysteine is in a transmembrane domain of the protein. Here we have analysed via fluorescence and NMR spectroscopy as well as molecular dynamics simulations whether glutathione is able to penetrate into the interior of a lipid membrane. No evidence for any penetration of glutathione into the membrane...

Thiol-Disulfide Exchange between Glutaredoxin and Glutathione

DEFF Research Database (Denmark)

Iversen, Rasmus; Andersen, Peter Anders; Jensen, Kristine Steen

2010-01-01

Glutaredoxins are ubiquitous thiol-disulfide oxidoreductases which catalyze the reduction of glutathione-protein mixed disulfides. Belonging to the thioredoxin family, they contain a conserved active site CXXC motif. The N-proximal active site cysteine can form a mixed disulfide with glutathione ...... has been replaced with serine. The exchange reaction between the reduced protein and oxidized glutathione leading to formation of the mixed disulfide could readily be monitored by isothermal titration calorimetry (ITC) due to the enthalpic contributions from the noncovalent interactions...

Glutathione Redox System in β-Thalassemia/Hb E Patients

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Ruchaneekorn W. Kalpravidh

2013-01-01

Full Text Available β-thalassemia/Hb E is known to cause oxidative stress induced by iron overload. The glutathione system is the major endogenous antioxidant that protects animal cells from oxidative damage. This study aimed to determine the effect of disease state and splenectomy on redox status expressed by whole blood glutathione (GSH/glutathione disulfide (GSSG and also to evaluate glutathione-related responses to oxidation in β-thalassemia/Hb E patients. Twenty-seven normal subjects and 25 β-thalassemia/Hb E patients were recruited and blood was collected. The GSH/GSSG ratio, activities of glutathione-related enzymes, hematological parameters, and serum ferritin levels were determined in individuals. Patients had high iron-induced oxidative stress, shown as significantly increased serum ferritin, a decreased GSH/GSSG ratio, and increased activities of glutathione-related enzymes. Splenectomy increased serum ferritin levels and decreased GSH levels concomitant with unchanged glutathione-related enzyme activities. The redox ratio had a positive correlation with hemoglobin levels and negative correlation with levels of serum ferritin. The glutathione system may be the body’s first-line defense used against oxidative stress and to maintain redox homeostasis in thalassemic patients based on the significant correlations between the GSH/GSSH ratio and degree of anemia or body iron stores.

Molecular-weight dependence of interchain polaron delocalization and exciton bandwidth in high-mobility conjugated polymers

DEFF Research Database (Denmark)

Chang, J.F.; Clark, J.; Zhao, N.

2006-01-01

delocalization with increasing conjugation length and crystalline quality. From comparative studies of field-effect transistor characteristics, film morphology, and optical properties our study provides a microscopic understanding of the factors which limit the charge transport in P3HT to field-effect mobilities......Interchain interactions have a profound effect on the optical as well as charge transport properties of conjugated polymer thin films. In contrast to oligomeric model systems in solution-deposited polymer thin films the study of such effects is complicated by the complex microstructure. We present...

Metabolism of N-methylformamide in mice: primary kinetic deuterium isotope effect and identification of S-(N-methylcarbamoyl)glutathione as a metabolite

International Nuclear Information System (INIS)

Threadgill, M.D.; Axworthy, D.B.; Baillie, T.A.; Farmer, P.B.; Farrow, K.C.; Gescher, A.; Kestell, P.; Pearson, P.G.; Shaw, A.J.

1987-01-01

S-(N-Methylcarbamoyl)glutathione has been identified by cesium ion liquid secondary ion mass spectrometry as a biliary metabolite in mice of the experimental antitumor agent and hepatotoxin N-methylformamide. Metabolism of N-methylformamide to urinary methylamine, urinary N-acetyl-S-(N-methylcarbamoyl)-cysteine and biliary S-(N-methylcarbamoyl)glutathione was found to be subject to large intermolecular primary kinetic isotope effects when hydrogen was replaced by deuterium in the formyl group (kH/kD = 5.5 +/- 0.2, 4.5 +/- 1.0 and 7 +/- 2, respectively), as shown by mass spectrometry of derivatives of these metabolites. These values indicate the existence of a common metabolic precursor for each of these metabolites. In particular, methylamine is shown not to arise from simple enzymatic hydrolysis of N-methylformamide but is associated with an oxidative process. Therefore, it is highly likely that N-methylformamide is oxidized and conjugated to form S-(N-methylcarbamoyl)glutathione which is metabolized further to N-acetyl-S-(N-methylcarbamoyl) cysteine. Either of these thiocarbamates could be hydrolyzed to give the parent thiol and the observed metabolic end products, methylamine and carbon dioxide. The presence of deuterium in the formyl moiety of N-methylformamide reduced markedly the hepatotoxicity of the compound, as shown by measurements of the activities of appropriate hepatic enzymes in plasma

Glutathione S-Transferases: Role in Combating Abiotic Stresses Including Arsenic Detoxification in Plants

Directory of Open Access Journals (Sweden)

Smita Kumar

2018-06-01

Full Text Available Arsenic (As, naturally occurring metalloid and a potential hazardous material, is found in low concentrations in the environment and emerges from natural sources and anthropogenic activities. The presence of As in ground water, which is used for irrigation, is a matter of great concern since it affects crop productivity and contaminates food chain. In plants, As alters various metabolic pathways in cells including the interaction of substrates/enzymes with the sulfhydryl groups of proteins and the replacement of phosphate in ATP for energy. In addition, As stimulates the generation of free radicals and reactive oxygen species (ROS, resulting in oxidative stress. Glutathione S-transferases (GSTs quench reactive molecules with the addition of glutathione (GSH and protect the cell from oxidative damage. GSTs are a multigene family of isozymes, known to catalyze the conjugation of GSH to miscellany of electrophilic and hydrophobic substrates. GSTs have been reported to be associated with plant developmental processes and are responsive to multitude of stressors. In past, several studies suggested involvement of plant GST gene family in As response due to the requirement of sulfur and GSH in the detoxification of this toxic metalloid. This review provides updated information about the role of GSTs in abiotic and biotic stresses with an emphasis on As uptake, metabolism, and detoxification in plants. Further, the genetic manipulations that helped in enhancing the understanding of the function of GSTs in abiotic stress response and heavy metal detoxification has been reviewed.

Functional role of oppA encoding an oligopeptide-binding protein from Lactobacillus salivarius Ren in bile tolerance.

Science.gov (United States)

Wang, Guohong; Li, Dan; Ma, Xiayin; An, Haoran; Zhai, Zhengyuan; Ren, Fazheng; Hao, Yanling

2015-08-01

Lactobacillus salivarius is a member of the indigenous microbiota of the human gastrointestinal tract (GIT), and some L. salivarius strains are considered as probiotics. Bile tolerance is a crucial property for probiotic bacteria to survive the transit through the GIT and exert their beneficial effects. In this work, the functional role of oppA encoding an oligopeptide transporter substrate-binding protein from L. salivarius Ren in bile salt tolerance was investigated. In silico analysis revealed that the oppA gene encodes a 61.7-kDa cell surface-anchored hydrophilic protein with a canonical lipoprotein signal peptide. Homologous overexpression of OppA was shown to confer 20-fold higher tolerance to 0.5 % oxgall in L. salivarius Ren. Furthermore, the recombinant strain exhibited 1.8-fold and 3.6-fold higher survival when exposed to the sublethal concentration of sodium taurocholate and sodium taurodeoxycholate, respectively, while no significant change was observed when exposed to sodium glycocholate and sodium glycodeoxycholate (GDCA). Our results indicate that OppA confers specific resistance to taurine-conjugated bile salts in L. salivarius Ren. In addition, the OppA overexpression strain also showed significant increased resistance to heat and salt stresses, suggesting the protective role of OppA against multiple stresses in L. salivarius Ren.

Upconversion Nanoparticles-Encoded Hydrogel Microbeads-Based Multiplexed Protein Detection

Science.gov (United States)

Shikha, Swati; Zheng, Xiang; Zhang, Yong

2018-06-01

Fluorescently encoded microbeads are in demand for multiplexed applications in different fields. Compared to organic dye-based commercially available Luminex's xMAP technology, upconversion nanoparticles (UCNPs) are better alternatives due to their large anti-Stokes shift, photostability, nil background, and single wavelength excitation. Here, we developed a new multiplexed detection system using UCNPs for encoding poly(ethylene glycol) diacrylate (PEGDA) microbeads as well as for labeling reporter antibody. However, to prepare UCNPs-encoded microbeads, currently used swelling-based encapsulation leads to non-uniformity, which is undesirable for fluorescence-based multiplexing. Hence, we utilized droplet microfluidics to obtain encoded microbeads of uniform size, shape, and UCNPs distribution inside. Additionally, PEGDA microbeads lack functionality for probe antibodies conjugation on their surface. Methods to functionalize the surface of PEGDA microbeads (acrylic acid incorporation, polydopamine coating) reported thus far quench the fluorescence of UCNPs. Here, PEGDA microbeads surface was coated with silica followed by carboxyl modification without compromising the fluorescence intensity of UCNPs. In this study, droplet microfluidics-assisted UCNPs-encoded microbeads of uniform shape, size, and fluorescence were prepared. Multiple color codes were generated by mixing UCNPs emitting red and green colors at different ratios prior to encapsulation. UCNPs emitting blue color were used to label the reporter antibody. Probe antibodies were covalently immobilized on red UCNPs-encoded microbeads for specific capture of human serum albumin (HSA) as a model protein. The system was also demonstrated for multiplexed detection of both human C-reactive protein (hCRP) and HSA protein by immobilizing anti-hCRP antibodies on green UCNPs.

The Human SLC25A33 and SLC25A36 Genes of Solute Carrier Family 25 Encode Two Mitochondrial Pyrimidine Nucleotide Transporters*

Science.gov (United States)

Di Noia, Maria Antonietta; Todisco, Simona; Cirigliano, Angela; Rinaldi, Teresa; Agrimi, Gennaro; Iacobazzi, Vito; Palmieri, Ferdinando

2014-01-01

The human genome encodes 53 members of the solute carrier family 25 (SLC25), also called the mitochondrial carrier family, many of which have been shown to transport inorganic anions, amino acids, carboxylates, nucleotides, and coenzymes across the inner mitochondrial membrane, thereby connecting cytosolic and matrix functions. Here two members of this family, SLC25A33 and SLC25A36, have been thoroughly characterized biochemically. These proteins were overexpressed in bacteria and reconstituted in phospholipid vesicles. Their transport properties and kinetic parameters demonstrate that SLC25A33 transports uracil, thymine, and cytosine (deoxy)nucleoside di- and triphosphates by an antiport mechanism and SLC25A36 cytosine and uracil (deoxy)nucleoside mono-, di-, and triphosphates by uniport and antiport. Both carriers also transported guanine but not adenine (deoxy)nucleotides. Transport catalyzed by both carriers was saturable and inhibited by mercurial compounds and other inhibitors of mitochondrial carriers to various degrees. In confirmation of their identity (i) SLC25A33 and SLC25A36 were found to be targeted to mitochondria and (ii) the phenotypes of Saccharomyces cerevisiae cells lacking RIM2, the gene encoding the well characterized yeast mitochondrial pyrimidine nucleotide carrier, were overcome by expressing SLC25A33 or SLC25A36 in these cells. The main physiological role of SLC25A33 and SLC25A36 is to import/export pyrimidine nucleotides into and from mitochondria, i.e. to accomplish transport steps essential for mitochondrial DNA and RNA synthesis and breakdown. PMID:25320081

Development and validation of the first assay method coupling liquid chromatography with chemiluminescence for the simultaneous determination of menadione and its thioether conjugates in rat plasma.

Science.gov (United States)

Elgawish, Mohamed Saleh; Shimomai, Chikako; Kishikawa, Naoya; Ohyama, Kaname; Wada, Mitsuhiro; Kuroda, Naotaka

2013-09-16

Menadione (2-methyl-1,4-naphthoquinone, MQ), a component of multivitamin drugs with antihemorrhagic, antineoplastic, and antimalarial activity, is frequently used to investigate quinone-induced cytotoxicity. The formation of MQ conjugates with glutathione (GSH) by Michael addition and subsequent biotransformation to yield N-acetyl-l-cysteine conjugates is believed to be an important detoxification process. However, the resulting conjugates, 2-methyl-3-(glutathione-S-yl)-1,4-naphthoquinone (MQ-GS) and 2-methyl-3-(N-acetyl-l-cysteine-S-yl)-1,4-naphthoquinone (MQ-NAC), retain the ability to redox cycle and to arylate cellular nucleophiles. Although the nephrotoxicity and hepatotoxicity of MQ-thiol conjugates have been reported in vitro, methods for their determination in vivo have yet to be published. Herein, a highly sensitive, simple, and selective HPLC-chemiluminescence (HPLC-CL) coupled method is reported, allowing for the first time the simultaneous determination of MQ, MQ-GS, and MQ-NAC in rat plasma after MQ administration. Our method exploits the unique redox characteristics of MQ, MQ-GS, and MQ-NAC to react with dithiothreitol (DTT) to liberate reactive oxygen species (ROS) which are detected by a CL assay using luminol as a CL probe. To verify the proposed mechanism, MQ-GS and MQ-NAC were synthetically prepared. Specimen preparation involved solid-phase extraction on an Oasis HLB cartridge followed by isocratic elution on an ODS column. No interference from endogenous substances was detected. Linearity was observed in the range of 5-120 nM for MQ-GS and MQ-NAC and 10-240 nM for MQ, with detection limits (S/N of 3) of 1.4, 0.8, and 128 fmol for MQ-GS, MQ-NAC, and MQ, respectively. The application of our method reported here is the first to extensively study the stability and reversibility of thiol-quinones.

Direct Cytoplasmic Delivery and Nuclear Targeting Delivery of HPMA-MT Conjugates in a Microtubules Dependent Fashion.

Science.gov (United States)

Zhong, Jiaju; Zhu, Xi; Luo, Kui; Li, Lian; Tang, Manlin; Liu, Yanxi; Zhou, Zhou; Huang, Yuan

2016-09-06

As the hearts of tumor cells, the nucleus is the ultimate target of many chemotherapeutic agents and genes. However, nuclear drug delivery is always hampered by multiple intracellular obstacles, such as low efficiency of lysosome escape and insufficient nuclear trafficking. Herein, an N-(2-hydroxypropyl) methacrylamide (HPMA) polymer-based drug delivery system was designed, which could achieve direct cytoplasmic delivery by a nonendocytic pathway and transport into the nucleus in a microtubules dependent fashion. A special targeting peptide (MT), derived from an endogenic parathyroid hormone-related protein, was conjugated to the polymer backbone, which could accumulate into the nucleus a by microtubule-mediated pathway. The in vitro studies found that low temperature and NaN3 could not influence the cell internalization of the conjugates. Besides, no obvious overlay of the conjugates with lysosome demonstrated that the polymer conjugates could enter the tumor cell cytoplasm by a nonendocytic pathway, thus avoiding the drug degradation in the lysosome. Furthermore, after suppression of the microtubule dynamics with microtubule stabilizing docetaxel (DTX) and destabilizing nocodazole (Noc), the nuclear accumulation of polymeric conjugates was significantly inhibited. Living cells fluorescence recovery after photobleaching study found that the nuclear import rate of conjugates was 2-fold faster compared with the DTX and Noc treated groups. These results demonstrated that the conjugates transported into the nucleus in a microtubules dependent way. Therefore, in addition to direct cytoplasmic delivery, our peptide conjugated polymeric platform could simultaneously mediate nuclear drug accumulation, which may open a new path for further intracellular genes/peptides delivery.

Blending genomes: distributive conjugal transfer in mycobacteria, a sexier form of HGT.

Science.gov (United States)

Gray, Todd A; Derbyshire, Keith M

2018-04-18

This review discusses a novel form of horizontal gene transfer (HGT) found in mycobacteria called Distributive Conjugal Transfer (DCT). While satisfying the criteria for conjugation, DCT occurs by a mechanism so distinct from oriT-mediated conjugation that it could be considered a fourth category of HGT. DCT involves the transfer of chromosomal DNA between mycobacteria and, most significantly, generates transconjugants with mosaic genomes of the parental strains. Multiple segments of donor chromosomal DNA can be co-transferred regardless of their location or the genetic selection and, as a result, the transconjugant genome contains many donor-derived segments; hence the name DCT. This distinguishing feature of DCT separates it from the other known mechanisms of HGT, which generally result in the introduction of a single, defined segment of DNA into the recipient chromosome (Fig. ). Moreover, these mosaic progeny are generated from a single conjugal event, which provides enormous capacity for rapid adaptation and evolution, again distinguishing it from the three classical modes of HGT. Unsurprisingly, the unusual mosaic products of DCT are generated by a conjugal mechanism that is also unusual. Here, we will describe the unique features of DCT and contrast those to other mechanisms of HGT, both from a mechanistic and an evolutionary perspective. Our focus will be on transfer of chromosomal DNA, as opposed to plasmid mobilization, because DCT mediates transfer of chromosomal DNA and is a chromosomally encoded process. © 2018 John Wiley & Sons Ltd.

Methionine sulfoximine supplementation enhances productivity in GS-CHOK1SV cell lines through glutathione biosynthesis.

Science.gov (United States)

Feary, Marc; Racher, Andrew J; Young, Robert J; Smales, C Mark

2017-01-01

In Lonza Biologics' GS Gene Expression System™, recombinant protein-producing GS-CHOK1SV cell lines are generated by transfection with an expression vector encoding both GS and the protein product genes followed by selection in MSX and glutamine-free medium. MSX is required to inhibit endogenous CHOK1SV GS, and in effect create a glutamine auxotrophy in the host that can be complemented by the expression vector encoded GS in selected cell lines. However, MSX is not a specific inhibitor of GS as it also inhibits the activity of GCL (a key enzyme in the glutathione biosynthesis pathway) to a similar extent. Glutathione species (GSH and GSSG) have been shown to provide both oxidizing and reducing equivalents to ER-resident oxidoreductases, raising the possibility that selection for transfectants with increased GCL expression could result in the isolation of GS-CHOKISV cell lines with improved capacity for recombinant protein production. In this study we have begun to address the relationship between MSX supplementation, the amount of intracellular GCL subunit and mAb production from a panel of GS-CHOK1SV cell lines. We then evaluated the influence of reduced GCL activity on batch culture of an industrially relevant mAb-producing GS-CHOK1SV cell line. To the best of our knowledge, this paper describes for the first time the change in expression of GCL subunits and recombinant mAb production in these cell lines with the degree of MSX supplementation in routine subculture. Our data also shows that partial inhibition of GCL activity in medium containing 75 µM MSX increases mAb productivity, and its more specific inhibitor BSO used at a concentration of 80 µM in medium increases the specific rate of mAb production eight-fold and the concentration in harvest medium by two-fold. These findings support a link between the inhibition of glutathione biosynthesis and recombinant protein production in industrially relevant systems and provide a process-driven method for

Glutathione system in Wolfram syndrome 1‑deficient mice.

Science.gov (United States)

Porosk, Rando; Kilk, Kalle; Mahlapuu, Riina; Terasmaa, Anton; Soomets, Ursel

2017-11-01

Wolfram syndrome 1 (WS) is a rare neurodegenerative disease that is caused by mutations in the Wolfram syndrome 1 (WFS1) gene, which encodes the endoplasmic reticulum (ER) glycoprotein wolframin. The pathophysiology of WS is ER stress, which is generally considered to induce oxidative stress. As WS has a well‑defined monogenetic origin and a model for chronic ER stress, the present study aimed to characterize how glutathione (GSH), a major intracellular antioxidant, was related to the disease and its progression. The concentration of GSH and the activities of reduction/oxidation system enzymes GSH peroxidase and GSH reductase were measured in Wfs1‑deficient mice. The GSH content was lower in most of the studied tissues, and the activities of antioxidative enzymes varied between the heart, kidneys and liver tissues. The results indicated that GSH may be needed for ER stress control; however, chronic ER stress from the genetic syndrome eventually depletes the cellular GSH pool and leads to increased oxidative stress.

The arabidopsis thaliana AGRAVITROPIC 1 gene encodes a component of the polar-auxin-transport efflux carrier

Science.gov (United States)

Chen, R.; Hilson, P.; Sedbrook, J.; Rosen, E.; Caspar, T.; Masson, P. H.

1998-01-01

Auxins are plant hormones that mediate many aspects of plant growth and development. In higher plants, auxins are polarly transported from sites of synthesis in the shoot apex to their sites of action in the basal regions of shoots and in roots. Polar auxin transport is an important aspect of auxin functions and is mediated by cellular influx and efflux carriers. Little is known about the molecular identity of its regulatory component, the efflux carrier [Estelle, M. (1996) Current Biol. 6, 1589-1591]. Here we show that mutations in the Arabidopsis thaliana AGRAVITROPIC 1 (AGR1) gene involved in root gravitropism confer increased root-growth sensitivity to auxin and decreased sensitivity to ethylene and an auxin transport inhibitor, and cause retention of exogenously added auxin in root tip cells. We used positional cloning to show that AGR1 encodes a putative transmembrane protein whose amino acid sequence shares homologies with bacterial transporters. When expressed in Saccharomyces cerevisiae, AGR1 promotes an increased efflux of radiolabeled IAA from the cells and confers increased resistance to fluoro-IAA, a toxic IAA-derived compound. AGR1 transcripts were localized to the root distal elongation zone, a region undergoing a curvature response upon gravistimulation. We have identified several AGR1-related genes in Arabidopsis, suggesting a global role of this gene family in the control of auxin-regulated growth and developmental processes.

Human glutathione transferases catalyzing the bioactivation of anticancer thiopurine prodrugs.

Science.gov (United States)

Eklund, Birgitta I; Gunnarsdottir, Sjofn; Elfarra, Adnan A; Mannervik, Bengt

2007-06-01

cis-6-(2-Acetylvinylthio)purine (cAVTP) and trans-6-(2-acetylvinylthio)guanine (tAVTG) are thiopurine prodrugs provisionally inactivated by an alpha,beta-unsaturated substituent on the sulfur of the parental thiopurines 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG). The active thiopurines are liberated intracellularly by glutathione (GSH) in reactions catalyzed by glutathione transferases (GSTs) (EC 2.5.1.18). Catalytic activities of 13 human GSTs representing seven distinct classes of soluble GSTs have been determined. The bioactivation of cAVTP and tAVTG occurs via a transient addition of GSH to the activated double bond of the S-substituent of the prodrug, followed by elimination of the thiopurine. The first of these consecutive reactions is rate-limiting for thiopurine release, but GST-activation of this first addition is shifting the rate limitation to the subsequent elimination. Highly active GSTs reveal the transient intermediate, which is detectable by UV spectroscopy and HPLC analysis. LC/MS analysis of the reaction products demonstrates that the primary GSH conjugate, 4-glutathionylbuten-2-one, can react with a second GSH molecule to form the 4-(bis-glutathionyl)butan-2-one. GST M1-1 and GST A4-4 were the most efficient enzymes with tAVTG, and GST M1-1 and GST M2-2 had highest activity with cAVTP. The highly efficient GST M1-1 is polymorphic and is absent in approximately half of the human population. GST P1-1, which is overexpressed in many cancer cells, had no detectable activity with cAVTP and only minor activity with tAVTG. Other GST-activated prodrugs have targeted GST P1-1-expressing cancer cells. Tumors expressing high levels of GST M1-1 or GST A4-4 can be predicted to be particularly vulnerable to chemotherapy with cAVTP or tAVTG.

Transport and biodistribution of dendrimers across human fetal membranes: implications for intravaginal administration of dendrimer-drug conjugates.

Science.gov (United States)

Menjoge, Anupa R; Navath, Raghavendra S; Asad, Abbas; Kannan, Sujatha; Kim, Chong J; Romero, Roberto; Kannan, Rangaramanujam M

2010-06-01

Dendrimers are emerging as promising topical antimicrobial agents, and as targeted nanoscale drug delivery vehicles. Topical intravaginal antimicrobial agents are prescribed to treat the ascending genital infections in pregnant women. The fetal membranes separate the extra-amniotic space and fetus. The purpose of the study is to determine if the dendrimers can be selectively used for local intravaginal application to pregnant women without crossing the membranes into the fetus. In the present study, the transport and permeability of PAMAM (poly (amidoamine)) dendrimers, across human fetal membrane (using a side by side diffusion chamber), and its biodistribution (using immunofluorescence) are evaluated ex-vivo. Transport across human fetal membranes (from the maternal side) was evaluated using Fluorescein (FITC), an established transplacental marker (positive control, size approximately 400 Da) and fluorophore-tagged G(4)-PAMAM dendrimers (approximately 16 kDa). The fluorophore-tagged G(4)-PAMAM dendrimers were synthesized and characterized using (1)H NMR, MALDI TOF MS and HPLC analysis. Transfer was measured across the intact fetal membrane (chorioamnion), and the separated chorion and amnion layers. Over a 5 h period, the dendrimer transport across all the three membranes was less than dendrimer (5.8 x 10(-8) cm(2)/s). The biodistribution showed that the dendrimers were largely present in interstitial spaces in the decidual stromal cells and the chorionic trophoblast cells (in 2.5-4 h) and surprisingly, to a smaller extent internalized in nuclei of trophoblast cells and nuclei and cytoplasm of stromal cells. Passive diffusion and paracellular transport appear to be the major route for dendrimer transport. The overall findings further suggest that entry of drugs conjugated to dendrimers would be restricted across the human fetal membranes when administered topically by intravaginal route, suggesting new ways of selectively delivering therapeutics to the mother

Modulation of xenobiotic metabolising enzymes by anticarcinogens-focus on glutathione S-transferases and their role as targets of dietary chemoprevention in colorectal carcinogenesis

Energy Technology Data Exchange (ETDEWEB)

Pool-Zobel, Beatrice [Department of Nutritional Toxicology, Institute for Nutrition, Friedrich Schiller University Jena, 07743 Jena (Germany)]. E-mail: b8pobe@uni-jena.de; Veeriah, Selvaraju [Department of Nutritional Toxicology, Institute for Nutrition, Friedrich Schiller University Jena, 07743 Jena (Germany); Boehmer, Frank-D. [Institute of Molecular Cell Biology, University Hospital, Friedrich Schiller University Jena, 07743 Jena (Germany)

2005-12-11

There is evidence that consumption of certain dietary ingredients may favourably modulate biotransformation of carcinogens. Associated with this is the hypothesis that the risk for developing colorectal cancer could be reduced, since its incidence is related to diet. Two main groups of biotransformation enzymes metabolize carcinogens, namely Phase I enzymes, which convert hydrophobic compounds to more water-soluble moieties, and Phase II enzymes (e.g. glutathione S-transferases [GST]), which primarily catalyze conjugation reactions. The conjugation of electrophilic Phase I intermediates with glutathione, for instance, frequently results in detoxification. Several possible colon carcinogens may serve as substrates for GST isoenzymes that can have marked substrate specificity. The conjugated products could be less toxic/genotoxic if GSTs are induced, thereby reducing exposure. Thus, numerous studies have shown that the induction of GSTs by antioxidants enables experimental animals to tolerate exposure to carcinogens. One important mechanism of GST induction involves an antioxidant-responsive response element (ARE) and the transcription factor nuclear factor E2-related factor 2 (Nrf2), which is bound to the Kelch-like ECH associated protein 1 (Keap1) in the cytoplasm. Antioxidants may disrupt the Keap-Nrf2 complex, allowing Nrf2 to translocate to the nucleus and mediate expression of Phase II genes via interaction with the ARE. GSTs are also induced by butyrate, a product of gut flora-derived fermentation of plant foods, which may act via different mechanisms, e.g. by increasing histone acetylation. GSTs are expressed with high inter-individual variability in human colonocytes, which points to large differences in cellular susceptibility to xenobiotics. Enhancing expression of GSTs in human colon tissue could therefore contribute to reducing cancer risks. However, it has not been demonstrated in humans that this mechanism is associated with cancer prevention. In the

Modulation of xenobiotic metabolising enzymes by anticarcinogens-focus on glutathione S-transferases and their role as targets of dietary chemoprevention in colorectal carcinogenesis

International Nuclear Information System (INIS)

Pool-Zobel, Beatrice; Veeriah, Selvaraju; Boehmer, Frank-D.

2005-01-01

There is evidence that consumption of certain dietary ingredients may favourably modulate biotransformation of carcinogens. Associated with this is the hypothesis that the risk for developing colorectal cancer could be reduced, since its incidence is related to diet. Two main groups of biotransformation enzymes metabolize carcinogens, namely Phase I enzymes, which convert hydrophobic compounds to more water-soluble moieties, and Phase II enzymes (e.g. glutathione S-transferases [GST]), which primarily catalyze conjugation reactions. The conjugation of electrophilic Phase I intermediates with glutathione, for instance, frequently results in detoxification. Several possible colon carcinogens may serve as substrates for GST isoenzymes that can have marked substrate specificity. The conjugated products could be less toxic/genotoxic if GSTs are induced, thereby reducing exposure. Thus, numerous studies have shown that the induction of GSTs by antioxidants enables experimental animals to tolerate exposure to carcinogens. One important mechanism of GST induction involves an antioxidant-responsive response element (ARE) and the transcription factor nuclear factor E2-related factor 2 (Nrf2), which is bound to the Kelch-like ECH associated protein 1 (Keap1) in the cytoplasm. Antioxidants may disrupt the Keap-Nrf2 complex, allowing Nrf2 to translocate to the nucleus and mediate expression of Phase II genes via interaction with the ARE. GSTs are also induced by butyrate, a product of gut flora-derived fermentation of plant foods, which may act via different mechanisms, e.g. by increasing histone acetylation. GSTs are expressed with high inter-individual variability in human colonocytes, which points to large differences in cellular susceptibility to xenobiotics. Enhancing expression of GSTs in human colon tissue could therefore contribute to reducing cancer risks. However, it has not been demonstrated in humans that this mechanism is associated with cancer prevention. In the

The antioxidant master glutathione and periodontal health

Directory of Open Access Journals (Sweden)

Vivek Kumar Bains

2015-01-01

Full Text Available Glutathione, considered to be the master antioxidant (AO, is the most-important redox regulator that controls inflammatory processes, and thus damage to the periodontium. Periodontitis patients have reduced total AO capacity in whole saliva, and lower concentrations of reduced glutathione (GSH in serum and gingival crevicular fluid, and periodontal therapy restores the redox balance. Therapeutic considerations for the adjunctive use of glutathione in management of periodontitis, in limiting the tissue damage associated with oxidative stress, and enhancing wound healing cannot be underestimated, but need to be evaluated further through multi-centered randomized controlled trials.

An ethylene-responsive enhancer element is involved in the senescence-related expression of the carnation glutathione-S-transferase (GST1) gene.

OpenAIRE

Itzhaki, H; Maxson, J M; Woodson, W R

1994-01-01

The increased production of ethylene during carnation petal senescence regulates the transcription of the GST1 gene encoding a subunit of glutathione-S-transferase. We have investigated the molecular basis for this ethylene-responsive transcription by examining the cis elements and trans-acting factors involved in the expression of the GST1 gene. Transient expression assays following delivery of GST1 5' flanking DNA fused to a beta-glucuronidase receptor gene were used to functionally define ...

Conjugates of a Photoactivated Rhodamine with Biopolymers for Cell Staining

Science.gov (United States)

Zaitsev, Sergei Yu.; Shaposhnikov, Mikhail N.; Solovyeva, Daria O.; Solovyeva, Valeria V.; Rizvanov, Albert A.

2014-01-01

Conjugates of the photoactivated rhodamine dyes with biopolymers (proteins, polysaccharides, and nucleic acids) are important tools for microscopic investigation of biological tissue. In this study, a precursor of the photoactivated fluorescent dye (PFD) has been successfully used for staining of numerous mammalian cells lines and for conjugate formation with chitosan (“Chitosan-PFD”) and histone H1 (“Histone H1.3-PFD”). The intensive fluorescence has been observed after photoactivation of these conjugates inside cells (A431, HaCaT, HEK239, HBL-100, and MDCK). Developed procedures and obtained data are important for further application of novel precursors of fluorescent dyes (“caged” dyes) for microscopic probing of biological objects. Thus, the synthesized “Chitosan-PFD” and “Histone H1-PFD” have been successfully applied in this study for intracellular transport visualization by fluorescent microscopy. PMID:25383365

Solvent-dependent self-assembly and ordering in slow-drying semi-crystalline conjugated polymer solutions

KAUST Repository

Zhao, Kui

2015-09-07

The mechanistic understanding of the intrinsic molecular self-assembly of conjugated polymers is of immense importance to controlling the microstructure development in organic semiconducting thin films, with meaningful impact on charge transport and optoelectronic properties. Yet, to date the vast majority of studies have focused on the fast solution process itself, with studies of slower intrinsic molecular self-assembly in formulations lagging behind. Here we have investigated molecular self-assembly during spontaneous organization and uncovered how changes in formulation influence the microstructure, morphology and transport properties of conjugated polymer thin films. Our results suggest that the polymer-solvent interaction is the key factor for the molecular self-assembly and changes in macroscopic charge transport, which is in contrast with most solution processes, such as spin-coating and blade coating, where solvent drying kinetics dominates the aggregation and crystallization processes. Energetically favourable interactions between the polymer and its solvent are shown to cause chain expansion, resulting in a large hydrodynamic volume and few chain entanglements in solution. This provides molecular freedom for self-assembly and is shown to greatly enhance the local and long range order of the polymer, intra-chain backbone planarity and crystallite size. These improvements, in turn, are shown to endow the conjugated polymer with high carrier transport, as demonstrated by organic thin film transistors.

The human SLC25A33 and SLC25A36 genes of solute carrier family 25 encode two mitochondrial pyrimidine nucleotide transporters.

Science.gov (United States)

Di Noia, Maria Antonietta; Todisco, Simona; Cirigliano, Angela; Rinaldi, Teresa; Agrimi, Gennaro; Iacobazzi, Vito; Palmieri, Ferdinando

2014-11-28

The human genome encodes 53 members of the solute carrier family 25 (SLC25), also called the mitochondrial carrier family, many of which have been shown to transport inorganic anions, amino acids, carboxylates, nucleotides, and coenzymes across the inner mitochondrial membrane, thereby connecting cytosolic and matrix functions. Here two members of this family, SLC25A33 and SLC25A36, have been thoroughly characterized biochemically. These proteins were overexpressed in bacteria and reconstituted in phospholipid vesicles. Their transport properties and kinetic parameters demonstrate that SLC25A33 transports uracil, thymine, and cytosine (deoxy)nucleoside di- and triphosphates by an antiport mechanism and SLC25A36 cytosine and uracil (deoxy)nucleoside mono-, di-, and triphosphates by uniport and antiport. Both carriers also transported guanine but not adenine (deoxy)nucleotides. Transport catalyzed by both carriers was saturable and inhibited by mercurial compounds and other inhibitors of mitochondrial carriers to various degrees. In confirmation of their identity (i) SLC25A33 and SLC25A36 were found to be targeted to mitochondria and (ii) the phenotypes of Saccharomyces cerevisiae cells lacking RIM2, the gene encoding the well characterized yeast mitochondrial pyrimidine nucleotide carrier, were overcome by expressing SLC25A33 or SLC25A36 in these cells. The main physiological role of SLC25A33 and SLC25A36 is to import/export pyrimidine nucleotides into and from mitochondria, i.e. to accomplish transport steps essential for mitochondrial DNA and RNA synthesis and breakdown. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

21 CFR 864.7375 - Glutathione reductase assay.

Science.gov (United States)

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Glutathione reductase assay. 864.7375 Section 864.7375 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7375 Glutathione...

The Human Gene SLC25A29, of Solute Carrier Family 25, Encodes a Mitochondrial Transporter of Basic Amino Acids*

Science.gov (United States)

Porcelli, Vito; Fiermonte, Giuseppe; Longo, Antonella; Palmieri, Ferdinando

2014-01-01

The human genome encodes 53 members of the solute carrier family 25 (SLC25), also called the mitochondrial carrier family, many of which have been shown to transport carboxylates, amino acids, nucleotides, and cofactors across the inner mitochondrial membrane, thereby connecting cytosolic and matrix functions. In this work, a member of this family, SLC25A29, previously reported to be a mitochondrial carnitine/acylcarnitine- or ornithine-like carrier, has been thoroughly characterized biochemically. The SLC25A29 gene was overexpressed in Escherichia coli, and the gene product was purified and reconstituted in phospholipid vesicles. Its transport properties and kinetic parameters demonstrate that SLC25A29 transports arginine, lysine, homoarginine, methylarginine and, to a much lesser extent, ornithine and histidine. Carnitine and acylcarnitines were not transported by SLC25A29. This carrier catalyzed substantial uniport besides a counter-exchange transport, exhibited a high transport affinity for arginine and lysine, and was saturable and inhibited by mercurial compounds and other inhibitors of mitochondrial carriers to various degrees. The main physiological role of SLC25A29 is to import basic amino acids into mitochondria for mitochondrial protein synthesis and amino acid degradation. PMID:24652292

The human gene SLC25A29, of solute carrier family 25, encodes a mitochondrial transporter of basic amino acids.

Science.gov (United States)

Porcelli, Vito; Fiermonte, Giuseppe; Longo, Antonella; Palmieri, Ferdinando

2014-05-09

The human genome encodes 53 members of the solute carrier family 25 (SLC25), also called the mitochondrial carrier family, many of which have been shown to transport carboxylates, amino acids, nucleotides, and cofactors across the inner mitochondrial membrane, thereby connecting cytosolic and matrix functions. In this work, a member of this family, SLC25A29, previously reported to be a mitochondrial carnitine/acylcarnitine- or ornithine-like carrier, has been thoroughly characterized biochemically. The SLC25A29 gene was overexpressed in Escherichia coli, and the gene product was purified and reconstituted in phospholipid vesicles. Its transport properties and kinetic parameters demonstrate that SLC25A29 transports arginine, lysine, homoarginine, methylarginine and, to a much lesser extent, ornithine and histidine. Carnitine and acylcarnitines were not transported by SLC25A29. This carrier catalyzed substantial uniport besides a counter-exchange transport, exhibited a high transport affinity for arginine and lysine, and was saturable and inhibited by mercurial compounds and other inhibitors of mitochondrial carriers to various degrees. The main physiological role of SLC25A29 is to import basic amino acids into mitochondria for mitochondrial protein synthesis and amino acid degradation.

Molecularly imprinted solid-phase extraction of glutathione from urine samples

International Nuclear Information System (INIS)

Song, Renyuan; Hu, Xiaoling; Guan, Ping; Li, Ji; Zhao, Na; Wang, Qiaoli

2014-01-01

Molecularly imprinted polymer (MIP) particles for glutathione were synthesized through iniferter-controlled living radical precipitation polymerization (IRPP) under ultraviolet radiation at ambient temperature. Static adsorption, solid-phase extraction, and high-performance liquid chromatography were carried out to evaluate the adsorption properties and selective recognition characteristics of the polymers for glutathione and its structural analogs. The obtained IRPP-MIP particles exhibited a regularly spherical shape, rapid binding kinetics, high imprinting factor, and high selectivity compared with the MIP particles prepared using traditional free-radical precipitation polymerization. The selective separation and enrichment of glutathione from the mixture of glycyl-glycine and glutathione disulfide could be achieved on the IRPP-MIP cartridge. The recoveries of glutathione, glycyl-glycine, and glutathione disulfide were 95.6% ± 3.65%, 29.5% ± 1.26%, and 49.9% ± 1.71%, respectively. The detection limit (S/N = 3) of glutathione was 0.5 mg·L −1 . The relative standard deviations (RSDs) for 10 replicate detections of 50 mg·L −1 of glutathione were 5.76%, and the linear range of the calibration curve was 0.5 mg·L −1 to 200 mg·L −1 under optimized conditions. The proposed approach was successfully applied to determine glutathione in spiked human urine samples with recoveries of 90.24% to 96.20% and RSDs of 0.48% to 5.67%. - Highlights: • Imprinted polymer particles were prepared by IRPP at ambient temperature. • High imprinting factor, high selectivity, and rapid binding kinetics were achieved. • Selective solid-phase extraction of glutathione from human urine samples

Effect of glutathione on phytochelatin synthesis in tomato cells. [Lycopersicon esculentum

Energy Technology Data Exchange (ETDEWEB)

Mendum, M.L.; Gupta, S.C.; Goldsbrough, P.B. (Purdue Univ., West Lafayette, IN (USA))

1990-06-01

Growth of cell suspension cultures of tomato, Lycopersicon esculentum Mill. cv VFNT-Cherry, in the presence of cadmium is inhibited by buthionine sulfoximine, an inhibitor of glutathione synthesis. Cell growth and phytochelatin synthesis are restored to cells treated with buthionine sulfoximine by the addition of glutathione to the medium. Glutathione stimulates the accumulation of phytochelatins in cadmium treated cells, indicating that availability of glutathione can limit synthesis of these peptides. Exogenous glutathione causes a disproportionate increase in the level of smaller phytochelatins, notably ({gamma}-Glu-Cys){sub 2}-Gly. In the presence of buthionine sulfoximine and glutathione, phytochelatins that are produced upon exposure to cadmium incorporate little ({sup 35}S)cysteine, indicating that these peptides are probably not synthesized by sequential addition of cysteine and glutamate to glutathione.

Defective canalicular transport and toxicity of dietary ursodeoxycholic acid in the abcb11-/- mouse: transport and gene expression studies.

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Wang, Renxue; Liu, Lin; Sheps, Jonathan A; Forrest, Dana; Hofmann, Alan F; Hagey, Lee R; Ling, Victor

2013-08-15

The bile salt export pump (BSEP), encoded by the abcb11 gene, is the major canalicular transporter of bile acids from the hepatocyte. BSEP malfunction in humans causes bile acid retention and progressive liver injury, ultimately leading to end-stage liver failure. The natural, hydrophilic, bile acid ursodeoxycholic acid (UDCA) is efficacious in the treatment of cholestatic conditions, such as primary biliary cirrhosis and cholestasis of pregnancy. The beneficial effects of UDCA include promoting bile flow, reducing hepatic inflammation, preventing apoptosis, and maintaining mitochondrial integrity in hepatocytes. However, the role of BSEP in mediating UDCA efficacy is not known. Here, we used abcb11 knockout mice (abcb11-/-) to test the effects of acute and chronic UDCA administration on biliary secretion, bile acid composition, liver histology, and liver gene expression. Acutely infused UDCA, or its taurine conjugate (TUDC), was taken up by the liver but retained, with negligible biliary output, in abcb11-/- mice. Feeding UDCA to abcb11-/- mice led to weight loss, retention of bile acids, elevated liver enzymes, and histological damage to the liver. Semiquantitative RT-PCR showed that genes encoding Mdr1a and Mdr1b (canalicular) as well as Mrp4 (basolateral) transporters were upregulated in abcb11-/- mice. We concluded that infusion of UDCA and TUDC failed to induce bile flow in abcb11-/- mice. UDCA fed to abcb11-/- mice caused liver damage and the appearance of biliary tetra- and penta-hydroxy bile acids. Supplementation with UDCA in the absence of Bsep caused adverse effects in abcb11-/- mice.

Do glutathione levels decline in aging human brain?

Science.gov (United States)

Tong, Junchao; Fitzmaurice, Paul S; Moszczynska, Anna; Mattina, Katie; Ang, Lee-Cyn; Boileau, Isabelle; Furukawa, Yoshiaki; Sailasuta, Napapon; Kish, Stephen J

2016-04-01

For the past 60 years a major theory of "aging" is that age-related damage is largely caused by excessive uncompensated oxidative stress. The ubiquitous tripeptide glutathione is a major antioxidant defense mechanism against reactive free radicals and has also served as a marker of changes in oxidative stress. Some (albeit conflicting) animal data suggest a loss of glutathione in brain senescence, which might compromise the ability of the aging brain to meet the demands of oxidative stress. Our objective was to establish whether advancing age is associated with glutathione deficiency in human brain. We measured reduced glutathione (GSH) levels in multiple regions of autopsied brain of normal subjects (n=74) aged one day to 99 years. Brain GSH levels during the infancy/teenage years were generally similar to those in the oldest examined adult group (76-99 years). During adulthood (23-99 years) GSH levels remained either stable (occipital cortex) or increased (caudate nucleus, frontal and cerebellar cortices). To the extent that GSH levels represent glutathione antioxidant capacity, our postmortem data suggest that human brain aging is not associated with declining glutathione status. We suggest that aged healthy human brains can maintain antioxidant capacity related to glutathione and that an age-related increase in GSH levels in some brain regions might possibly be a compensatory response to increased oxidative stress. Since our findings, although suggestive, suffer from the generic limitations of all postmortem brain studies, we also suggest the need for "replication" investigations employing the new (1)H MRS imaging procedures in living human brain. Copyright © 2016 Elsevier Inc. All rights reserved.

Mechanisms of radiosensitization and protection studied with glutathione-deficient human cell lines

International Nuclear Information System (INIS)

Revesz, L.; Edgren, M.

1982-01-01

Glutathione-deficient fibroblasts and lymphoblastoid cells, derived from patients with an inborn error of glutathione synthetase activity, and glutathione-proficient cells, derived from clinically healthy individuals, were used to investigate the importance of glutathione for radiosensitization by misonidazole. With single-strand DNA breaks as an end point, misonidazole as well as oxygen was found to lack any sensitizing effect on cells deficient in glutathione. The post-irradiation repair of single-strand breaks induced by hypoxic irradiation of misonidazole treated cells was found to be a great extent glutathione dependent, like the repair of breaks induced by oxic irradiation. Naturally occurring aminothiols in glutathione-deficient cells appeared to be in efficient as substitutes for glutatione. Artificial aminothiols, such as cysteamine or dithiothreitol, were found to effectively replace glutathione

Glutathione, cell proliferation and differentiation | Ashtiani | African ...

African Journals Online (AJOL)

All organisms require an equivalent source for living. Reduced glutathione is the most abundant thiol containing protein in mammalian cells and organs. Glutathione was discovered by Hopkins in 1924 who published his findings in JBC. It is a three peptide containing glutamic acid, cystein and glycin and is found in reduced ...

The biochemistry of acetaminophen hepatotoxicity and rescue: a mathematical model

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Ben-Shachar Rotem

2012-12-01

Full Text Available Abstract Background Acetaminophen (N-acetyl-para-aminophenol is the most widely used over-the-counter or prescription painkiller in the world. Acetaminophen is metabolized in the liver where a toxic byproduct is produced that can be removed by conjugation with glutathione. Acetaminophen overdoses, either accidental or intentional, are the leading cause of acute liver failure in the United States, accounting for 56,000 emergency room visits per year. The standard treatment for overdose is N-acetyl-cysteine (NAC, which is given to stimulate the production of glutathione. Methods We have created a mathematical model for acetaminophen transport and metabolism including the following compartments: gut, plasma, liver, tissue, urine. In the liver compartment the metabolism of acetaminophen includes sulfation, glucoronidation, conjugation with glutathione, production of the toxic metabolite, and liver damage, taking biochemical parameters from the literature whenever possible. This model is then connected to a previously constructed model of glutathione metabolism. Results We show that our model accurately reproduces published clinical and experimental data on the dose-dependent time course of acetaminophen in the plasma, the accumulation of acetaminophen and its metabolites in the urine, and the depletion of glutathione caused by conjugation with the toxic product. We use the model to study the extent of liver damage caused by overdoses or by chronic use of therapeutic doses, and the effects of polymorphisms in glucoronidation enzymes. We use the model to study the depletion of glutathione and the effect of the size and timing of N-acetyl-cysteine doses given as an antidote. Our model accurately predicts patient death or recovery depending on size of APAP overdose and time of treatment. Conclusions The mathematical model provides a new tool for studying the effects of various doses of acetaminophen on the liver metabolism of acetaminophen and

Effects of reduced glutathion and vitamin c on cisplatin-induced ...

African Journals Online (AJOL)

glutathione peroxidase [GSHPx], catalase [CAT], glutathione reductase [GSHR] activities and gene expression, glutathione [GSH] content) and lipid peroxidation products (malondialdehyde, MDA) in rat liver tissue were measured. CDDP hepatotoxicity was manifested by an increase in serum ALT and AST, elevation of MDA ...

Glutathione Metabolism and Parkinson’s Disease

OpenAIRE

Smeyne, Michelle; Smeyne, Richard Jay

2013-01-01

It has been established that oxidative stress, defined as the condition when the sum of free radicals in a cell exceeds the antioxidant capacity of the cell, contributes to the pathogenesis of Parkinson’s disease. Glutathione is a ubiquitous thiol tripeptide that acts alone, or in concert with enzymes within cells to reduce superoxide radicals, hydroxyl radicals and peroxynitrites. In this review, we examine the synthesis, metabolism and functional interactions of glutathione, and discuss how...

Application of superparamagnetic microspheres for affinity adsorption and purification of glutathione

International Nuclear Information System (INIS)

Wang Qiang; Guan Yueping; Yang Mingzhu

2012-01-01

The superparamagnetic poly-(MA–DVB) microspheres with micron size were synthesized by the modified suspension polymerization method. Adsorption of glutathione by magnetic poly-(MA–DVB) microspheres with IDA-copper was investigated. The effect of solution pH value, affinity adsorption and desorption of glutathione was studied. The results showed that the optimum pH value for glutathione adsorption was found at pH=3.5, the maximum capacity for glutathione of magnetic poly-(MA–DVB) microspheres was estimated at 42.4 mg/g by fitting the experimental data to the Langmuir equation. The adsorption equilibrium of glutathione was obtained in about 10 min and the adsorbed glutathione was desorbed from the magnetic microspheres in about 30 min using NaCl buffer solution. The magnetic microspheres could be repeatedly utilized for the affinity adsorption of glutathione. - Highlights: ► The magnetic microsphere with surface IDA–Cu groups was synthesized. ► The magnetic microspheres were applied for adsorption of GSH. ► The adsorption–desorption of glutathione was investigated. ► The maximum adsorption capacity of GSH was fitted at 42.4 mg/g.

Effect of Vitamin C on Glutathione Peroxidase Activities in Pregnant ...

African Journals Online (AJOL)

Glutathione peroxidase is one of the most important antioxidant enzymes in humans. We studied the relationship between serum glutathione peroxidase activity and vitamin C ingestion during normal pregnancy in women attending antenatal clinic in the University of Ilorin Teaching Hospital, Ilorin. Glutathione peroxidase ...

Compartment specific importance of glutathione during abiotic and biotic stress

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Bernd eZechmann

2014-10-01

Full Text Available The tripeptide thiol glutathione (γ-L-glutamyl-L-cysteinyl-glycine is the most important sulfur containing antioxidant in plants and essential for plant defense against abiotic and biotic stress conditions. It is involved in the detoxification of reactive oxygen species, redox signaling, the modulation of defense gene expression and important for the regulation of enzymatic activities. Even though changes in glutathione contents are well documented in plants and its roles in plant defense are well established, still too little is known about its compartment specific importance during abiotic and biotic stress conditions. Due to technical advances in the visualization of glutathione and the redox state of plants through microscopical methods some progress was made in the last few years in studying the importance of subcellular glutathione contents during stress conditions in plants. This review summarizes the data available on compartment specific importance of glutathione in the protection against abiotic and biotic stress conditions such as high light stress, exposure to cadmium, drought, and pathogen attack (Pseudomonas, Botrytis, Tobacco Mosaic Virus. The data will be discussed in connection with the subcellular accumulation of ROS during these conditions and glutathione synthesis which are both highly compartment specific (e.g. glutathione synthesis takes place in chloroplasts and the cytosol. Thus this review will reveal the compartment specific importance of glutathione during abiotic and biotic stress conditions.

Superoxide radical (O2-) reactivity with respect to glutathione

International Nuclear Information System (INIS)

Sekaki, A.; Gardes-Albert, M.; Ferradini, C.

1984-01-01

Influence of superoxide radicals formed during gamma irradiation of glutathione in aerated aqueous solutions is examined. Solutions are buffered at pH7 and contain sodium formate for capture of H and OH radicals which are transformed in COO - radicals and then O 2 - radicals. G value of glutathione disparition vs glutathione concentration are given with and without enzyme or catalase. Reaction mechanism are interpreted [fr

Requirement for Vibrio cholerae Integration Host Factor in Conjugative DNA Transfer

OpenAIRE

McLeod, Sarah M.; Burrus, Vincent; Waldor, Matthew K.

2006-01-01

The requirement for host factors in the transmission of integrative and conjugative elements (ICEs) has not been extensively explored. Here we tested whether integration host factor (IHF) or Fis, two host-encoded nucleoid proteins, are required for transfer of SXT, a Vibrio cholerae-derived ICE that can be transmitted to many gram-negative species. Fis did not influence the transfer of SXT to or from V. cholerae. In contrast, IHF proved to be required for V. cholerae to act as an SXT donor. I...

Cotransporting Ion is a Trigger for Cellular Endocytosis of Transporter-Targeting Nanoparticles: A Case Study of High-Efficiency SLC22A5 (OCTN2)-Mediated Carnitine-Conjugated Nanoparticles for Oral Delivery of Therapeutic Drugs.

Science.gov (United States)

Kou, Longfa; Yao, Qing; Sun, Mengchi; Wu, Chunnuan; Wang, Jia; Luo, Qiuhua; Wang, Gang; Du, Yuqian; Fu, Qiang; Wang, Jian; He, Zhonggui; Ganapathy, Vadivel; Sun, Jin

2017-09-01

OCTN2 (SLC22A5) is a Na + -coupled absorption transporter for l-carnitine in small intestine. This study tests the potential of this transporter for oral delivery of therapeutic drugs encapsulated in l-carnitine-conjugated poly(lactic-co-glycolic acid) (PLGA) nanoparticles (LC-PLGA NPs) and discloses the molecular mechanism for cellular endocytosis of transporter-targeting nanoparticles. Conjugation of l-carnitine to a surface of PLGA-NPs enhances the cellular uptake and intestinal absorption of encapsulated drug. In both cases, the uptake process is dependent on cotransporting ion Na + . Computational OCTN2 docking analysis shows that the presence of Na + is important for the formation of the energetically stable intermediate complex of transporter-Na + -LC-PLGA NPs, which is also the first step in cellular endocytosis of nanoparticles. The transporter-mediated intestinal absorption of LC-PLGA NPs occurs via endocytosis/transcytosis rather than via the traditional transmembrane transport. The portal blood versus the lymphatic route is evaluated by the plasma appearance of the drug in the control and lymph duct-ligated rats. Absorption via the lymphatic system is the predominant route in the oral delivery of the NPs. In summary, LC-PLGA NPs can effectively target OCTN2 on the enterocytes for enhancing oral delivery of drugs and the critical role of cotransporting ions should be noticed in designing transporter-targeting nanoparticles. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Targeting Aberrant Glutathione Metabolism to Eradicate Human Acute Myelogenous Leukemia Cells*

Science.gov (United States)

Pei, Shanshan; Minhajuddin, Mohammad; Callahan, Kevin P.; Balys, Marlene; Ashton, John M.; Neering, Sarah J.; Lagadinou, Eleni D.; Corbett, Cheryl; Ye, Haobin; Liesveld, Jane L.; O'Dwyer, Kristen M.; Li, Zheng; Shi, Lei; Greninger, Patricia; Settleman, Jeffrey; Benes, Cyril; Hagen, Fred K.; Munger, Joshua; Crooks, Peter A.; Becker, Michael W.; Jordan, Craig T.

2013-01-01

The development of strategies to eradicate primary human acute myelogenous leukemia (AML) cells is a major challenge to the leukemia research field. In particular, primitive leukemia cells, often termed leukemia stem cells, are typically refractory to many forms of therapy. To investigate improved strategies for targeting of human AML cells we compared the molecular mechanisms regulating oxidative state in primitive (CD34+) leukemic versus normal specimens. Our data indicate that CD34+ AML cells have elevated expression of multiple glutathione pathway regulatory proteins, presumably as a mechanism to compensate for increased oxidative stress in leukemic cells. Consistent with this observation, CD34+ AML cells have lower levels of reduced glutathione and increased levels of oxidized glutathione compared with normal CD34+ cells. These findings led us to hypothesize that AML cells will be hypersensitive to inhibition of glutathione metabolism. To test this premise, we identified compounds such as parthenolide (PTL) or piperlongumine that induce almost complete glutathione depletion and severe cell death in CD34+ AML cells. Importantly, these compounds only induce limited and transient glutathione depletion as well as significantly less toxicity in normal CD34+ cells. We further determined that PTL perturbs glutathione homeostasis by a multifactorial mechanism, which includes inhibiting key glutathione metabolic enzymes (GCLC and GPX1), as well as direct depletion of glutathione. These findings demonstrate that primitive leukemia cells are uniquely sensitive to agents that target aberrant glutathione metabolism, an intrinsic property of primary human AML cells. PMID:24089526

Redox and pH Responsive Poly (Amidoamine Dendrimer-Heparin Conjugates via Disulfide Linkages for Letrozole Delivery

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Thanh Luan Nguyen

2017-01-01

Full Text Available Heparin (Hep conjugated to poly (amidoamine dendrimer G3.5 (P via redox-sensitive disulfide bond (P-SS-Hep was studied. The redox and pH dual-responsive nanocarriers were prepared by a simple method that minimized many complex steps as previous studies. The functional characterization of G3.5 coated Hep was investigated by the proton nuclear magnetic resonance spectroscopy. The size and formation were characterized by the dynamic light scattering, zeta potential, and transmission electron microscopy. P-SS-Hep was spherical in shape with average diameter about 11 nm loaded with more than 20% letrozole. This drug carrier could not only eliminate toxicity to cells and improve the drugs solubility but also increase biocompatibility of the system under reductive environment of glutathione. In particular, P-SS-Hep could enhance the effectiveness of cancer therapy after removing Hep from the surface. These results demonstrated that the P-SS-Hep conjugates could be a promising candidate as redox and pH responsive nanocarriers for cancer chemotherapy.

Glutathione level after long-term occupational elemental mercury exposure

International Nuclear Information System (INIS)

Kobal, Alfred Bogomir; Prezelj, Marija; Horvat, Milena; Krsnik, Mladen; Gibicar, Darija; Osredkar, Josko

2008-01-01

Many in vitro and in vivo studies have elucidated the interaction of inorganic mercury (Hg) and glutathione. However, human studies are limited. In this study, we investigated the potential effects of remote long-term intermittent occupational elemental Hg vapour (Hg o ) exposure on erythrocyte glutathione levels and some antioxidative enzyme activities in ex-mercury miners in the period after exposure. The study included 49 ex-mercury miners divided into subgroups of 28 still active, Hg o -not-exposed miners and 21 elderly retired miners, and 41 controls, age-matched to the miners subgroup. The control workers were taken from 'mercury-free works'. Reduced glutathione (GSH) and oxidized disulphide glutathione (GSSG) concentrations in haemolysed erythrocytes were determined by capillary electrophoresis, while total glutathione (total GSH) and the GSH/GSSG ratio were calculated from the determined values. Catalase (CAT), glutathione peroxidase (GPx), and glutathione reductase (GR) activities in erythrocytes were measured using commercially available reagent kits, while urine Hg (U-Hg) concentrations were determined by cold vapour atomic absorption (CVAAS). No correlation of present U-Hg levels, GSH, GSSG, and antioxidative enzymes with remote occupational biological exposure indices were found. The mean CAT activity in miners and retired miners was significantly higher (p o could be an inductive and additive response to maintain the balance between GSH and antioxidative enzymes in interaction with the Hg body burden accumulated during remote occupational exposure, which does not represent a severely increased oxidative stress

Effect of rosella ( Hibiscus sabdariffa L ) extract on glutathione-S ...

African Journals Online (AJOL)

Purpose: To determine the effect of rosella (Hibiscus sabdariffa L) extract on glutathione-S-trasferase (GST) activity and its hepatoprotective effect. Methods: A total of 25 rats were divided randomly into 5 groups (5 rats per group). Group I served as the baseline, group II was the negative control group, while groups III, IV and ...

DISORDERS OF CARNITINE TRANSPORT AND THE CARNITINE CYCLE

OpenAIRE

Longo, Nicola; di San Filippo, Cristina Amat; Pasquali, Marzia

2006-01-01

Carnitine plays an essential role in the transfer of long-chain fatty acids across the inner mitochondrial membrane. This transfer requires enzymes and transporters that accumulate carnitine within the cell (OCTN2 carnitine transporter), conjugate it with long chain fatty acids (carnitine palmitoyl transferase 1, CPT1), transfer the acylcarnitine across the inner plasma membrane (carnitine-acylcarnitine translocase, CACT), and conjugate the fatty acid back to Coenzyme A for subsequent beta ox...

Differential Roles for VviGST1, VviGST3, and VviGST4 in Proanthocyanidin and Anthocyanin Transport in Vitis vinífera.

Science.gov (United States)

Pérez-Díaz, Ricardo; Madrid-Espinoza, José; Salinas-Cornejo, Josselyn; González-Villanueva, Enrique; Ruiz-Lara, Simón

2016-01-01

In plant cells, flavonoids are synthesized in the cytosol and then are transported and accumulated in the vacuole. Glutathione S-transferase-mediated transport has been proposed as a mechanism involved in flavonoid transport, however, whether binding of flavonoids to glutathione S-transferase (GST) or their transport is glutathione-dependent is not well understood. Glutathione S-transferases from Vitis vinífera (VviGSTs) have been associated with the transport of anthocyanins, however, their ability to transport other flavonoids such as proanthocyanidins (PAs) has not been established. Following bioinformatics approaches, we analyzed the capability of VviGST1, VviGST3, VviGST4, and Arabidopsis TT19 to bind different flavonoids. Analyses of protein-ligand interactions indicate that these GSTs can bind glutathione and monomers of anthocyanin, PAs and flavonols. A total or partial overlap of the binding sites for glutathione and flavonoids was found in VviGST1, and a similar condition was observed in VviGST3 using anthocyanin and flavonols as ligands, whereas VviGST4 and TT19 have both sites for GSH and flavonoids separated. To validate the bioinformatics predictions, functional complementation assays using the Arabidopsis tt19 mutant were performed. Overexpression of VviGST3 in tt19-1 specifically rescued the dark seed coat phenotype associated to correct PA transport, which correlated with higher binding affinity for PA precursors. VviGST4, originally characterized as an anthocyanin-related GST, complemented both the anthocyanin and PA deposition, resembling the function of TT19. By contrast, VviGST1 only partially rescued the normal seed color. Furthermore the expression pattern of these VviGSTs showed that each of these genes could be associated with the accumulation of different flavonoids in specific tissues during grapevine fruit development. These results provide new insights into GST-mediated PA transport in grapevine and suggest that VviGSTs present

Physiological impact of transposable elements encoding DDE transposases in the environmental adaptation of Streptococcus agalactiae.

Science.gov (United States)

Fléchard, Maud; Gilot, Philippe

2014-07-01

We have referenced and described Streptococcus agalactiae transposable elements encoding DDE transposases. These elements belonged to nine families of insertion sequences (ISs) and to a family of conjugative transposons (TnGBSs). An overview of the physiological impact of the insertion of all these elements is provided. DDE-transposable elements affect S. agalactiae in a number of aspects of its capability to adapt to various environments and modulate the expression of several virulence genes, the scpB-lmB genomic region and the genes involved in capsule expression and haemolysin transport being the targets of several different mobile elements. The referenced mobile elements modify S. agalactiae behaviour by transferring new gene(s) to its genome, by modifying the expression of neighbouring genes at the integration site or by promoting genomic rearrangements. Transposition of some of these elements occurs in vivo, suggesting that by dynamically regulating some adaptation and/or virulence genes, they improve the ability of S. agalactiae to reach different niches within its host and ensure the 'success' of the infectious process. © 2014 The Authors.

Reduction-sensitive micelles self-assembled from amphiphilic chondroitin sulfate A-deoxycholic acid conjugate for triggered release of doxorubicin.

Science.gov (United States)

Liu, Hongxia; Wu, Shuqin; Yu, Jingmou; Fan, Dun; Ren, Jin; Zhang, Lei; Zhao, Jianguo

2017-06-01

Reduction-sensitive chondroitin sulfate A (CSA)-based micelles were developed. CSA was conjugated with deoxycholic acid (DOCA) via a disulfide linkage. The bioreducible conjugate (CSA-ss-DOCA) can form self-assembled micelles in aqueous medium. The critical micelle concentration (CMC) of CSA-ss-DOCA conjugate is 0.047mg/mL, and its mean diameter is 387nm. The anticancer drug doxorubicin (DOX) was chosen as a model drug, and was effectively encapsulated into the micelles with high loading efficiency. Reduction-sensitive micelles and reduction-insensitive control micelles displayed similar DOX release behavior in phosphate buffered saline (PBS, pH7.4). Notably, DOX release from the reduction-sensitive micelles in vitro was accelerated in the presence of 20mM glutathione-containing PBS environment. Moreover, DOX-loaded CSA-ss-DOCA (CSA-ss-DOCA/DOX) micelles exhibited intracellular reduction-responsive characteristics in human gastric cancer HGC-27 cells determined by confocal laser scanning microscopy (CLSM). Furthermore, CSA-ss-DOCA/DOX micelles demonstrated higher antitumor efficacy than reduction-insensitive control micelles in HGC-27 cells. These results suggested that reduction-sensitive CSA-ss-DOCA micelles had the potential as intracellular targeted carriers of anticancer drugs. Copyright © 2017 Elsevier B.V. All rights reserved.

Entanglements in Conjugated Polymers

Science.gov (United States)

Xie, Renxuan; Lee, Youngmin; Aplan, Melissa; Caggiano, Nick; Gomez, Enrique; Colby, Ralph

Conjugated polymers, such as poly(3-hexylthiophene-2,5-diyl) (P3HT) and poly-((9,9-dioctylfluorene)-2,7-diyl-alt-[4,7-bis(thiophen-5-yl)-2,1,3-benzothiadiazole]-2',2''-diyl) (PFTBT), are widely used as hole and electron transport materials in a variety of electronic devices. However, fundamental knowledge regarding chain entanglements and nematic-to-isotropic transition is still lacking and are crucial to maximize charge transport properties. A systematic melt rheology study on P3HT with various molecular weights and regio regularities was performed. We find that the entanglement molecular weight Me is 5.0 kg/mol for regiorandom P3HT, but the apparent Me for regioregular P3HT is significantly higher. The difference is postulated to arise from the presence of a nematic phase only in regioregular P3HT. Analogously, PFTBT shows a clear rheological signature of the nematic-to-isotropic transition as a reversible sharp transition at 278 C. Shearing of this nematic phase leads to anisotropic crystalline order in PFTBT. We postulate that aligning the microstructure will impact charge transport and thereby advance the field of conducting polymers. National Science Foundation.

Implications of Glutathione Levels in the Plasmodium berghei Response to Chloroquine and Artemisinin.

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Joel Vega-Rodríguez

Full Text Available Malaria is one of the most devastating parasitic diseases worldwide. Plasmodium drug resistance remains a major challenge to malaria control and has led to the re-emergence of the disease. Chloroquine (CQ and artemisinin (ART are thought to exert their anti-malarial activity inducing cytotoxicity in the parasite by blocking heme degradation (for CQ and increasing oxidative stress. Besides the contribution of the CQ resistance transporter (PfCRT and the multidrug resistant gene (pfmdr, CQ resistance has also been associated with increased parasite glutathione (GSH levels. ART resistance was recently shown to be associated with mutations in the K13-propeller protein. To analyze the role of GSH levels in CQ and ART resistance, we generated transgenic Plasmodium berghei parasites either deficient in or overexpressing the gamma-glutamylcysteine synthetase gene (pbggcs encoding the rate-limiting enzyme in GSH biosynthesis. These lines produce either lower (pbggcs-ko or higher (pbggcs-oe levels of GSH than wild type parasites. In addition, GSH levels were determined in P. berghei parasites resistant to CQ and mefloquine (MQ. Increased GSH levels were detected in both, CQ and MQ resistant parasites, when compared to the parental sensitive clone. Sensitivity to CQ and ART remained unaltered in both pgggcs-ko and pbggcs-oe parasites when tested in a 4 days drug suppressive assay. However, recrudescence assays after the parasites have been exposed to a sub-lethal dose of ART showed that parasites with low levels of GSH are more sensitive to ART treatment. These results suggest that GSH levels influence Plasmodium berghei response to ART treatment.

Transport of surface engineered polyamidoamine (PAMAM) dendrimers across IPEC-J2 cell monolayers.

Science.gov (United States)

Pisal, Dipak S; Yellepeddi, Venkata K; Kumar, Ajay; Palakurthi, Srinath

2008-11-01

The aim of our study was to prepare arginine-and ornithine-conjugated Polyamidoamine (PAMAM) dendrimers and study their permeability across IPEC-J2 cell monolayers, a new intestinal cell line model for drug absorption studies. Arginine and ornithine were conjugated to the amine terminals of the PAMAM(G4) dendrimers by Fmoc synthesis. The apical-to-basolateral (AB) and basolateral-to-apical (BA) apparent permeability coefficients (P(app)) for the PAMAM dendrimers increased by conjugating the dendrimers with both of these polyamines. The enhancement in permeability was dependent on the dendrimer concentration and duration of incubation. Correlation between monolayer permeability and the decrease in transepithelial electrical resistance (TEER) with the PAMAM dendrimers and the polyamine-conjugated dendrimers suggests that paracellular transport is one of the mechanisms of transport across the epithelial cells. Cytotoxicity of these surface-modified dendrimers was evaluated in IPEC-J2 cells by MTT (methylthiazoletetrazolium) assay. Arginine-conjugated dendrimers were insignificantly more toxic than PAMAM dendrimer as well as ornithine-conjugated dendrimers. Though investigations on the possible involvement of other transport mechanisms are in progress, results of the present study suggest the potential of dendrimer-polyamine conjugates as the carriers for antigen/drug delivery through the oral mucosa.

Effect of glutathione aerosol on oxidant-antioxidant imbalance in idiopathic pulmonary fibrosis.

Science.gov (United States)

Borok, Z; Buhl, R; Grimes, G J; Bokser, A D; Hubbard, R C; Holroyd, K J; Roum, J H; Czerski, D B; Cantin, A M; Crystal, R G

1991-07-27

Idiopathic pulmonary fibrosis (IPF) is characterised by alveolar inflammation, exaggerated release of oxidants, and subnormal concentrations of the antioxidant glutathione in respiratory epithelial lining fluid (ELF). Glutathione (600 mg twice daily for 3 days) was given by aerosol to 10 patients with IPF. Total ELF glutathione rose transiently, ELF oxidised glutathione concentrations increased, and there was a decrease in spontaneous superoxide anion release by alveolar macrophages. Thus, glutathione by aerosol could be a means of reversing the oxidant-antioxidant imbalance in IPF.

The IRC7 gene encodes cysteine desulphydrase activity and confers on yeast the ability to grow on cysteine as a nitrogen source.

Science.gov (United States)

Santiago, Margarita; Gardner, Richard C

2015-07-01

Although cysteine desulphydrase activity has been purified and characterized from Saccharomyces cerevisiae, the gene encoding this activity in vivo has never been defined. We show that the full-length IRC7 gene, encoded by the YFR055W open reading frame, encodes a protein with cysteine desulphydrase activity. Irc7p purified to homogeneity is able to utilize l-cysteine as a substrate, producing pyruvate and hydrogen sulphide as products of the reaction. Purified Irc7p also utilized l-cystine and some other cysteine conjugates, but not l-cystathionine or l-methionine, as substrates. We further show that, in vivo, the IRC7 gene is both necessary and sufficient for yeast to grow on l-cysteine as a nitrogen source, and that overexpression of the gene results in increased H2 S production. Strains overexpressing IRC7 are also hypersensitive to a toxic analogue, S-ethyl-l-cysteine. While IRC7 has been identified as playing a critical role in converting cysteine conjugates to volatile thiols that are important in wine aroma, its biological role in yeast cells is likely to involve regulation of cysteine and redox homeostasis. Copyright © 2015 John Wiley & Sons, Ltd.

A Naturally Encoded Dipeptide Handle for Bioorthogonal Chan-Lam Coupling.

Science.gov (United States)

Ohata, Jun; Zeng, Yimeng; Segatori, Laura; Ball, Zachary T

2018-04-03

Manipulation of biomacromolecules is ideally achieved through unique and bioorthogonal chemical reactions of genetically encoded, naturally occurring functional groups. The toolkit of methods for site-specific conjugation is limited by selectivity concerns and a dearth of naturally occurring functional groups with orthogonal reactivity. We report that pyroglutamate amide N-H bonds exhibit bioorthogonal copper-catalyzed Chan-Lam coupling at pyroglutamate-histidine dipeptide sequences. The pyroglutamate residue is readily incorporated into proteins of interest by natural enzymatic pathways, allowing specific bioconjugation at a minimalist dipeptide tag. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

The role of nuclear factor E2-Related factor 2 and uncoupling protein 2 in glutathione metabolism: Evidence from an in vivo gene knockout study

International Nuclear Information System (INIS)

Chen, Yanyan; Xu, Yuanyuan; Zheng, Hongzhi; Fu, Jingqi; Hou, Yongyong; Wang, Huihui; Zhang, Qiang; Yamamoto, Masayuki; Pi, Jingbo

2016-01-01

Nuclear factor E2-related factor 2 (NRF2) and uncoupling protein 2 (UCP2) are indicated to protect from oxidative stress. They also play roles in the homeostasis of glutathione. However, the detailed mechanisms are not well understood. In the present study, we found Nrf2-knockout (Nrf2-KO) mice exhibited altered glutathione homeostasis and reduced expression of various genes involved in GSH biosynthesis, regeneration, utilization and transport in the liver. Ucp2-knockout (Ucp2-KO) mice exhibited altered glutathione homeostasis in the liver, spleen and blood, as well as increased transcript of cystic fibrosis transmembrane conductance regulator in the liver, a protein capable of mediating glutathione efflux. Nrf2-Ucp2-double knockout (DKO) mice showed characteristics of both Nrf2-KO and Ucp2-KO mice. But no significant difference was observed in DKO mice when compared with Nrf2-KO or Ucp2-KO mice, except in blood glutathione levels. These data suggest that ablation of Nrf2 and Ucp2 leads to disrupted GSH balance, which could result from altered expression of genes involved in GSH metabolism. DKO may not evoke more severe oxidative stress than the single gene knockout. - Highlights: • Nrf2/Ucp2 deficiency leads to alteration of glutathione homeostasis. • Nrf2 regulates expression of genes in glutathione generation and utilization. • Ucp2 affects glutathione metabolism by regulating hepatic efflux of glutathione. • Nrf2 deficiency may not aggravate oxidative stress in Ucp2-deficient mice.

The role of nuclear factor E2-Related factor 2 and uncoupling protein 2 in glutathione metabolism: Evidence from an in vivo gene knockout study

Energy Technology Data Exchange (ETDEWEB)

Chen, Yanyan [The First Affiliated Hospital, China Medical University, Shenyang, Liaoning (China); The Hamner Institutes for Health Sciences, Research Triangle Park, NC (United States); Xu, Yuanyuan, E-mail: yyxu@cmu.edu.cn [School of Public Health, China Medical University, Shenyang, Liaoning (China); Zheng, Hongzhi [The First Affiliated Hospital, China Medical University, Shenyang, Liaoning (China); The Hamner Institutes for Health Sciences, Research Triangle Park, NC (United States); Fu, Jingqi; Hou, Yongyong; Wang, Huihui [School of Public Health, China Medical University, Shenyang, Liaoning (China); Zhang, Qiang [Rollins School of Public Health, Emory University, Atlanta, GA (United States); Yamamoto, Masayuki [Graduate School of Medicine, Tohoku University, Sendai (Japan); Pi, Jingbo, E-mail: jbpi@cmu.edu.cn [School of Public Health, China Medical University, Shenyang, Liaoning (China); The Hamner Institutes for Health Sciences, Research Triangle Park, NC (United States)

2016-09-09

Nuclear factor E2-related factor 2 (NRF2) and uncoupling protein 2 (UCP2) are indicated to protect from oxidative stress. They also play roles in the homeostasis of glutathione. However, the detailed mechanisms are not well understood. In the present study, we found Nrf2-knockout (Nrf2-KO) mice exhibited altered glutathione homeostasis and reduced expression of various genes involved in GSH biosynthesis, regeneration, utilization and transport in the liver. Ucp2-knockout (Ucp2-KO) mice exhibited altered glutathione homeostasis in the liver, spleen and blood, as well as increased transcript of cystic fibrosis transmembrane conductance regulator in the liver, a protein capable of mediating glutathione efflux. Nrf2-Ucp2-double knockout (DKO) mice showed characteristics of both Nrf2-KO and Ucp2-KO mice. But no significant difference was observed in DKO mice when compared with Nrf2-KO or Ucp2-KO mice, except in blood glutathione levels. These data suggest that ablation of Nrf2 and Ucp2 leads to disrupted GSH balance, which could result from altered expression of genes involved in GSH metabolism. DKO may not evoke more severe oxidative stress than the single gene knockout. - Highlights: • Nrf2/Ucp2 deficiency leads to alteration of glutathione homeostasis. • Nrf2 regulates expression of genes in glutathione generation and utilization. • Ucp2 affects glutathione metabolism by regulating hepatic efflux of glutathione. • Nrf2 deficiency may not aggravate oxidative stress in Ucp2-deficient mice.

Phosphorus acquisition efficiency in arbuscular mycorrhizal maize is correlated with the abundance of root-external hyphae and the accumulation of transcripts encoding PHT1 phosphate transporters

DEFF Research Database (Denmark)

Sawers, Ruairidh J H; Svane, Simon F; Quan, Clement

2016-01-01

content, abundance of intra-radical and root-external fungal structures, mycorrhizal phosphorus uptake, and accumulation of transcripts encoding plant PHT1 family phosphate transporters varied among lines. Larger growth responses in Oh43 were correlated with extensive development of root-external hyphae...

Arabidopsis GLUTATHIONE REDUCTASE1 plays a crucial role in leaf responses to intracellular hydrogen peroxide and in ensuring appropriate gene expression through both salicylic acid and jasmonic acid signaling pathways.

Science.gov (United States)

Mhamdi, Amna; Hager, Jutta; Chaouch, Sejir; Queval, Guillaume; Han, Yi; Taconnat, Ludivine; Saindrenan, Patrick; Gouia, Houda; Issakidis-Bourguet, Emmanuelle; Renou, Jean-Pierre; Noctor, Graham

2010-07-01

Glutathione is a major cellular thiol that is maintained in the reduced state by glutathione reductase (GR), which is encoded by two genes in Arabidopsis (Arabidopsis thaliana; GR1 and GR2). This study addressed the role of GR1 in hydrogen peroxide (H(2)O(2)) responses through a combined genetic, transcriptomic, and redox profiling approach. To identify the potential role of changes in glutathione status in H(2)O(2) signaling, gr1 mutants, which show a constitutive increase in oxidized glutathione (GSSG), were compared with a catalase-deficient background (cat2), in which GSSG accumulation is conditionally driven by H(2)O(2). Parallel transcriptomics analysis of gr1 and cat2 identified overlapping gene expression profiles that in both lines were dependent on growth daylength. Overlapping genes included phytohormone-associated genes, in particular implicating glutathione oxidation state in the regulation of jasmonic acid signaling. Direct analysis of H(2)O(2)-glutathione interactions in cat2 gr1 double mutants established that GR1-dependent glutathione status is required for multiple responses to increased H(2)O(2) availability, including limitation of lesion formation, accumulation of salicylic acid, induction of pathogenesis-related genes, and signaling through jasmonic acid pathways. Modulation of these responses in cat2 gr1 was linked to dramatic GSSG accumulation and modified expression of specific glutaredoxins and glutathione S-transferases, but there is little or no evidence of generalized oxidative stress or changes in thioredoxin-associated gene expression. We conclude that GR1 plays a crucial role in daylength-dependent redox signaling and that this function cannot be replaced by the second Arabidopsis GR gene or by thiol systems such as the thioredoxin system.

Solvent-dependent self-assembly and ordering in slow-drying semi-crystalline conjugated polymer solutions

KAUST Repository

Zhao, Kui; Yu, Xinhong; Li, Ruipeng; Amassian, Aram; Han, Yanchun

2015-01-01

The mechanistic understanding of the intrinsic molecular self-assembly of conjugated polymers is of immense importance to controlling the microstructure development in organic semiconducting thin films, with meaningful impact on charge transport

Two pear glutathione S-transferases genes are regulated during fruit development and involved in response to salicylic acid, auxin, and glucose signaling.

Directory of Open Access Journals (Sweden)

Hai-Yan Shi

Full Text Available Two genes encoding putative glutathione S-transferase proteins were isolated from pear (Pyrus pyrifolia and designated PpGST1 and PpGST2. The deduced PpGST1 and PpGST2 proteins contain conserved Glutathione S-transferase N-terminal domain (GST_N and Glutathione S-transferase, C-terminal domain (GST_C. Using PCR amplification technique, the genomic clones corresponding to PpGST1 and PpGST2 were isolated and shown to contain two introns and a singal intron respectively with typical GT/AG boundaries defining the splice junctions. Phylogenetic analysis clearly demonstrated that PpGST1 belonged to Phi class of GST superfamilies and had high homology with apple MdGST, while PpGST2 was classified into the Tau class of GST superfamilies. The expression of PpGST1 and PpGST2 genes was developmentally regulated in fruit. Further study demonstrated that PpGST1 and PpGST2 expression was remarkably induced by glucose, salicylic acid (SA and indole-3-aceticacid (IAA treatments in pear fruit, and in diseased fruit. These data suggested that PpGST1 and PpGST2 might be involved in response to sugar, SA, and IAA signaling during fruit development of pear.

Antimicrobial Peptide-PNA Conjugates Selectively Targeting Bacterial Genes

Science.gov (United States)

2013-07-22

antibacterial therapy. Initial publications suggest that conjugates of cell penetrating peptides and PNA’s can overcome the barrier in transporting ...Zhou, Y., Hou, Z., Meng, J., and Luo, X. Targeting RNA polymerase primary σ70 as a therapeutic strategy against methicillin - resistant ... Staphylococcus aureus by antisense peptide nucleic acid. PLoS One. 2012; 7(1):e29886. 2. Good, L., Sandberg, R., Larsson, O., Nielsen, P.E., and Wahlestedt, C

Numerical Simulation of Solid Combustion with a Robust Conjugate-Gradient Solution for Pressure

National Research Council Canada - National Science Library

Wang, Yi-Zun

2002-01-01

A Bi-Conjugate Gradient method (Bi-CGSTAB) is studied and tested for solid combustion in which the gas and solid phases are coupled by a set of conditions describing mass, momentum and heat transport across the interface...

Quantum key distribution using basis encoding of Gaussian-modulated coherent states

Science.gov (United States)

Huang, Peng; Huang, Jingzheng; Zhang, Zheshen; Zeng, Guihua

2018-04-01

The continuous-variable quantum key distribution (CVQKD) has been demonstrated to be available in practical secure quantum cryptography. However, its performance is restricted strongly by the channel excess noise and the reconciliation efficiency. In this paper, we present a quantum key distribution (QKD) protocol by encoding the secret keys on the random choices of two measurement bases: the conjugate quadratures X and P . The employed encoding method can dramatically weaken the effects of channel excess noise and reconciliation efficiency on the performance of the QKD protocol. Subsequently, the proposed scheme exhibits the capability to tolerate much higher excess noise and enables us to reach a much longer secure transmission distance even at lower reconciliation efficiency. The proposal can work alternatively to strengthen significantly the performance of the known Gaussian-modulated CVQKD protocol and serve as a multiplier for practical secure quantum cryptography with continuous variables.

Genes Encoding Aluminum-Activated Malate Transporter II and their Association with Fruit Acidity in Apple

Directory of Open Access Journals (Sweden)

Baiquan Ma

2015-11-01

Full Text Available A gene encoding aluminum-activated malate transporter (ALMT was previously reported as a candidate for the locus controlling acidity in apple ( × Borkh.. In this study, we found that apple genes can be divided into three families and the gene belongs to the family. Duplication of genes in apple is related to the polyploid origin of the apple genome. Divergence in expression has occurred between the gene and its homologs in the family and only the gene is significantly associated with malic acid content. The locus consists of two alleles, and . resides in the tonoplast and its ectopic expression in yeast was found to increase the influx of malic acid into yeast cells significantly, suggesting it may function as a vacuolar malate channel. In contrast, encodes a truncated protein because of a single nucleotide substitution of G with A in the last exon. As this truncated protein resides within the cell membrane, it is deemed to be nonfunctional as a vacuolar malate channel. The frequency of the genotype is very low in apple cultivars but is high in wild relatives, which suggests that apple domestication may be accompanied by selection for the gene. In addition, variations in the malic acid content of mature fruits were also observed between accessions with the same genotype in the locus. This suggests that the gene is not the only genetic determinant of fruit acidity in apple.

Histochemical Localization of Glutathione Dependent NBT-Reductase in Mouse Skin

Institute of Scientific and Technical Information of China (English)

无

2001-01-01

Objective　Localization of the glutathione dependent Nitroblue tetrazolium (NBT) reductase in fresh frozen sections of mouse skin and possible dependence of NBT reductase on tissue thiol levels has been investigated. Methods　The fresh frozen tissue sections (8m thickness) were prepared and incubated in medium containing NBT, reduced glutathione (GSH) and phosphate buffer. The staining for GSH was performed with mercury orange. Results　 The activity of the NBT-reductase in mouse skin has been found to be localized in the areas rich in glutathione and actively proliferating area of the skin. Conclusion　The activity of the NBT-reductase seems to be dependent on the glutathione contents.

[Alternative nutrition and glutathione levels].

Science.gov (United States)

Krajcovicová-Kudlácková, M; Simoncic, R; Béderová, A; Brtková, A; Magálová, T; Barteková, S

1999-08-30

Low protein quality and quantity is reported to be a possible risk of alternative nutrition. Pulses contain 18-41% of methionine in relation to reference protein, moreover, its content in cereals is by one half lower. Therefore vegetarians and vegans may have an insufficient intake of sulphur-containing amino acids that may subsequently affect glutathione values (precursors of its synthesis). In groups of adults on an alternative diet--lactoovovegetarians (n = 47) and vegans (n = 44) aged 19-62 years with average duration on a vegetarian or vegan diet of 7.6 and 4.9 years, respectively, glutathione levels (GSH) were measured in erythrocytes (spectrophotometrically), as well as the activity of GSH-dependent enzymes. As nutritional control (n = 42) served an average sample of omnivores selected from a group of 489 examined, apparently healthy subjects of the same age range living in the same region. One to low protein intake (56% of RDA) exclusively of plant origin significantly lower levels of total proteins were observed in vegans with a 16% frequency of hypoproteinaemia (vs 0% in omnivores). In comparison to omnivores a significantly lower glutathione level was found (4.28 +/- 0.12 vs 4.84 +/- 0.14 mumol/g Hb, P vegan diet also in adult age.

Avocado oil induces long-term alleviation of oxidative damage in kidney mitochondria from type 2 diabetic rats by improving glutathione status.

Science.gov (United States)

Ortiz-Avila, Omar; Figueroa-García, María Del Consuelo; García-Berumen, Claudia Isabel; Calderón-Cortés, Elizabeth; Mejía-Barajas, Jorge A; Rodriguez-Orozco, Alain R; Mejía-Zepeda, Ricardo; Saavedra-Molina, Alfredo; Cortés-Rojo, Christian

2017-04-01

Hyperglycemia and mitochondrial ROS overproduction have been identified as key factors involved in the development of diabetic nephropathy. This has encouraged the search for strategies decreasing glucose levels and long-term improvement of redox status of glutathione, the main antioxidant counteracting mitochondrial damage. Previously, we have shown that avocado oil improves redox status of glutathione in liver and brain mitochondria from streptozotocin-induced diabetic rats; however, the long-term effects of avocado oil and its hypoglycemic effect cannot be evaluated because this model displays low survival and insulin depletion. Therefore, we tested during 1 year the effects of avocado oil on glycemia, ROS levels, lipid peroxidation and glutathione status in kidney mitochondria from type 2 diabetic Goto-Kakizaki rats. Diabetic rats exhibited glycemia of 120-186 mg/dL the first 9 months with a further increase to 250-300 mg/dL. Avocado oil decreased hyperglycemia at intermediate levels between diabetic and control rats. Diabetic rats displayed augmented lipid peroxidation and depletion of reduced glutathione throughout the study, while increased ROS generation was observed at the 3rd and 12th months along with diminished content of total glutathione at the 6th and 12th months. Avocado oil ameliorated all these defects and augmented the mitochondrial content of oleic acid. The beneficial effects of avocado oil are discussed in terms of the hypoglycemic effect of oleic acid and the probable dependence of glutathione transport on lipid peroxidation and thiol oxidation of mitochondrial carriers.

Phosphorus acquisition efficiency in arbuscular mycorrhizal maize is correlated with the abundance of root-external hyphae and the accumulation of transcripts encoding PHT1 phosphate transporters

DEFF Research Database (Denmark)

Sawers, Ruairidh J. H.; Svane, Simon; Quan, Clement

2017-01-01

-internal and -external fungal structures, mycorrhizal phosphorus uptake, and accumulation of transcripts encoding plant PHT1 family phosphate transporters varied among lines. Superior response in Oh43 is correlated with extensive development of root-external hyphae, accumulation of specific Pht1 transcripts and high...

Glutathione-dependent responses of plants to drought: a review

Directory of Open Access Journals (Sweden)

Mateusz Labudda

2014-02-01

Full Text Available Water is a renewable resource. However, with the human population growth, economic development and improved living standards, the world’s supply of fresh water is steadily decreasing and consequently water resources for agricultural production are limited and diminishing. Water deficiency is a significant problem in agriculture and increasing efforts are currently being made to understand plant tolerance mechanisms and to develop new tools (especially molecular that could underpin plant breeding and cultivation. However, the biochemical and molecular mechanisms of plant water deficit tolerance are not fully understood, and the data available is incomplete. Here, we review the significance of glutathione and its related enzymes in plant responses to drought. Firstly, the roles of reduced glutathione and reduced/oxidized glutathione ratio, are discussed, followed by an extensive discussion of glutathione related enzymes, which play an important role in plant responses to drought. Special attention is given to the S-glutathionylation of proteins, which is involved in cell metabolism regulation and redox signaling in photosynthetic organisms subjected to abiotic stress. The review concludes with a brief overview of future perspectives for the involvement of glutathione and related enzymes in drought stress responses.

Photoaffinity labelling of the active site of the rat glutathione transferases 3-3 and 1-1 and human glutathione transferase A1-1.

OpenAIRE

Cooke, R J; Björnestedt, R; Douglas, K T; McKie, J H; King, M D; Coles, B; Ketterer, B; Mannervik, B

1994-01-01

The glutathione transferases (GSTs) form a group of enzymes responsible for a wide range of molecular detoxications. The photoaffinity label S-(2-nitro-4-azidophenyl)glutathione was used to study the hydrophobic region of the active site of the rat liver GST 1-1 and 2-2 isoenzymes (class Alpha) as well as the rat class-Mu GST 3-3. Photoaffinity labelling was carried out using a version of S-(2-nitro-4-azidophenyl)glutathione tritiated in the arylazido ring. The labelling occurred with higher ...

Response of Glutathione and Glutathione S-transferase in Rice Seedlings Exposed to Cadmium Stress

Directory of Open Access Journals (Sweden)

Chun-hua ZHANG

2008-03-01

Full Text Available A hydroponic culture experiment was done to investigate the effect of Cd stress on glutathione content (GSH and glutathione S-transferase (GST, EC 2.5.1.18 activity in rice seedlings. The rice growth was severely inhibited when Cd level in the solution was higher than 10 mg/L. In rice shoots, GSH content and GST activity increased with the increasing Cd level, while in roots, GST was obviously inhibited by Cd treatments. Compared with shoots, the rice roots had higher GSH content and GST activity, indicating the ability of Cd detoxification was much higher in roots than in shoots. There was a significant correlation between Cd level and GSH content or GST activity, suggesting that both parameters may be used as biomarkers of Cd stress in rice.

Evaluation of iodovinyl antibody conjugates: Comparison with a p-iodobenzoyl conjugate and direct radioiodination

International Nuclear Information System (INIS)

Hadley, S.W.; Wilbur, D.S.

1990-01-01

The preparations and conjugations of 2,3,5,6-tetrafluorophenyl 5-[125I/131I]iodo-4-pentenoate (7a) and 2,3,5,6-tetrafluorophenyl 3,3-dimethyl-5-[125I/131I]iodo-4-pentenoate (7b) to monoclonal antibodies are reported. Reagents 7a and 7b were prepared in high radiochemical yield by iododestannylation of their corresponding 5-tri-n-butylstannyl precursors. Radioiodinated antibody conjugates were prepared by reaction of 7a or 7b with the protein at basic pH. Evaluation of these conjugates by several in vitro procedures demonstrated that the radiolabel was attached to the antibody in a stable manner and that the conjugates maintained immunoreactivity. Comparative dual-isotope biodistribution studies of a monoclonal antibody Fab fragment conjugate of 7a and 7b with the same Fab fragment labeled with N-succinimidyl p-[131I]iodobenzoate (PIB, p-iodobenzoate, 2) or directly radioiodinated have been carried out in tumor-bearing nude mice. Coinjection of the Fab conjugate of 7a with the Fab conjugate of 2 demonstrated that the biodistributions were similar in most organs, except the neck tissue (thyroid-containing) and the stomach, which contained substantially increased levels of the 7a label. Coinjection of the Fab conjugate of 7a with the Fab fragment radioiodinated by using the chloramine-T method demonstrated that the biodistributions were remarkably similar, suggesting roughly equivalent in vivo deiodination of these labeled antibody fragments. Coinjection of the Fab conjugate of 7a with the Fab conjugate of 7b indicated that there was ∼ a 2-fold reduction in the amount of in vivo deiodination of the 7b conjugate as compared to the 7a conjugate

Redox regulation of photosynthetic gene expression.

Science.gov (United States)

Queval, Guillaume; Foyer, Christine H

2012-12-19

Redox chemistry and redox regulation are central to the operation of photosynthesis and respiration. However, the roles of different oxidants and antioxidants in the regulation of photosynthetic or respiratory gene expression remain poorly understood. Leaf transcriptome profiles of a range of Arabidopsis thaliana genotypes that are deficient in either hydrogen peroxide processing enzymes or in low molecular weight antioxidant were therefore compared to determine how different antioxidant systems that process hydrogen peroxide influence transcripts encoding proteins targeted to the chloroplasts or mitochondria. Less than 10 per cent overlap was observed in the transcriptome patterns of leaves that are deficient in either photorespiratory (catalase (cat)2) or chloroplastic (thylakoid ascorbate peroxidase (tapx)) hydrogen peroxide processing. Transcripts encoding photosystem II (PSII) repair cycle components were lower in glutathione-deficient leaves, as were the thylakoid NAD(P)H (nicotinamide adenine dinucleotide (phosphate)) dehydrogenases (NDH) mRNAs. Some thylakoid NDH mRNAs were also less abundant in tAPX-deficient and ascorbate-deficient leaves. Transcripts encoding the external and internal respiratory NDHs were increased by low glutathione and low ascorbate. Regulation of transcripts encoding specific components of the photosynthetic and respiratory electron transport chains by hydrogen peroxide, ascorbate and glutathione may serve to balance non-cyclic and cyclic electron flow pathways in relation to oxidant production and reductant availability.

Entanglements in Marginal Solutions: A Means of Tuning Pre-Aggregation of Conjugated Polymers with Positive Implications for Charge Transport

KAUST Repository

Hu, Hanlin

2015-06-17

The solution-processing of conjugated polymers, just like commodity polymers, is subject to solvent and molecular weight-dependent solubility, interactions and chain entanglements within the polymer, all of which can influence the crystallization and microstructure development in semi-crystalline polymers and consequently affect charge transport and optoelectronic properties. Disentanglement of polymer chains in marginal solvents was reported to work via ultrasonication, facilitating the formation of photophysically ordered polymer aggregates. In this contribution, we explore how a wide range of technologically relevant solvents and formulations commonly used in organic electronics influence chain entanglement and the aggregation behaviour of P3HT using a combination of rheological and spectrophotometric measurements. The specific viscosity of the solution offers an excellent indication of the degree of entanglements in the solution, which is found to be related to the solubility of P3HT in a given solvent. Moreover, deliberately disentangling the solution in the presence of solvophobic driving forces, leads consistently to formation of photophysically visible aggregates which is indicative of local and perhaps long range order in the solute. We show for a broad range of solvents and molecular weights that disentanglement ultimately leads to significant ordering of the polymer in the solid state and a commensurate increase in charge transport properties. In doing so we demonstrate a remarkable ability to tune the microstructure which has important implications for transport properties. We discuss its potential implications in the context of organic photovoltaics.

Entanglements in Marginal Solutions: A Means of Tuning Pre-Aggregation of Conjugated Polymers with Positive Implications for Charge Transport

KAUST Repository

Hu, Hanlin; Zhao, Kui; Fernandes, Nikhil J.; Boufflet, Pierre; Bannock, James Henry; Yu, Liyang; de Mello, John C; Stingelin, Natalie; Heeney, Martin; Giannelis, Emmanuel P.; Amassian, Aram

2015-01-01

The solution-processing of conjugated polymers, just like commodity polymers, is subject to solvent and molecular weight-dependent solubility, interactions and chain entanglements within the polymer, all of which can influence the crystallization and microstructure development in semi-crystalline polymers and consequently affect charge transport and optoelectronic properties. Disentanglement of polymer chains in marginal solvents was reported to work via ultrasonication, facilitating the formation of photophysically ordered polymer aggregates. In this contribution, we explore how a wide range of technologically relevant solvents and formulations commonly used in organic electronics influence chain entanglement and the aggregation behaviour of P3HT using a combination of rheological and spectrophotometric measurements. The specific viscosity of the solution offers an excellent indication of the degree of entanglements in the solution, which is found to be related to the solubility of P3HT in a given solvent. Moreover, deliberately disentangling the solution in the presence of solvophobic driving forces, leads consistently to formation of photophysically visible aggregates which is indicative of local and perhaps long range order in the solute. We show for a broad range of solvents and molecular weights that disentanglement ultimately leads to significant ordering of the polymer in the solid state and a commensurate increase in charge transport properties. In doing so we demonstrate a remarkable ability to tune the microstructure which has important implications for transport properties. We discuss its potential implications in the context of organic photovoltaics.

Glutathione S-transferase genes and the risk of type 2 diabetes mellitus: Role of sexual dimorphism, gene-gene and gene-smoking interactions in disease susceptibility.

Science.gov (United States)

Azarova, Iuliia; Bushueva, Olga; Konoplya, Alexander; Polonikov, Alexey

2018-05-01

Compromised defense against reactive oxygen species (ROS) is considered important in the pathogenesis of type 2 diabetes mellitus (T2DM); therefore, genes encoding antioxidant defense enzymes may contribute to disease susceptibility. This study investigated whether polymorphisms in genes encoding glutathione S-transferase M1 (GSTM1), T1 (GSTT1), and P1 (GSTP1) jointly contribute to the risk of T2DM. In all, 1120 unrelated Russian subjects (600 T2DM patients, 520 age- and sex-matched healthy subjects), were recruited to the study. Genotyping was performed by multiplex polymerase chain reaction (PCR; del/del polymorphisms of GSTM1 and GSTT1) and TaqMan-based PCR (polymorphisms I105V and A114V of GSTP1). Plasma ROS and glutathione levels in study subjects were analyzed by fluorometric and colorimetric assays, respectively. Genotype del/del GSTT1 was significantly associated with the risk of T2DM (odds ratio [OR] 1.60, 95% confidence interval [CI] 1.17-2.21, P = 0.003). Gender-stratified analysis showed that the deletion genotypes of GSTM1 (OR 1.99, 95% CI 1.30-3.05; P = 0.0002, Q = 0.016) and GSTT1 (OR 2.23, 95% CI 1.22-4.09; P = 0.008, Q = 0.0216), as well as genotype 114A/V of GSTP1 (OR 2.85, 95% CI 1.44-5.62; P = 0.005, Q = 0.02) were associated with an increased risk of T2DM exclusively in males. Three genotype combinations (i.e. GSTM1+ × GSTT1+, GSTM1+ × GSTP1 114A/A and GSTT1+ × GSTP1 114A/A) showed significant associations with a decreased risk of T2DM in males. This study demonstrates, for the first time, that genes encoding glutathione S-transferases jointly contribute to the risk of T2DM, and that their effects on disease susceptibility are gender specific. © 2017 Ruijin Hospital, Shanghai Jiaotong University School of Medicine and John Wiley & Sons Australia, Ltd.

Differential roles for VviGST1, VviGST3 and VviGST4 in proanthocyanidin and anthocyanin transport in Vitis vinífera

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Ricardo Pérez-Díaz

2016-08-01

Full Text Available In plant cells, flavonoids are synthesized in the cytosol and then are transported and accumulated in the vacuole. Glutathione S-transferase-mediated transport has been proposed as a mechanism involved in flavonoid transport however whether binding of flavonoids to GST or their transport is glutathione-dependent is not well understood. Glutathione S-transferases from Vitis vinífera (VviGSTs have been associated with the transport of anthocyanins, however their ability to transport other flavonoids such as proanthocyanidins (PAs has not been established. Following bioinformatics approaches, we analyzed the capability of VviGST1, VviGST3, VviGST4 and Arabidopsis TT19 to bind different flavonoids. Analyses of protein-ligand interactions indicate that these GSTs can bind glutathione and monomers of anthocyanin, PAs and flavonols. A total or partial overlap of the binding sites for glutathione and flavonoids was found in VviGST1, and a similar condition was observed in VviGST3 using anthocyanin and flavonols as ligands, whereas VviGST4 and TT19 have both sites for GSH and flavonoids separated. To validate the bioinformatics predictions, functional complementation assays using the Arabidopsis tt19 mutant were performed. Overexpression of VviGST3 in tt19-1 specifically rescued the dark seed coat phenotype associated to correct PA transport, which correlated with higher binding affinity for PA precursors. VviGST4, originally characterized as an anthocyanin-related GST, complemented both the anthocyanin and PA deposition, resembling the function of TT19. By contrast, VviGST1 only partially rescued the normal seed color. Furthermore the expression pattern of these VviGSTs showed that each of these genes could be associated with the accumulation of different flavonoids in specific tissues during grapevine fruit development. These results provide new insights into GST-mediated PA transport in grapevine and suggest that VviGSTs present different

Asymmetric Synthesis and Evaluation of Danshensu-Cysteine Conjugates as Novel Potential Anti-Apoptotic Drug Candidates

Science.gov (United States)

Pan, Li-Long; Wang, Jie; Jia, Yao-Ling; Zheng, Hong-Ming; Wang, Yang; Zhu, Yi-Zhun

2014-01-01

We have previously reported that the danshensu-cysteine conjugate N-((R)-3-benzylthio-1-methoxy-1-oxo-2-propanyl)-2-acetoxy-3-(3,4-diacetoxyphenyl) propanamide (DSC) is a potent anti-oxidative and anti-apoptotic agent. Herein, we further design and asymmetrically synthesize two diastereoisomers of DSC and explore their potential bioactivities. Our results show that DSC and its two diastereoisomers exert similar protective effects in hydrogen peroxide (H2O2)-induced cellular injury in SH-SY5Y cells, as evidenced by the increase of cell viability, superoxide dismutase (SOD), and reduced glutathione (GSH) activity, and glutathione peroxidase (GPx) expression, and the decrease of cellular morphological changes and nuclear condensation, lactate dehydrogenase (LDH) release, and malondialdehyde (MDA) production. In H2O2-stimulated human umbilical vein endothelial cells (HUVEC), DSC concentration-dependently attenuates H2O2-induced cell death, LDH release, mitochondrial membrane potential collapse, and modulates the expression of apoptosis-related proteins (Bcl-2, Bax, caspase-3, and caspase-9). Our results provide strong evidence that DSC and its two diastereoisomers have similar anti-oxidative activity and that DSC exerts significant vascular-protective effects, at least in part, through inhibition of apoptosis and modulation of endogenous antioxidant enzymes. PMID:25551606

Hemoglobin-catalyzed fluorometric method for the determination of glutathione

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Wang, Ruiqiang; Tang, Lin; Li, Hua; Wang, Yi; Gou, Rong; Guo, Yuanyuan; Fang, Yudong; Chen, Fengmei

2016-01-01

A new spectrofluorometric method for the determination of glutathione based on the reaction catalyzed by hemoglobin was reported. The reaction product gave a highly fluorescent intensity with the excitation and emission wavelengths of 320.0 nm and 413.0 nm, respectively. The optimum experimental conditions were investigated. Results showed that low concentration glutathione enhanced the fluorescence intensity significantly. The line ranges were 1.0 × 10-6-1.0 × 10-5 mol L-1 of glutathione and 6.0 × 10-10 mol L-1-1.0 × 10-8 mol L-1, respectively. The detection limit was calculated to be 1.1 × 10-11 mol L-1. The recovery test by the standard addition method gave values in the range of 90.78%-102.20%. This method was used for the determination of glutathione in synthetic and real samples with satisfactory results.

Charge transport behavior of benodithiophene-diketopyrrololpyrrole-based conjugated polymer in organic field-effect transistors

Energy Technology Data Exchange (ETDEWEB)

Park, Jin Kuen [Dept. of Chemistry, Hankuk University of Foreign Studies, Yongin (Korea, Republic of)

2015-07-15

Organic optoelectronic devices, such as light-emitting diodes, organic solar cells (OSCs), and organic field effect transistors (OFETs), have emerged due to the development of π-conjugated polymers. Because the delocalized π-framework can significantly reduce the energy gap between the highest-occupied molecular orbital (HOMO) and the lowest-unoccupied molecular orbital (LUMO), their intrinsic optoelectronic properties can be tunable with their conjugation length in terms of average molecular weights and their π-backbone structures. The new type of low bandgap conjugated polymer (P1) has been successively polymerized via a palladium- catalyzed Stille cross-coupling reaction with bis-ethylhexyl BDT and bis-n-decane DPP. With a linear alkyl chain in the DPP units, the intermolecular packing structure was thought to be enhanced by proving the UV–Vis and UPS spectra. In addition, the electronic properties of P1 via field-effect transistors well illustrate the typical p-type semiconducting property without showing the significant improvement by thermal annealing. From a broader perspective, this research indicates that a wider choice of linear alkyl chain length in DPP units and modification of the interface between dielectric and active layers should be sought to further optimize device performance. Hence, progressive works with the strategy presented in this report will be pursued to address the different challenges in attaining target OFET performances.

Hepatobiliary transport kinetics of the conjugated bile acid tracer 11C-CSar quantified in healthy humans and patients by positron emission tomography.

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Ørntoft, Nikolaj Worm; Munk, Ole Lajord; Frisch, Kim; Ott, Peter; Keiding, Susanne; Sørensen, Michael

2017-08-01

Hepatobiliary secretion of bile acids is an important liver function. Here, we quantified the hepatic transport kinetics of conjugated bile acids using the bile acid tracer [N-methyl- 11 C]cholylsarcosine ( 11 C-CSar) and positron emission tomography (PET). Nine healthy participants and eight patients with varying degrees of cholestasis were examined with 11 C-CSar PET and measurement of arterial and hepatic venous blood concentrations of 11 C-CSar. Results are presented as median (range). The hepatic intrinsic clearance was 1.50 (1.20-1.76) ml blood/min/ml liver tissue in healthy participants and 0.46 (0.13-0.91) in patients. In healthy participants, the rate constant for secretion of 11 C-CSar from hepatocytes to bile was 0.36 (0.30-0.62)min -1 , 20 times higher than the rate constant for backflux from hepatocytes to blood (0.02, 0.005-0.07min -1 ). In the patients, rate constant for transport from hepatocyte to bile was reduced to 0.12 (0.006-0.27)min -1 , 2.3times higher than the rate constant for backflux to blood (0.05, 0.04-0.09). The increased backflux did not fully normalize exposure of the hepatocyte to bile acids as mean hepatocyte residence time of 11 C-CSar was 2.5 (1.6-3.1)min in healthy participants and 6.4 (3.1-23.7)min in patients. The rate constant for transport of 11 C-CSar from intrahepatic to extrahepatic bile was 0.057 (0.023-0.11)min -1 in healthy participants and only slightly reduced in patients 0.039 (0.017-0.066). This first in vivo quantification of individual steps involved in the hepatobiliary secretion of a conjugated bile acid in humans provided new insight into cholestatic disease. Positron emission tomography (PET) using the radiolabelled bile acid ( 11 C-CSar) enabled quantification of the individual steps of the hepatic transport of bile acids from blood to bile in man. Cholestasis reduced uptake and secretion and increased backflux to blood. These findings improve our understanding of cholestatic liver diseases and may support

Extraction of glutathione from EFB fermentation waste using methanol with sonication process

Science.gov (United States)

Muryanto, Muryanto; Alvin, Nurdin, Muhammad; Hanifah, Ummu; Sudiyani, Yanni

2017-11-01

Glutathione is important compound on the human body. Glutathione have a widely use at pharmacy and cosmetics as detoxification, skin whitening agent, antioxidant and many other. This study aims to obtain glutathione from Saccharomyces cerevisiae in fermentation waste of second generation bioethanol. The remaining yeast in the empty fruit bunch (EFB) fermentation was separated from the fermentation solution use centrifugation process and then extracted using a methanol-water solution. The extraction process was done by maceration which was assisted by sonication process. Solvent concentration and time of sonication were varied to see its effect on glutathione concentration. The concentration of glutathione from the extraction process was analyzed using alloxan method with UV-Vis spectrophotometer. The results show that the highest glutathione concentration was approximately 1.32 g/L obtained with methanol solvent at 90 minutes of maceration following with 15 minutes sonication.

Co-conjugation vis-à-vis individual conjugation of α-amylase and glucoamylase for hydrolysis of starch.

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Jadhav, Swati B; Singhal, Rekha S

2013-10-15

Two enzymes, α-amylase and glucoamylase have been individually and co-conjugated to pectin by covalent binding. Both the enzyme systems showed better thermal and pH stability over the free enzyme system with the complete retention of original activities. Mixture of individually conjugated enzymes showed lower inactivation rate constant with longer half life than the co-conjugated enzyme system. Individually conjugated enzymes showed an increase of 56.48 kJ/mole and 38.22 kJ/mole in activation energy for denaturation than the free enzymes and co-conjugated enzymes, respectively. Km as well as Vmax of individually and co-conjugated enzymes was found to be higher than the free enzymes. SDS-polyacrylamide gel electrophoresis confirmed the formation of conjugate and co-conjugate as evident by increased molecular weight. Both the enzyme systems were used for starch hydrolysis where individually conjugated enzymes showed highest release of glucose at 60 °C and pH 5.0 as compared to free and co-conjugated enzyme. Copyright © 2013 Elsevier Ltd. All rights reserved.

Albumin-gold-glutathione is a probable auranofin metabolite

International Nuclear Information System (INIS)

Shaw, C.F. III; Coffer, M.; Isab, A.A.

1989-01-01

The newly licensed gold drug, auranofin ((2,3,4,6-tetra-O-acetyl-β-1-D-gluco-pyranosato-S-)triethylphoshine-gold(I)) crosses cell membranes and enters cells which are inaccessible to parenteral gold drugs. In vivo, the triethylphosphine ligand and gold of auranofin, but not the thio-sugar moiety, accumulate in and subsequently efflux from red blood cells (RBCs). Extracellular albumin increases in the extent of gold efflux and acts as a gold binding site. The rate of efflux is first-order in RBC gold concentration. Studies using RBCs in which labelled [ 14 C]-glutathione is generated in situ incorporation of [ 14 C]- glycine demonstrate that glutathione also effluxes from the RBCs and forms a gold-glutathione-albumin complex. This may be the immunopharmacologically active complex

Expression of Glutathione Peroxidase and Glutathione Reductase and Level of Free Radical Processes under Toxic Hepatitis in Rats

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Igor Y. Iskusnykh

2013-01-01

Full Text Available Correlation between intensity of free radical processes estimated by biochemiluminesce parameters, content of lipoperoxidation products, and changes of glutathione peroxidase (GP, EC 1.11.1.9 and glutathione reductase (GR, EC 1.6.4.2 activities at rats liver injury, after 12, 36, 70, 96, 110, and 125 hours & tetrachloromethane administration have been investigated. The histological examination of the liver sections of rats showed that prominent hepatocytes with marked vacuolisation and inflammatory cells which were arranged around the necrotic tissue are more at 96 h after exposure to CCl4. Moreover maximum increase in GR and GP activities, 2.1 and 2.5 times, respectively, was observed at 96 h after exposure to CCl4, what coincided with the maximum of free radical oxidation processes. Using a combination of reverse transcription and real-time polymerase chain reaction, expression of the glutathione peroxidase and glutathione reductase genes (Gpx1 and Gsr was analyzed by the determination of their respective mRNAs in the rat liver tissue under toxic hepatitis conditions. The analyses of Gpx1 and Gsr expression revealed that the transcript levels increased in 2.5- and 3.0-folds, respectively. Western blot analysis revealed that the amounts of hepatic Gpx1 and Gsr proteins increased considerably after CCl4 administration. It can be proposed that the overexpression of these enzymes could be a mechanism of enhancement of hepatocytes tolerance to oxidative stress.

Transcriptional regulation of Saccharomyces cerevisiaeCYS3 encoding cystathionine γ-lyase

Science.gov (United States)

Hiraishi, Hiroyuki; Miyake, Tsuyoshi

2008-01-01

In studying the regulation of GSH11, the structural gene of the high-affinity glutathione transporter (GSH-P1) in Saccharomyces cerevisiae, a cis-acting cysteine responsive element, CCGCCACAC (CCG motif), was detected. Like GSH-P1, the cystathionine γ-lyase encoded by CYS3 is induced by sulfur starvation and repressed by addition of cysteine to the growth medium. We detected a CCG motif (−311 to −303) and a CGC motif (CGCCACAC; −193 to −186), which is one base shorter than the CCG motif, in the 5′-upstream region of CYS3. One copy of the centromere determining element 1, CDE1 (TCACGTGA; −217 to −210), being responsible for regulation of the sulfate assimilation pathway genes, was also detected. We tested the roles of these three elements in the regulation of CYS3. Using a lacZ-reporter assay system, we found that the CCG/CGC motif is required for activation of CYS3, as well as for its repression by cysteine. In contrast, the CDE1 motif was responsible for only activation of CYS3. We also found that two transcription factors, Met4 and VDE, are responsible for activation of CYS3 through the CCG/CGC and CDE1 motifs. These observations suggest a dual regulation of CYS3 by factors that interact with the CDE1 motif and the CCG/CGC motifs. PMID:18317767

The SXT conjugative element and linear prophage N15 encode toxin-antitoxin-stabilizing systems homologous to the tad-ata module of the Paracoccus aminophilus plasmid pAMI2.

Science.gov (United States)

Dziewit, Lukasz; Jazurek, Magdalena; Drewniak, Lukasz; Baj, Jadwiga; Bartosik, Dariusz

2007-03-01

A group of proteic toxin-antitoxin (TA) cassettes whose representatives are widely distributed among bacterial genomes has been identified. These cassettes occur in chromosomes, plasmids, bacteriophages, and noncomposite transposons, as well as in the SXT conjugative element of Vibrio cholerae. The following four homologous loci were subjected to detailed comparative studies: (i) tad-ata from plasmid pAMI2 of Paracoccus aminophilus (the prototype of this group), (ii) gp49-gp48 from the linear bacteriophage N15 of Escherichia coli, (iii) s045-s044 from SXT, and (iv) Z3230-Z3231 from the genomic island of enterohemorrhagic Escherichia coli O157:H7 strain EDL933. Functional analysis revealed that all but one of these loci (Z3230-Z3231) are able to stabilize heterologous replicons, although the host ranges varied. The TA cassettes analyzed have the following common features: (i) the toxins are encoded by the first gene of each operon; (ii) the antitoxins contain a predicted helix-turn-helix motif of the XRE family; and (iii) the cassettes have two promoters that are different strengths, one which is located upstream of the toxin gene and one which is located upstream of the antitoxin gene. All four toxins tested are functional in E. coli; overexpression of the toxins (in the absence of antitoxin) results in a bacteriostatic effect manifested by elongation of bacterial cells and growth arrest. The toxins have various effects on cell viability, which suggests that they may recognize different intracellular targets. Preliminary data suggest that different cellular proteases are involved in degradation of antitoxins encoded by the loci analyzed.

Molecular Orientation of Conjugated Polymer Chains in Nanostructures and Thin Films: Review of Processes and Application to Optoelectronics

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Varun Vohra

2017-01-01

Full Text Available Semiconducting polymers are composed of elongated conjugated polymer backbones and side chains with high solubility and mechanical properties. The combination of these two features results in a high processability and a potential to orient the conjugated backbones in thin films and nanofibers. The thin films and nanofibers are usually composed of highly crystalline (high charge transport and amorphous parts. Orientation of conjugated polymer can result in enhanced charge transport or optical properties as it induces increased crystallinity or preferential orientation of the crystallites. After summarizing the potential strategies to exploit molecular order in conjugated polymer based optoelectronic devices, we will review some of the fabrication processes to induce molecular orientation. In particular, we will review the cases involving molecular and interfacial interactions, unidirectional deposition processes, electrospinning, and postdeposition mechanical treatments. The studies presented here clearly demonstrate that process-controlled molecular orientation of the conjugated polymer chains can result in high device performances (mobilities over 40 cm2·V−1·s−1 and solar cells with efficiencies over 10%. Furthermore, the peculiar interactions between molecularly oriented polymers and polarized light have the potential not only to generate low-cost and low energy consumption polarized light sources but also to fabricate innovative devices such as solar cell integrated LCDs or bipolarized LEDs.

Evaluation of chemical fluorescent dyes as a protein conjugation partner for live cell imaging.

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Yoko Hayashi-Takanaka

Full Text Available To optimize live cell fluorescence imaging, the choice of fluorescent substrate is a critical factor. Although genetically encoded fluorescent proteins have been used widely, chemical fluorescent dyes are still useful when conjugated to proteins or ligands. However, little information is available for the suitability of different fluorescent dyes for live imaging. We here systematically analyzed the property of a number of commercial fluorescent dyes when conjugated with antigen-binding (Fab fragments directed against specific histone modifications, in particular, phosphorylated H3S28 (H3S28ph and acetylated H3K9 (H3K9ac. These Fab fragments were conjugated with a fluorescent dye and loaded into living HeLa cells. H3S28ph-specific Fab fragments were expected to be enriched in condensed chromosomes, as H3S28 is phosphorylated during mitosis. However, the degree of Fab fragment enrichment on mitotic chromosomes varied depending on the conjugated dye. In general, green fluorescent dyes showed higher enrichment, compared to red and far-red fluorescent dyes, even when dye:protein conjugation ratios were similar. These differences are partly explained by an altered affinity of Fab fragment after dye-conjugation; some dyes have less effect on the affinity, while others can affect it more. Moreover, red and far-red fluorescent dyes tended to form aggregates in the cytoplasm. Similar results were observed when H3K9ac-specific Fab fragments were used, suggesting that the properties of each dye affect different Fab fragments similarly. According to our analysis, conjugation with green fluorescent dyes, like Alexa Fluor 488 and Dylight 488, has the least effect on Fab affinity and is the best for live cell imaging, although these dyes are less photostable than red fluorescent dyes. When multicolor imaging is required, we recommend the following dye combinations for optimal results: Alexa Fluor 488 (green, Cy3 (red, and Cy5 or CF640 (far-red.

Internalization and Subcellular Trafficking of Poly-l-lysine Dendrimers Are Impacted by the Site of Fluorophore Conjugation.

Science.gov (United States)

Avaritt, Brittany R; Swaan, Peter W

2015-06-01

Internalization and intracellular trafficking of dendrimer-drug conjugates play an important role in achieving successful drug delivery. In this study, we aimed to elucidate the endocytosis mechanisms and subcellular localization of poly-l-lysine (PLL) dendrimers in Caco-2 cells. We also investigated the impact of fluorophore conjugation on cytotoxicity, uptake, and transepithelial transport. Oregon green 514 (OG) was conjugated to PLL G3 at either the dendrimer periphery or the core. Chemical inhibitors of clathrin-, caveolin-, cholesterol-, and dynamin-mediated endocytosis pathways and macropinocytosis were employed to establish internalization mechanisms, while colocalization with subcellular markers was used to determine dendrimer trafficking. Cell viability, internalization, and uptake were all influenced by the site of fluorophore conjugation. Uptake was found to be highly dependent on cholesterol- and dynamin-mediated endocytosis as well as macropinocytosis. Dendrimers were trafficked to endosomes and lysosomes, and subcellular localization was impacted by the fluorophore conjugation site. The results of this study indicate that PLL dendrimers exploit multiple pathways for cellular entry, and internalization and trafficking can be impacted by conjugation. Therefore, design of dendrimer-drug conjugates requires careful consideration to achieve successful drug delivery.

Role of glutathione in tolerance to arsenite in Salvinia molesta, an aquatic fern

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Adinan Alves da Silva

2017-09-01

Full Text Available ABSTRACT In many plant species, tolerance to toxic metals is highly dependent on glutathione, an essential metabolite for cellular detoxification. We evaluated the responses of glutathione metabolism to arsenite (AsIII in Salvinia molesta, an aquatic fern that has unexplored phytoremediation potential. Plants were exposed to different AsIII concentrations in nutrient solution for 24 h. AsIII caused cell membrane damage to submerged leaves, indicating oxidative stress. There was an increase in the glutathione content and ϒ-glutamylcysteine synthetase enzyme activity in the submerged and floating leaves. The glutathione peroxidase and glutathione sulfotransferase enzymes also showed increased activity in both plant parts, whereas glutathione reductase only showed increased activity in the submerged leaves. These findings suggest an important role for glutathione in the protection of S. molesta against the toxic effects of AsIII, with more effective tolerance responses in the floating leaves.

Effect of exogenous glutathione in medium fertilization to improve blastocyst rates of bovine embryos

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E Triwulaninngsih

2002-06-01

Full Text Available Glutathione (C10H17N3O6S is a tripeptide (γ-Glu-Cys-Gly widespread in living organism. Glutathione (GSH at the 5 mM concentration increased the motility and fertility of frozen-thawed sperm. Intracellulair glutathione improved the cleavage rate and embryo development to the blastocyst rate. Research on in vitro embryos production through the implementation of GSH during IVF (in vitro fertilization on embryo development has been conducted at the Laboratorium Reproductive of Physiology, Research Institute for Animal Production. Ovaries of beef cattle as source of oocytes were collected from the slaughterhouse in a thermo flask with 350C PBS as medium and transported to the laboratory. The oocytes were fertilized in vitro with selected motile sperm using Percoll gradient (90 and 45%. Ten COCs (cumulus oocytes complexes were transfered to 44 μl of fertilization medium (mTALP was performed with either 0; 0.25; 0.50; 0.75 and 1.00 mM of glutathione as treatment A, B, C, D and E respectively, and inseminated with 2 μl of capacitated sperm and added 2 μl of heparin and 2 μl of PHE (consisting of 20 μM penicillamine, 10 μM hypotaurine, 1 μM epinephrine. Incubation between sperm and oocytes in the 5% CO2 incubator and RH 90% in fertilization media (TALP for 20 hours. All zygotes were cultured in modification of CR1aa medium up to blastocyst and were fed serum 5 μl/50μl medium on day 6. Results of this experiment showed that the effect of concentration of glutathione (0, 0.25; 0.50; 0.75 and 1.00 mM on fertilization medium to the percentage of cleavage rates were 69.35 + 29.40; 79.07 + 13.17; 67.88 + 10.65; 98.10 + 3.30 and 82.62 + 24.19% not significant different (P>0.05 among treatments A, B, C, D dan E respectively.The precentages of morula and blastocyst for treatment D were 50.45 + 42.64% and 31.28 + 24.28%; while 36.55 + 24.13% and 17.74 + 17.86% for treatment E respectively.

Modular evolution of glutathione peroxidase genes in association with different biochemical properties of their encoded proteins in invertebrate animals

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Zo Young-Gun

2009-04-01

Full Text Available Abstract Background Phospholipid hydroperoxide glutathione peroxidases (PHGPx, the most abundant isoforms of GPx families, interfere directly with hydroperoxidation of lipids. Biochemical properties of these proteins vary along with their donor organisms, which has complicated the phylogenetic classification of diverse PHGPx-like proteins. Despite efforts for comprehensive analyses, the evolutionary aspects of GPx genes in invertebrates remain largely unknown. Results We isolated GPx homologs via in silico screening of genomic and/or expressed sequence tag databases of eukaryotic organisms including protostomian species. Genes showing strong similarity to the mammalian PHGPx genes were commonly found in all genomes examined. GPx3- and GPx7-like genes were additionally detected from nematodes and platyhelminths, respectively. The overall distribution of the PHGPx-like proteins with different biochemical properties was biased across taxa; selenium- and glutathione (GSH-dependent proteins were exclusively detected in platyhelminth and deuterostomian species, whereas selenium-independent and thioredoxin (Trx-dependent enzymes were isolated in the other taxa. In comparison of genomic organization, the GSH-dependent PHGPx genes showed a conserved architectural pattern, while their Trx-dependent counterparts displayed complex exon-intron structures. A codon for the resolving Cys engaged in reductant binding was found to be substituted in a series of genes. Selection pressure to maintain the selenocysteine codon in GSH-dependent genes also appeared to be relaxed during their evolution. With the dichotomized fashion in genomic organizations, a highly polytomic topology of their phylogenetic trees implied that the GPx genes have multiple evolutionary intermediate forms. Conclusion Comparative analysis of invertebrate GPx genes provides informative evidence to support the modular pathways of GPx evolution, which have been accompanied with sporadic

Uranyl complexes of glutathione

Energy Technology Data Exchange (ETDEWEB)

Marzotto, A [Consiglio Nazionale delle Ricerche, Padua (Italy). Lab. di Chimica e Tecnologia dei Radioelementi

1977-01-01

Dioxouranium(VI) complexes of the tripeptide glutathione having different molar ratios were prepared and studied by IR, PMR, electronic absorption and circular dichroism spectra. The results indicate that coordination occurs at the carboxylato groups, acting as monodentate ligands, whereas no significant interaction with the amino and sulfhydrylic groups takes place.

Identification and characterisation of multiple glutathione S-transferase genes from the diamondback moth, Plutella xylostella.

Science.gov (United States)

Chen, Xi'en; Zhang, Ya-lin

2015-04-01

The diamondback moth (DBM), Plutella xylostella, is one of the most harmful insect pests on crucifer crops worldwide. In this study, 19 cDNAs encoding glutathione S-transferases (GSTs) were identified from the genomic and transcriptomic database for DBM (KONAGAbase) and further characterized. Phylogenetic analysis showed that the 19 GSTs were classified into six different cytosolic classes, including four in delta, six in epsilon, three in omega, two in sigma, one in theta and one in zeta. Two GSTs were unclassified. RT-PCR analysis revealed that most GST genes were expressed in all developmental stages, with higher expression in the larval stages. Six DBM GSTs were expressed at the highest levels in the midgut tissue. Twelve purified recombinant GSTs showed varied enzymatic properties towards 1-chloro-2,4-dinitrobenzene and glutathione, whereas rPxGSTo2, rPxGSTz1 and rPxGSTu2 had no activity. Real-time quantitative PCR revealed that expression levels of the 19 DBM GST genes were varied and changed after exposure to acephate, indoxacarb, beta-cypermethrin and spinosad. PxGSTd3 was significantly overexpressed, while PxGSTe3 and PxGSTs2 were significantly downregulated by all four insecticide exposures. The changes in DBM GST gene expression levels exposed to different insecticides indicate that they may play individual roles in tolerance to insecticides and xenobiotics. © 2014 Society of Chemical Industry.

Reduced glutathione as a persistence indicator of alien plants of the Amelancheir family

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L. G. Dolgova

2009-04-01

Full Text Available It was proved that glutathione is an important indicator of the vegetation condition and persistence. According to the amount of glutathione the studied mespilus species are adapted to the environmental conditions. Increase of the glutathione amount is caused by some abiotic factors, e.g. temperature. Some differences of the glutathione content may be explained by the plants species patterns.

Molecular cloning of a cDNA and chromosomal localization of a human theta-class glutathione S-transferase gene (GSTT2) to chromosome 22

Energy Technology Data Exchange (ETDEWEB)

Tan, K.L.; Baker, R.T.; Board, P.G. [Australian National Univ., Canberra (Australia)] [and others

1995-01-20

Until recently the Theta-class glutathione S-transferases (GSTs) were largely overlooked due to their low activity with the model substrate 1-chloro-2,4-dinitrobenzene (CDNB) and their failure to bind to immobilized glutathione affinity matrices. Little is known about the number of genes in this class. Recently, Pemble et al. reported the cDNA cloning of a human Theta-class GST, termed GSTT1. In this study, we describe the molecular cloning of a cDNA encoding a second human Theta-class GST (GSTT2) from a {lambda}gt11 human liver 5{prime}-stretch cDNA library. The encoded protein contains 244 amino acids and has 78.3% sequence identity with the rat subunit 12 and only 55.0% identity with human GSTT1. GSTT2 has been mapped to chromosome 22 by somatic cell hybrid analysis. The precise position of the gene was localized to subband 22q11.2 by in situ hybridization. The absence of other regions of hybridization suggests that there are no closely related sequences (e.g., reverse transcribed pseudogenes) scattered throughout the genome and that if there are closely related genes, they must be clustered near GSTT2. Southern blot analysis of human DNA digested with BamHI shows that the size of the GSTT2 gene is relatively small, as the coding sequence falls within a 3.6-kb BamHI fragment. 35 refs., 6 figs.

Glutathione level and its relation to radiation therapy in patients with cancer of uterine cervix

International Nuclear Information System (INIS)

Mukundan, H.; Bahadur, A.K.; Kumar, A.; Sardana, S.; Naik, S.L.D.; Ray, A.; Sharma, B.K.

1999-01-01

Glutathione functions as an important antioxidant in the destruction of hydrogen peroxide and lipid peroxides by providing substrate for the glutathione peroxidase and also promotes the ascorbic acid. Glutathione plays a vital role in detoxification of xenobiotics, carcinogens, free radicals and maintenance of immune functions. The study was aimed to determine plasma glutathione as well as erythrocyte glutathione and glutathione peroxidase in patients with invasive cervical carcinoma (n=30) before initiation and after completion of radiotherapy and subsequently, at the time of first three monthly follow-up visit. The levels of plasma glutathione, erythrocyte glutathione and glutathione peroxidase activity were found to be lower in all cervical cancer patients as compared to age matched normal control women. The study indicates a change in antioxidant status in relation with the glutathione system among patients with invasive carcinoma of the uterine cervix. This study also demonstrates the effect of radiation therapy on this antioxidant system. (author)

Reduced glutathione concentration and glutathione reductase activity in various rat tissues after the administration of some radioprotective agents

International Nuclear Information System (INIS)

Pulpanova, J.; Kovarova, H.; Ledvina, M.

1982-01-01

The concentrations of reduced glutathione (GSH) and activity of glutathione reductase were investigated in rat liver, kidney and spleen after intraperitoneal administration of cystamine (50 mg/kg), mexamine (10 mg/kg), or a mixture of cystamine with mexamine (20 + 10 mg/kg). The GSH concentration increased after the administration of cystamine in the liver (maximum between the 20th and 30th min), in the kidney and spleen (maximum after 60 min). The cystamine + mexamine mixture also caused a significant increase of the GSH concentration in all the organs investigated; however, the values increased at earlier intervals as after the cystamine administration. No substantial effect was shown in the case of the mexamine administration, only 30 min after the administration the values were higher. The activity of glutathione reductase was significantly lower over the entire period examined. This was found in the liver and kidney as after the administration of cystamine, as after the radioprotective mixture. There was also a less pronounced inhibition of the enzyme activity in the spleen. Mexamine as a single radioprotector had practically no influence on the activity. (author)

Electric-Field Control of Interfering Transport Pathways in a Single-Molecule Anthraquinone Transistor

Science.gov (United States)

Koole, Max; Thijssen, Jos M.; Valkenier, Hennie; Hummelen, Jan C.; Zant, Herre S. J. van der

2015-08-01

It is understood that molecular conjugation plays an important role in charge transport through single-molecule junctions. Here, we investigate electron transport through an anthraquinone based single-molecule three-terminal device. With the use of an electric-field induced by a gate electrode, the molecule is reduced resulting into a ten-fold increase in the off-resonant differential conductance. Theoretical calculations link the change in differential conductance to a reduction-induced change in conjugation, thereby lifting destructive interference of transport pathways.

An encoding device and a method of encoding

DEFF Research Database (Denmark)

2012-01-01

The present invention relates to an encoding device, such as an optical position encoder, for encoding input from an object, and a method for encoding input from an object, for determining a position of an object that interferes with light of the device. The encoding device comprises a light source...... in the area in the space and may interfere with the light, which interference may be encoded into a position or activation....

Interactions of [alpha,beta]-unsaturated carbonyl compounds with the glutathione-related biotransformation system

NARCIS (Netherlands)

Iersel, van M.L.P.S.

1998-01-01

Introduction
Modulation of glutathione-related biotransformation steps may play a role in important phenomena as anticarcinogenicity and multidrug resistance. Glutathione-related biotransformation comprises three main aspects i.e. glutathione, the

Liver ischemia and ischemia-reperfusion induces and trafficks the multi-specific metal transporter Atp7b to bile duct canaliculi: possible preferential transport of iron into bile.

Science.gov (United States)

Goss, John A; Barshes, Neal R; Karpen, Saul J; Gao, Feng-Qin; Wyllie, Samuel

2008-04-01

Both Atp7b (Wilson disease gene) and Atp7a (Menkes disease gene) have been reported to be trafficked by copper. Atp7b is trafficked to the bile duct canaliculi and Atp7a to the plasma membrane. Whether or not liver ischemia or ischemia-reperfusion modulates Atp7b expression and trafficking has not been reported. In this study, we report for the first time that the multi-specific metal transporter Atp7b is significantly induced and trafficked by both liver ischemia alone and liver ischemia-reperfusion, as judged by immunohistochemistry and Western blot analyses. Although hepatocytes also stained for Atp7b, localized intense staining of Atp7b was found on bile duct canaliculi. Inductive coupled plasma-mass spectrometry analysis of bile copper, iron, zinc, and manganese found a corresponding significant increase in biliary iron. In our attempt to determine if the increased biliary iron transport observed may be a result of altered bile flow, lysosomal trafficking, or glutathione biliary transport, we measured bile flow, bile acid phosphatase activity, and glutathione content. No significant difference was found in bile flow, bile acid phosphatase activity, and glutathione, between control livers and livers subjected to ischemia-reperfusion. Thus, we conclude that liver ischemia and ischemia-reperfusion induction and trafficking Atp7b to the bile duct canaliculi may contribute to preferential iron transport into bile.

Multiscale modelling approach combining a kinetic model of glutathione metabolism with PBPK models of paracetamol and the potential glutathione-depletion biomarkes ophthalmic acid and 5-oxoproline in humans and rats

NARCIS (Netherlands)

Geenen, S.; Yates, J.W.T.; Kenna, J.G.; Bois, F.Y.; Wilson, I.D.; Westerhoff, H.V.

2014-01-01

A key role of the antioxidant glutathione is detoxification of chemically reactive electrophilic drug metabolites within the liver. Therefore glutathione depletion can have severe toxic consequences. Ophthalmic acid and 5-oxoproline are metabolites involved in glutathione metabolism, which can be

Glutathione transferases are structural and functional outliers in the thioredoxin fold.

Science.gov (United States)

Atkinson, Holly J; Babbitt, Patricia C

2009-11-24

Glutathione transferases (GSTs) are ubiquitous scavengers of toxic compounds that fall, structurally and functionally, within the thioredoxin fold suprafamily. The fundamental catalytic capability of GSTs is catalysis of the nucleophilic addition or substitution of glutathione at electrophilic centers in a wide range of small electrophilic compounds. While specific GSTs have been studied in detail, little else is known about the structural and functional relationships between different groupings of GSTs. Through a global analysis of sequence and structural similarity, it was determined that variation in the binding of glutathione between the two major subgroups of cytosolic (soluble) GSTs results in a different mode of glutathione activation. Additionally, the convergent features of glutathione binding between cytosolic GSTs and mitochondrial GST kappa are described. The identification of these structural and functional themes helps to illuminate some of the fundamental contributions of the thioredoxin fold to catalysis in the GSTs and clarify how the thioredoxin fold can be modified to enable new functions.

Glutathione reductase: solvent equilibrium and kinetic isotope effects

International Nuclear Information System (INIS)

Wong, K.K.; Vanoni, M.A.; Blanchard, J.S.

1988-01-01

Glutathione reductase catalyzes the NADPH-dependent reduction of oxidized glutathione (GSSG). The kinetic mechanism is ping-pong, and we have investigated the rate-limiting nature of proton-transfer steps in the reactions catalyzed by the spinach, yeast, and human erythrocyte glutathione reductases using a combination of alternate substrate and solvent kinetic isotope effects. With NADPH or GSSG as the variable substrate, at a fixed, saturating concentration of the other substrate, solvent kinetic isotope effects were observed on V but not V/K. Plots of Vm vs mole fraction of D 2 O (proton inventories) were linear in both cases for the yeast, spinach, and human erythrocyte enzymes. When solvent kinetic isotope effect studies were performed with DTNB instead of GSSG as an alternate substrate, a solvent kinetic isotope effect of 1.0 was observed. Solvent kinetic isotope effect measurements were also performed on the asymmetric disulfides GSSNB and GSSNP by using human erythrocyte glutathione reductase. The Km values for GSSNB and GSSNP were 70 microM and 13 microM, respectively, and V values were 62 and 57% of the one calculated for GSSG, respectively. Both of these substrates yield solvent kinetic isotope effects greater than 1.0 on both V and V/K and linear proton inventories, indicating that a single proton-transfer step is still rate limiting. These data are discussed in relationship to the chemical mechanism of GSSG reduction and the identity of the proton-transfer step whose rate is sensitive to solvent isotopic composition. Finally, the solvent equilibrium isotope effect measured with yeast glutathione reductase is 4.98, which allows us to calculate a fractionation factor for the thiol moiety of GSH of 0.456

Direct Electrochemistry of Horseradish Peroxidase-Gold Nanoparticles Conjugate

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Chanchal K. Mitra

2009-02-01

Full Text Available We have studied the direct electrochemistry of horseradish peroxidase (HRP coupled to gold nanoparticles (AuNP using electrochemical techniques, which provide some insight in the application of biosensors as tools for diagnostics because HRP is widely used in clinical diagnostics kits. AuNP capped with (i glutathione and (ii lipoic acid was covalently linked to HRP. The immobilized HRP/AuNP conjugate showed characteristic redox peaks at a gold electrode. It displayed good electrocatalytic response to the reduction of H2O2, with good sensitivity and without any electron mediator. The covalent linking of HRP and AuNP did not affect the activity of the enzyme significantly. The response of the electrode towards the different concentrations of H2O2 showed the characteristics of Michaelis Menten enzyme kinetics with an optimum pH between 7.0 to 8.0. The preparation of the sensor involves single layer of enzyme, which can be carried out efficiently and is also highly reproducible when compared to other systems involving the layer-by-layer assembly, adsorption or encapsulation of the enzyme. The immobilized AuNP-HRP can be used for immunosensor applications

Glutathione mediation of papain inactivation by hydrogen peroxide and hydroxyl radicals

International Nuclear Information System (INIS)

Lin, W.S.; Armstrong, D.A.

1977-01-01

Glutathione reacts with papainCys 25 SOH, formed by the reaction of papain with hydrogen peroxide, to give papainCys 25 SSG. Subsequent reaction of this mixed disulfide with glutathione is slow (k -1 sec -1 ). However, at 30 0 C it is readily cleaved by cysteine to form active papain, i.e., papainCys 25 SH. Glutathione resembles cysteine in protecting papain by the scavenging of .OH radicals, but, unlike cysteine, glutathione gave no evidence for the repair of enzyme radical lesions or for the conversion of papainCys 25 S. radicals to repairable derivatives. Its overall effectiveness for reducing the radiation inactivation of papain in aqueous solution is much less than that of cysteine

Hepatic and erythrocytic glutathione peroxidase activity in liver diseases.

Science.gov (United States)

Cordero, R; Ortiz, A; Hernández, R; López, V; Gómez, M M; Mena, P

1996-09-01

Hepatic and erythrocytic glutathione peroxidase activity, together with malondialdehyde levels, were determined as indicators of peroxidation in 83 patients from whom liver biopsies had been taken for diagnostic purposes. On histological study, the patients were classified into groups as minimal changes (including normal liver), steatosis, alcoholic hepatitis, hepatic cirrhosis, light to moderately active chronic hepatitis, and severe chronic active hepatitis. The glutathione peroxidase activity in erythrocytes showed no significant changes in any liver disease group. In the hepatic study, an increased activity was observed in steatosis with respect to the minimal changes group, this increased activity induced by the toxic agent in the initial stages of the alcoholic hepatic disease declining as the hepatic damage progressed. There was a negative correlation between the levels of hepatic malondialdehyde and hepatic glutathione peroxidase in subjects with minimal changes. This suggested the existence of an oxidative equilibrium in this group. This equilibrium is broken in the liver disease groups as was manifest in a positive correlation between malondialdehyde and glutathione peroxidase activity.

Subcellular distribution of glutathione and cysteine in cyanobacteria

OpenAIRE

Zechmann, Bernd; Tomašić, Ana; Horvat, Lucija; Fulgosi, Hrvoje

2010-01-01

Glutathione plays numerous important functions in eukaryotic and prokaryotic cells. Whereas it can be found in virtually all eukaryotic cells, its production in prokaryotes is restricted to cyanobacteria and proteobacteria and a few strains of gram-positive bacteria. In bacteria, it is involved in the protection against reactive oxygen species (ROS), osmotic shock, acidic conditions, toxic chemicals, and heavy metals. Glutathione synthesis in bacteria takes place in two steps out of cysteine,...

Bending of conjugated molecular wires and its effect on electron conduction properties

International Nuclear Information System (INIS)

Das, Bidisa

2010-01-01

The electronic structure and electron transport properties of simple conjugated molecular wires like oligophenylene ethynylene (OPE) and oligophenylene vinylene (OPV) are studied under compression. If artificially confined to a given shorter length, the oligomers tend to bend and bending causes a loss in the overlap of the conjugated molecular orbitals. Theoretical modeling of electronic transport has been carried out for all undistorted and compressed OPE/OPV oligomers. OPV exists in step-like or V-like conformations and they have the same stability with very similar frontier molecular orbitals. The conductances of these molecular wires are calculated when inserted between two gold probes and the conductances for OPV are found to be comparable to OPE when the interfaces are same. The conductance decreases with bending due to the gradual loss in overlap of the molecular orbitals. It is also found that the conductances of the molecular wires decrease very strongly if the terminal sulfur atom is simultaneously bonded to hydrogen and a gold surface, thus reflecting the importance of the interface in determining the conductance in two-probe systems. From the conductance studies it may be concluded that if one or more benzene rings of OPE are rotated from coplanar conditions, the orthogonal molecular orbitals may completely block the electronic transport, rendering the molecule insulating.

Control of charge carrier dynamics in disordered conjugated polymers

Energy Technology Data Exchange (ETDEWEB)

Hertel, Dirk [Physical Chemistry, University of Cologne, Luxemburgerstr. 116, 50939 Cologne, Germany, (Germany)

2011-07-01

We developed a new method to probe charge carrier mobility on ultrafast time scale. It is based on electric field induced second harmonic generation. The method is applied to prototypical amorphous conjugated polymers of the polyphenylene- and polyfluorene-type. Typically the carrier mobility in these organic polymers decreases with time in a power law fashion from about 1 cm{sup 2}Vs{sup -1} at 1 ps to its stationary value of about 10{sup -6} cm{sup 2}Vs{sup -1} in hundreds of ns. The dynamics of the mobility is discussed. It is shown, that in nanoscale devices the macroscopic mobility is not adequate to describe charge transport. We study the influence of disorder, morphology and temperature on ultrafast transport. At early times the transport is dominated by tunneling and disorder plays already an essential role. Comparison of transient photocurrents with Monte-Carlo simulation reveals that on-chain transport has to be invoked to rationalize our results. The hopping rates for intrachain transport are much larger compared with interchain transport. The results give access to essential transport properties for the development of advanced theoretical models and may help to design improved solar cells.

Cytotoxicity of S-conjugates of the sevoflurane degradation product fluoromethyl-2,2-difluoro-1-(trifluoromethyl) vinyl ether (Compound A) in a human proximal tubular cell line

International Nuclear Information System (INIS)

Altuntas, T. Gul; Zager, Richard A.; Kharasch, Evan D.

2003-01-01

Fluoromethyl-2,2-difluoro-1-(trifluoromethyl)vinyl ether (FDVE) is a fluorinated alkene formed by degradation of the volatile anesthetic sevoflurane in anesthesia machines. FDVE is nephrotoxic in rats but not humans. Rat FDVE nephrotoxicity is attributed to FDVE glutathione conjugation and bioactivation of subsequent FDVE-cysteine S-conjugates, in part by renal β-lyase. Although FDVE conjugation and metabolism occur in both rats and humans, the mechanism for selective toxicity in rats and lack of effect in humans is incompletely elucidated. This investigation measured FDVE S-conjugate cytotoxicity in cultured human proximal tubular HK-2 cells, and compared this with known cytotoxic S-conjugates. HK-2 cells were incubated with FDVE and its GSH, cysteine S-mercapturic acid, cysteine S-sulfoxide, and mercapturic acid sulfoxide conjugates (0.1-2.7 mM) for 24 h. Cytotoxicity was determined by lactate dehydrogenase (LDH) release, total LDH, and the ability of viable cells to reduce a tetrazolium-based compound (MTT). FDVE was cytotoxic only at concentrations ≥0.9 mM. No increase in LDH release was observed with either FDVE-GSH conjugate. The FDVE-cysteine conjugates S-(1,1-difluoro-2-fluoromethoxy-2-(trifluoromethyl) ethyl)-L-cysteine (DFEC) and (Z)-S-(1-fluoro-2-fluoromethoxy-2-(trifluoromethyl) vinyl)-L-cysteine ((Z)-FFVC) caused significant differences in LDH release and MTT reduction only at 2.7 mM; (Z)-FFVC was slightly more cytotoxic. Both S-(1,1-difluoro-2-fluoromethoxy-2-(trifluoromethyl) ethyl)-L-cysteine sulfoxide (DFEC-SO) and (Z)-N-acetyl-S-(1-fluoro-2-fluoromethoxy-2-(trifluoromethyl) vinyl)-L-cysteine sulfoxide ((Z)-N-Ac-FFVC-SO) caused slightly greater changes in LDH release or total LDH than the corresponding equimolar DFEC and (Z)-N-acetyl-S-(1-fluoro-2-fluoromethoxy-2-(trifluoromethyl) vinyl)-L-cysteine ((Z)-N-Ac-FFVC) conjugates. In contrast to FDVE S-conjugates, S-(1,2-dichlorovinyl)-L-cysteine was markedly cytotoxic, at concentrations as low as 0

Bacteriophytochromes control conjugation in Agrobacterium fabrum.

Science.gov (United States)

Bai, Yingnan; Rottwinkel, Gregor; Feng, Juan; Liu, Yiyao; Lamparter, Tilman

2016-08-01

Bacterial conjugation, the transfer of single stranded plasmid DNA from donor to recipient cell, is mediated through the type IV secretion system. We performed conjugation assays using a transmissible artificial plasmid as reporter. With this assay, conjugation in Agrobacterium fabrum was modulated by the phytochromes Agp1 and Agp2, photoreceptors that are most sensitive in the red region of visible light. In conjugation studies with wild-type donor cells carrying a pBIN-GUSINT plasmid as reporter that lacked the Ti (tumor inducing) plasmid, no conjugation was observed. When either agp1(-) or agp2(-) knockout donor strains were used, plasmid DNA was delivered to the recipient, indicating that both phytochromes suppress conjugation in the wild type donor. In the recipient strains, the loss of Agp1 or Agp2 led to diminished conjugation. When wild type cells with Ti plasmid and pBIN-GUS reporter plasmid were used as donor, a high rate of conjugation was observed. The DNA transfer was down regulated by red or far-red light by a factor of 3.5. With agp1(-) or agp2(-) knockout donor cells, conjugation in the dark was about 10 times lower than with the wild type donor, and with the double knockout donor no conjugation was observed. These results imply that the phytochrome system has evolved to inhibit conjugation in the light. The decrease of conjugation under different temperature correlated with the decrease of phytochrome autophosphorylation. Copyright © 2015 Elsevier B.V. All rights reserved.

No net splanchnic release of glutathione in man during N-acetylcysteine infusion

DEFF Research Database (Denmark)

Poulsen, H E; Vilstrup, H; Almdal, T

1993-01-01

Glutathione and amino acid concentrations were measured in arterial and hepatic vein plasma in four healthy volunteers and two patients with cirrhosis. There was no significant splanchnic efflux of glutathione (95% confidence limits, -0.501 to 0.405 mumol/min). After infusion of N...... to 0.97 +/- 0.11 (mean +/- SEM; p amino acids corresponded to an increased load on hepatic metabolic N conversion and transamination among nonessential amino acids. Splanchnic uptake of serine, alanine, cystine, isoleucine, and phenylalanine increased...... after NAC compatible with stimulated hepatic glutathione synthesis. In contrast to the rat, plasma glutathione in man probably originates mainly from extrahepatic tissues....

Polymers for Protein Conjugation

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Gianfranco Pasut

2014-01-01

Full Text Available Polyethylene glycol (PEG at the moment is considered the leading polymer for protein conjugation in view of its unique properties, as well as to its low toxicity in humans, qualities which have been confirmed by its extensive use in clinical practice. Other polymers that are safe, biodegradable and custom-designed have, nevertheless, also been investigated as potential candidates for protein conjugation. This review will focus on natural polymers and synthetic linear polymers that have been used for protein delivery and the results associated with their use. Genetic fusion approaches for the preparation of protein-polypeptide conjugates will be also reviewed and compared with the best known chemical conjugation ones.

Determination of Glutathione and Its Redox Status in Isolated Vacuoles of Red Beetroot Cells

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E.V. Pradedova

2016-02-01

Full Text Available The glutathione of the red beetroot vacuoles (Beta vulgaris L. was measured using three well-known methods: the spectrofluorimetric method with orthophthalic aldehyde (OPT; the spectrophotometric method with 5.5'-dithiobis-2-nitrobenzoic acid (DTNB; the high-performance liquid chromatography (HPLC. The content of reduced (GSH and oxidized glutathione (GSSG differed depending on the research method. With OPT the concentration of glutathione was: GSH – 0.059 µmol /mg protein; GSSG – 0.019 µmol/mg protein and total glutathione (GSHtotal – 0.097 µmol/mg protein. In the case of determining with DTNB the concentration of glutathione was: GSH – 0.091 µmol/mg protein; GSSG – 0.031 µmol/mg protein; GSHtotal – 0.153 µmol/mg protein. HPLC-defined concentration of glutathione was lower: GSH – 0.039 µmol/mg protein; GSSG – 0.007 µmol/mg protein; GSHtotal – 0.053 µmol/mg protein. Redox ratio of GSH/GSSG was also dependent on the method of determination: with OPT – 3.11; with DTNB – 2.96 and HPLC – 5.57. Redox ratio of glutathione in vacuoles was much lower than the tissue extracts of red beetroot, which, depending on the method of determination, was: 7.23, 7.16 and 9.22. The results showed the vacuoles of red beetroot parenchyma cells contain glutathione. Despite the low value of the redox ratio GSH/GSSG, in vacuoles the pool of reduced glutathione prevailed over the pool of oxidized glutathione.

Different roles of glutathione in copper and zinc chelation in Brassica napus roots.

Science.gov (United States)

Zlobin, Ilya E; Kartashov, Alexander V; Shpakovski, George V

2017-09-01

We investigated the specific features of copper and zinc excess action on the roots of canola (Brassica napus L.) plants. Copper rapidly accumulated in canola root cells and reached saturation during several hours of treatment, whereas the root zinc content increased relatively slowly. Excessive copper and zinc entry inside the cell resulted in significant cell damage, as evidenced by alterations in plasmalemma permeability and decreases in cellular enzymatic activity. Zinc excess specifically damaged root hair cells, which correlated with a pronounced elevation of their labile zinc level. In vitro, we showed that reduced glutathione (GSH) readily reacted with copper ions to form complexes with blocked sulfhydryl groups. In contrast, zinc ions were ineffective as glutathione blockers, and glutathione molecules did not lose their specific chemical activity in the presence of Zn 2+ ions. The effect of copper and zinc excess on the glutathione pool in canola root cells was analysed by a combination of biochemical determination of total and oxidized glutathione contents and fluorescent staining of free reduced glutathione with monochlorobimane dye. Excess copper led to dose-dependent diminution of free reduced glutathione contents in the root cells, which could not be explained by the loss of total cellular glutathione or its oxidation. In contrast, we observed little effect of much higher intracellular zinc concentrations on the free reduced glutathione content. We concluded that GSH plays an important role in copper excess, but not zinc excess chelation, in canola root cells. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

A S-cysteine conjugate, precursor of aroma of White Sauvignon

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Takatoshi Tominaga

1995-12-01

Full Text Available 4-mercapto-4-methylpentan-2-one (4-MMP, a strongly odorant compound responsible for the « boxtree » or « broom plant » odour of the Sauvignon wines, can be enzymaticaly released in vitro from an odourless must extract. The enzyme source used is a cell-free extract of the gastrointestinal bacterium Eubacterium limosum. This crude preparation exhibits a cysteine β-lyase activity which requires the presence of pyridoxal phosphate. The release of 4-MMP is inhibited when the substrate is previously treated with N-hydroxysuccimide acetate which reacts with a primary amine. The same bacterial extract is also able to release 4-MMP, pyruvic acid and ammonium, from S-(4-méthylpentan-2-one-L-cysteine. On the other hand, the cleavage of S-(4-méthylpentan-2-oneD,L-homocysteine and S-(4-méthylpentan-2-one- glutathione is very limited. These results suggest that the precursor of 4-MMP in Sauvignon must is a S-cysteine conjugate. Such an aroma precursor in grapes or in other fruits has never been round berore.

Lack of oxygen effect in glutathione-deficient human cells in culture

International Nuclear Information System (INIS)

Edgren, M.; Larsson, A.; Nilsson, K.; Revesz, L.; Scott, O.C.A.

1980-01-01

The frequency of X-ray-induced DNA breaks was determined in human cell lines which are deficient in glutathione synthetase and have a greatly reduced glutathione content. Hydroxyapatite chromatography was used for the estimation of the DNA breaks in cell cultures, which were derived either from lymphoblasts transformed by infection with EB virus or from fibroblasts. The dose-effect relationship for the induction of breaks when radiation exposure was made in argon, was similar to that found when exposure was made in air. In control cultures with normal glutathione content, the induction of breaks was enhanced when irradiation was made under aerobic, instead of anaerobic, conditions. Treatment of the glutathione-deficient cells with the hypoxic radiosensitizer misonidazole did not enhance the induction of breaks by radiation delivered either in air or in argon. In control cultures, radiation induction of breaks was enhanced by misonidazole under anaerobic but not under aerobic conditions. When the glutathione-deficient cells were pretreated with cysteamine however, irradiation in the absence of oxygen resulted in a decreased frequency of DNA breaks. (author)

Imaging Mass Spectrometry Revealed the Accumulation Characteristics of the 2-Nitroimidazole-Based Agent "Pimonidazole" in Hypoxia.

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Yukiko Masaki

Full Text Available Hypoxia, or low oxygen concentration, is a key factor promoting tumor progression and angiogenesis and resistance of cancer to radiotherapy and chemotherapy. 2-Nitroimidazole-based agents have been widely used in pathological and nuclear medicine examinations to detect hypoxic regions in tumors; in particular, pimonidazole is used for histochemical staining of hypoxic regions. It is considered to accumulate in hypoxic cells via covalent binding with macromolecules or by forming reductive metabolites after reduction of its nitro group. However, the detailed mechanism of its accumulation remains unknown. In this study, we investigated the accumulation mechanism of pimonidazole in hypoxic tumor tissues in a mouse model by mass spectrometric analyses including imaging mass spectrometry (IMS. Pimonidazole and its reductive metabolites were observed in the tumor tissues. However, their locations in the tumor sections were not similar to the positively stained areas in pimonidazole-immunohistochemistry, an area considered hypoxic. The glutathione conjugate of reduced pimonidazole, a low-molecular-weight metabolite of pimonidazole, was found in tumor tissues by LC-MS analysis, and our IMS study determined that the intratumor localization of the glutathione conjugate was consistent with the area positively immunostained for pimonidazole. We also found complementary localization of the glutathione conjugate and reduced glutathione (GSH, implying that formation of the glutathione conjugate occurred in the tumor tissue. These results suggest that in hypoxic tumor cells, pimonidazole is reduced at its nitro group, followed by conjugation with GSH.

Conjugative Plasmid Transfer in Xylella fastidiosa Is Dependent on tra and trb Operon Functions.

Science.gov (United States)

Burbank, Lindsey P; Van Horn, Christopher R

2017-11-01

The insect-transmitted plant pathogen Xylella fastidiosa is capable of efficient horizontal gene transfer (HGT) and recombination. Natural transformation occurs at high rates in X. fastidiosa , but there also is evidence that certain strains of X. fastidiosa carry native plasmids equipped with transfer and mobilization genes, suggesting conjugation as an additional mechanism of HGT in some instances. Two operons, tra and trb , putatively encoding a conjugative type IV secretion system, are found in some but not all X. fastidiosa isolates, often on native plasmids. X. fastidiosa strains that carry the conjugative transfer genes can belong to different subspecies and frequently differ in host ranges. Using X. fastidiosa strain M23 ( X. fastidiosa subsp. fastidiosa ) or Dixon ( X. fastidiosa subsp. multiplex ) as the donor strain and Temecula ( X. fastidiosa subsp. fastidiosa ) as the recipient strain, plasmid transfer was characterized using the mobilizable broad-host-range vector pBBR5pemIK. Transfer of plasmid pBBR5pemIK was observed under in vitro conditions with both donor strains and was dependent on both tra and trb operon functions. A conjugative mechanism likely contributes to gene transfer between diverse strains of X. fastidiosa , possibly facilitating adaptation to new environments or different hosts. IMPORTANCE Xylella fastidiosa is an important plant pathogen worldwide, infecting a wide range of different plant species. The emergence of new diseases caused by X. fastidiosa , or host switching of existing strains, is thought to be primarily due to the high frequency of HGT and recombination in this pathogen. Transfer of plasmids by a conjugative mechanism enables movement of larger amounts of genetic material at one time, compared with other routes of gene transfer such as natural transformation. Establishing the prevalence and functionality of this mechanism in X. fastidiosa contributes to a better understanding of HGT, adaptation, and disease emergence

Glutathione treatment of hepatocellular carcinoma

DEFF Research Database (Denmark)

Dalhoff, K; Ranek, L; Mantoni, M

1992-01-01

This prospective study was undertaken to substantiate observations that glutathione (GSH) inhibits or reverses tumor growth in humans with hepatocellular carcinoma (HCC), a neoplasm with an extremely poor prognosis. Eight patients with biopsy-proven HCC not amenable to surgery were given 5 g of GSH...

The depletion of nuclear glutathione impairs cell proliferation in 3t3 fibroblasts.

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Jelena Markovic

2009-07-01

Full Text Available Glutathione is considered essential for survival in mammalian cells and yeast but not in prokaryotic cells. The presence of a nuclear pool of glutathione has been demonstrated but its role in cellular proliferation and differentiation is still a matter of debate.We have studied proliferation of 3T3 fibroblasts for a period of 5 days. Cells were treated with two well known depleting agents, diethyl maleate (DEM and buthionine sulfoximine (BSO, and the cellular and nuclear glutathione levels were assessed by analytical and confocal microscopic techniques, respectively. Both agents decreased total cellular glutathione although depletion by BSO was more sustained. However, the nuclear glutathione pool resisted depletion by BSO but not with DEM. Interestingly, cell proliferation was impaired by DEM, but not by BSO. Treating the cells simultaneously with DEM and with glutathione ethyl ester to restore intracellular GSH levels completely prevented the effects of DEM on cell proliferation.Our results demonstrate the importance of nuclear glutathione in the control of cell proliferation in 3T3 fibroblasts and suggest that a reduced nuclear environment is necessary for cells to progress in the cell cycle.

Plaque formation reduction with glutathione monoester in mice fed on atherogenic diet

International Nuclear Information System (INIS)

Iqbal, M.; Mehboobali, N.; Pervez, S.

2006-01-01

To determine the role of glutathione monoester on reducing the development of plaque formation in an animal model. Twenty-four Balb/c mice were divided into 3 equal groups. First group was fed on atherogenic diet alone, while the second group received atherogenic diet plus twice weekly injections of glutathione monoester. The third group was fed on normal diet for mice. After one year, the animals were sacrificed. Blood was analyzed for lipid levels, while liver, kidney, spleen, heart and aorta were removed to study morphological changes. Results: In the groups of mice receiving atherogenic diet (with and without glutathione monoesters), there was significant increase in levels of total cholesterol (p=0.011) and LDL cholesterol (p=0.001) compared to levels of these lipids in mice on normal diet. However, a significant decrease in levels of triglycerides (p=0.01) was observed in the group receiving atherogenic diet along with glutathione monoester. Supplementation with glutathione monoester had the most pronounced effect only on triglyceride levels. Atherosclerotic plaques were seen in heart and/or aorta of mice receiving atherogenic diet. However, such plaques were either totally absent or if seen in an animal, were extremely small and diffuse in the group receiving glutathione monoester along with atherogenic diet. Mice on normal diet had no evidence of any plaque formation. Cholesterol granuloma was seen in liver of mice on atherogenic diet alone. In mice receiving atherogenic diet plus glutathione monoester, no cholesterol granuloma was found in liver. There were no remarkable morphological changes in spleen and kidney in the three groups of mice. Glutathione monoester appears to inhibit or reduce the development of plaque formation in mice. (author)

Thioredoxin and glutathione systems differ in parasitic and free-living platyhelminths

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Salinas Gustavo

2010-04-01

Full Text Available Abstract Background The thioredoxin and/or glutathione pathways occur in all organisms. They provide electrons for deoxyribonucleotide synthesis, function as antioxidant defenses, in detoxification, Fe/S biogenesis and participate in a variety of cellular processes. In contrast to their mammalian hosts, platyhelminth (flatworm parasites studied so far, lack conventional thioredoxin and glutathione systems. Instead, they possess a linked thioredoxin-glutathione system with the selenocysteine-containing enzyme thioredoxin glutathione reductase (TGR as the single redox hub that controls the overall redox homeostasis. TGR has been recently validated as a drug target for schistosomiasis and new drug leads targeting TGR have recently been identified for these platyhelminth infections that affect more than 200 million people and for which a single drug is currently available. Little is known regarding the genomic structure of flatworm TGRs, the expression of TGR variants and whether the absence of conventional thioredoxin and glutathione systems is a signature of the entire platyhelminth phylum. Results We examine platyhelminth genomes and transcriptomes and find that all platyhelminth parasites (from classes Cestoda and Trematoda conform to a biochemical scenario involving, exclusively, a selenium-dependent linked thioredoxin-glutathione system having TGR as a central redox hub. In contrast, the free-living platyhelminth Schmidtea mediterranea (Class Turbellaria possesses conventional and linked thioredoxin and glutathione systems. We identify TGR variants in Schistosoma spp. derived from a single gene, and demonstrate their expression. We also provide experimental evidence that alternative initiation of transcription and alternative transcript processing contribute to the generation of TGR variants in platyhelminth parasites. Conclusions Our results indicate that thioredoxin and glutathione pathways differ in parasitic and free-living flatworms and

Thioredoxin and glutathione systems differ in parasitic and free-living platyhelminths

Science.gov (United States)

2010-01-01

Background The thioredoxin and/or glutathione pathways occur in all organisms. They provide electrons for deoxyribonucleotide synthesis, function as antioxidant defenses, in detoxification, Fe/S biogenesis and participate in a variety of cellular processes. In contrast to their mammalian hosts, platyhelminth (flatworm) parasites studied so far, lack conventional thioredoxin and glutathione systems. Instead, they possess a linked thioredoxin-glutathione system with the selenocysteine-containing enzyme thioredoxin glutathione reductase (TGR) as the single redox hub that controls the overall redox homeostasis. TGR has been recently validated as a drug target for schistosomiasis and new drug leads targeting TGR have recently been identified for these platyhelminth infections that affect more than 200 million people and for which a single drug is currently available. Little is known regarding the genomic structure of flatworm TGRs, the expression of TGR variants and whether the absence of conventional thioredoxin and glutathione systems is a signature of the entire platyhelminth phylum. Results We examine platyhelminth genomes and transcriptomes and find that all platyhelminth parasites (from classes Cestoda and Trematoda) conform to a biochemical scenario involving, exclusively, a selenium-dependent linked thioredoxin-glutathione system having TGR as a central redox hub. In contrast, the free-living platyhelminth Schmidtea mediterranea (Class Turbellaria) possesses conventional and linked thioredoxin and glutathione systems. We identify TGR variants in Schistosoma spp. derived from a single gene, and demonstrate their expression. We also provide experimental evidence that alternative initiation of transcription and alternative transcript processing contribute to the generation of TGR variants in platyhelminth parasites. Conclusions Our results indicate that thioredoxin and glutathione pathways differ in parasitic and free-living flatworms and that canonical enzymes

Effect of Glutathione on the Taste and Texture of Type I Sourdough Bread.

Science.gov (United States)

Tang, Kai Xing; Zhao, Cindy J; Gänzle, Michael G

2017-05-31

Type I sourdough fermentations with Lactobacillus sanfranciscensis as predominant organism accumulate reduced glutathione through glutathione reductase (GshR) activity of L. sanfranciscensis. Reduced glutathione acts as chain terminator for gluten polymerization but is also kokumi-active and may thus enhance bread taste. This study implemented a type I model sourdough fermentations to quantitate glutathione accumulation sourdough, bread dough, and bread and to assess the effect of L. sanfranciscensis GshR on bread volume by comparison of L. sanfranciscensis and an isogenic strain devoid of GshR. L. sanfranciscensis sourdough accumulated the highest amount of reduced glutathione during proofing. Bread produced with the wild type strain had a lower volume when compared to the gshR deficient mutant. The accumulation of γ-glutamyl-cysteine was also higher in L. sanfranciscensis sourdoughs when compared to doughs fermented with the gshR mutant strain. The accumulation of reduced glutathione in L. sanfranciscensis bread did not enhance the saltiness of bread.

Anisotropic charge transport in large single crystals of π-conjugated organic molecules.

Science.gov (United States)

Hourani, Wael; Rahimi, Khosrow; Botiz, Ioan; Koch, Felix Peter Vinzenz; Reiter, Günter; Lienerth, Peter; Heiser, Thomas; Bubendorff, Jean-Luc; Simon, Laurent

2014-05-07

The electronic properties of organic semiconductors depend strongly on the nature of the molecules, their conjugation and conformation, their mutual distance and the orientation between adjacent molecules. Variations of intramolecular distances and conformation disturb the conjugation and perturb the delocalization of charges. As a result, the mobility considerably decreases compared to that of a covalently well-organized crystal. Here, we present electrical characterization of large single crystals made of the regioregular octamer of 3-hexyl-thiophene (3HT)8 using a conductive-atomic force microscope (C-AFM) in air. We find a large anisotropy in the conduction with charge mobility values depending on the crystallographic orientation of the single crystal. The smaller conduction is in the direction of π-π stacking (along the long axis of the single crystal) with a mobility value in the order of 10(-3) cm(2) V(-1) s(-1), and the larger one is along the molecular axis (in the direction normal to the single crystal surface) with a mobility value in the order of 0.5 cm(2) V(-1) s(-1). The measured current-voltage (I-V) curves showed that along the molecular axis, the current followed an exponential dependence corresponding to an injection mode. In the π-π stacking direction, the current exhibits a space charge limited current (SCLC) behavior, which allows us to estimate the charge carrier mobility.

Exploring the origin of high optical absorption in conjugated polymers

KAUST Repository

Vezie, Michelle S.; Few, Sheridan; Meager, Iain; Pieridou, Galatia; Dö rling, Bernhard; Ashraf, Raja Shahid; Goñ i, Alejandro R.; Bronstein, Hugo; McCulloch, Iain; Hayes, Sophia C.; Campoy-Quiles, Mariano; Nelson, Jenny

2016-01-01

The specific optical absorption of an organic semiconductor is critical to the performance of organic optoelectronic devices. For example, higher light-harvesting efficiency can lead to higher photocurrent in solar cells that are limited by sub-optimal electrical transport. Here, we compare over 40 conjugated polymers, and find that many different chemical structures share an apparent maximum in their extinction coefficients. However, a diketopyrrolopyrrole-thienothiophene copolymer shows remarkably high optical absorption at relatively low photon energies. By investigating its backbone structure and conformation with measurements and quantum chemical calculations, we find that the high optical absorption can be explained by the high persistence length of the polymer. Accordingly, we demonstrate high absorption in other polymers with high theoretical persistence length. Visible light harvesting may be enhanced in other conjugated polymers through judicious design of the structure.

Exploring the origin of high optical absorption in conjugated polymers

KAUST Repository

Vezie, Michelle S.

2016-05-16

The specific optical absorption of an organic semiconductor is critical to the performance of organic optoelectronic devices. For example, higher light-harvesting efficiency can lead to higher photocurrent in solar cells that are limited by sub-optimal electrical transport. Here, we compare over 40 conjugated polymers, and find that many different chemical structures share an apparent maximum in their extinction coefficients. However, a diketopyrrolopyrrole-thienothiophene copolymer shows remarkably high optical absorption at relatively low photon energies. By investigating its backbone structure and conformation with measurements and quantum chemical calculations, we find that the high optical absorption can be explained by the high persistence length of the polymer. Accordingly, we demonstrate high absorption in other polymers with high theoretical persistence length. Visible light harvesting may be enhanced in other conjugated polymers through judicious design of the structure.

Functional analysis and localisation of a delta-class glutathione S-transferase from Sarcoptes scabiei.

Science.gov (United States)

Pettersson, Eva U; Ljunggren, Erland L; Morrison, David A; Mattsson, Jens G

2005-01-01

The mite Sarcoptes scabiei causes sarcoptic mange, or scabies, a disease that affects both animals and humans worldwide. Our interest in S. scabiei led us to further characterise a glutathione S-transferase. This multifunctional enzyme is a target for vaccine and drug development in several parasitic diseases. The S. scabiei glutathione S-transferase open reading frame reported here is 684 nucleotides long and yields a protein with a predicted molecular mass of 26 kDa. Through phylogenetic analysis the enzyme was classified as a delta-class glutathione S-transferase, and our paper is the first to report that delta-class glutathione S-transferases occur in organisms other than insects. The recombinant S. scabiei glutathione S-transferase was expressed in Escherichia coli via three different constructs and purified for biochemical analysis. The S. scabiei glutathione S-transferase was active towards the substrate 1-chloro-2,4-dinitrobenzene, though the positioning of fusion partners influenced the kinetic activity of the enzyme. Polyclonal antibodies raised against S. scabiei glutathione S-transferase specifically localised the enzyme to the integument of the epidermis and cavities surrounding internal organs in adult parasites. However, some minor staining of parasite intestines was observed. No staining was seen in host tissues, nor could we detect any antibody response against S. scabiei glutathione S-transferase in sera from naturally S. scabiei infected dogs or pigs. Additionally, the polyclonal sera raised against recombinant S. scabiei glutathione S-transferase readily detected a protein from mites, corresponding to the predicted size of native glutathione S-transferase.

Effects of Transportation on Antioxidant Status in Cynomolgus Macaques (Macaca fascicularis).

Science.gov (United States)

Pan, Xueying; Lu, Liang; Zeng, Xiancheng; Chang, Yan; Hua, Xiuguo

2016-01-01

To evaluate the effects of transportation on oxidative stress in cynomolgus monkeys, we measured serum levels of reduced glutathione (GSH), malondialdehyde, and protein carbonyl (PC) and the activities of total antioxidant capacity (TAOC), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and catalase in cynomolgus macaques before transportation (day 0), on the day of arrival (day 1), and on days 7, 14, and 21 after transportation. Compared with that on day 0, TAOC and catalase activities on days 1, 7, and 14 after transportation were significantly decreased, reached their nadirs on day 7, and increased thereafter to reach their pretransportation levels by day 21 after transportation. Compared with day 0 levels, mean SOD activity and GSH concentration were decreased significantly on day 1; they thereafter increased to reach their pretransportation measures by day 7 after transportation. In contrast, PC and malondialdehyde concentrations in serum and the activity of GSH-Px were increased on day 1 compared with day 0 and thereafter decreased to reach their pretransportation levels by day 14 after transportation. In summary, GSH, TAOC, catalase, and SOD levels decreased and malondialdehyde, PC, and GSH-Px concentrations increased in cynomolgus macaques after transportation. These results suggest that transportation might imbalance oxidant and antioxidant levels to create excess oxidative stress in cynomolgus macaques. Therefore, cynomolgus macaques should have at least 21 d to recover after transportation and regain their healthy status.

Glutathione and its antiaging and antimelanogenic effects

Directory of Open Access Journals (Sweden)

Weschawalit S

2017-04-01

Full Text Available Sinee Weschawalit,1 Siriwan Thongthip,2 Phanupong Phutrakool,3 Pravit Asawanonda1 1Department of Medicine, Division of Dermatology, 2Chula Clinical Research Center, 3Chula Data Management Center, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand Background: Previous studies showed that supplementation of reduced form of glutathione (GSH, 500 mg/d has a skin-lightening efficacy in humans. This study was designed to evaluate the influences of both GSH and oxidized form (GSSG, at doses lower than 500 mg/d, on improving skin properties. Patients and methods: A randomized, double-blind, placebo-controlled, parallel, three-arm study was conducted. Healthy female subjects were equally randomized into three groups and took GSH (250 mg/d, GSSG (250 mg/d, or placebo orally for 12 weeks. At each visit at baseline and for 12 weeks, skin features including melanin index, wrinkles, and other relevant biophysical properties were measured. Blood samples were collected for safety monitoring. Results: In generalized estimating equation analyses, melanin index and ultraviolet spots of all sites including face and arm when given GSH and GSSG tended to be lower than placebo. At some sites evaluated, subjects who received GSH showed a significant reduction in wrinkles compared with those taking placebo. A tendency toward increased skin elasticity was observed in GSH and GSSG compared with placebo. There were no serious adverse effects throughout the study. Conclusion: We showed that oral glutathione, 250 mg/d, in both reduced and oxidized forms effectively influences skin properties. Overall, glutathione in both forms are well tolerated. Keywords: glutathione, melanin, pigment, aging, wrinkle, whitening

Pharmacokinetic analysis of trichloroethylene metabolism in male B6C3F1 mice: Formation and disposition of trichloroacetic acid, dichloroacetic acid, S-(1,2-dichlorovinyl)glutathione and S-(1,2-dichlorovinyl)-L-cysteine

Science.gov (United States)

Kim, Sungkyoon; Kim, David; Pollack, Gary M.; Collins, Leonard B.; Rusyn, Ivan

2009-01-01

Trichloroethylene (TCE) is a well-known carcinogen in rodents and concerns exist regarding its potential carcinogenicity in humans. Oxidative metabolites of TCE, such as dichloroacetic acid (DCA) and trichloroacetic acid (TCA), are thought to be hepatotoxic and carcinogenic in mice. The reactive products of glutathione conjugation, such as S-(1,2-dichlorovinyl)-L-cysteine (DCVC), and S-(1,2-dichlorovinyl) glutathione (DCVG), are associated with renal toxicity in rats. Recently, we developed a new analytical method for simultaneous assessment of these TCE metabolites in small-volume biological samples. Since important gaps remain in our understanding of the pharmacokinetics of TCE and its metabolites, we studied a time-course of DCA, TCA, DCVG and DCVG formation and elimination after a single oral dose of 2100 mg/kg TCE in male B6C3F1 mice. Based on systemic concentration-time data, we constructed multi-compartment models to explore the kinetic properties of the formation and disposition of TCE metabolites, as well as the source of DCA formation. We conclude that TCE-oxide is the most likely source of DCA. According to the best-fit model, bioavailability of oral TCE was ~74%, and the half-life and clearance of each metabolite in the mouse were as follows: DCA: 0.6 hr, 0.081 ml/hr; TCA: 12 hr, 3.80 ml/hr; DCVG: 1.4 hr, 16.8 ml/hr; DCVC: 1.2 hr, 176 ml/hr. In B6C3F1 mice, oxidative metabolites are formed in much greater quantities (~3600 fold difference) than glutathione-conjugative metabolites. In addition, DCA is produced to a very limited extent relative to TCA, while most of DCVG is converted into DCVC. These pharmacokinetic studies provide insight into the kinetic properties of four key biomarkers of TCE toxicity in the mouse, representing novel information that can be used in risk assessment. PMID:19409406

Pharmacokinetic analysis of trichloroethylene metabolism in male B6C3F1 mice: Formation and disposition of trichloroacetic acid, dichloroacetic acid, S-(1,2-dichlorovinyl)glutathione and S-(1,2-dichlorovinyl)-L-cysteine

International Nuclear Information System (INIS)

Kim, Sungkyoon; Kim, David; Pollack, Gary M.; Collins, Leonard B.; Rusyn, Ivan

2009-01-01

Trichloroethylene (TCE) is a well-known carcinogen in rodents and concerns exist regarding its potential carcinogenicity in humans. Oxidative metabolites of TCE, such as dichloroacetic acid (DCA) and trichloroacetic acid (TCA), are thought to be hepatotoxic and carcinogenic in mice. The reactive products of glutathione conjugation, such as S-(1,2-dichlorovinyl)-L-cysteine (DCVC), and S-(1,2-dichlorovinyl) glutathione (DCVG), are associated with renal toxicity in rats. Recently, we developed a new analytical method for simultaneous assessment of these TCE metabolites in small-volume biological samples. Since important gaps remain in our understanding of the pharmacokinetics of TCE and its metabolites, we studied a time-course of DCA, TCA, DCVG and DCVG formation and elimination after a single oral dose of 2100 mg/kg TCE in male B6C3F1 mice. Based on systemic concentration-time data, we constructed multi-compartment models to explore the kinetic properties of the formation and disposition of TCE metabolites, as well as the source of DCA formation. We conclude that TCE-oxide is the most likely source of DCA. According to the best-fit model, bioavailability of oral TCE was ∼ 74%, and the half-life and clearance of each metabolite in the mouse were as follows: DCA: 0.6 h, 0.081 ml/h; TCA: 12 h, 3.80 ml/h; DCVG: 1.4 h, 16.8 ml/h; DCVC: 1.2 h, 176 ml/h. In B6C3F1 mice, oxidative metabolites are formed in much greater quantities (∼ 3600 fold difference) than glutathione-conjugative metabolites. In addition, DCA is produced to a very limited extent relative to TCA, while most of DCVG is converted into DCVC. These pharmacokinetic studies provide insight into the kinetic properties of four key biomarkers of TCE toxicity in the mouse, representing novel information that can be used in risk assessment.

Optical phase conjugation

CERN Document Server

Fisher, Robert A

1983-01-01

This book appears at a time of intense activity in optical phase conjugation. We chose not to await the maturation of the field, but instead to provide this material in time to be useful in its development. We have tried very hard to elucidate and interrelate the various nonlinear phenomena which can be used for optical phase conjugation.

Intracellular thiol levels and radioresistance: Studies with glutathione and glutathione mono ethyl ester

International Nuclear Information System (INIS)

Astor, M.B.; Meister, A.; Anderson, M.E.

1987-01-01

Intracellular thiols such as glutathione (GSH) protect cells against free radicals formed during oxidative metabolism or from exposure to drugs or ionizing radiation. The role of intracellular GSH in the repair of radiation induced free radical damage was studied using GSH or its analog glutathione mono ethyl ester (GEE), which readily penetrates into the cell. Chinese hamster V79 cells with normal GSH levels were afforded equal protection under aerated and hypoxic conditions (DMF = 1.2 OER = 3.7) by both 10 mM GSH and GEE although GEE had raised interacellular GSH levels three-fold. Growth of V79 cells in cysteine free media resulted in undetectable levels of GSH and OER of 2.2 with no change in aerated survival. Restoration of intracellular GSH by 10 mM GEE resulted in an increase of the OER from 2.2. to 3.8 (DMF = 1.7). Only 14% of the intracellular GSH needs to be repleted to give an OER of 3.0. These experiments provide evidence that thiols do play a role in the oxygen effect and are present at levels in excess of what is necessary for maximal radioprotection

Single-bilayer graphene oxide sheet tolerance and glutathione redox system significance assessment in faba bean (Vicia faba L.)

International Nuclear Information System (INIS)

Anjum, Naser A.; Singh, Neetu; Singh, Manoj K.; Shah, Zahoor A.; Duarte, Armando C.; Pereira, Eduarda; Ahmad, Iqbal

2013-01-01

Adsorbents based on single-bilayer graphene oxide sheet (hereafter termed “graphene oxide”) are widely used in contaminated environments cleanup which may easily open the avenues for their entry to different environmental compartments, exposure to organisms and their subsequent transfer to human/animal food chain. Considering a common food crop—faba bean (Vicia faba L.) germinating seedlings as a model plant system, this study assesses the V. faba-tolerance to different concentrations (0, 100, 200, 400, 800, and 1600 mg L −1 ) of graphene oxide (0.5–5 μm) and evaluates glutathione (γ-glutamyl-cysteinyl-glycine) redox system significance in this context. The results showed significantly increased V. faba sensitivity under three graphene oxide concentrations (in order of impact: 1,600 > 200 > 100 mg graphene oxide L −1 ), which was accompanied by decreased glutathione redox (reduced glutathione-to-oxidized glutathione) ratio, reduced glutathione pool, as well as significant and equally elevated activities of glutathione-regenerating (glutathione reductase) and glutathione-metabolizing (glutathione peroxidase; glutathione sulfo-transferase) enzymes. Contrarily, the two graphene oxide concentrations (in order of impact: 800 > 400 graphene oxide mg L −1 ) yielded promising results; where, significant improvements in V. faba health status (measured as increased graphene oxide tolerance) were clearly perceptible with increased ratio of the reduced glutathione-to-oxidized glutathione, reduced glutathione pool and glutathione reductase activity but decreased activities of glutathione-metabolizing enzymes. It is inferred that V. faba seedlings-sensitivity and/or tolerance to graphene oxide concentrations depends on both the cellular redox state (reduced glutathione-to-oxidized glutathione ratio) and the reduced glutathione pool which in turn are controlled by a finely tuned modulation of the coordination between glutathione-regenerating and glutathione

Single-bilayer graphene oxide sheet tolerance and glutathione redox system significance assessment in faba bean (Vicia faba L.)

Energy Technology Data Exchange (ETDEWEB)

Anjum, Naser A. [University of Aveiro, Centre for Environmental and Marine Studies (CESAM) and Department of Chemistry (Portugal); Singh, Neetu; Singh, Manoj K. [University of Aveiro, Center for Mechanical Technology and Automation (TEMA) and Department of Mechanical Engineering (Portugal); Shah, Zahoor A. [University of Toledo, Department of Medicinal and Biological Chemistry, College of Pharmacy and Pharmaceutical Sciences (United States); Duarte, Armando C.; Pereira, Eduarda; Ahmad, Iqbal, E-mail: ahmadr@ua.pt [University of Aveiro, Centre for Environmental and Marine Studies (CESAM) and Department of Chemistry (Portugal)

2013-07-15

Adsorbents based on single-bilayer graphene oxide sheet (hereafter termed 'graphene oxide') are widely used in contaminated environments cleanup which may easily open the avenues for their entry to different environmental compartments, exposure to organisms and their subsequent transfer to human/animal food chain. Considering a common food crop-faba bean (Vicia faba L.) germinating seedlings as a model plant system, this study assesses the V. faba-tolerance to different concentrations (0, 100, 200, 400, 800, and 1600 mg L{sup -1}) of graphene oxide (0.5-5 {mu}m) and evaluates glutathione ({gamma}-glutamyl-cysteinyl-glycine) redox system significance in this context. The results showed significantly increased V. faba sensitivity under three graphene oxide concentrations (in order of impact: 1,600 > 200 > 100 mg graphene oxide L{sup -1}), which was accompanied by decreased glutathione redox (reduced glutathione-to-oxidized glutathione) ratio, reduced glutathione pool, as well as significant and equally elevated activities of glutathione-regenerating (glutathione reductase) and glutathione-metabolizing (glutathione peroxidase; glutathione sulfo-transferase) enzymes. Contrarily, the two graphene oxide concentrations (in order of impact: 800 > 400 graphene oxide mg L{sup -1}) yielded promising results; where, significant improvements in V. faba health status (measured as increased graphene oxide tolerance) were clearly perceptible with increased ratio of the reduced glutathione-to-oxidized glutathione, reduced glutathione pool and glutathione reductase activity but decreased activities of glutathione-metabolizing enzymes. It is inferred that V. faba seedlings-sensitivity and/or tolerance to graphene oxide concentrations depends on both the cellular redox state (reduced glutathione-to-oxidized glutathione ratio) and the reduced glutathione pool which in turn are controlled by a finely tuned modulation of the coordination between glutathione-regenerating and

Transmission of electric fields and photoelectron fluxes between conjugate ionospheric F2-regions

International Nuclear Information System (INIS)

Petelski, E.F.

1975-01-01

The dynamic behaviour of the ionospheric F2-layer requires considerable vertical transport of ionization. Possible causes of such transport are ambipolar diffusion, neutral air winds and electric fields. Here mid-latitude electric fields are investigated. Real height variations of the F2-layer indicate that the phases and amplitudes of these fields are similar at well conjugate points and that the field strengths can become unexpectedly high. It is further shown that photoelectrons can migrate between the two hemispheres along the geomagnetic field lines. (orig.) [de

Gene cloning and overexpression of two conjugated polyketone reductases, novel aldo-keto reductase family enzymes, of Candida parapsilosis.

Science.gov (United States)

Kataoka, M; Delacruz-Hidalgo, A-R G; Akond, M A; Sakuradani, E; Kita, K; Shimizu, S

2004-04-01

The genes encoding two conjugated polyketone reductases (CPR-C1, CPR-C2) of Candida parapsilosis IFO 0708 were cloned and sequenced. The genes encoded a total of 304 and 307 amino acid residues for CPR-C1 and CPR-C2, respectively. The deduced amino acid sequences of the two enzymes showed high similarity to each other and to several proteins of the aldo-keto reductase (AKR) superfamily. However, several amino acid residues in putative active sites of AKRs were not conserved in CPR-C1 and CPR-C2. The two CPR genes were overexpressed in Escherichia coli. The E. coli transformant bearing the CPR-C2 gene almost stoichiometrically reduced 30 mg ketopantoyl lactone/ml to D-pantoyl lactone.

Genetic encoding of a bicyclo[6.1.0]nonyne-charged amino acid enables fast cellular protein imaging by metal-free ligation.

Science.gov (United States)

Borrmann, Annika; Milles, Sigrid; Plass, Tilman; Dommerholt, Jan; Verkade, Jorge M M; Wiessler, Manfred; Schultz, Carsten; van Hest, Jan C M; van Delft, Floris L; Lemke, Edward A

2012-09-24

Visualizing biomolecules by fluorescent tagging is a powerful method for studying their behaviour and function inside cells. We prepared and genetically encoded an unnatural amino acid (UAA) that features a bicyclononyne moiety. This UAA offered exceptional reactivity in strain-promoted azide-alkyne cycloadditions. Kinetic measurements revealed that the UAA reacted also remarkably fast in the inverse-electron-demand Diels-Alder cycloaddition with tetrazine-conjugated dyes. Genetic encoding of the new UAA inside mammalian cells and its subsequent selective labeling at low dye concentrations demonstrate the usefulness of the new amino acid for future imaging studies. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Real-time quantification of subcellular H2O2 and glutathione redox potential in living cardiovascular tissues.

Science.gov (United States)

Panieri, Emiliano; Millia, Carlo; Santoro, Massimo M

2017-08-01

Detecting and measuring the dynamic redox events that occur in vivo is a prerequisite for understanding the impact of oxidants and redox events in normal and pathological conditions. These aspects are particularly relevant in cardiovascular tissues wherein alterations of the redox balance are associated with stroke, aging, and pharmacological intervention. An ambiguous aspect of redox biology is how redox events occur in subcellular organelles including mitochondria, and nuclei. Genetically-encoded Rogfp2 fluorescent probes have become powerful tools for real-time detection of redox events. These probes detect hydrogen peroxide (H 2 O 2 ) levels and glutathione redox potential (E GSH ), both with high spatiotemporal resolution. By generating novel transgenic (Tg) zebrafish lines that express compartment-specific Rogfp2-Orp1 and Grx1-Rogfp2 sensors we analyzed cytosolic, mitochondrial, and the nuclear redox state of endothelial cells and cardiomyocytes of living zebrafish embryos. We provide evidence for the usefulness of these Tg lines for pharmacological compounds screening by addressing the blocking of pentose phosphate pathways (PPP) and glutathione synthesis, thus altering subcellular redox state in vivo. Rogfp2-based transgenic zebrafish lines represent valuable tools to characterize the impact of redox changes in living tissues and offer new opportunities for studying metabolic driven antioxidant response in biomedical research. Copyright © 2017 Elsevier Inc. All rights reserved.

Glutathione metabolism modelling: a mechanism for liver drug-robustness and a new biomarker strategy

NARCIS (Netherlands)

Geenen, S.; du Preez, F.B.; Snoep, J.L.; Foster, A.J.; Sarda, S.; Kenna, J.G.; Wilson, I.D.; Westerhoff, H.V.

2013-01-01

Background Glutathione metabolism can determine an individual's ability to detoxify drugs. To increase understanding of the dynamics of cellular glutathione homeostasis, we have developed an experiment-based mathematical model of the kinetics of the glutathione network. This model was used to

MMS2, Encoding a ubiquitin-conjugating-enzyme-like protein, is a member of the yeast error-free postreplication repair pathway

International Nuclear Information System (INIS)

Broomfield, S.; Chow, B.L.; Xiao, W.

1998-01-01

Among the three Saccharomyces cerevisiae DNA repair epistasis groups, the RAD6 group is the most complicated and least characterized, primarily because it consists of two separate repair pathways: an error-free postreplication repair pathway, and a mutagenesis pathway. The rad6 and rad18 mutants are defective in both pathways, and the rev3 mutant affects only the mutagenesis pathway, but a yeast gene that is involved only in error-free postreplication repair has not been reported. We cloned the MMS2 gene from a yeast genomic library by functional complementation of the mms2-1 mutant [Prakash, L. and Prakash, S. (1977) Genetics 86, 33-55]. MMS2 encodes a 137-amino acid, 15.2-kDa protein with significant sequence homology to a conserved family of ubiquitin-conjugating (Ubc) proteins. However, Mms2 does not appear to possess Ubc activity. Genetic analyses indicate that the mms2 mutation is hypostatic to rad6 and rad18 but is synergistic with the rev3 mutation, and the mms2 mutant is proficient in UV-induced mutagenesis. These phenotypes are reminiscent of a pol30-46 mutant known to be impaired in postreplication repair. The mms2 mutant also displayed a REV3-dependent mutator phenotype, strongly suggesting that the MMS2 gene functions in the error-free postreplication repair pathway, parallel to the REV3 mutagenesis pathway. Furthermore, with respect to UV sensitivity, mms2 was found to be hypostatic to the rad6 delta 1-9 mutation, which results in the absence of the first nine amino acids of Rad6. On the basis of these collective results, we propose that the mms2 null mutation and two other allele-specific mutations, rad6 delta 1-9 and pol30-46, define the error-free mode of DNA postreplication repair, and that these mutations may enhance both spontaneous and DNA damage-induced mutagenesis

Unique Helicase Determinants in the Essential Conjugative TraI Factor from Salmonella enterica Serovar Typhimurium Plasmid pCU1

Energy Technology Data Exchange (ETDEWEB)

McLaughlin, Krystle J.; Nash, Rebekah P.; Redinbo, Mathew R. (UNC)

2014-06-16

The widespread development of multidrug-resistant bacteria is a major health emergency. Conjugative DNA plasmids, which harbor a wide range of antibiotic resistance genes, also encode the protein factors necessary to orchestrate the propagation of plasmid DNA between bacterial cells through conjugative transfer. Successful conjugative DNA transfer depends on key catalytic components to nick one strand of the duplex DNA plasmid and separate the DNA strands while cell-to-cell transfer occurs. The TraI protein from the conjugative Salmonella plasmid pCU1 fulfills these key catalytic roles, as it contains both single-stranded DNA-nicking relaxase and ATP-dependent helicase domains within a single, 1,078-residue polypeptide. In this work, we unraveled the helicase determinants of Salmonella pCU1 TraI through DNA binding, ATPase, and DNA strand separation assays. TraI binds DNA substrates with high affinity in a manner influenced by nucleic acid length and the presence of a DNA hairpin structure adjacent to the nick site. TraI selectively hydrolyzes ATP, and mutations in conserved helicase motifs eliminate ATPase activity. Surprisingly, the absence of a relatively short (144-residue) domain at the extreme C terminus of the protein severely diminishes ATP-dependent strand separation. Collectively, these data define the helicase motifs of the conjugative factor TraI from Salmonella pCU1 and reveal a previously uncharacterized C-terminal functional domain that uncouples ATP hydrolysis from strand separation activity.

Identification and characterization of an oleate hydratase-encoding gene from Bifidobacterium breve.

Science.gov (United States)

O'Connell, Kerry Joan; Motherway, Mary O'Connell; Hennessey, Alan A; Brodhun, Florian; Ross, R Paul; Feussner, Ivo; Stanton, Catherine; Fitzgerald, Gerald F; van Sinderen, Douwe

2013-01-01

Bifidobacteria are common commensals of the mammalian gastrointestinal tract. Previous studies have suggested that a bifidobacterial myosin cross reactive antigen (MCRA) protein plays a role in bacterial stress tolerance, while this protein has also been linked to the biosynthesis of conjugated linoleic acid (CLA) in bifidobacteria. In order to increase our understanding on the role of MCRA in bifidobacteria we created and analyzed an insertion mutant of the MCRA-encoding gene of B. breve NCFB 2258. Our results demonstrate that the MCRA protein of B. breve NCFB 2258 does not appear to play a role in CLA production, yet is an oleate hydratase, which contributes to bifidobacterial solvent stress protection.

Qualidade conjugal: mapeando conceitos

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Clarisse Mosmann

2006-12-01

Full Text Available Apesar da ampla utilização do conceito de qualidade conjugal, identifica-se falta de clareza conceitual acerca das variáveis que o compõem. Esse artigo apresenta revisão da literatura na área com o objetivo de mapear o conceito de qualidade conjugal. Foram analisadas sete principais teorias sobre o tema: Troca Social, Comportamental, Apego, Teoria da Crise, Interacionismo Simbólico. Pelos postulados propostos nas diferentes teorias, podem-se identificar três grupos de variáveis fundamentais na definição da qualidade conjugal: recursos pessoais dos cônjuges, contexto de inserção do casal e processos adaptativos. Neste sentido, a qualidade conjugal é resultado do processo dinâmico e interativo do casal, razão deste caráter multidimensional.

Newly identified protein Imi1 affects mitochondrial integrity and glutathione homeostasis in Saccharomyces cerevisiae.

Science.gov (United States)

Kowalec, Piotr; Grynberg, Marcin; Pająk, Beata; Socha, Anna; Winiarska, Katarzyna; Fronk, Jan; Kurlandzka, Anna

2015-09-01

Glutathione homeostasis is crucial for cell functioning. We describe a novel Imi1 protein of Saccharomyces cerevisiae affecting mitochondrial integrity and involved in controlling glutathione level. Imi1 is cytoplasmic and, except for its N-terminal Flo11 domain, has a distinct solenoid structure. A lack of Imi1 leads to mitochondrial lesions comprising aberrant morphology of cristae and multifarious mtDNA rearrangements and impaired respiration. The mitochondrial malfunctioning is coupled to significantly decrease the level of intracellular reduced glutathione without affecting oxidized glutathione, which decreases the reduced/oxidized glutathione ratio. These defects are accompanied by decreased cadmium sensitivity and increased phytochelatin-2 level. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

CHANGES IN THE GLUTATHIONE SYSTEM IN P19 EMBRYONAL CARCINOMA CELLS UNDER HYPOXIC CONDITIONS

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D. S. Orlov

2015-01-01

Full Text Available Introduction. According to modern perceptions, tumor growth, along with oxidative stress formation, is accompanied by hypoxia. Nowadays studying the regulation of cellular molecular system functioning by conformational changes in proteins appears to be a topical issue. Research goal was to evaluate the state of the glutathione system and the level of protein glutathionylation in P19 embryonal carcinoma (EC cells under hypoxic conditions.Material and methods. P19 EC cells (mouse embryonal carcinoma cultured under normoxic and hypox-ic conditions served the research material.The concentration of total, oxidized, reduced and protein-bound glutathione, the reduced to oxidized thiol ratio as well as glutathione peroxidase and glutathione reductase activity were determined by spectropho-tometry.Results. Glutathione imbalance was accompanied by a decrease in P19 EC cell redox status under hypox-ic conditions against the backdrop of a rise in protein-bound glutathione.Conclusions. As a result of the conducted study oxidative stress formation was identified when modeling hypoxia in P19 embryonal carcinoma cells. The rise in the concentration of protein-bound glutathione may indicate the role of protein glutathionylation in regulation of P19 cell metabolism and functions un-der hypoxia. 

Glutathione system participation in thoracic aneurysms from patients with Marfan syndrome.

Science.gov (United States)

Zúñiga-Muñoz, Alejandra María; Pérez-Torres, Israel; Guarner-Lans, Verónica; Núñez-Garrido, Elías; Velázquez Espejel, Rodrigo; Huesca-Gómez, Claudia; Gamboa-Ávila, Ricardo; Soto, María Elena

2017-05-01

Aortic dilatation in Marfan syndrome (MFS) is progressive. It is associated with oxidative stress and endothelial dysfunction that contribute to the early acute dissection of the vessel and can result in rupture of the aorta and sudden death. We evaluated the participation of the glutathione (GSH) system, which could be involved in the mechanisms that promote the formation and progression of the aortic aneurysms in MFS patients. Aortic aneurysm tissue was obtained during chest surgery from eight control subjects and 14 MFS patients. Spectrophotometrical determination of activity of glutathione peroxidase (GPx), glutathione-S-transferase (GST), glutathione reductase (GR), lipid peroxidation (LPO) index, carbonylation, total antioxidant capacity (TAC), and concentration of reduced and oxidized glutathione (GSH and GSSG respectively), was performed in the homogenate from aortic aneurysm tissue. LPO index, carbonylation, TGF-β1, and GR activity were increased in MFS patients (p < 0.04), while TAC, GSH/GSSG ratio, GPx, and GST activity were significantly decreased (p < 0.04). The depletion of GSH, in spite of the elevated activity of GR, not only diminished the activity of GSH-depend GST and GPx, but increased LPO, carbonylation and decreased TAC. These changes could promote the structural and functional alterations in the thoracic aorta of MFS patients.

Cross-Conjugated n-Dopable Aromatic Polyketone

NARCIS (Netherlands)

Voortman, Thomas P.; Bartesaghi, Davide; Koster, L. Jan Anton; Chiechi, Ryan C.

2015-01-01

This paper describes the synthesis and characterization of a high molecular weight cross-conjugated polyketone synthesized via scalable Friedel Crafts chemistry. Cross-conjugated polyketones are precursors to conjugated polyions; they become orders of magnitude more conductive after a two-electron

The effect of excimer laser keratectomy on corneal glutathione-related enzymes in rabbits.

Science.gov (United States)

Bilgihan, Ayşe; Bilgihan, Kamil; Yis, Ozgür; Yis, Nilgün Safak; Hasanreisoglu, Berati

2003-04-01

Glutathione related enzymes are involved in the metabolism and detoxification of cytotoxic and carcinogenic compounds as well as reactive oxygen species. Excimer laser is a very useful tool for the treatment of refractive errors and removing superficial corneal opacities. Previous studies have shown that excimer laser may initiate free radical formation in the cornea. In the present study, we evaluated the effect of excimer laser keratectomy on corneal glutathione-related enzyme activities in rabbits. Animals were divided into five groups, and all groups were compared with the controls (group 1), after epithelial scraping (group 2), transepithelial photorefractive keratectomy (PRK) (group 3), traditional PRK (group 4) and deep traditional PRK (group 5). Corneal glutathione peroxidase (GPx), glutathione S-transferase (GST) and glutathione reductase (GR) activities were measured after 24h. Corneal GPx and GR activities significantly decreased only in group 5 (p < 0.05) but GST activities significantly decreased in all groups when compared with the control group (p < 0.05). In conclusion, excimer laser inhibits the glutathione dependent defense system in the cornea, this effect becomes more prominent after high doses of excimer laser energy and antioxidants may be useful to reduce free radical mediated complications.

Glutathione modulation in cancer treatment: will it work

International Nuclear Information System (INIS)

Mitchell, J.B.; Cook, J.A.; DeGraff, W.; Glatstein, E.; Russo, A.

1989-01-01

Glutathione (GSH) assumes a pivotal role in numerous cellular functions including bioreductive reactions, maintenance of enzyme activity, amino acid transport, protection from harmful oxidative species, and detoxification of xenobiotics. The importance of GSH in modifying the cellular response to several anti-cancer treatment modalities has become better appreciated with the introduction of agents which can either decrease or elevate GSH levels in cells and tissues. In general, GSH depletion has been demonstrated to further enhance the cytotoxicity of several chemotherapy drugs and nitroimidazole hypoxic cell radiosensitizers. Conversely, GSH elevation affords varying degrees of protection. Whether or not GSH modulating agents will be useful as an adjuvant to selected cancer treatment modalities will depend on whether differential levels of GSH can be achieved in tumor versus normal tissues. Accurate GSH measurements in tumor and normal tissues will be required to adequately use and interpret the results of clinical studies where GSH modulating agents are employed. Precise tumor GSH measurements pose a considerable challenge due to the complicated cellular makeup of tumors.44 references

Block-conjugate-gradient method

International Nuclear Information System (INIS)

McCarthy, J.F.

1989-01-01

It is shown that by using the block-conjugate-gradient method several, say s, columns of the inverse Kogut-Susskind fermion matrix can be found simultaneously, in less time than it would take to run the standard conjugate-gradient algorithm s times. The method improves in efficiency relative to the standard conjugate-gradient algorithm as the fermion mass is decreased and as the value of the coupling is pushed to its limit before the finite-size effects become important. Thus it is potentially useful for measuring propagators in large lattice-gauge-theory calculations of the particle spectrum

Unraveling the regulatory network of IncA/C plasmid mobilization: When genomic islands hijack conjugative elements.

Science.gov (United States)

Carraro, Nicolas; Matteau, Dominick; Burrus, Vincent; Rodrigue, Sébastien

2015-01-01

Conjugative plasmids of the A/C incompatibility group (IncA/C) have become substantial players in the dissemination of multidrug resistance. These large conjugative plasmids are characterized by their broad host-range, extended spectrum of antimicrobials resistance, and prevalence in enteric bacteria recovered from both environmental and clinical settings. Until recently, relatively little was known about the basic biology of IncA/C plasmids, mostly because of the hindrance of multidrug resistance for molecular biology experiments. To circumvent this issue, we previously developed pVCR94ΔX, a convenient prototype that codes for a reduced set of antibiotic resistances. Using pVCR94ΔX, we then characterized the regulatory pathway governing IncA/C plasmid dissemination. We found that the expression of roughly 2 thirds of the genes encoded by this plasmid, including large operons involved in the conjugation process, depends on an FlhCD-like master activator called AcaCD. Beyond the mobility of IncA/C plasmids, AcaCD was also shown to play a key role in the mobilization of different classes of genomic islands (GIs) identified in various pathogenic bacteria. By doing so, IncA/C plasmids can have a considerable impact on bacterial genomes plasticity and evolution.

Correlation between serum and peritoneal fluid glutathione S-transferases T1 concentration with different stages of endometriosis

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Sohail Mashayekhi

2018-03-01

Full Text Available Endometriosis is a gynecological disease defined by the histological presence of endometrial glands and stroma outside the uterine cavity. Ectopic endometrial cell proliferation and chronic inflammation in endometriosis were shown to be associated with oxidative stress (OS induction. OS is a condition in which reactive oxygen species (ROS overproduction and antioxidant deficiency cause a shift in oxidant/antioxidant balance. Glutathione S-transferases (GSTs comprise a family of eukaryotic and prokaryotic phase II metabolic isozymes best known for their ability to catalyze the conjugation of the reduced form of glutathione (GSH to xenobiotic substrates for the purpose of detoxification. The aim of this project was to study the concentrations of GSTT1 in the serum and peritoneal fluid (PF of patients with different stages of endometriosis. Frothy two PF and serum from normal and 152 from different stages of patients with endometriosis (stage I: n = 30, stage II: n = 39, stage III: n = 43 and stage IV: n = 40 were included in this study. The level of GSTT1 in the serum was determined by enzyme linked immunosorbent assay (ELISA. The results showed the presence of GSTT1 in all serum and peritoneal fluid samples, while, starting from stages I to IV endometriosis, a significant decrease in GSTT1 concentration was seen as compared to controls. It is concluded that levels of GSTT1 is negatively correlated with advanced stages of endometriosis. It is also suggested that the detection of serum and/or peritoneal fluid GSTT1 concentration may be valuable in the classifying of endometriosis.

Stress hormones promote growth of B16-F10 melanoma metastases: an interleukin 6- and glutathione-dependent mechanism

OpenAIRE

Valles, Soraya L; Benlloch, Mar?a; Rodriguez, Mar?a L; Mena, Salvador; Pellicer, Jos? A; Asensi, Miguel; Obrador, Elena; Estrela, Jos? M

2013-01-01

[EN] Background: Interleukin (IL)-6 (mainly of tumor origin) activates glutathione (GSH) release from hepatocytes and its interorgan transport to B16-F10 melanoma metastatic foci. We studied if this capacity to overproduce IL-6 is regulated by cancer cell-independent mechanisms. Methods: Murine B16-F10 melanoma cells were cultured, transfected with red fluorescent protein, injected i.v. into syngenic C57BL/6J mice to generate lung and liver metastases, and isolated from metastatic f...

Reduced glutathione and glutathione disulfide in the blood of glucose-6-phosphate dehydrogenase-deficient newborns.

Science.gov (United States)

Gong, Zhen-Hua; Tian, Guo-Li; Huang, Qi-Wei; Wang, Yan-Min; Xu, Hong-Ping

2017-07-20

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is commonly detected during mass screening for neonatal disease. We developed a method to measure reduced glutathione (GSH) and glutathione disulfide (GSSG) using tandem mass spectrometry (MS/MS) for detecting G6PD deficiency. The concentration of GSH and the GSH/GSSG ratio in newborn dry-blood-spot (DBS) screening and in blood plus sodium citrate for test confirmation were examined by MS/MS using labeled glycine as an internal standard. G6PD-deficient newborns had a lower GSH content (242.9 ± 15.9 μmol/L)and GSH/GSSG ratio (14.9 ± 7.2) than neonatal controls (370.0 ± 53.2 μmol/L and 46.7 ± 19.6, respectively). Although the results showed a significance of P blood measured using MS/MS on the first day of sample preparation are consistent with G6PD activity and are helpful for diagnosing G6PD deficiency.

Protein carriers of conjugate vaccines

Science.gov (United States)

Pichichero, Michael E

2013-01-01

The immunogenicity of polysaccharides as human vaccines was enhanced by coupling to protein carriers. Conjugation transformed the T cell-independent polysaccharide vaccines of the past to T cell-dependent antigenic vaccines that were much more immunogenic and launched a renaissance in vaccinology. This review discusses the conjugate vaccines for prevention of infections caused by Hemophilus influenzae type b, Streptococcus pneumoniae, and Neisseria meningitidis. Specifically, the characteristics of the proteins used in the construction of the vaccines including CRM, tetanus toxoid, diphtheria toxoid, Neisseria meningitidis outer membrane complex, and Hemophilus influenzae protein D are discussed. The studies that established differences among and key features of conjugate vaccines including immunologic memory induction, reduction of nasopharyngeal colonization and herd immunity, and antibody avidity and avidity maturation are presented. Studies of dose, schedule, response to boosters, of single protein carriers with single and multiple polysaccharides, of multiple protein carriers with multiple polysaccharides and conjugate vaccines administered concurrently with other vaccines are discussed along with undesirable consequences of conjugate vaccines. The clear benefits of conjugate vaccines in improving the protective responses of the immature immune systems of young infants and the senescent immune systems of the elderly have been made clear and opened the way to development of additional vaccines using this technology for future vaccine products. PMID:23955057

Guanidinylated Neomycin Conjugation Enhances Intranasal Enzyme Replacement in the Brain.

Science.gov (United States)

Tong, Wenyong; Dwyer, Chrissa A; Thacker, Bryan E; Glass, Charles A; Brown, Jillian R; Hamill, Kristina; Moremen, Kelley W; Sarrazin, Stéphane; Gordts, Philip L S M; Dozier, Lara E; Patrick, Gentry N; Tor, Yitzhak; Esko, Jeffrey D

2017-12-06

Iduronidase (IDUA)-deficient mice accumulate glycosaminoglycans in cells and tissues and exhibit many of the same neuropathological symptoms of patients suffering from Mucopolysaccharidosis I. Intravenous enzyme-replacement therapy for Mucopolysaccharidosis I ameliorates glycosaminoglycan storage and many of the somatic aspects of the disease but fails to treat neurological symptoms due to poor transport across the blood-brain barrier. In this study, we examined the delivery of IDUA conjugated to guanidinoneomycin (GNeo), a molecular transporter. GNeo-IDUA and IDUA injected intravenously resulted in reduced hepatic glycosaminoglycan accumulation but had no effect in the brain due to fast clearance from the circulation. In contrast, intranasally administered GNeo-IDUA entered the brain rapidly. Repetitive intranasal treatment with GNeo-IDUA reduced glycosaminoglycan storage, lysosome size and number, and neurodegenerative astrogliosis in the olfactory bulb and primary somatosensory cortex, whereas IDUA was less effective. The enhanced efficacy of GNeo-IDUA was not the result of increased nose-to-brain delivery or enzyme stability, but rather due to more efficient uptake into neurons and astrocytes. GNeo conjugation also enhanced glycosaminoglycan clearance by intranasally delivered sulfamidase to the brain of sulfamidase-deficient mice, a model of Mucopolysaccharidosis IIIA. These findings suggest the general utility of the guanidinoglycoside-based delivery system for restoring missing lysosomal enzymes in the brain. Copyright © 2017 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.

Conjugated polymer zwitterions and solar cells comprising conjugated polymer zwitterions

Science.gov (United States)

Emrick, Todd; Russell, Thomas; Page, Zachariah; Liu, Yao

2018-06-05

A conjugated polymer zwitterion includes repeating units having structure (I), (II), or a combination thereof ##STR00001## wherein Ar is independently at each occurrence a divalent substituted or unsubstituted C3-30 arylene or heteroarylene group; L is independently at each occurrence a divalent C1-16 alkylene group, C6-30arylene or heteroarylene group, or alkylene oxide group; and R1 is independently at each occurrence a zwitterion. A polymer solar cell including the conjugated polymer zwitterion is also disclosed.

Efficacy of peptide nucleic acid and selected conjugates against specific cellular pathologies of amyotrophic lateral sclerosis.

Science.gov (United States)

Browne, Elisse C; Parakh, Sonam; Duncan, Luke F; Langford, Steven J; Atkin, Julie D; Abbott, Belinda M

2016-04-01

Cellular studies have been undertaken on a nonamer peptide nucleic acid (PNA) sequence, which binds to mRNA encoding superoxide dismutase 1, and a series of peptide nucleic acids conjugated to synthetic lipophilic vitamin analogs including a recently prepared menadione (vitamin K) analog. Reduction of both mutant superoxide dismutase 1 inclusion formation and endoplasmic reticulum stress, two of the key cellular pathological hallmarks in amyotrophic lateral sclerosis, by two of the prepared PNA oligomers is reported for the first time. Crown Copyright © 2016. Published by Elsevier Ltd. All rights reserved.

Research study of conjugate materials; Conjugate material no chosa kenkyu

Energy Technology Data Exchange (ETDEWEB)

NONE

1998-03-01

The paper reported an introductory research on possibilities of new glass `conjugate materials.` The report took up the structure and synthetic process of conjugate materials to be researched/developed, classified them according to structural elements on molecular, nanometer and cluster levels, and introduced the structures and functions. Further, as glasses with new functions to be proposed, the paper introduced transparent and high-strength glass used for houses and vehicles, light modulation glass which realizes energy saving and optical data processing, and environmentally functional glass which realizes environmental cleaning or high performance biosensor. An initial survey was also conducted on rights of intellectual property to be taken notice of in Japan and abroad in the present situation. Reports were summed up and introduced of Osaka National Research Institute, Electrotechnical Laboratory, and National Industrial Research Institute of Nagoya which are all carrying out leading studies of conjugate materials. 235 refs., 135 figs., 6 tabs.

Protocol for Evaluating the Permissiveness of Bacterial Communities Toward Conjugal Plasmids by Quantification and Isolation of Transconjugants

DEFF Research Database (Denmark)

Klümper, Uli; Dechesne, Arnaud; Smets, Barth F.

2014-01-01

may encode catabolic pathways, virulence factors, and antibiotic or metal resistances, it is of environmental, evolutionary, and medical relevance to track and monitor the fate of plasmids in mixed microbial community. When assessing the short-term and long-term implications of conjugal plasmid...... a gfp-tagged plasmid in a mCherry red fluorescently tagged donor strain repressing gfp expression. We take advantage of fluorescent marker genes to microscopically detect plasmid transfer events and use subsequent high-throughput fluorescence-activated cell sorting (FACS) to isolate...

Site-directed Mutagenesis of Cysteine Residues in Phi-class Glutathione S-transferase F3 from Oryza sativa

Energy Technology Data Exchange (ETDEWEB)

Jo, Hyunjoo; Lee, Juwon; Noh, Jinseok; Kong, Kwanghoon [Chung-Ang Univ., Seoul (Korea, Republic of)

2012-12-15

To elucidate the roles of cysteine residues in rice Phi-class GST F3, in this study, all three cysteine residues were replaced with alanine by site-directed mutagenesis in order to obtain mutants C22A, C73A and C77A. Three mutant enzymes were expressed in Escherichia coli and purified to electrophoretic homogeneity by affinity chromatography on immobilized GSH. The substitutions of Cys73 and Cys77 residues in OsGSTF3 with alanine did not affect the glutathione conjugation activities, showing non-essentiality of these residues. On the other hand, the substitution of Cys22 residue with alanine resulted in approximately a 60% loss of specific activity toward ethacrynic acid. Moreover, the K{sub m}{sup CDNB} value of the mutant C22A was approximately 2.2 fold larger than that of the wild type. From these results, the evolutionally conserved cysteine 22 residue seems to participate rather in the structural stability of the active site in OsGSTF3 by stabilizing the electrophilic substrates-binding site's conformation than in the substrate binding directly.

SnoVault and encodeD: A novel object-based storage system and applications to ENCODE metadata.

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Benjamin C Hitz

Full Text Available The Encyclopedia of DNA elements (ENCODE project is an ongoing collaborative effort to create a comprehensive catalog of functional elements initiated shortly after the completion of the Human Genome Project. The current database exceeds 6500 experiments across more than 450 cell lines and tissues using a wide array of experimental techniques to study the chromatin structure, regulatory and transcriptional landscape of the H. sapiens and M. musculus genomes. All ENCODE experimental data, metadata, and associated computational analyses are submitted to the ENCODE Data Coordination Center (DCC for validation, tracking, storage, unified processing, and distribution to community resources and the scientific community. As the volume of data increases, the identification and organization of experimental details becomes increasingly intricate and demands careful curation. The ENCODE DCC has created a general purpose software system, known as SnoVault, that supports metadata and file submission, a database used for metadata storage, web pages for displaying the metadata and a robust API for querying the metadata. The software is fully open-source, code and installation instructions can be found at: http://github.com/ENCODE-DCC/snovault/ (for the generic database and http://github.com/ENCODE-DCC/encoded/ to store genomic data in the manner of ENCODE. The core database engine, SnoVault (which is completely independent of ENCODE, genomic data, or bioinformatic data has been released as a separate Python package.

role of conjugated linoleic acid in the prevention of radiation hazard in male rats

International Nuclear Information System (INIS)

Hussien, E.M.; Osman, N.N.; Haggag, A.M.

2009-01-01

the objective of the present study was to examine the effect of conjugated linoleic acid (CLA) as a natural product in minimizing the radiation hazards. male rats were assigned to six groups each of 7 animals throughout six weeks, fed 1% CLA (wt/wt)added to commercial diet in the form of milk powder 182 g/kg diet. rats exposed to 6 Gy whole body gamma irradiation showed significant increase in total cholesterol (TC), low density lipoprotein cholesterol (LDL-C).triglycerides (TG), atherosclerosis index, total lipid (TL), phospholipids (ph-lipids), malondialdehyde (MDA), urea,creatinine, uric acid, calcium (Ca) and phosphorous levels associated with decrease in high density lipoprotein-cholesterol (HDL-C), activity of reduced glutathione (GSH), superoxide dismutase (SOD), catalase (CAT), total antioxidant status, body weight, testes weight and testosterone both irradiated and non-irradiated milk powder administrated to irradiated rat groups minimized the radiation damage in the assayed parameters indicating its beneficial role as a promising antioxidant in scavenging free radicals and reactive oxygen species

Cadmium induced changes in subcellular glutathione contents within glandular trichomes of Cucurbita pepo L.

Science.gov (United States)

Kolb, Dagmar; Müller, Maria; Zellnig, Günther; Zechmann, Bernd

2010-07-01

Plants cope with cadmium (Cd) stress by complexation with phytochelatins (Pc), metallothioneins and glutathione and sequestration within vacuoles. Especially glutathione was found to play a major role in Cd detoxification as Cd shows a high binding affinity towards thiols and as glutathione is a precursor for Pc synthesis. In the present study, we have used an immunohistochemical approach combined with computer-supported transmission electron microscopy in order to measure changes in the subcellular distribution of glutathione during Cd-stress in mesophyll cells and cells of different glandular trichomes (long and short stalked) of Cucurbita pepo L. subsp. pepo var. styriaca GREB: . Even though no ultrastructural alterations were observed in leaf and glandular trichome cells after the treatment of plants with 50 microM cadmium chloride (CdCl(2)) for 48 h, all cells showed a large decrease in glutathione contents. The strongest decrease was found in nuclei and the cytosol (up to 76%) in glandular trichomes which are considered as a major side of Cd accumulation in leaves. The ratio of glutathione between the cytosol and nuclei and the other cell compartments was strongly decreased only in glandular trichomes (more than 50%) indicating that glutathione in these two cell compartments is especially important for the detoxification of Cd in glandular trichomes. Additionally, these data indicate that large amounts of Cd are withdrawn from nuclei during Cd exposure. The present study gives a detailed insight into the compartment-specific importance of glutathione during Cd exposure in mesophyll cells and glandular trichomes of C. pepo L. plants.

Glutathione provides a source of cysteine essential for intracellular multiplication of Francisella tularensis.

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Khaled Alkhuder

2009-01-01

Full Text Available Francisella tularensis is a highly infectious bacterium causing the zoonotic disease tularemia. Its ability to multiply and survive in macrophages is critical for its virulence. By screening a bank of HimarFT transposon mutants of the F. tularensis live vaccine strain (LVS to isolate intracellular growth-deficient mutants, we selected one mutant in a gene encoding a putative gamma-glutamyl transpeptidase (GGT. This gene (FTL_0766 was hence designated ggt. The mutant strain showed impaired intracellular multiplication and was strongly attenuated for virulence in mice. Here we present evidence that the GGT activity of F. tularensis allows utilization of glutathione (GSH, gamma-glutamyl-cysteinyl-glycine and gamma-glutamyl-cysteine dipeptide as cysteine sources to ensure intracellular growth. This is the first demonstration of the essential role of a nutrient acquisition system in the intracellular multiplication of F. tularensis. GSH is the most abundant source of cysteine in the host cytosol. Thus, the capacity this intracellular bacterial pathogen has evolved to utilize the available GSH, as a source of cysteine in the host cytosol, constitutes a paradigm of bacteria-host adaptation.

Modelling conjugation with stochastic differential equations

DEFF Research Database (Denmark)

Philipsen, Kirsten Riber; Christiansen, Lasse Engbo; Hasman, Henrik

2010-01-01

Enterococcus faecium strains in a rich exhaustible media. The model contains a new expression for a substrate dependent conjugation rate. A maximum likelihood based method is used to estimate the model parameters. Different models including different noise structure for the system and observations are compared......Conjugation is an important mechanism involved in the transfer of resistance between bacteria. In this article a stochastic differential equation based model consisting of a continuous time state equation and a discrete time measurement equation is introduced to model growth and conjugation of two...... using a likelihood-ratio test and Akaike's information criterion. Experiments indicating conjugation on the agar plates selecting for transconjugants motivates the introduction of an extended model, for which conjugation on the agar plate is described in the measurement equation. This model is compared...

Structures of a putative ζ-class glutathione S-transferase from the pathogenic fungus Coccidioides immitis

International Nuclear Information System (INIS)

Edwards, Thomas E.; Bryan, Cassie M.; Leibly, David J.; Dieterich, Shellie H.; Abendroth, Jan; Sankaran, Banumathi; Sivam, Dhileep; Staker, Bart L.; Van Voorhis, Wesley C.; Myler, Peter J.; Stewart, Lance J.

2011-01-01

The pathogenic fungus C. immitis causes coccidioidomycosis, a potentially fatal disease. Here, apo and glutathione-bound crystal structures of a previously uncharacterized protein from C. immitis that appears to be a ζ-class glutathione S-transferase are presented. Coccidioides immitis is a pathogenic fungus populating the southwestern United States and is a causative agent of coccidioidomycosis, sometimes referred to as Valley Fever. Although the genome of this fungus has been sequenced, many operons are not properly annotated. Crystal structures are presented for a putative uncharacterized protein that shares sequence similarity with ζ-class glutathione S-transferases (GSTs) in both apo and glutathione-bound forms. The apo structure reveals a nonsymmetric homodimer with each protomer comprising two subdomains: a C-terminal helical domain and an N-terminal thioredoxin-like domain that is common to all GSTs. Half-site binding is observed in the glutathione-bound form. Considerable movement of some components of the active site relative to the glutathione-free form was observed, indicating an induced-fit mechanism for cofactor binding. The sequence homology, structure and half-site occupancy imply that the protein is a ζ-class glutathione S-transferase, a maleylacetoacetate isomerase (MAAI)

Parts of Speech in Non-typical Function: (Asymmetrical Encoding of Non-verbal Predicates in Erzya

Directory of Open Access Journals (Sweden)

Rigina Turunen

2011-01-01

Full Text Available Erzya non-verbal conjugation refers to symmetric paradigms in which non-verbal predicates behave morphosyntactically in a similar way to verbal predicates. Notably, though, non-verbal conjugational paradigms are asymmetric, which is seen as an outcome of paradigmatic neutralisation in less frequent/less typical contexts. For non-verbal predicates it is not obligatory to display the same amount of behavioural potential as it is for verbal predicates, and the lexical class of non-verbal predicate operates in such a way that adjectival predicates are more likely to be conjugated than nominals. Further, besides symmetric paradigms and constructions, in Erzya there are non-verbal predicate constructions which display a more overt structural encoding than do verbal ones, namely, copula constructions. Complexity in the domain of non-verbal predication in Erzya decreases the symmetry of the paradigms. Complexity increases in asymmetric constructions, as well as in paradigmatic neutralisation when non-verbal predicates cannot be inflected in all the tenses and moods occurring in verbal predication. The results would be the reverse if we were to measure complexity in terms of the morphological structure. The asymmetric features in non-verbal predication are motivated language-externally, because non-verbal predicates refer to states and occur less frequently as predicates than verbal categories. The symmetry of the paradigms and constructions is motivated language-internally: a grammatical system with fewer rules is economical.

Cosensitization Properties of Glutathione-Protected Au25 Cluster on Ruthenium Dye-Sensitized TiO2 Photoelectrode

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Kazuya Nakata

2013-01-01

Full Text Available Cosensitization by glutathione-protected Au25 clusters on Ru complex, N719-sensitized TiO2 photoelectrodes is demonstrated. Glutathione-protected Au25 clusters showed no significant changes in properties after adsorption onto TiO2 particles, as confirmed by optical absorption spectroscopy, transmission electron microscopy, and laser desorption/ionization mass spectrometry. Adsorption property of the glutathione-protected Au25 clusters depends on the pH, which affects the incident photon-to-current conversion efficiency (IPCE of the TiO2 photoelectrode containing Au25 clusters. When pH 7. The IPCE of a TiO2 photoelectrode sensitized by both glutathione-protected Au25 clusters and N719 was increased compared with photoelectrodes containing either glutathione-protected Au25 clusters or N719, which suggests that glutathione-protected Au25 clusters act as a coadsorbent for N719 on TiO2 photoelectrodes. This is also supported by the results that the IPCE of N719-sensitized TiO2 photoelectrodes increased upon addition of glutathione. Furthermore, cosensitization by glutathione-protected Au25 clusters on N719-sensitized TiO2 photoelectrodes allows that wavelength of photoelectric conversion was extended to the near infrared (NIR region. These results suggest that glutathione-protected Au25 clusters act not only as a coadsorbent to increase IPCE but also as an NIR-active sensitizer.

The maize glossy13 gene, cloned via BSR-Seq and Seq-walking encodes a putative ABC transporter required for the normal accumulation of epicuticular waxes.

Directory of Open Access Journals (Sweden)

Li Li

Full Text Available Aerial plant surfaces are covered by epicuticular waxes that among other purposes serve to control water loss. Maize glossy mutants originally identified by their "glossy" phenotypes exhibit alterations in the accumulation of epicuticular waxes. By combining data from a BSR-Seq experiment and the newly developed Seq-Walking technology, GRMZM2G118243 was identified as a strong candidate for being the glossy13 gene. The finding that multiple EMS-induced alleles contain premature stop codons in GRMZM2G118243, and the one knockout allele of gl13, validates the hypothesis that gene GRMZM2G118243 is gl13. Consistent with this, GRMZM2G118243 is an ortholog of AtABCG32 (Arabidopsis thaliana, HvABCG31 (barley and OsABCG31 (rice, which encode ABCG subfamily transporters involved in the trans-membrane transport of various secondary metabolites. We therefore hypothesize that gl13 is involved in the transport of epicuticular waxes onto the surfaces of seedling leaves.

Poly(2-oxazoline)-Antibiotic Conjugates with Penicillins.

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Schmidt, Martin; Bast, Livia K; Lanfer, Franziska; Richter, Lena; Hennes, Elisabeth; Seymen, Rana; Krumm, Christian; Tiller, Joerg C

2017-09-20

The conjugation of antibiotics with polymers is rarely done, but it might be a promising alternative to low-molecular-weight derivatization. The two penicillins penicillin G (PenG) and penicillin V (PenV) were attached to the end groups of different water-soluble poly(2-oxazoline)s (POx) via their carboxylic acid function. This ester group was shown to be more stable against hydrolysis than the β-lactam ring of the penicillins. The conjugates are still antimicrobially active and up to 20 times more stable against penicillinase catalyzed hydrolysis. The antibiotic activity of the conjugates against Staphylococcus aureus in the presence of penicillinase is up to 350 times higher compared with the free antibiotics. Conjugates with a second antimicrobial function, a dodecyltrimethylammonium group (DDA-X), at the starting end of the PenG and PenV POx conjugates are more antimicrobially active than the conjugates without DDA-X and show high activity in the presence of penicillinase. For example, the conjugates DDA-X-PEtOx-PenG and DDA-X-PEtOx-PenV are 200 to 350 times more active against S. aureus in the presence of penicillinase and almost as effective as the penicillinase stable cloxacollin (Clox) under these conditions. These conjugates show even greater activity compared to cloxacollin without this enzyme present. Further, both conjugates kill Escherichia coli more effectively than PenG and Clox.

Vectorization of agrochemicals: amino acid carriers are more efficient than sugar carriers to translocate phenylpyrrole conjugates in the Ricinus system.

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Wu, Hanxiang; Marhadour, Sophie; Lei, Zhi-Wei; Yang, Wen; Marivingt-Mounir, Cécile; Bonnemain, Jean-Louis; Chollet, Jean-François

2018-05-01

Producing quality food in sufficient quantity while using less agrochemical inputs will be one of the great challenges of the twenty-first century. One way of achieving this goal is to greatly reduce the doses of plant protection compounds by improving the targeting of pests to eradicate. Therefore, we developed a vectorization strategy to confer phloem mobility to fenpiclonil, a contact fungicide from the phenylpyrrole family used as a model molecule. It consists in coupling the antifungal compound to an amino acid or a sugar, so that the resulting conjugates are handled by active nutrient transport systems. The method of click chemistry was used to synthesize three conjugates combining fenpiclonil to glucose or glutamic acid with a spacer containing a triazole ring. Systemicity tests with the Ricinus model have shown that the amino acid promoiety was clearly more favorable to phloem mobility than that of glucose. In addition, the transport of the amino acid conjugate is carrier mediated since the derivative of the L series was about five times more concentrated in the phloem sap than its counterpart of the D series. The systemicity of the L-derivative is pH dependent and almost completely inhibited by the protonophore carbonyl cyanide 3-chlorophenylhydrazone (CCCP). These data suggest that the phloem transport of the L-derivative is governed by a stereospecific amino acid carrier system energized by the proton motive force.

A perillyl alcohol-conjugated analog of 3-bromopyruvate without cellular uptake dependency on monocarboxylate transporter 1 and with activity in 3-BP-resistant tumor cells.

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Chen, Thomas C; Yu, Jiali; Nouri Nigjeh, Eslam; Wang, Weijun; Myint, Phyo Thazin; Zandi, Ebrahim; Hofman, Florence M; Schönthal, Axel H

2017-08-01

The anticancer agent 3-bromopyruvate (3-BP) is viewed as a glycolytic inhibitor that preferentially kills glycolytic cancer cells through energy depletion. However, its cytotoxic activity is dependent on cellular drug import through transmembrane monocarboxylate transporter 1 (MCT-1), which restricts its anticancer potential to MCT-1-positive tumor cells. We created and characterized an MCT-1-independent analog of 3-BP, called NEO218. NEO218 was synthesized by covalently conjugating 3-BP to perillyl alcohol (POH), a natural monoterpene. The responses of various tumor cell lines to treatment with either compound were characterized in the presence or absence of supplemental pyruvate or antioxidants N-acetyl-cysteine (NAC) and glutathione (GSH). Drug effects on glyceraldehyde 3-phosphate dehydrogenase (GAPDH) enzyme activity were investigated by mass spectrometric analysis. The development of 3-BP resistance was investigated in MCT-1-positive HCT116 colon carcinoma cells in vitro. Our results show that NEO218: (i) pyruvylated GAPDH on all 4 of its cysteine residues and shut down enzymatic activity; (ii) severely lowered cellular ATP content below life-sustaining levels, and (iii) triggered rapid necrosis. Intriguingly, supplemental antioxidants effectively prevented cytotoxic activity of NEO218 as well as 3-BP, but supplemental pyruvate powerfully protected cells only from 3-BP, not from NEO218. Unlike 3-BP, NEO218 exerted its potent cytotoxic activity irrespective of cellular MCT-1 status. Treatment of HCT116 cells with 3-BP resulted in prompt development of resistance, based on the emergence of MCT-1-negative cells. This was not the case with NEO218, and highly 3-BP-resistant cells remained exquisitely sensitive to NEO218. Thus, our study identifies a mechanism by which tumor cells develop rapid resistance to 3-BP, and presents NEO218 as a superior agent not subject to this cellular defense. Furthermore, our results offer alternative interpretations of previously

Site-selective conjugation of an anticoagulant aptamer to recombinant albumins and maintenance of neonatal Fc receptor binding

DEFF Research Database (Denmark)

Schmøkel, Julie; Voldum, Anders; Tsakiridou, Georgia

2017-01-01

-linked aptamer to that of aptamer alone was found using an anticoagulant activity assay measuring temporal levels of activated partial thrombin. Covalent albumin-aptamer conjugation, however, substantially compromized binding to hFcRn, to 10% affinity of that of non-conjugated WT, determined by biolayer......-life, predominately facilitated by engagement with the cellular recycling neonatal Fc receptor (FcRn), and ligand transport properties of albumin promote it as an attractive candidate to improve the pharmacokinetic profile of aptamers. This study investigates the effect of Cys34 site-selective covalent attachment...... of a factor IXa anticoagulant aptamer on aptamer functionality and human FcRn (hFcRn) engagement using recombinant human albumin (rHA) of either a wild type (WT) or an engineered human FcRn high binding variant (HB). Albumin-aptamer conjugates, connected covalently through a heterobifunctional succinimidyl 4...

The influence of heroin abuse on glutathione-dependent enzymes in human brain.

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Gutowicz, Marzena; Kaźmierczak, Beata; Barańczyk-Kuźma, Anna

2011-01-01

Heroin is an illicit narcotic abused by millions of people worldwide. In our earlier studies we have shown that heroin intoxication changes the antioxidant status in human brain. In the present work we continued our studies by estimating the effect of heroin abuse on reduced glutathione (GSH) and enzymes related to this cofactor, such as glutathione S-transferase detoxifying electrophilics (GST) and organic peroxides (as Se-independent glutathione peroxidase-GSHPx), and Se-dependent glutathione peroxidase (Se-GSHPx) specific mainly for hydrogen peroxide. Studies were conducted on human brains obtained from autopsy of 9 heroin abusers and 8 controls. The level of GSH and the activity of glutathione-related enzymes were determined spectrophotometrically. The expression of GST pi on mRNA and protein level was studied by RT-PCR and Western blotting, respectively. The results indicated significant increase of GST and GSHPx activities, unchanged Se-GSHPx activity, and decreased level of GSH in frontal, temporal, parietal and occipital cortex, brain stem, hippocampus, and white matter of heroin abusers. GST pi expression was increased on both mRNA and protein levels, however the increase was lower in brain stem than in other regions. Heroin affects all regions of human brain, and especially brain stem. Its intoxication leads to an increase of organic rather then inorganic peroxides in various brain regions. Glutathione S-transferase plays an important role during heroin intoxication, however its protective effect is lower in brain stem than in brain cortex or hippocampus. Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.

Rhodamine B-conjugated encrypted vipericidin nonapeptide is a potent toxin to zebrafish and associated with in vitro cytotoxicity.

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Wang, Liang; Chan, Judy Y W; Rêgo, Juciane V; Chong, Cheong-Meng; Ai, Nana; Falcão, Cláudio B; Rádis-Baptista, Gandhi; Lee, Simon M Y

2015-06-01

Animal venoms contain a diverse array of proteins and enzymes that are toxic toward various physiological systems. However, there are also some practical medicinal uses for these toxins including use as anti-bacterial and anti-tumor agents. In this study, we identified a nine-residue cryptic oligopeptide, KRFKKFFKK (EVP50) that is repeatedly encoded in tandem within vipericidin sequences. EVP50 displayed in vivo potent lethal toxicity to zebrafish larvae (LD50=6 μM) when the peptide's N-terminus was chemically conjugated to rhodamine B (RhoB). In vitro, RhoB-conjugated EVP50 (RhoB-EVP50) exhibited a concentration-dependent cytotoxic effect toward MCF-7 and MDA-MB-231 breast cancer cells. In MCF-7 cells, the RhoB-EVP50 nonapeptide accumulated inside the cells within minutes. In the cytoplasm, the RhoB-EVP50 induced extracellular calcium influx and intracellular calcium release. Membrane budding was also observed after incubation with micromolar concentrations of the fluorescent EVP50 conjugate. The conjugate's interference with calcium homeostasis, its intracellular accumulation and its induced membrane dysfunction (budding and vacuolization) seem to act in concert to disrupt the cell circuitry. Contrastively, unconjugated EVP50 peptide did not display neither toxic nor cytotoxic activities in our in vivo and in vitro models. The synergic mechanism of toxicity was restricted to the structurally modified encrypted vipericidin nonapeptide. Copyright © 2015 Elsevier B.V. All rights reserved.

Monitoring thioredoxin redox with a genetically encoded red fluorescent biosensor.

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Fan, Yichong; Makar, Merna; Wang, Michael X; Ai, Hui-Wang

2017-09-01

Thioredoxin (Trx) is one of the two major thiol antioxidants, playing essential roles in redox homeostasis and signaling. Despite its importance, there is a lack of methods for monitoring Trx redox dynamics in live cells, hindering a better understanding of physiological and pathological roles of the Trx redox system. In this work, we developed the first genetically encoded fluorescent biosensor for Trx redox by engineering a redox relay between the active-site cysteines of human Trx1 and rxRFP1, a redox-sensitive red fluorescent protein. We used the resultant biosensor-TrxRFP1-to selectively monitor perturbations of Trx redox in various mammalian cell lines. We subcellularly localized TrxRFP1 to image compartmentalized Trx redox changes. We further combined TrxRFP1 with a green fluorescent Grx1-roGFP2 biosensor to simultaneously monitor Trx and glutathione redox dynamics in live cells in response to chemical and physiologically relevant stimuli.

Approximate error conjugation gradient minimization methods

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Kallman, Jeffrey S

2013-05-21

In one embodiment, a method includes selecting a subset of rays from a set of all rays to use in an error calculation for a constrained conjugate gradient minimization problem, calculating an approximate error using the subset of rays, and calculating a minimum in a conjugate gradient direction based on the approximate error. In another embodiment, a system includes a processor for executing logic, logic for selecting a subset of rays from a set of all rays to use in an error calculation for a constrained conjugate gradient minimization problem, logic for calculating an approximate error using the subset of rays, and logic for calculating a minimum in a conjugate gradient direction based on the approximate error. In other embodiments, computer program products, methods, and systems are described capable of using approximate error in constrained conjugate gradient minimization problems.

CHARACTERIZATION OF DANSYLATED CYSTEINE, CYSTINE, GLUTATHIONE, AND GLUTATHIONE DISULFIDE BY NARROW BORE LIQUID CHROMATOGRAPHY - ELECTROSPRAY IONIZATION MASS SPECTROMETRY

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A method using reversed phase high performance liquid chromtography/electrospray ionization-mass spectrometry (RP-LC/ESI-MS) has been developed to confirm the dientity of dansylated derivatives of cysteine (C) and glutathione (GSH), and their respective dimers, cystine (CSSC) and...

Ursolic Acid-enriched herba cynomorii extract induces mitochondrial uncoupling and glutathione redox cycling through mitochondrial reactive oxygen species generation: protection against menadione cytotoxicity in h9c2 cells.

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Chen, Jihang; Wong, Hoi Shan; Ko, Kam Ming

2014-01-27

Herba Cynomorii (Cynomorium songaricum Rupr., Cynomoriaceae) is one of the most commonly used 'Yang-invigorating' tonic herbs in Traditional Chinese Medicine (TCM). An earlier study in our laboratory has demonstrated that HCY2, an ursolic acid-enriched fraction derived from Herba Cynomorii, increased mitochondrial ATP generation capacity (ATP-GC) and induced mitochondrial uncoupling as well as a cellular glutathione response, thereby protecting against oxidant injury in H9c2 cells. In this study, we demonstrated that pre-incubation of H9c2 cells with HCY2 increased mitochondrial reactive oxygen species (ROS) generation in these cells, which is likely an event secondary to the stimulation of the mitochondrial electron transport chain. The suppression of mitochondrial ROS by the antioxidant dimethylthiourea abrogated the HCY2-induced enhancement of mitochondrial uncoupling and glutathione reductase (GR)-mediated glutathione redox cycling, and also protected against menadione-induced cytotoxicity. Studies using specific inhibitors of uncoupling protein and GR suggested that the HCY2-induced mitochondrial uncoupling and glutathione redox cycling play a determining role in the cytoprotection against menadione-induced oxidant injury in H9c2 cells. Experimental evidence obtained thus far supports the causal role of HCY2-induced mitochondrial ROS production in eliciting mitochondrial uncoupling and glutathione antioxidant responses, which offer cytoprotection against oxidant injury in H9c2 cells.

Microbial cycling of isoprene, the most abundantly produced biological volatile organic compound on Earth.

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McGenity, Terry J; Crombie, Andrew T; Murrell, J Colin

2018-04-01

Isoprene (2-methyl-1,3-butadiene), the most abundantly produced biogenic volatile organic compound (BVOC) on Earth, is highly reactive and can have diverse and often detrimental atmospheric effects, which impact on climate and health. Most isoprene is produced by terrestrial plants, but (micro)algal production is important in aquatic environments, and the relative bacterial contribution remains unknown. Soils are a sink for isoprene, and bacteria that can use isoprene as a carbon and energy source have been cultivated and also identified using cultivation-independent methods from soils, leaves and coastal/marine environments. Bacteria belonging to the Actinobacteria are most frequently isolated and identified, and Proteobacteria have also been shown to degrade isoprene. In the freshwater-sediment isolate, Rhodococcus strain AD45, initial oxidation of isoprene to 1,2-epoxy-isoprene is catalyzed by a multicomponent isoprene monooxygenase encoded by the genes isoABCDEF. The resultant epoxide is converted to a glutathione conjugate by a glutathione S-transferase encoded by isoI, and further degraded by enzymes encoded by isoGHJ. Genome sequence analysis of actinobacterial isolates belonging to the genera Rhodococcus, Mycobacterium and Gordonia has revealed that isoABCDEF and isoGHIJ are linked in an operon, either on a plasmid or the chromosome. In Rhodococcus strain AD45 both isoprene and epoxy-isoprene induce a high level of transcription of 22 contiguous genes, including isoABCDEF and isoGHIJ. Sequence analysis of the isoA gene, encoding the large subunit of the oxygenase component of isoprene monooxygenase, from isolates has facilitated the development of PCR primers that are proving valuable in investigating the ecology of uncultivated isoprene-degrading bacteria.

Hydroquinone PBPK model refinement and application to dermal exposure.

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Poet, Torka S; Carlton, Betsy D; Deyo, James A; Hinderliter, Paul M

2010-11-01

A physiologically based pharmacokinetic (PBPK) model for hydroquinone (HQ) was refined to include an expanded description of HQ-glucuronide metabolites and a description of dermal exposures to support route-to-route and cross-species extrapolation. Total urinary excretion of metabolites from in vivo rat dermal exposures was used to estimate a percutaneous permeability coefficient (K(p); 3.6×10(-5) cm/h). The human in vivo K(p) was estimated to be 1.62×10(-4) cm/h, based on in vitro skin permeability data in rats and humans and rat in vivo values. The projected total multi-substituted glutathione (which was used as an internal dose surrogate for the toxic glutathione metabolites) was modeled following an exposure scenario based on submersion of both hands in a 5% aqueous solution of HQ (similar to black and white photographic developing solution) for 2 h, a worst-case exposure scenario. Total multi-substituted glutathione following this human dermal exposure scenario was several orders of magnitude lower than the internal total glutathione conjugates in rats following an oral exposure to the rat NOEL of 20 mg/kg. Thus, under more realistic human dermal exposure conditions, it is unlikely that toxic glutathione conjugates (primarily the di- and, to a lesser degree, the tri-glutathione conjugate) will reach significant levels in target tissues. Copyright © 2010. Published by Elsevier Ltd.

Fluorescence detection of glutathione and oxidized glutathione in blood with a NIR-excitable cyanine probe

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Liu, Chang-hui; Qi, Feng-pei; Wen, Fu-bin; Long, Li-ping; Liu, Ai-juan; Yang, Rong-hua

2018-04-01

Cyanine has been widely utilized as a near infrared (NIR) fluorophore for detection of glutathione (GSH). However, the excitation of most of the reported cyanine-based probes was less than 800 nm, which inevitably induce biological background absorption and lower the sensitivity, limiting their use for detection of GSH in blood samples. To address this issue, here, a heptamethine cyanine probe (DNIR), with a NIR excitation wavelength at 804 nm and a NIR emission wavelength at 832 nm, is employed for the detection of GSH and its oxidized form (GSSG) in blood. The probe displays excellent selectivity for GSH over GSSG and other amino acids, and rapid response to GSH, in particular a good property for indirect detection of GSSG in the presence of enzyme glutathione reductase and the reducing agent nicotinamideadenine dinucleotide phosphate, without further separation prior to fluorescent measurement. To the best of our knowledge, this is the first attempt to explore NIR fluorescent approach for the simultaneous assay of GSH and GSSG in blood. As such, we expect that our fluorescence sensors with both NIR excitation and NIR emission make this strategy suitable for the application in complex physiological systems.

High performance liquid chromatographic assay for the quantitation of total glutathione in plasma

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Abukhalaf, Imad K.; Silvestrov, Natalia A.; Menter, Julian M.; von Deutsch, Daniel A.; Bayorh, Mohamed A.; Socci, Robin R.; Ganafa, Agaba A.

2002-01-01

A simple and widely used homocysteine HPLC procedure was applied for the HPLC identification and quantitation of glutathione in plasma. The method, which utilizes SBDF as a derivatizing agent utilizes only 50 microl of sample volume. Linear quantitative response curve was generated for glutathione over a concentration range of 0.3125-62.50 micromol/l. Linear regression analysis of the standard curve exhibited correlation coefficient of 0.999. Limit of detection (LOD) and limit of quantitation (LOQ) values were 5.0 and 15 pmol, respectively. Glutathione recovery using this method was nearly complete (above 96%). Intra-assay and inter-assay precision studies reflected a high level of reliability and reproducibility of the method. The applicability of the method for the quantitation of glutathione was demonstrated successfully using human and rat plasma samples.

Identification and characterization of a selenium-dependent glutathione peroxidase in Setaria cervi

International Nuclear Information System (INIS)

Singh, Anchal; Rathaur, Sushma

2005-01-01

Setaria cervi a bovine filarial parasite secretes selenium glutathione peroxidase during in vitro cultivation. A significant amount of enzyme activity was detected in the somatic extract of different developmental stages of the parasite. Among different stages, microfilariae showed a higher level of selenium glutathione peroxidase activity followed by males then females. However, when the activity was compared in excretory secretory products of these stages males showed higher activity than microfilariae and female worms. The enzyme was purified from female somatic extract using a combination of glutathione agarose and gel filtration chromatography, which migrated as a single band of molecular mass ∼20 kDa. Selenium content of purified enzyme was estimated by atomic absorption spectroscopy and found to be 3.5 ng selenium/μg of protein. Further, inhibition of enzyme activity by potassium cyanide suggested the presence of selenium at the active site of enzyme. This is the first report of identification of selenium glutathione peroxidase from any filarial parasite

Effect of cystamine on rat tissue GSH level and glutathione reductase activity

International Nuclear Information System (INIS)

Kovarova, H.; Pulpanova, J.

1979-01-01

Reduced glutathione (GSH) level and glutathione reductase activity were determined by means of the spectrophotometric method in various rat tissues after i.p. administration of cystamine (50 mg/kg and 20 mg/kg). GSH amount dropped in the spleen and kidney at 10 and 20 min; following this interval, an increase of GSH level was observed in the liver at 20-30 min, in the spleen and kidney at 60 min after the treatment with a radioprotective cystamine dose (50 mg/kg). The changes in GSH level induced by a non-radioprotective cystamine dose (20 mg/kg) had an opposite tendency. The activity of glutathione reductase was decreased in all tissues studied. As to the mechanism of the radioprotective action, both the inactivation of glutathione reductase activity and the changes in GSH level seem to be the factors contributing to the radioprotective effect of cystamine by strengthening the cellular radioresistance. (orig.) 891 MG/orig. 892 RKD [de

A multidomain fusion protein in Listeria monocytogenes catalyzes the two primary activities for glutathione biosynthesis.

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Gopal, Shubha; Borovok, Ilya; Ofer, Amos; Yanku, Michaela; Cohen, Gerald; Goebel, Werner; Kreft, Jürgen; Aharonowitz, Yair

2005-06-01

Glutathione is the predominant low-molecular-weight peptide thiol present in living organisms and plays a key role in protecting cells against oxygen toxicity. Until now, glutathione synthesis was thought to occur solely through the consecutive action of two physically separate enzymes, gamma-glutamylcysteine ligase and glutathione synthetase. In this report we demonstrate that Listeria monocytogenes contains a novel multidomain protein (termed GshF) that carries out complete synthesis of glutathione. Evidence for this comes from experiments which showed that in vitro recombinant GshF directs the formation of glutathione from its constituent amino acids and the in vivo effect of a mutation in GshF that abolishes glutathione synthesis, results in accumulation of the intermediate gamma-glutamylcysteine, and causes hypersensitivity to oxidative agents. We identified GshF orthologs, consisting of a gamma-glutamylcysteine ligase (GshA) domain fused to an ATP-grasp domain, in 20 gram-positive and gram-negative bacteria. Remarkably, 95% of these bacteria are mammalian pathogens. A plausible origin for GshF-dependent glutathione biosynthesis in these bacteria was the recruitment by a GshA ancestor gene of an ATP-grasp gene and the subsequent spread of the fusion gene between mammalian hosts, most likely by horizontal gene transfer.

Preparation, structural analysis and bioactivity of ribonuclease A-albumin conjugate: tetra-conjugation or PEG as the linker.

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Li, Chunju; Lin, Qixun; Wang, Jun; Shen, Lijuan; Ma, Guanghui; Su, Zhiguo; Hu, Tao

2012-12-31

Ribonuclease A (RNase A) is a therapeutic enzyme with cytotoxic action against tumor cells. Its clinical application is limited by the short half-life and insufficient stability. Conjugation of albumin can overcome the limitation, whereas dramatically decrease the enzymatic activity of RNase A. Here, three strategies were proposed to prepare the RNase A-bovine serum albumin (BSA) conjugates. R-SMCC-B (a conjugate of four RNase A attached with one BSA) and R-PEG-B (a mono-conjugate) were prepared using Sulfo-SMCC (a short bifunctional linker) and mal-PEG-NHS (a bifunctional PEG), respectively. Mal-PEG-NHS and hexadecylamine (HDA) were used to prepare the mono-conjugate, R-HDA-B, where HDA was adopted to bind BSA. The PEG linker can elongate the proximity between RNase A and BSA. In contrast, four RNase A were closely located on BSA in R-SMCC-B. R-SMCC-B showed the lowest K(m) and the highest relative enzymatic activity and k(cat)/K(m) in the three conjugates. Presumably, the tetravalent interaction of RNase A in R-SMCC-B can increase the binding affinity to its substrate. In addition, the slow release of BSA from R-HDA-B may increase the enzymatic activity of R-HDA-B. Our study is expected to provide strategies to develop protein-albumin conjugate with high therapeutic potential. Copyright © 2012 Elsevier B.V. All rights reserved.

Elevated oxidized glutathione in cystinotic proximal tubular epithelial cells.

Science.gov (United States)

Wilmer, Martijn J G; de Graaf-Hess, Adriana; Blom, Henk J; Dijkman, Henry B P M; Monnens, Leo A; van den Heuvel, Lambertus P; Levtchenko, Elena N

2005-11-18

Cystinosis, the most frequent cause of inborn Fanconi syndrome, is characterized by the lysosomal cystine accumulation, caused by mutations in the CTNS gene. To elucidate the pathogenesis of cystinosis, we cultured proximal tubular cells from urine of cystinotic patients (n = 9) and healthy controls (n = 9), followed by immortalization with human papilloma virus (HPV E6/E7). Obtained cell lines displayed basolateral polarization, alkaline phosphatase activity, and presence of aminopeptidase N (CD-13) and megalin, confirming their proximal tubular origin. Cystinotic cell lines exhibited elevated cystine levels (0.86 +/- 0.95 nmol/mg versus 0.09 +/- 0.01 nmol/mg protein in controls, p = 0.03). Oxidized glutathione was elevated in cystinotic cells (1.16 +/- 0.83 nmol/mg versus 0.29 +/- 0.18 nmol/mg protein, p = 0.04), while total glutathione, free cysteine, and ATP contents were normal in these cells. In conclusion, elevated oxidized glutathione in cystinotic proximal tubular epithelial cell lines suggests increased oxidative stress, which may contribute to tubular dysfunction in cystinosis.

Bipolar and Unipolar Silylene-Diphenylene σ-π Conjugated Polymer Route for Highly Efficient Electrophosphorescence

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Chang, Yao-Tang; Sharma, Sunil; Hung, Miao-Ken; Lee, Yu-Hsuan; Chen, Show-An

2016-12-01

σ-π conjugated polymer strategy is proposed for designing electroluminescent host polymers with silylene-diphenylene as the backbone repeat unit giving a high triplet energy (ET = 2.67 eV). By incorporation of high ET (3.0 eV) electron (oxadiazole, OXD) and hole (triphenyl amine, TPA) transport moieties, or TPA alone (in this case, the main chain acts as electron transport channel) as side arms on the silylene, the high ET bipolar and unipolar polymers are formed, allowing a use of iridium green phosphor (Ir(ppy)2(acac), Ir-G) (ET = 2.40 eV) as the dopant. The matching of energy levels of the dopant with the hosts, leading to charge trapping into it; and singlets and triplets of the exciplex and excimer can be harvested via energy transfer to the dopant. Using these host-guest systems as the emitting layer, chlorinated indium-tin-oxide (Cl-ITO) as the anode, and benzimidazole derivative (TPBI) as the electron transport layer, this two-layer device gives the high luminance efficiency 80.1 cd/A and external quantum efficiency 21.2%, which is the best among the report values for polymer light emitting diode (PLED) in the literatures. This example manifests that σ-π conjugated polymer strategy is a promising route for designing polymer host for efficient electrophosphorescence.

Differential substrate behaviours of ethylene oxide and propylene oxide towards human glutathione transferase theta hGSTT1-1.

Science.gov (United States)

Thier, R; Wiebel, F A; Bolt, H M

1999-11-01

The transformation of ethylene oxide (EO), propylene oxide (PO) and 1-butylene oxide (1-BuO) by human glutathione transferase theta (hGSTT1-1) was studied comparatively using 'conjugator' (GSTT1 + individuals) erythrocyte lysates. The relative sequence of velocity of enzymic transformation was PO > EO > 1-BuO. The faster transformation of PO compared to EO was corroborated in studies with human and rat GSTT1-1 (hGSTT1-1 and rGSTT1-1, respectively) expressed by Salmonella typhimurium TA1535. This sequence of reactivities of homologous epoxides towards GSTT1-1 contrasts to the sequence observed in homologous alkyl halides (methyl bromide, MBr; ethyl bromide, EtBr; n-propyl bromide, PrBr) where the relative sequence MeBr > EtBr > PrBr is observed. The higher reactivity towards GSTT1-1 of propylene oxide compared to ethylene oxide is consistent with a higher chemical reactivity. This is corroborated by experimental data of acid-catalysed hydrolysis of a number of aliphatic epoxides, including ethylene oxide and propylene oxide and consistent with semi-empirical molecular orbital modelings.

Prolonged fasting increases glutathione biosynthesis in postweaned northern elephant seals

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Vázquez-Medina, José Pablo; Zenteno-Savín, Tania; Forman, Henry Jay; Crocker, Daniel E.; Ortiz, Rudy M.

2011-01-01

SUMMARY Northern elephant seals experience prolonged periods of absolute food and water deprivation (fasting) while breeding, molting or weaning. The postweaning fast in elephant seals is characterized by increases in the renin–angiotensin system, expression of the oxidant-producing protein Nox4, and NADPH oxidase activity; however, these increases are not correlated with increased oxidative damage or inflammation. Glutathione (GSH) is a potent reductant and a cofactor for glutathione peroxidases (GPx), glutathione-S transferases (GST) and 1-cys peroxiredoxin (PrxVI) and thus contributes to the removal of hydroperoxides, preventing oxidative damage. The effects of prolonged food deprivation on the GSH system are not well described in mammals. To test our hypothesis that GSH biosynthesis increases with fasting in postweaned elephant seals, we measured circulating and muscle GSH content at the early and late phases of the postweaning fast in elephant seals along with the activity/protein content of glutamate-cysteine ligase [GCL; catalytic (GCLc) and modulatory (GCLm) subunits], γ-glutamyl transpeptidase (GGT), glutathione disulphide reductase (GR), glucose-6-phosphate dehydrogenase (G6PDH), GST and PrxVI, as well as plasma changes in γ-glutamyl amino acids, glutamate and glutamine. GSH increased two- to four-fold with fasting along with a 40–50% increase in the content of GCLm and GCLc, a 75% increase in GGT activity, a two- to 2.5-fold increase in GR, G6PDH and GST activities and a 30% increase in PrxVI content. Plasma γ-glutamyl glutamine, γ-glutamyl isoleucine and γ-glutamyl methionine also increased with fasting whereas glutamate and glutamine decreased. Results indicate that GSH biosynthesis increases with fasting and that GSH contributes to counteracting hydroperoxide production, preventing oxidative damage in fasting seals. PMID:21430206

Chemical de-conjugation for investigating the stability of small molecule drugs in antibody-drug conjugates.

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Chen, Tao; Su, Dian; Gruenhagen, Jason; Gu, Christine; Li, Yi; Yehl, Peter; Chetwyn, Nik P; Medley, Colin D

2016-01-05

Antibody-drug conjugates (ADCs) offer new therapeutic options for advanced cancer patients through precision killing with fewer side effects. The stability and efficacy of ADCs are closely related, emphasizing the urgency and importance of gaining a comprehensive understanding of ADC stability. In this work, a chemical de-conjugation approach was developed to investigate the in-situ stability of the small molecule drug while it is conjugated to the antibody. This method involves chemical-mediated release of the small molecule drug from the ADC and subsequent characterization of the released small molecule drug by HPLC. The feasibility of this technique was demonstrated utilizing a model ADC containing a disulfide linker that is sensitive to the reducing environment within cancer cells. Five reducing agents were screened for use in de-conjugation; tris(2-carboxyethyl) phosphine (TCEP) was selected for further optimization due to its high efficiency and clean impurity profile. The optimized de-conjugation assay was shown to have excellent specificity and precision. More importantly, it was shown to be stability indicating, enabling the identification and quantification of the small molecule drug and its degradation products under different formulation pHs and storage temperatures. In summary, the chemical de-conjugation strategy demonstrated here offers a powerful tool to assess the in-situ stability of small molecule drugs on ADCs and the resulting information will shed light on ADC formulation/process development and storage condition selection. Copyright © 2015 Elsevier B.V. All rights reserved.

Depression of biliary glutathione excretion by chronic ethanol feeding in the rat

International Nuclear Information System (INIS)

Vendemiale, G.; Jayatilleke, E.; Shaw, S.; Lieber, C.S.

1984-01-01

The effects of chronic alcohol feeding on biliary glutathione excretion were studied in rats pair fed diets containing either ethanol (36% of total energy) or isocaloric carbohydrate for 4-6 weeks. An exteriorized biliary-duodenal fistula was established and total glutathione (GSH) and oxidized glutathione (GSSG) were measured. A significant decrease was observed in rats fed alcohol chronically compared to their pair fed controls in the biliary excretion of GSH (55.7 +/- 37.0 vs 243.1 +/- 29.0 μg/ml bile, p 35 -L-methionine incorporation into hepatic and biliary GSH was unchanged or even increased after chronic ethanol feeding. 22 references, 4 figures

ABC transporters in Arthropods: genomic comparison and role in insecticide transport and resistance

NARCIS (Netherlands)

Dermauw, W.; Van Leeuwen, T.

2014-01-01

About a 100 years ago, the Drosophila white mutant marked the birth of Drosophila genetics. The white gene turned out to encode the first well studied ABC transporter in arthropods. The ABC gene family is now recognized as one of the largest transporter families in all kingdoms of life. The majority

Auranofin-induced oxidative stress causes redistribution of the glutathione pool in Taenia crassiceps cysticerci.

Science.gov (United States)

Martínez-González, J J; Guevara-Flores, A; Rendón, J L; Arenal, I P Del

2015-05-01

Previously, we have studied the effect of the gold-compound auranofin (AF) on both thioredoxin-glutathione reductasa (TGR) activity and viability of Taenia crassiceps cysticerci. It was demonstrated that micromolar concentrations of AF were high enough to fully inhibit TGR and kill the parasites. In this work, the dynamics of changes in the glutathione pool of T. crassiceps cysticerci following the addition of AF, was analyzed. A dose-dependent decrease in the internal glutathione concentration, concomitant with an increase in ROS production was observed. These changes were simultaneous with the formation of glutathione-protein complexes and the export of glutathione disulfide (GSSG) to the culture medium. Incubation of cysticerci in the presence of both AF and N-acetyl cysteine (NAC) prevents all the above changes, maintaining cysticerci viability. By contrast, the presence of both AF and buthionine sulfoximine (BSO) resulted in a potentiation of the effects of the gold compound, jeopardizing cysticerci viability. These results suggest the lethal effect of AF on T. crassiceps cysticerci, observed at micromolar concentrations, can be explained as a consequence of major changes in the glutathione status, which results in a significant increase in the oxidative stress of the parasites. Copyright © 2015 Elsevier B.V. All rights reserved.

Photoluminescence in conjugated polymers

International Nuclear Information System (INIS)

Furst, J.E.; Laugesen, R.; Dastoor, P.; McNeill, C.

2002-01-01

Full text: Conjugated polymers combine the electronic and optical properties of semiconductors with the processability of polymers. They contain a sequence of alternate single and double carbon bonds so that the overlap of unhybridised p z orbitals creates a delocalised ρ system which gives semiconducting properties with p-bonding (valence) and p* -antibonding (conduction) bands. Photoluminesence (PL) in conjugated polymers results from the radiative decay of singlet excitons confined to a single chain. The present work is the first in a series of studies in our laboratory that will characterize the optical properties of conjugated polymers. The experiment involves the illumination of thin films of conjugated polymer with UV light (I=360 nm) and observing the subsequent fluorescence using a custom-built, fluorescence spectrometer. Photoluminesence spectra provide basic information about the structure of the polymer film. A typical spectrum is shown in the accompanying figure. The position of the first peak is related to the polymer chain length and resolved multiple vibronic peaks are an indication of film structure and morphology. We will also present results related to the optical degradation of these materials when exposed to air and UV light

Bio-Conjugates for Nanoscale Applications

DEFF Research Database (Denmark)

Villadsen, Klaus

Bio-conjugates for Nanoscale Applications is the title of this thesis, which covers three different projects in chemical bio-conjugation research, namely synthesis and applications of: Lipidated fluorescent peptides, carbohydrate oxime-azide linkers and N-aryl O-R2 oxyamine derivatives. Lipidated...

From Semi- to Full-Two-Dimensional Conjugated Side-Chain Design: A Way toward Comprehensive Solar Energy Absorption

Energy Technology Data Exchange (ETDEWEB)

Chao, Pengjie [Department; School; Wang, Huan [Department; Qu, Shiwei [Department; Mo, Daize [Department; Meng, Hong [School; Chen, Wei [Materials; Institute; He, Feng [Department

2017-12-05

Two polymers with fully two-dimensional (2D) conjugated side chains, 2D-PTB-Th and 2D-PTB-TTh, were synthesized and characterized through simultaneously integrating the 2D-TT and the 2D-BDT monomers onto the polymer backbone. Resulting from the synergistic effect from the conjugated side chains on both monomers, the two polymers showed remarkably efficient absorption of the sunlight and improved pi-pi intermolecular interactions for efficient charge carrier transport. The optimized bulk heterojunction device based on 2D-PTB-Th and PC71BM shows a higher PCE of 9.13% compared to PTB7-Th with a PCE of 8.26%, which corresponds to an approximately 10% improvement in solar energy conversion. The fully 2D-conjugated side-chain concept reported here developed a new molecular design strategy for polymer materials with enhanced sunlight absorption and efficient solar energy conversion.

Interplay of alternative conjugated pathways and steric interactions on the electronic and optical properties of donor-acceptor conjugated polymers

KAUST Repository

Lima, Igo T.; Risko, Chad; Aziz, Saadullah Gary; Da Silva Filho, Demé trio A Da Silva; Bredas, Jean-Luc

2014-01-01

Donor-acceptor π-conjugated copolymers are of interest for a wide range of electronic applications, including field-effect transistors and solar cells. Here, we present a density functional theory (DFT) study of the impact of varying the conjugation pathway on the geometric, electronic, and optical properties of donor-acceptor systems. We consider both linear ("in series"), traditional conjugation among the donor-acceptor moieties versus structures where the acceptor units are appended orthogonally to the linear, donor-only conjugated backbone. Long-range-corrected hybrid functionals are used in the investigation with the values of the tuned long-range separation parameters providing an estimate of the extent of conjugation as a function of the oligomer architecture. Considerable differences in the electronic and optical properties are determined as a function of the nature of the conjugation pathway, features that should be taken into account in the design of donor-acceptor copolymers.

Identification of arsenite-and arsenic diglutathione-binding proteins in human hepatocarcinoma cells

Energy Technology Data Exchange (ETDEWEB)

Mizumura, Ayano; Watanabe, Takayuki [Graduate School of Pharmaceutical Sciences, Chiba University, Yayoi, Inage, Chiba 263-8522 (Japan); Kobayashi, Yayoi [Graduate School of Pharmaceutical Sciences, Chiba University, Yayoi, Inage, Chiba 263-8522 (Japan); Environmental Health Sciences Division, National Institute for Environmental Studies, 16-2 Onogawa, Tsukuba, Ibaraki 305-8506 (Japan); Hirano, Seishiro [Graduate School of Pharmaceutical Sciences, Chiba University, Yayoi, Inage, Chiba 263-8522 (Japan); Research Center for Environmental Risk, National Institute for Environmental Studies, 16-2 Onogawa, Tsukuba, Ibaraki 305-8506 (Japan)

2010-01-15

It is generally accepted that trivalent arsenicals are more toxic than the corresponding pentavalent arsenicals, since trivalent arsenicals bind the thiol groups of biomolecules, leading to a deterioration in cellular functions. In the present study, we prepared three different arsenic-bound sepharoses and investigated the binding of hepatic cytosolic proteins to pentavalent, trivalent, and glutathione-conjugated trivalent arsenicals. SDS-PAGE showed no proteins bound to pentavalent arsenic specifically. In contrast, we found a number of proteins that have specific and high affinity for trivalent arsenic. Two of those proteins were identified: protein disulfide isomerase-related protein 5 (PDSIRP5) and peroxiredoxin 1/enhancer protein (PRX1/EP). These proteins have vicinal cysteines, as previously reported. In contrast, one of the prominent proteins that did not bind to trivalent arsenic was identified as calreticulin precursor. Although there are 3 cysteines in calreticulin precursor, two of the cysteines are spaced more than 25 amino acids apart. Five synthetic peptides containing 2 vicinal cysteines were prepared to study whether they would inhibit the binding of PDSIRP5, PRX1/EP, and other arsenic-binding proteins to trivalent arsenicals. Only two of the five peptides effectively inhibited binding, suggesting that other amino acids besides the 2 vicinal cysteines may modulate the affinity of cysteine-rich proteins for trivalent arsenicals. We further investigated hepatic cytosolic proteins that bound specifically to glutathione-conjugated trivalent arsenic, which is the most abundant form of arsenical in bile fluid. Four proteins that bound specifically to glutathione-conjugated trivalent arsenic were identified; interestingly, these proteins were different from the trivalent arsenic-binding proteins. These results suggest that although glutathione-conjugation is an important process in the metabolism, excretion, and detoxification of arsenicals, glutathione-conjugated

Glutathione--linking cell proliferation to oxidative stress.

Science.gov (United States)

Diaz-Vivancos, Pedro; de Simone, Ambra; Kiddle, Guy; Foyer, Christine H

2015-12-01

The multifaceted functions of reduced glutathione (gamma-glutamyl-cysteinyl-glycine; GSH) continue to fascinate plants and animal scientists, not least because of the dynamic relationships between GSH and reactive oxygen species (ROS) that underpin reduction/oxidation (redox) regulation and signalling. Here we consider the respective roles of ROS and GSH in the regulation of plant growth, with a particular focus on regulation of the plant cell cycle. Glutathione is discussed not only as a crucial low molecular weight redox buffer that shields nuclear processes against oxidative challenge but also a flexible regulator of genetic and epigenetic functions. The intracellular compartmentalization of GSH during the cell cycle is remarkably consistent in plants and animals. Moreover, measurements of in vivo glutathione redox potentials reveal that the cellular environment is much more reducing than predicted from GSH/GSSG ratios measured in tissue extracts. The redox potential of the cytosol and nuclei of non-dividing plant cells is about -300 mV. This relatively low redox potential maintained even in cells experiencing oxidative stress by a number of mechanisms including vacuolar sequestration of GSSG. We propose that regulated ROS production linked to glutathione-mediated signalling events are the hallmark of viable cells within a changing and challenging environment. The concept that the cell cycle in animals is subject to redox controls is well established but little is known about how ROS and GSH regulate this process in plants. However, it is increasingly likely that redox controls exist in plants, although possibly through different pathways. Moreover, redox-regulated proteins that function in cell cycle checkpoints remain to be identified in plants. While GSH-responsive genes have now been identified, the mechanisms that mediate and regulate protein glutathionylation in plants remain poorly defined. The nuclear GSH pool provides an appropriate redox environment

Highly conductive carbon nanotube buckypapers with improved doping stability via conjugational cross-linking.

Science.gov (United States)

Chen, I-Wen Peter; Liang, Richard; Zhao, Haibo; Wang, Ben; Zhang, Chuck

2011-12-02

Carbon nanotube (CNT) sheets or buckypapers have demonstrated promising electrical conductivity and mechanical performance. However, their electrical conductivity is still far below the requirements for engineering applications, such as using as a substitute for copper mesh, which is currently used in composite aircraft structures for lightning strike protection. In this study, different CNT buckypapers were stretched to increase their alignment, and then subjected to conjugational cross-linking via chemical functionalization. The conjugationally cross-linked buckypapers (CCL-BPs) demonstrated higher electrical conductivity of up to 6200 S cm( - 1), which is more than one order increase compared to the pristine buckypapers. The CCL-BPs also showed excellent doping stability in over 300 h in atmosphere and were resistant to degradation at elevated temperatures. The tensile strength of the stretched CCL-BPs reached 220 MPa, which is about three times that of pristine buckypapers. We attribute these property improvements to the effective and stable conjugational cross-links of CNTs, which can simultaneously improve the electrical conductivity, doping stability and mechanical properties. Specifically, the electrical conductivity increase resulted from improving the CNT alignment and inter-tube electron transport capability. The conjugational cross-links provide effective 3D conductive paths to increase the mobility of electrons among individual nanotubes. The stable covalent bonding also enhances the thermal stability and load transfer. The significant electrical and mechanical property improvement renders buckypaper a multifunctional material for various applications, such as conducting composites, battery electrodes, capacitors, etc.

The crucial protective role of glutathione against tienilic acid hepatotoxicity in rats

International Nuclear Information System (INIS)

Nishiya, Takayoshi; Mori, Kazuhiko; Hattori, Chiharu; Kai, Kiyonori; Kataoka, Hiroko; Masubuchi, Noriko; Jindo, Toshimasa; Manabe, Sunao

2008-01-01

To investigate the hepatotoxic potential of tienilic acid in vivo, we administered a single oral dose of tienilic acid to Sprague-Dawley rats and performed general clinicopathological examinations and hepatic gene expression analysis using Affymetrix microarrays. No change in the serum transaminases was noted at up to 1000 mg/kg, although slight elevation of the serum bile acid and bilirubin, and very mild hepatotoxic changes in morphology were observed. In contrast to the marginal clinicopathological changes, marked upregulation of the genes involved in glutathione biosynthesis [glutathione synthetase and glutamate-cysteine ligase (Gcl)], oxidative stress response [heme oxygenase-1 and NAD(P)H dehydrogenase quinone 1] and phase II drug metabolism (glutathione S-transferase and UDP glycosyltransferase 1A6) were noted after 3 or 6 h post-dosing. The hepatic reduced glutathione level decreased at 3-6 h, and then increased at 24 or 48 h, indicating that the upregulation of NF-E2-related factor 2 (Nrf2)-regulated gene and the late increase in hepatic glutathione are protective responses against the oxidative and/or electrophilic stresses caused by tienilic acid. In a subsequent experiment, tienilic acid in combination with L-buthionine-(S,R)-sulfoximine (BSO), an inhibitor of Gcl caused marked elevation of serum alanine aminotransferase (ALT) with extensive centrilobular hepatocyte necrosis, whereas BSO alone showed no hepatotoxicity. The elevation of ALT by this combination was observed at the same dose levels of tienilic acid as the upregulation of the Nrf2-regulated genes by tienilic acid alone. In conclusion, these results suggest that the impairment of glutathione biosynthesis may play a critical role in the development of tienilic acid hepatotoxicity through extensive oxidative and/or electrophilic stresses

Organometallic B12-DNA conjugate

DEFF Research Database (Denmark)

Hunger, Miriam; Mutti, Elena; Rieder, Alexander

2014-01-01

Design, synthesis, and structural characterization of a B12-octadecanucleotide are presented herein, a new organometallic B12-DNA conjugate. In such covalent conjugates, the natural B12 moiety may be a versatile vector for controlled in vivo delivery of oligonucleotides to cellular targets in hum...

The Conjugate Acid-Base Chart.

Science.gov (United States)

Treptow, Richard S.

1986-01-01

Discusses the difficulties that beginning chemistry students have in understanding acid-base chemistry. Describes the use of conjugate acid-base charts in helping students visualize the conjugate relationship. Addresses chart construction, metal ions, buffers and pH titrations, and the organic functional groups and nonaqueous solvents. (TW)

REVIEW ARTICLE Conjugated Hyperbilirubinaemia in Early Infancy ...

African Journals Online (AJOL)

REVIEW ARTICLE Conjugated Hyperbilirubinaemia in Early Infancy. AOK Johnson. Abstract. Conjugated hyperbilirubinaemia exists when the conjugated serum bilirubin level is more than 2 mg/dl or more than 20 per cent of the total serum bilirubin. It is always pathological in early infancy. The causes are many and diverse ...

Glutathione S-transferase gene polymorphisms in presbycusis.

Science.gov (United States)

Ateş, Nurcan Aras; Unal, Murat; Tamer, Lülüfer; Derici, Ebru; Karakaş, Sevim; Ercan, Bahadir; Pata, Yavuz Selim; Akbaş, Yücel; Vayisoğlu, Yusuf; Camdeviren, Handan

2005-05-01

Glutathione and glutathione-related antioxidant enzymes are involved in the metabolism and detoxification of cytotoxic and carcinogenic compounds as well as reactive oxygen species. Reactive oxygen species generation occurs in prolonged relative hypoperfusion conditions such as in aging. The etiology of presbycusis is much less certain; however, a complex genetic cause is most likely. The effect of aging shows a wide interindividual range; we aimed to investigate whether profiles of (glutathione S-transferase (GST) M1, T1 and P1 genotypes may be associated with the risk of age-related hearing loss. We examined 68 adults with presbycusis and 69 healthy controls. DNA was extracted from whole blood, and the GSTM1, GSTT1 and GSTP1 polymorphisms were determined using a real-time polymerase chain reaction and fluorescence resonance energy transfer with a Light-Cycler Instrument. Associations between specific genotypes and the development of presbycusis were examined by use of logistic regression analyses to calculate odds ratios and 95% confidence intervals. Gene polymorphisms at GSTM1, GSTT1, and GSTP1 in subjects with presbycusis were not significantly different than in the controls (p > 0.05). Also, the combinations of different GSTM1, GSTT1, and GSTP1 genotypes were not an increased risk of presbycusis (p > 0.05). We could not demonstrate any significant association between the GSTM1, GSTT1, and GSTP1 polymorphism and age-related hearing loss in this population. This may be because of our sample size, and further studies need to investigate the exact role of GST gene polymorphisms in the etiopathogenesis of the presbycusis.

Glutathione oxidation in response to intracellular H2O2: Key but overlapping roles for dehydroascorbate reductases.

Science.gov (United States)

Rahantaniaina, Marie-Sylviane; Li, Shengchun; Chatel-Innocenti, Gilles; Tuzet, Andrée; Mhamdi, Amna; Vanacker, Hélène; Noctor, Graham

2017-08-03

Glutathione is a pivotal molecule in oxidative stress, during which it is potentially oxidized by several pathways linked to H 2 O 2 detoxification. We have investigated the response and functional importance of 3 potential routes for glutathione oxidation pathways mediated by glutathione S-transferases (GST), glutaredoxin-dependent peroxiredoxins (PRXII), and dehydroascorbate reductases (DHAR) in Arabidopsis during oxidative stress. Loss-of-function gstU8, gstU24, gstF8, prxIIE and prxIIF mutants as well as double gstU8 gstU24, gstU8 gstF8, gstU24 gstF8, prxIIE prxIIF mutants were obtained. No mutant lines showed marked changes in their phenotype and glutathione profiles in comparison to the wild-type plants in either optimal conditions or oxidative stress triggered by catalase inhibition. By contrast, multiple loss of DHAR functions markedly decreased glutathione oxidation triggered by catalase deficiency. To assess whether this effect was mediated directly by loss of DHAR enzyme activity, or more indirectly by upregulation of other enzymes involved in glutathione and ascorbate recycling, we measured expression of glutathione reductase (GR) and expression and activity of monodehydroascorbate reductases (MDHAR). No evidence was obtained that either GRs or MDHARs were upregulated in plants lacking DHAR function. Hence, interplay between different DHARs appears to be necessary to couple ascorbate and glutathione pools and to allow glutathione-related signaling during enhanced H 2 O 2 metabolism.

Radioprotective effect of cysteamine in glutathione synthetase-deficient cells

International Nuclear Information System (INIS)

Deschavanne, P.J.; Debieu, D.; Malaise, E.P.; Midander, J.; Revesz, L.

1986-01-01

The radioprotective role of endogenous and exogenous thiols was investigated, with survival as the end-point, after radiation exposure of cells under oxic and hypoxic conditions. Human cell strains originating from a 5-oxoprolinuria patient and from a related control were used. Due to a genetic deficiency in glutathione synthetase, the level of free SH groups, and in particular that of glutathione, is decreased in 5-oxoprolinuria cells. The glutathione synthetase deficient cells have a reduced oxygen enhancement ratio (1.5) compared to control cells (2.7). The radiosensitivity was assessed for both cell strains in the presence of different concentrations of an exogenous radioprotector:cysteamine. At concentrations varying between 0.1 and 20 mM, cysteamine protected the two cell strains to the same extent when irradiated under oxic and hypoxic conditions. The protective effect of cysteamine was lower under hypoxia than under oxic conditions for both cell strains. Consequently, the oxygen enhancement ratio decreased for both cell strains when cysteamine concentration increased. These results suggest that cysteamine cannot replace endogenous thiols as far as they are implicated in the radiobiological oxygen effect. (author)

Application of optical phase conjugation to plasma diagnostics (invited)

International Nuclear Information System (INIS)

Jahoda, F.C.; Anderson, B.T.; Forman, P.R.; Weber, P.G.

1985-01-01

Several possibilities for plasma diagnostics provided by optical phase conjugation and, in particular, self-pumped phase conjugation in barium titanate (BaTiO 3 ) are discussed. These include placing a plasma within a dye laser cavity equipped with a phase conjugate mirror for intracavity absorption measurements, time differential refractometry with high spatial resolution, and simplified real-time holographic interferometry. The principles of phase conjugation with particular reference to photorefractive media and the special advantages of self-pumped phase conjugation are reviewed prior to the discussion of the applications. Distinctions are made in the applications between those for which photorefractive conjugators are essential and those for which they only offer experimental simplification relative to other types of phase conjugators

Selenium concentrations and enzyme activities of glutathione metabolism in wild long-tailed ducks and common eiders

Science.gov (United States)

Franson, J. Christian; Hoffman, David J.; Flint, Paul L.

2011-01-01

The relationships of selenium (Se) concentrations in whole blood with plasma activities of total glutathione peroxidase, Se-dependent glutathione peroxidase, and glutathione reductase were studied in long-tailed ducks (Clangula hyemalis) and common eiders (Somateria mollissima) sampled along the Beaufort Sea coast of Alaska, USA. Blood Se concentrations were >8 μg/g wet weight in both species. Linear regression revealed that the activities of total and Se-dependent glutathione peroxidase were significantly related to Se concentrations only in long-tailed ducks, raising the possibility that these birds were experiencing early oxidative stress.

Current status and emerging role of glutathione in food grade lactic acid bacteria

Directory of Open Access Journals (Sweden)

Pophaly Sarang

2012-08-01

Full Text Available Abstract Lactic acid bacteria (LAB have taken centre stage in perspectives of modern fermented food industry and probiotic based therapeutics. These bacteria encounter various stress conditions during industrial processing or in the gastrointestinal environment. Such conditions are overcome by complex molecular assemblies capable of synthesizing and/or metabolizing molecules that play a specific role in stress adaptation. Thiols are important class of molecules which contribute towards stress management in cell. Glutathione, a low molecular weight thiol antioxidant distributed widely in eukaryotes and Gram negative organisms, is present sporadically in Gram positive bacteria. However, new insights on its occurrence and role in the latter group are coming to light. Some LAB and closely related Gram positive organisms are proposed to possess glutathione synthesis and/or utilization machinery. Also, supplementation of glutathione in food grade LAB is gaining attention for its role in stress protection and as a nutrient and sulfur source. Owing to the immense benefits of glutathione, its release by probiotic bacteria could also find important applications in health improvement. This review presents our current understanding about the status of glutathione and its role as an exogenously added molecule in food grade LAB and closely related organisms.

Differential activation of diverse glutathione transferases of Clonorchis sinensis in response to the host bile and oxidative stressors.

Directory of Open Access Journals (Sweden)

Young-An Bae

Full Text Available BACKGROUND: Clonorchis sinensis causes chronic cumulative infections in the human hepatobiliary tract and is intimately associated with cholangiocarcinoma. Approximately 35 million people are infected and 600 million people are at risk of infections worldwide. C. sinensis excretory-secretory products (ESP constitute the first-line effector system affecting the host-parasite interrelationship by interacting with bile fluids and ductal epithelium. However, the secretory behavior of C. sinensis in an environment close to natural host conditions is unclear. C. sinensis differs from Fasciola hepatica in migration to, and maturation in, the hepatic bile duct, implying that protein profile of the ESP of these two trematodes might be different from each other. METHODOLOGY/PRINCIPAL FINDINGS: We conducted systemic approaches to analyze the C. sinensis ESP proteome and the biological reactivity of C. sinensis glutathione transferases (GSTs, such as global expression patterns and induction profiles under oxidative stress and host bile. When we observed ex host excretion behavior of C. sinensis in the presence of 10% host bile, the global proteome pattern was not significantly altered, but the amount of secretory proteins was increased by approximately 3.5-fold. Bioactive molecules secreted by C. sinensis revealed universal/unique features in relation to its intraluminal hydrophobic residing niche. A total of 38 protein spots identified abundantly included enzymes involved in glucose metabolism (11 spots, 28.9% and diverse-classes of glutathione transferases (GSTs; 10 spots, 26.3%. Cathepsin L/F (four spots, 10.5% and transporter molecules (three spots, 7.9% were also recognized. The universal secretory proteins found in other parasites, such as several enzymes involved in glucose metabolism and oxygen transporters, were commonly detected. C. sinensis secreted less cysteine proteases and fatty acid binding proteins compared to other tissue-invading or

Differential Activation of Diverse Glutathione Transferases of Clonorchis sinensis in Response to the Host Bile and Oxidative Stressors

Science.gov (United States)

Bae, Young-An; Ahn, Do-Whan; Lee, Eung-Goo; Kim, Seon-Hee; Cai, Guo-Bin; Kang, Insug; Sohn, Woon-Mok; Kong, Yoon

2013-01-01

Background Clonorchis sinensis causes chronic cumulative infections in the human hepatobiliary tract and is intimately associated with cholangiocarcinoma. Approximately 35 million people are infected and 600 million people are at risk of infections worldwide. C. sinensis excretory-secretory products (ESP) constitute the first-line effector system affecting the host-parasite interrelationship by interacting with bile fluids and ductal epithelium. However, the secretory behavior of C. sinensis in an environment close to natural host conditions is unclear. C. sinensis differs from Fasciola hepatica in migration to, and maturation in, the hepatic bile duct, implying that protein profile of the ESP of these two trematodes might be different from each other. Methodology/Principal Findings We conducted systemic approaches to analyze the C. sinensis ESP proteome and the biological reactivity of C. sinensis glutathione transferases (GSTs), such as global expression patterns and induction profiles under oxidative stress and host bile. When we observed ex host excretion behavior of C. sinensis in the presence of 10% host bile, the global proteome pattern was not significantly altered, but the amount of secretory proteins was increased by approximately 3.5-fold. Bioactive molecules secreted by C. sinensis revealed universal/unique features in relation to its intraluminal hydrophobic residing niche. A total of 38 protein spots identified abundantly included enzymes involved in glucose metabolism (11 spots, 28.9%) and diverse-classes of glutathione transferases (GSTs; 10 spots, 26.3%). Cathepsin L/F (four spots, 10.5%) and transporter molecules (three spots, 7.9%) were also recognized. The universal secretory proteins found in other parasites, such as several enzymes involved in glucose metabolism and oxygen transporters, were commonly detected. C. sinensis secreted less cysteine proteases and fatty acid binding proteins compared to other tissue-invading or intravascular

In vivo evaluation of an oral salmon calcitonin-delivery system based on a thiolated chitosan carrier matrix.

Science.gov (United States)

Guggi, Davide; Kast, Constantia E; Bernkop-Schnürch, Andreas

2003-12-01

To develop and evaluate an oral delivery system for salmon calcitonin. 2-Iminothiolane was covalently bound to chitosan in order to improve the mucoadhesive and cohesive properties of the polymer. The resulting chitosan-TBA conjugate (chitosan-4-thiobutylamidine conjugate) was homogenized with salmon calcitonin. mannitol, and a chitosan-Bowman-Birk inhibitor conjugate and a chitosan-elastatinal conjugate (6.75 + 0.25 + 1 + 1 + 1). Optionally 0.5% (m/m) reduced glutathione. used as permeation mediator, was added. Each mixture was compressed to 2 mg microtablets and enteric coated with a polymethacrylate. Biofeedback studies were performed in rats by oral administration of the delivery system and determination of the decrease in plasma calcium level as a function of time. Test formulations led to a significant (p thiolated chitosan, chitosan-enzyme-inhibitor conjugates and the permeation mediator glutathione seems to represent a promising strategy for the oral delivery of salmon calcitonin.

Conformation sensitive charge transport in conjugated polymers

International Nuclear Information System (INIS)

Mattias Andersson, L.; Hedström, Svante; Persson, Petter

2013-01-01

Temperature dependent charge carrier mobility measurements using field effect transistors and density functional theory calculations are combined to show how the conformation dependent frontier orbital delocalization influences the hole- and electron mobilities in a donor-acceptor based polymer. A conformationally sensitive lowest unoccupied molecular orbital results in an electron mobility that decreases with increasing temperature above room temperature, while a conformationally stable highest occupied molecular orbital is consistent with a conventional hole mobility behavior and also proposed to be one of the reasons for why the material works well as a hole transporter in amorphous bulk heterojunction solar cells

The tomato Fni3 lysine-63-specific ubiquitin-conjugating enzyme and suv ubiquitin E2 variant positively regulate plant immunity.

Science.gov (United States)

Mural, Ravi V; Liu, Yao; Rosebrock, Tracy R; Brady, Jennifer J; Hamera, Sadia; Connor, Richard A; Martin, Gregory B; Zeng, Lirong

2013-09-01

The activation of an immune response in tomato (Solanum lycopersicum) against Pseudomonas syringae relies on the recognition of E3 ligase-deficient forms of AvrPtoB by the host protein kinase, Fen. To investigate the mechanisms by which Fen-mediated immunity is regulated, we characterize in this study a Fen-interacting protein, Fni3, and its cofactor, S. lycoperiscum Uev (Suv). Fni3 encodes a homolog of the Ubc13-type ubiquitin-conjugating enzyme that catalyzes exclusively Lys-63-linked ubiquitination, whereas Suv is a ubiquitin-conjugating enzyme variant. The C-terminal region of Fen was necessary for interaction with Fni3, and this interaction was required for cell death triggered by overexpression of Fen in Nicotiana benthamiana leaves. Fni3 was shown to be an active E2 enzyme, but Suv displayed no ubiquitin-conjugating activity; Fni3 and Suv together directed Lys-63-linked ubiquitination. Decreased expression of Fni3, another tomato Ubc13 homolog, Sl-Ubc13-2, or Suv in N. benthamiana leaves diminished cell death associated with Fen-mediated immunity and cell death elicited by several other resistance (R) proteins and their cognate effectors. We also discovered that coexpression of Fen and other R proteins/effectors with a Fni3 mutant that is compromised for ubiquitin-conjugating activity diminished the cell death. These results suggest that Fni3/Sl-Ubc13-2 and Suv regulate the immune response mediated by Fen and other R proteins through Lys-63-linked ubiquitination.

Glutathione protects liver and kidney tissue from cadmium- and lead-provoked lipid peroxidation

Directory of Open Access Journals (Sweden)

Jovanović Jasmina M.

2013-01-01

Full Text Available Cd and Pb represent a serious ecological problem due to their soluble nature, their mobility and ability to accumulate in the soil. The exposure to these heavy metals can originate from different sources (drinking water, food, air, and they can make their way into the human body through the respiratory and digestive system. We investigated the effects of glutathione on Cd and Pb accumulation and lipid peroxidation effects in the liver and kidneys of heavy metal intoxicated rats. The content of the marker of lipid peroxidation - malondialdehyde was increased several fold the in tissues of exposed animals, the effects being more pronounced in liver. The treatment of intoxicated animals with glutathione drastically suppressed lipid peroxidation. Our results imply that the application of glutathione may have protective role in heavy metal intoxication by inhibiting lipid peroxidation. However, precaution should be made when it comes to Cd, since it seems that glutathione promoted Cd accumulation in the liver.

Magnetic and transport properties of conjugated and cumulated molecules: the π-system enlightens part of the story

DEFF Research Database (Denmark)

Sarbadhikary, Prodipta; Shil, Suranjan; Misra, Anirban

2018-01-01

We have investigated the intramolecular magnetic exchange coupling constants (J) for a series of nitronyl nitroxide diradicals connected by a range of linear conjugated and cumulene couplers focusing on the unusual π-interaction properties within the couplers. Distance between radical centers, sp...

Targeting brain cells with glutathione-modulated nanoliposomes: in vitro and in vivo study

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Salem HF

2015-07-01

Full Text Available Heba F Salem,1 Sayed M Ahmed,2 Ashraf E Hassaballah,3 Mahmoud M Omar1,4 1Department of Pharmaceutics and Industrial Pharmacy, Beni-suef University, 2Department of Industrial Pharmacy, Assiut University, 3Department of Clinical Pathology, Faculty of Medicine, Assiut University, Assuit, 4Department of Pharmaceutics and Industrial Pharmacy, Deraya University, Egypt Background: The blood–brain barrier prevents many drug moieties from reaching the central nervous system. Therefore, glutathione-modulated nanoliposomes have been engineered to enhance the targeting of flucytosine to the brain. Methods: Glutathione-modulated nanoliposomes were prepared by thin-film hydration technique and evaluated in the primary brain cells of rats. Lecithin, cholesterol, and span 65 were mixed at 1:1:1 molar ratio. The molar percentage of PEGylated glutathione varied from 0 mol% to 0.75 mol%. The cellular binding and the uptake of the targeted liposomes were both monitored by epifluorescent microscope and flow cytometry techniques. A biodistribution and a pharmacokinetic study of flucytosine and flucytosine-loaded glutathione–modulated liposomes was carried out to evaluate the in vivo brain-targeting efficiency. Results: The size of glutathione-modulated nanoliposomes was <100 nm and the zeta potential was more than -65 mV. The cumulative release reached 70% for certain formulations. The cellular uptake increased as molar percent of glutathione increased to reach the maximum at 0.75 mol%. The uptake of the targeted liposomes by brain cells of the rats was three times greater than that of the nontargeted liposomes. An in vivo study showed that the relative efficiency was 2.632±0.089 and the concentration efficiency was 1.590±0.049, and also, the drug-targeting index was 3.670±0.824. Conclusion: Overall, these results revealed that glutathione-PEGylated nanoliposomes enhance the effective delivery of flucytosine to brain and could become a promising new

Activation of the microsomal glutathione-S-transferase and reduction of the glutathione dependent protection against lipid peroxidation by acrolein

NARCIS (Netherlands)

Haenen, G R; Vermeulen, N P; Tai Tin Tsoi, J N; Ragetli, H M; Timmerman, H; Blast, A

1988-01-01

Allyl alcohol is hepatotoxic. It is generally believed that acrolein, generated out of allyl alcohol by cytosolic alcohol dehydrogenase, is responsible for this toxicity. The effect of acrolein in vitro and in vivo on the glutathione (GSH) dependent protection of liver microsomes against lipid

Oxidative Stress Markers and Genetic Polymorphisms of Glutathione ...

African Journals Online (AJOL)

2017-10-26

Oct 26, 2017 ... stress such as asthma, lung cancer, and type 2 diabetes mellitus.[11-13]. Although ... epigenetic, and environmental factors. Little is known ..... glutathione Stransferase genes increase risk of prostate cancer biochemical ...

An MCBJ case study : The influence of π-conjugation on the single-molecule conductance at a solid/liquid interface

NARCIS (Netherlands)

Hong, Wenjing; Valkenier, Hennie; Meszaros, Gabor; Manrique, David Zsolt; Mishchenko, Artem; Putz, Alexander; Garcia, Pavel Moreno; Lambert, Colin J.; Hummelen, Jan C.; Wandlowski, Thomas

2011-01-01

π-Conjugation plays an important role in charge transport through single molecular junctions. We describe in this paper the construction of a mechanically controlled break-junction setup (MCBJ) equipped with a highly sensitive log I–V converter in order to measure ultralow conductances of molecular

Transepithelial transport of PEGylated anionic poly(amidoamine) dendrimers: implications for oral drug delivery.

Science.gov (United States)

Sweet, Deborah M; Kolhatkar, Rohit B; Ray, Abhijit; Swaan, Peter; Ghandehari, Hamidreza

2009-08-19

The purpose of this work was to assess the impact of PEGylation on transepithelial transport of anionic poly(amidoamine) dendrimers. Cytotoxicity, uptake and transport across Caco-2 cells of PEGylated G3.5 and G4.5 PAMAM dendrimers were studied. Methoxy polyethylene glycol (750 Da) was conjugated to carboxylic acid-terminated PAMAM dendrimers at feed ratios of 1, 2 and 4 PEG per dendrimer. Compared to the control, PEGylation of anionic dendrimers did not significantly alter cytotoxicity up to a concentration of 0.1 mM. PEGylation of G3.5 dendrimers significantly decreased cellular uptake and transepithelial transport while PEGylation of G4.5 dendrimers led to a significant increase in uptake, but also a significant decrease in transport. Dendrimer PEGylation reduced the opening of tight junctions as evidenced by confocal microscopy techniques. Modulation of the tight junctional complex correlated well with changes in PEGylated dendrimer transport and suggests that anionic dendrimers are transported primarily through the paracellular route. PEGylated dendrimers show promise in oral delivery applications where increased functionality for drug conjugation and release is desired.

Degree of glutathione deficiency and redox imbalance depend on subtype of mitochondrial disease and clinical status.

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Gregory M Enns

Full Text Available Mitochondrial disorders are associated with decreased energy production and redox imbalance. Glutathione plays a central role in redox signaling and protecting cells from oxidative damage. In order to understand the consequences of mitochondrial dysfunction on in vivo redox status, and to determine how this varies by mitochondrial disease subtype and clinical severity, we used a sensitive tandem mass spectrometry assay to precisely quantify whole blood reduced (GSH and oxidized (GSSG glutathione levels in a large cohort of mitochondrial disorder patients. Glutathione redox potential was calculated using the Nernst equation. Compared to healthy controls (n = 59, mitochondrial disease patients (n = 58 as a group showed significant redox imbalance (redox potential -251 mV ± 9.7, p<0.0001 with an increased level of oxidation by ∼ 9 mV compared to controls (-260 mV ± 6.4. Underlying this abnormality were significantly lower whole blood GSH levels (p = 0.0008 and GSH/GSSG ratio (p = 0.0002, and significantly higher GSSG levels (p<0.0001 in mitochondrial disease patients compared to controls. Redox potential was significantly more oxidized in all mitochondrial disease subgroups including Leigh syndrome (n = 15, electron transport chain abnormalities (n = 10, mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (n = 8, mtDNA deletion syndrome (n = 7, mtDNA depletion syndrome (n = 7, and miscellaneous other mitochondrial disorders (n = 11. Patients hospitalized in metabolic crisis (n = 7 showed the greatest degree of redox imbalance at -242 mV ± 7. Peripheral whole blood GSH and GSSG levels are promising biomarkers of mitochondrial dysfunction, and may give insights into the contribution of oxidative stress to the pathophysiology of the various mitochondrial disorders. In particular, evaluation of redox potential may be useful in monitoring of clinical status or response to redox-modulating therapies in clinical trials.

Methotrexate and epirubicin conjugates as potential antitumor drugs

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Szymon Wojciech Kmiecik

2017-07-01

Full Text Available Introduction: The use of hybrid molecules has become one of the most significant approaches in new cytotoxic drug design. This study describes synthesis and characterization of conjugates consisting of two well-known and characterized chemotherapeutic agents: methotrexate (MTX and epirubicin (EPR. The synthesized conjugates combine two significant anticancer strategies: combinatory therapy and targeted therapy. These two drugs were chosen because they have different mechanisms of action, which can increase the anticancer effect of the obtained conjugates. MTX, which is a folic acid analog, has high cytotoxic properties and can serve as a targeting moiety that can reach folate receptors (FRs overexpresing tumor cells. Combination of nonselective drugs such as EPR with MTX can increase the selectivity of the obtained conjugates, while maintaining the high cytotoxic properties.Materials and methods: Conjugates were purified by RP-HPLC and the structure was investigated by MS and MS/MS methods. The effect of the conjugates on proliferation of LoVo, LoVo/Dx, MCF-7 and MV-4-11 human cancer cell lines was determined by SRB or MTT assay.Results: The conjugation reaction results in the formation of monosubstituted (α, γ and disubstituted MTX derivatives. In vitro proliferation data demonstrate that the conjugates synthesized in our study show lower cytotoxic properties than both chemotherapeutics used alone.Discussion: Epirubicin cytotoxicity was not observed in obtained conjugates. Effective drugs release after internalization needs further investigation.

Blood selenium concentrations and enzyme activities related to glutathione metabolism in wild emperor geese

Science.gov (United States)

Franson, J. Christian; Hoffman, David J.; Schmutz, Joel A.

2002-01-01

In 1998, we collected blood samples from 63 emperor geese (Chen canagica) on their breeding grounds on the Yukon-Kuskokwim Delta (YKD) in western Alaska, USA. We studied the relationship between selenium concentrations in whole blood and the activities of glutathione peroxidase and glutathione reductase in plasma. Experimental studies have shown that plasma activities of these enzymes are useful biomarkers of selenium-induced oxidative stress, but little information is available on their relationship to selenium in the blood of wild birds. Adult female emperor geese incubating their eggs in mid-June had a higher mean concentration of selenium in their blood and a greater activity of glutathione peroxidase in their plasma than adult geese or goslings that were sampled during the adult flight feathermolting period in late July and early August. Glutathione peroxidase activity was positively correlated with the concentration of selenium in the blood of emperor geese, and the rate of increase relative to selenium was greater in goslings than in adults. The activity of glutathione reductase was greatest in the plasma of goslings and was greater in molting adults than incubating females but was not significantly correlated with selenium in the blood of adults or goslings. Incubating female emperor geese had high selenium concentrations in their blood, accompanied by increased glutathione peroxidase activity consistent with early oxidative stress. These findings indicate that further study of the effects of selenium exposure, particularly on reproductive success, is warranted in this species.

Balneotherapy and platelet glutathione metabolism in type II diabetic patients

Science.gov (United States)

Ohtsuka, Yoshinori; Yabunaka, Noriyuki; Watanabe, Ichiro; Noro, Hiroshi; Agishi, Yuko

1996-09-01

Effects of balneotherapy on platelet glutathione metabolism were investigated in 12 type II (non-insulin-dependent) diabetic patients. Levels of the reduced form of glutathione (GSH) on admission were well correlated with those of fasting plasma glucose (FPG; r=0.692, Pbalneotherapy, the mean level of GSH showed no changes; however, in well-controlled patients (FPG 150 mg/dl), the value decreased ( Pbalneotherapy, the activity increased in 5 patients, decreased in 3 patients and showed no changes (alteration within ±3%) in all the other patients. From these findings in diabetic patients we concluded: (1) platelet GSH synthesis appeared to be induced in response to oxidative stress; (2) lowered GPX activities indicated that the antioxidative defense system was impaired; and (3) platelet glutathione metabolism was partially improved by 4 weeks balneotherapy, an effect thought to be dependent on the control status of plasma glucose levels. It is suggested that balneotherapy is beneficial for patients whose platelet antioxidative defense system is damaged, such as those with diabetes mellitus and coronary heart disease.

Analysis of glutathione S-transferase (M1, T1 and P1) gene ...

African Journals Online (AJOL)

Glutathione S-transferase enzymes are active in detoxifying a wide number of endogenous and exogenous chemical carcinogens and subsequently, are crucial in protecting the DNA. Several studies show some differences in association of glutathione S-transferase M1, T1 and P1 genetic polymorphisms with the risk of ...

The cataract and glucosuria associated monocarboxylate transporter MCT12 is a new creatine transporter

Science.gov (United States)

Abplanalp, Jeannette; Laczko, Endre; Philp, Nancy J.; Neidhardt, John; Zuercher, Jurian; Braun, Philipp; Schorderet, Daniel F.; Munier, Francis L.; Verrey, François; Berger, Wolfgang; Camargo, Simone M.R.; Kloeckener-Gruissem, Barbara

2013-01-01

Creatine transport has been assigned to creatine transporter 1 (CRT1), encoded by mental retardation associated SLC6A8. Here, we identified a second creatine transporter (CRT2) known as monocarboxylate transporter 12 (MCT12), encoded by the cataract and glucosuria associated gene SLC16A12. A non-synonymous alteration in MCT12 (p.G407S) found in a patient with age-related cataract (ARC) leads to a significant reduction of creatine transport. Furthermore, Slc16a12 knockout (KO) rats have elevated creatine levels in urine. Transport activity and expression characteristics of the two creatine transporters are distinct. CRT2 (MCT12)-mediated uptake of creatine was not sensitive to sodium and chloride ions or creatine biosynthesis precursors, breakdown product creatinine or creatine phosphate. Increasing pH correlated with increased creatine uptake. Michaelis–Menten kinetics yielded a Vmax of 838.8 pmol/h/oocyte and a Km of 567.4 µm. Relative expression in various human tissues supports the distinct mutation-associated phenotypes of the two transporters. SLC6A8 was predominantly found in brain, heart and muscle, while SLC16A12 was more abundant in kidney and retina. In the lens, the two transcripts were found at comparable levels. We discuss the distinct, but possibly synergistic functions of the two creatine transporters. Our findings infer potential preventive power of creatine supplementation against the most prominent age-related vision impaired condition. PMID:23578822

ERP Correlates of Encoding Success and Encoding Selectivity in Attention Switching

Science.gov (United States)

Yeung, Nick

2016-01-01

Long-term memory encoding depends critically on effective processing of incoming information. The degree to which participants engage in effective encoding can be indexed in electroencephalographic (EEG) data by studying event-related potential (ERP) subsequent memory effects. The current study investigated ERP correlates of memory success operationalised with two different measures—memory selectivity and global memory—to assess whether previously observed ERP subsequent memory effects reflect focused encoding of task-relevant information (memory selectivity), general encoding success (global memory), or both. Building on previous work, the present study combined an attention switching paradigm—in which participants were presented with compound object-word stimuli and switched between attending to the object or the word across trials—with a later recognition memory test for those stimuli, while recording their EEG. Our results provided clear evidence that subsequent memory effects resulted from selective attentional focusing and effective top-down control (memory selectivity) in contrast to more general encoding success effects (global memory). Further analyses addressed the question of whether successful encoding depended on similar control mechanisms to those involved in attention switching. Interestingly, differences in the ERP correlates of attention switching and successful encoding, particularly during the poststimulus period, indicated that variability in encoding success occurred independently of prestimulus demands for top-down cognitive control. These results suggest that while effects of selective attention and selective encoding co-occur behaviourally their ERP correlates are at least partly dissociable. PMID:27907075

Controlled release of β-carotene in β-lactoglobulin-dextran-conjugated nanoparticles' in vitro digestion and transport with Caco-2 monolayers.

Science.gov (United States)

Yi, Jiang; Lam, Tina I; Yokoyama, Wallace; Cheng, Luisa W; Zhong, Fang

2014-09-03

Undesirable aggregation of nanoparticles stabilized by proteins may occur at the protein's isoelectric point when the particle has zero net charge. Stability against aggregation of nanoparticles may be improved by reacting free amino groups with reducing sugars by the Maillard reaction. β-Lactoglobulin (BLG)-dextran conjugates were characterized by SDS-PAGE and CD. Nanoparticles (60-70 nm diameter) of β-carotene (BC) encapsulated by BLG or BLG-dextran were prepared by the homogenization-evaporation method. Both BLG and BLG-dextran nanoparticles appeared to be spherically shaped and uniformly dispersed by TEM. The stability and release of BC from the nanoparticles under simulated gastrointestinal conditions were evaluated. Dextran conjugation prevented the flocculation or aggregation of BLG-dextran particles at pH ∼4-5 compared to very large sized aggregates of BLG nanoparticles. The released contents of BC from BLG and BLG-dextran nanoparticles under acidic gastric conditions were 6.2 ± 0.9 and 5.4 ± 0.3%, respectively. The release of BC from BLG-dextran nanoparticles by trypsin digestion was 51.8 ± 4.3% of total encapsulated BC, and that from BLG nanoparticles was 60.9 ± 2.9%. Neither BLG-BC nanoparticles nor the Maillard-reacted BLG-dextran conjugates were cytotoxic to Caco-2 cells, even at 10 mg/mL. The apparent permeability coefficient (Papp) of Caco-2 cells to BC was improved by nanoencapsulation, compared to free BC suspension. The results indicate that BC-encapsulated β-lactoglobulin-dextran-conjugated nanoparticles are more stable to aggregation under gastric pH conditions with good release and permeability properties.

Transport processes in pea seed coats

NARCIS (Netherlands)

Dongen, Joost Thomas van

2001-01-01

The research described in this thesis concerns transport processes in coats of developing pea seeds. The scope of the investigation ranges from seed coat anatomy, via transport studies to the cloning of cDNA encoding proteinaceous membrane pores, and the heterologous expression of these

Interplay of a non-conjugative integrative element and a conjugative plasmid in the spread of antibiotic resistance via suicidal plasmid transfer from an aquaculture Vibrio isolate.

Science.gov (United States)

Nonaka, Lisa; Yamamoto, Tatsuya; Maruyama, Fumito; Hirose, Yuu; Onishi, Yuki; Kobayashi, Takeshi; Suzuki, Satoru; Nomura, Nobuhiko; Masuda, Michiaki; Yano, Hirokazu

2018-01-01

The capture of antimicrobial resistance genes (ARGs) by mobile genetic elements (MGEs) plays a critical role in resistance acquisition for human-associated bacteria. Although aquaculture environments are recognized as important reservoirs of ARGs, intra- and intercellular mobility of MGEs discovered in marine organisms is poorly characterized. Here, we show a new pattern of interspecies ARGs transfer involving a 'non-conjugative' integrative element. To identify active MGEs in a Vibrio ponticus isolate, we conducted whole-genome sequencing of a transconjugant obtained by mating between Escherichia coli and Vibrio ponticus. This revealed integration of a plasmid (designated pSEA1) into the chromosome, consisting of a self-transmissible plasmid backbone of the MOBH group, ARGs, and a 13.8-kb integrative element Tn6283. Molecular genetics analysis suggested a two-step gene transfer model. First, Tn6283 integrates into the recipient chromosome during suicidal plasmid transfer, followed by homologous recombination between the Tn6283 copy in the chromosome and that in the newly transferred pSEA1. Tn6283 is unusual among integrative elements in that it apparently does not encode transfer function and its excision barely generates unoccupied donor sites. Thus, its movement is analogous to the transposition of insertion sequences rather than to that of canonical integrative and conjugative elements. Overall, this study reveals the presence of a previously unrecognized type of MGE in a marine organism, highlighting diversity in the mode of interspecies gene transfer.

Electric-Field Control of Interfering Transport Pathways in a Single-Molecule Anthraquinone Transistor

NARCIS (Netherlands)

Koole, Max; Thijssen, Jos M.; Valkenier, Hennie; Hummelen, Jan C.; van der Zant, Herre S. J.

It is understood that molecular conjugation plays an important role in charge transport through single-molecule junctions. Here, we investigate electron transport through an anthraquinone based single-molecule three-terminal device. With the use of an electric-field induced by a gate electrode, the

OXIDATIVE MODIFICATION OF PROTEINS AND GLUTATHIONE SYSTEM IN ADIPOCYTES UNDER DIABETES

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Ye. V. Shakhristova

2014-01-01

Full Text Available Currently, diabetes ranks third in relation to medical and social significance after cardiovascular diseases and cancer and is the leading cause of blindness; it greatly increases the risk of myocardial infarction, coronary heart disease, nephropathy and hypertension in patients with this disorder; therefore clinical and experimental studies aimed at investigation of diabetes emergence and development mechanisms are urgent.The aim of the study was to investigate the status of oxidative modification of proteins and glutathionedependent antioxidant defense system in adipocytes of rats with alloxan diabetes under conditions of oxidative stress.Material and methods. Development of type 1 diabetes was induced in rats by alloxan administration (90 mg/kg of body mass. Adipocytes were obtained from epididymal adipose tissue of rats. The level of carbonyl derivatives of proteins, oxidized tryptophan, bityrosine, general, reduced, oxygenated and protein-bound glutathione, as well as glutathione peroxidase activity in adipocytes of rats was determined.Results. In adipocytes of rats with alloxan diabetes, concentration of carbonyl derivatives of proteins, bityrosine and oxidized tryptophan increased on the background of redox-potential of glutathione system and glutathione peroxidase activity decrease.Conclusion. The obtained data indicate the activation of free-radical oxidation of proteins and reduction of antioxidant defense under conditions of oxidative stress in the adipose tissue of rats with alloxan diabetes; this process plays an important role in pathogenesis of diabetes and its complications development.

Calixarene-mediated liquid membrane transport of choline conjugates 3: The effect of handle variation on neurotransmitter transport.

Science.gov (United States)

Collins, James L; Fujii, Ayu; Roshandel, Sahar; To, Cuong-Alexander; Schramm, Michael P

2017-07-01

Upper rim phosphonic acid functionalized calix[4]arene affects selective transport of multiple molecular payloads through a liquid membrane. The secret is in the attachment of a receptor-complementary handle to the payload. We find that the trimethylammonium ethylene group present in choline is one of several general handles for the transport of drug and drug-like species. Herein we compare the effect of handle variation against the transport of serotonin and dopamine. We find that several ionizable amine termini handles are sufficient for transport and identify two ideal candidates. Their performance is significantly enhanced in HEPES buffered solutions. This inquiry completes a series of 3 studies aimed at optimization of this strategy. In completion a new approach towards synthetic receptor mediated selective small molecule transport has emerged; future work in vesicular and cellular systems will follow. Copyright © 2017 Elsevier Ltd. All rights reserved.

The synthesis of taurine-conjugated bile acids and bile acid sulfates labeled with 14C or 3H in the taurine moiety

International Nuclear Information System (INIS)

Jie Zhang; Griffiths, W.J.; Sjoevall, Jan

1997-01-01

Studies of bile acid transport systems require radio-labeled taurine-conjugated bile acids with high specific activity. An established procedure was optimized to provide mild, fast, and effective conjugation of radio-labeled taurine with different types of bile acids, including those with labile 7α-hydroxy-3-oxo-Δ 4 or 3β, 7α-dihydroxy-Δ 5 structures. Taurine labeled with 14 C or 3 H was reacted with excess bile acid anhydride formed from the tributylamine salt and ethylchloroformate (2/1 M/M) in aqueous dioxane for 15 min at room temperature. The yields were higher than 95% and less than 2% side products were formed. Bile acid sulfates were conjugated with 14 C- or 3 H-labeled taurine by using N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline as the coupling reagent. The products were effectively purified by chromatography of the sodium salts on Sephadex LH-20. The yields of taurine-conjugated bile acid sulfates were 65-70%. (author)

The Triple Roles of Glutathione for a DNA-Cleaving DNAzyme and Development of a Fluorescent Glutathione/Cu2+-Dependent DNAzyme Sensor for Detection of Cu2+ in Drinking Water.

Science.gov (United States)

Wang, Shijin; Liu, Chengcheng; Li, Guiying; Sheng, Yongjie; Sun, Yanhong; Rui, Hongyue; Zhang, Jin; Xu, Jiacui; Jiang, Dazhi

2017-03-24

Pistol-like DNAzyme (PLDz) is an oxidative DNA-cleaving catalytic DNA with ascorbic acid as cofactor. Herein, glutathione was induced into the reaction system to maintain reduced ascorbic acid levels for higher efficient cleavage. However, data indicated that glutathione played triple roles in PLDz-catalyzed reactions. Glutathione alone had no effect on PLDz, and showed inhibitory effect on ascorbic acid-induced PLDz catalysis, but exhibited stimulating effect on Cu 2+ -promoted self-cleavage of PLDz. Further analysis of the effect of glutathione/Cu 2+ on PLDz indicated that H 2 O 2 played a key role in PLDz catalysis. Finally, we developed a fluorescent Cu 2+ sensor (PL-Cu 1.0) based on the relationship between glutathione/Cu 2+ and catalytic activity of PLDz. The fluorescent intensity showed a linear response toward the logarithm concentration of Cu 2+ over the range from 80 nM to 30 μM, with a detection limit of 21.1 nM. PL-Cu 1.0 provided only detection of Cu 2+ over other divalent metal ions. Ca 2+ and Mg 2+ could not interfere with Cu 2+ detection even at a 1000-fold concentration. We further applied PL-Cu 1.0 for Cu 2+ detection in tap and bottled water. Water stored in copper taps overnight had relatively high Cu 2+ concentrations, with a maximum 22.3 μM. Trace Cu 2+ (52.2 nM) in deep spring was detected among the tested bottled water. Therefore, PL-Cu 1.0 is feasible to detect Cu 2+ in drinking water, with a practical application.

Synthesis and Characterization of Anti-HER2 Antibody Conjugated CdSe/CdZnS Quantum Dots for Fluorescence Imaging of Breast Cancer Cells

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Takashi Jin

2009-11-01

Full Text Available The early detection of HER2 (human epidermal growth factor receptor 2 status in breast cancer patients is very important for the effective implementation of anti-HER2 antibody therapy. Recently, HER2 detections using antibody conjugated quantum dots (QDs have attracted much attention. QDs are a new class of fluorescent materials that have superior properties such as high brightness, high resistance to photo-bleaching, and multi-colored emission by a single-light source excitation. In this study, we synthesized three types of anti-HER2 antibody conjugated QDs (HER2Ab-QDs using different coupling agents (EDC/sulfo-NHS, iminothiolane/sulfo-SMCC, and sulfo-SMCC. As water-soluble QDs for the conjugation of antibody, we used glutathione coated CdSe/CdZnS QDs (GSH-QDs with fluorescence quantum yields of 0.23~0.39 in aqueous solution. Dispersibility, hydrodynamic size, and apparent molecular weights of the GSH-QDs and HER2Ab-QDs were characterized by using dynamic light scattering, fluorescence correlation spectroscopy, atomic force microscope, and size-exclusion HPLC. Fluorescence imaging of HER2 overexpressing cells (KPL-4 human breast cancer cell line was performed by using HER2Ab-QDs as fluorescent probes. We found that the HER2Ab-QD prepared by using SMCC coupling with partially reduced antibody is a most effective probe for the detection of HER2 expression in KPL-4 cells. We have also studied the size dependency of HER2Ab-QDs (with green, orange, and red emission on the fluorescence image of KPL-4 cells.