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The fruit fly Drosophila melanogaster GSTE6 and E7; characterization, immobilization and transgenic overexpression
Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.ORCID-id: 0000-0001-6258-1443
2014 (engelsk)Licentiatavhandling, med artikler (Annet vitenskapelig)
Abstract [en]

Glutathione transferases (GSTs) are multifunctional enzymes that are universally distributed in most eukaryotes and prokaryotes. They play a pivotal role in the metabolism and detoxication of numerous endogenous and exogenous electrophiles by conjugating them with ubiquitous tripeptide glutathione. In this study we have immobilized two heterologously expressed and purified Epsilon-class enzymes, GSTE6 and GSTE7, from of Drosophila melanogaster on nanoporous alumina membranes. The membranes were derivatized with 3-aminopropyl-triethoxysilane and the amino groups were activated with carbonyldiimidazole to allow coupling of the enzymes. Kinetic analyses of the immobilized enzymes were carried out in a circulating flow system using CDNB (1-chloro-2,4-dinitrobenzene) as substrate, followed by specificity screening with alternative substrates. A good correlation was observed between the substrate screening data for immobilized enzyme and corresponding data for the enzymes in solution. The stability of the immobilized enzymes was virtually identical to that for the enzymes in solution and no leakage of enzyme from the matrix could be observed.

Additionally, we have investigated the catalytic activities of GSTE7 with organic isothiocyanates (ITCs). These reactive compounds are strong electrophilic molecules produced in plants by the hydrolysis of glucosinolates and exert toxicity in biological tissues.  Our in vitro studies, showed high catalytic activity of GSTE7 towards ITCs. We have then explored the in vivo effect of phenethyl isothiocyanate (PEITC) and allyl isothiocyanate (AITC) in transgenic fruit flies overexpressing GSTE7. A concentration of 0.25 mM PEITC in standard fly food was shown to be toxic to flies and significantly shortened the lifespan. We noticed that overexpression of GSTE7 could protect females from the initial acute toxic effects, but had no positive effect on long term exposure. The effect on males seems to be the opposite to that of females, where a higher mortality was seen in fly males overexpressing GST E7 after one week of exposure.  On the other hand 1mM concentration of AITC showed no toxic effects, but dramatically reduced the oviposition activity of wild-type flies in comparison to the transgenic flies.

sted, utgiver, år, opplag, sider
Stockholm: Department of Neurochemistry, Stockholm University , 2014.
Emneord [en]
Glutathione transferases, drosophila melanogaster, isothiocyanates, toxicity, immobilization, kinetics
HSV kategori
Forskningsprogram
neurokemi med molekylär neurobiologi
Identifikatorer
URN: urn:nbn:se:su:diva-102003ISBN: 978-91-7447-888-4 (tryckt)OAI: oai:DiVA.org:su-102003DiVA, id: diva2:706542
Presentation
2014-04-08, Heilbronnsalen, C 458, Svante Arrhenius väg 16 B, Stockholm, 12:15 (engelsk)
Opponent
Veileder
Merknad

At the time of the seminar the following paper was unpublished and had a status as follows: Paper 2: Submitted manuscript.

Tilgjengelig fra: 2014-04-01 Laget: 2014-03-20 Sist oppdatert: 2018-01-11bibliografisk kontrollert
Delarbeid
1. Glutathione transferases immobilized on nanoporous alumina: Flow system kinetics, screening, and stability
Åpne denne publikasjonen i ny fane eller vindu >>Glutathione transferases immobilized on nanoporous alumina: Flow system kinetics, screening, and stability
Vise andre…
2014 (engelsk)Inngår i: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 446, s. 59-63Artikkel i tidsskrift (Fagfellevurdert) Published
Abstract [en]

The previously uncharacterized Drosophila melanogaster Epsilon-class glutathione transferases E6 and E7 were immobilized on nanoporous alumina. The nanoporous anodized alumina membranes were derivatized with 3-aminopropyl-triethoxysilane, and the amino groups were activated with carbonyldiimidazole to allow coupling of the enzymes via c-amino groups. Kinetic analyses of the immobilized enzymes were carried out in a circulating flow system using CDNB (1-chloro-2,4-dinitrobenzene) as substrate, followed by specificity screening with alternative substrates. A good correlation was observed between the substrate screening data for immobilized enzyme and corresponding data for the enzyme in solution. A limited kinetic study was also carried out on immobilized human GST S1-1 (also known as hematopoietic prostaglandin D synthase). The stability of the immobilized enzymes was virtually identical to that of enzymes in solution, and no leakage of enzyme from the matrix could be observed.

Emneord
Immobilization, Glutathione transferase, Kinetics, Screening, Enzyme reactor, Drosophila
HSV kategori
Forskningsprogram
neurokemi med molekylär neurobiologi
Identifikatorer
urn:nbn:se:su:diva-100853 (URN)10.1016/j.ab.2013.10.004 (DOI)000329949500010 ()
Merknad

AuthorCount:5;

Tilgjengelig fra: 2014-02-21 Laget: 2014-02-17 Sist oppdatert: 2017-12-05bibliografisk kontrollert
2. Transgenic Overexpression of Glutathione Transferase E7 in Drosophila Attenuates Toxicity of Organic Isothiocyanates Affecting Survival and Oviposition
Åpne denne publikasjonen i ny fane eller vindu >>Transgenic Overexpression of Glutathione Transferase E7 in Drosophila Attenuates Toxicity of Organic Isothiocyanates Affecting Survival and Oviposition
(engelsk)Manuskript (preprint) (Annet vitenskapelig)
Abstract [en]

Organic isothiocyanates (ITCs) are allelochemicals produced by plants in order to combat insects and other herbivores. The compounds are toxic electrophiles that can be inactivated and conjugated with intracellular glutathione in reactions catalyzed by glutathione transferases (GSTs). The Drosophila melanogaster GSTE7 was heterologously expressed in Escherichia coli and purified for functional studies. The enzyme showed high catalytic activity with various isothiocyanates including phenethyl isothiocyanate (PEITC) and allyl isothiocyanate (AITC), which in millimolar dietary concentrations conferred toxicity to adult D. melanogaster leading to death or a shortened life-span of the flies. In situ hybridization revealed a maternal contribution of GSTE7 transcripts to embryos, and strongest zygotic expression in the digestive tract.  Transgenesis involving the GSTE7 gene controlled by an actin promoter produced viable flies expressing the GSTE7 transcript ubiquitously. Transgenic females show a significant extension in life-span when subjected to the same PEITC treatment as the wild-type flies. By contrast, transgenic male flies showed no significant effect in the first few days, and subsequently showed a somewhat lower survival rate. At 1 mM AITC concentration, no toxicity was noted. However, the oviposition activity was dramatically enhanced from a very low level in wild-type flies reared in the presence of 1 mM AITC to values an order of magnitude higher for the transgenic flies. The results demonstrate a clear protective effect of GSTE7 against exposure to ITC allelochemicals which can affect both life-span and fecundity of female flies.

Emneord
Glutathione transferase, isothiocyanate toxicity, oviposition, GST transgene, insect
HSV kategori
Identifikatorer
urn:nbn:se:su:diva-102001 (URN)
Tilgjengelig fra: 2014-03-20 Laget: 2014-03-20 Sist oppdatert: 2018-01-11bibliografisk kontrollert

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