1,721,033 research outputs found
FLUORESCENT ASSAY TO TEST PROTEIN DISULFIDE ISOMERASE ACTIVITY INDUCED BY THIOREDOXIN REDUCTASE.
No full textIn eukaryotic cells the ubiquitous thioredoxin reductase-thioredoxin system (TrxR-Trx) catalyses substrate disulfide reduction using NADPH as a source of reducing equivalents. Thioredoxin forms a superfamily with several other proteins sharing little sequence similarity but possessing a common active site. Among these, protein disulfide isomerase (PDI) is colocalized, in cells,with TrxR and there are now some in vitro evidences that PDI can be a substrate of TrxR1. To further demonstrate this interaction we have now produced a fluorogenic substrate suitable to test TrxR-dependent PDI activity. The substrate was formed by two identical cys-containing tri-peptides linked at the N-terminus to a molecule of fluorescein-5-isothiocyanate (FITC) and held together by a disulfide bridge between the cysteines; it was called pep-FITC. The reduction of pep-FITC was measured at 25°C in a Fluoromax spectrofluorimeter and was demonstrated by an increase of the FITC fluorescence at 520nm with excitation at 494nm. We could show that the TrxR-PDI system successfully reduced pep-FITC, while the isolated components did not. Further proofs of the interaction between PDI and TrxR were obtained by fluorescence resonant energy transfer (FRET). In this case we labelled the enzymes with Alexa Fluor 546 and Alexa Fluor 488 respectively, two dyes forming a donor/acceptor pair with a Forster distance of 58 Å.A FRET was observed under suitable conditions confirming tight interaction
Synthesis of liquid organic compounds from CH4 and CO2 in a dielectric barrier discharge operating at atmospheric pressure
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Changes in membrane lipid composition in marine species of Euplotes adapted to different thermal environments
No full textCell integrity and activities are largely influenced by membrane fluidity, which is temperature-dependent. Below the so-called transition temperature, phospholipids mobility is drastically reduced. Therefore, life adaptation to extreme environments is supposed to involve significant variations in membrane lipids.
We determined the phospholipid composition in three ecologically separated species of Euplotes: one (E. raikovi) from temperate areas, one (E. focardii) endemic to Antarctic, and one (E. polaris) colonizer of both Arctic and Antarctic.
Lipids were extracted from cell cultures and analyzed by RPLC-ESI-QqQ-MS in positive mode. E. polaris and E. raikovi contained several species of phosphocholines (PC), lyso-PC (L-PC), phosphatidylethanol-amines (PE), lyso-PE, sphingomyelins (SM), phosphatydilinositol (PI) and phosphatidylglycerol (PG). E. focardii contained all the classes but PE and SM. Very interestingly, E. focardii contained the lowest number of lipid species and showed a greater unsaturation index, while total PE of E. polaris were highly unsaturated compared to PE the other two species
Raman scattering investigation on the inclusion complex of anti-inflammatory drug indomethacin with cyclodextrin
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Chemical defence by mono-prenyl hydroquinone in a freshwater ciliate, Spirostomum ambiguum
No full textSeveral species of ciliates produce and accumulate low molecular weight toxic compounds in specialised membrane-bound ejectable organelles: extrusomes. These compounds can be used in predator–prey interaction for killing prey as well as for chemical defence. Here, we describe the isolation and characterisation of 2-(3-methylbut-2-enyl)benzene-1,4-diol(mono-prenyl hydroquinone), the extrusomal defensive toxin of the freshwater heterotrich ciliate Spirostomum ambiguum. The toxin was purified at homogeneity by RP-HPLC, and its structural characterisation was carried out through NMR and MS measurements. In vivo experiments involving S. ambiguum and Climacostomum virens in predator–prey interaction, and the analysis of cytotoxic activity of mono-prenyl hydroquinone on a panel of free-living freshwater ciliates, indicated that the toxin is very effective in S. ambiguum’s chemical defence
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