| 1. |
- Monteiro, Ana C. S., et al.
(author)
-
Identification, characterization and localization of chagasin, a tight-binding cysteine protease inhibitor in Trypanosoma cruzi
- 2001
-
In: Journal of Cell Science. - 0021-9533. ; 114:21, s. 3933-3942
-
Journal article (peer-reviewed)abstract
- Lysosomal cysteine proteases from mammalian cells and plants are regulated by endogenous tight-binding inhibitors from the cystatin superfamily. The presence of cystatin-like inhibitors in lower eukaryotes such as protozoan parasites has not yet been demonstrated, although these cells express large quantities of cysteine proteases and may also count on endogenous inhibitors to regulate cellular proteolysis. Trypanosoma cruzi, the causative agent of Chagas heart disease, is a relevant model to explore this possibility because these intracellular parasites rely on their major lysosomal cysteine protease (cruzipain) to invade and multiply in mammalian host cells. Here we report the isolation, biochemical characterization, developmental stage distribution and subcellular localization of chagasin, an endogenous cysteine protease inhibitor in T. cruzi. We used high temperature induced denaturation to isolate a heat-stable cruzipain-binding protein (apparent molecular mass, 12 kDa) from epimastigote lysates. This protein was subsequently characterized as a tight-binding and reversible inhibitor of papain-like cysteine proteases. Immunoblotting indicated that the expression of chagasin is developmentally regulated and inversely correlated with that of cruzipain. Gold-labeled antibodies localized chagasin to the flagellar pocket and cytoplasmic vesicles of trypomastigotes and to the cell surface of amastigotes. Binding assays performed by probing living parasites with fluorescein (FITC)-cruzipain or FITC-chagasin revealed the presence of both inhibitor and protease at the cell surface of amastigotes. The intersection of chagasin and cruzipain trafficking pathways may represent a checkpoint for downstream regulation of proteolysis in trypanosomatid protozoa.
|
|
| 2. |
|
|
| 3. |
- Santos, F J, et al.
(author)
-
Analysis of heterocyclic amines in food products: interlaboratory studies
- 2004
-
In: Journal of Chromatography. B. - : Elsevier BV. - 1873-376X .- 1570-0232. ; 802:1, s. 69-78
-
Journal article (peer-reviewed)abstract
- A feasibility study and two interlaboratory exercises on the determination of selected heterocyclic amines (HAs) in beef extract, organised in the framework of a European project, are presented. The aim of these exercises was to improve the quality of the laboratories and to evaluate the performance of a standardised analytical method and also the methods currently used by each of the participants for the analysis of these compounds. Three lyophilised portions of a commercial beef material previously spiked with HAs at different concentration levels ranging from 10 to 75 ng g(-1) were used as laboratory reference materials (lot A, B and C). Firstly, a feasibility study was carried out using a test standard solution and the beef extract (lot A), which contained only five HAs. Then, two interlaboratory exercises were carried out using the laboratory reference materials lot B and lot C, containing 10 selected HAs at two different concentration levels, 75 and 10ng/g, respectively. The results obtained by all participant laboratories using the proposed method showed satisfactory agreement and the CV(%) between-laboratories obtained were from 8.3 to 24.1% for lot B and from 8.7 to 44.5% for lot C. The standardised method evaluated in these collaborative studies is therefore proposed for the analysis of HAs in food material. Moreover, LC-MS is recommended as the most suitable technique for the analysis of a large number of HAs in food samples. (C) 2003 Elsevier B.V All rights reserved.
|
|
| 4. |
- Saraiva, Jorge M., et al.
(author)
-
Issues in Human GenEthics
- 2001
-
In: Genetics in Medicine. - 1098-3600. ; 3:3, s. 218-218
-
Journal article (peer-reviewed)
|
|
