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- Ahmad, Nafees, et al.
(författare)
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Pitx3 directly regulates Foxe3 during early lens development.
- 2013
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Ingår i: The International journal of developmental biology. - : UPV/EHU Press. - 1696-3547 .- 0214-6282. ; 57, s. 741-751
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Tidskriftsartikel (refereegranskat)abstract
- Pitx3 is a bicoid-related homeodomain transcription factor critical for the development of the ocular lens, mesencephalic dopaminergic neurons and skeletal muscle. In humans, mutations in PITX3 are responsible for cataracts and anterior segment abnormalities of varying degree; polymorphisms are associated with Parkinsons disease. In aphakia (ak) mice, two deletions in the promoter region of Pitx3 cause abnormal lens development. Here, we investigated systematically the role of Pitx3 in lens development including its molecular targets responsible for the ak phenotype. We have shown that ak lenses exhibit reduced proliferation and aberrant fiber cell differentiation. This was associated with loss of Foxe3 expression, complete absence of Prox1 expression, reduced expression of epsilon-tubulin and earlier expression of gamma-crystallin during lens development. Using EMSA and ChIP assays, we demonstrated that Pitx3 binds to an evolutionary conserved bicoid-binding site on the 5-upstream region of Foxe3. Finally, Pitx3 binding to 5-upstream region of Foxe3 increased transcriptional activity significantly in a cell-based reporter assay. Identification of Foxe3 as a transcriptional target of Pitx3 explains at least in part some of the phenotypic similarities of the ak and dyl mice (dysgenic lens, a Foxe3 allele). These findings enhance our understanding of the molecular cascades which subserve lens development.
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| 2. |
- Aslam, Muhammad
(författare)
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The fruit fly Drosophila melanogaster GSTE6 and E7; characterization, immobilization and transgenic overexpression
- 2014
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Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- 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.
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