Abstract: Endocrine disrupting compounds (EDCs) have been studied due to their impact on human health and increasing awareness of their impact on wildlife species. Studies concerning the organ-specific molecular effects of EDC in invertebrates are important to understand the mechanisms of action of this class of toxicants but are scarce in the literature. We have used a dose/response approach to unravel the protein expression in different organs of isopods exposed to bisphenol A (BPA) and vinclozolin (Vz) and assess their potential use as surrogate species. Male isopods were exposed to a range of Vz or of BPA concentrations. After animal dissection, proteins were extracted from gut, hepatopancreas and testes. Protein profiles were analysed by electrophoresis and differentially expressed proteins were identified by MALDI mass spectrometry. EDCs affected proteins involved in the energy metabolism (arginine kinase), proteins of the heat shock protein family (Hsp70 and GRP78) and most likely microtubule dynamics (tubulin). Different proteins expressed at different concentrations in different organs are indicative of the organ-specific effects of BPA and Vz. Additionally, several proteins were up-regulated at lower but not higher BPA or Vz concentrations, bringing new data to the non-monotonic response curve controversy. Furthermore, our findings suggest that some common responses to EDCs in both vertebrates and invertebrates may exist.
Abstract: The growing interest in the application of proteomic technologies to solve toxicology issues and its relevance in ecotoxicology research has resulted in the emergence of 'ecotoxicoproteomics'. There is a general consensus that ecotoxicoproteomics is a powerful tool to spot early molecular events involved in toxicant responses, which are responsible for the adverse effects observed at higher levels of biological organization, thus contributing to elucidate the mode of action of stressors and to identify specific biomarkers. Ultimately, early-warning indicators can then be developed and deployed in 'in situ' bioassays and in environmental risk assessment.The number of field experiments or laboratory trials using ecologically relevant test-species and involving proteomics has been until recently, insufficient to allow a critical analysis of the real benefits of the application of this approach to ecotoxicology.This article intends to present an overview on the applications of proteomics in the context of ecotoxicology, focusing mainly the prospective research to be done in invertebrates. Although these represent around 95% of all animal species and in spite of the key structural and functional roles they play in ecosystems, proteomic research in invertebrates is still in an incipient stage. We will review applications of ecotoxicoproteomics by evaluating the technical methods employed, the organisms and the contexts studied, the advances achieved until now and lastly the limitations yet to overcome will be discussed.
Abstract: The antioxidant activity of different edible mushrooms was evaluated considering the different contribution of individual and combined extracts. The radical scavenging capacity was evaluated through hydrogen atom transfer and single electron transfer reaction-based assays: DPPH radical scavenging activity and reducing power, respectively. The inhibition of lipid peroxidation was studied in lipossomes solutions by the beta-carotene-linoleate system. Three types of interactions (synergistic, additive and negative synergistic effects) were observed, synergism being the most abundant effect. Marasmius oreades is present in the mixtures with higher antioxidant properties and synergistic effects, while Cantharellus cibarius is present in the mixtures with lowest antioxidant properties and negative synergist effects. Two discriminant analyses were performed considering individual species in one case and mushroom mixtures in the other. The five mushroom species were clustered in five individual groups, but a similar result could not be obtained for the combined mushrooms, for which only the cases containing C. cibarius were separated in individual clusters.
Abstract: Aspartic proteinases (AP) play major roles in physiologic and pathologic scenarios in a wide range of organisms from vertebrates to plants or viruses. The present work deals with the purification and characterisation of four new APs from the cardoon Cynara cardunculus L., bringing the number of APs that have been isolated, purified and biochemically characterised from this organism to nine. This is, to our knowledge, one of the highest number of APs purified from a single organism, consistent with a specific and important biological function of these protein within C. cardunculus. These enzymes, cardosins E, F, G and H, are dimeric, glycosylated, pepstatin-sensitive APs, active at acidic pH, with a maximum activity around pH 4.3. Their primary structures were partially determined by N- and C-terminal sequence analysis, peptide mass fingerprint analysis on a MALDI-TOF/TOF instrument and by LC-MS/MS analysis on a Q-TRAP instrument. All four enzymes are present on C. cardunculus L. pistils, along with cyprosins and cardosins A and B. Their micro-heterogeneity was detected by 2D-electrophoresis and mass spectrometry. The enzymes resemble cardosin A more than they resemble cardosin B or cyprosin, with cardosin E and cardosin G being more active than cardosin A, towards the synthetic peptide KPAEFF(NO(2))AL. The specificity of these enzymes was investigated and it is shown that cardosin E, although closely related to cardosin A, exhibits different specificity.
Abstract: In order to increase understanding of the basis of the stability of the native conformational state of porcine pepsin A, a strategy based on induction and monitoring of protein denaturation was developed. Structural perturbation was achieved by adding acetonitrile (MeCN) to the protein-solvent system. MeCN was found to induce non-coincident disruption of the secondary and tertiary structural features of pepsin A. It is proposed that gross unfolding is prompted by disruption of the protein hydration pattern induced by the organic co-solvent. It should be noted that the functional properties and thermal stability of the protein were already impaired before the onset of global unfolding. Low and intermediate contents of MeCN in the protein-solvent system affected the sharpness of the thermal transition and the degree of residual structure of the heat-denatured state. The importance of hydration to the conformational stability of pepsin A in its biologically active state is discussed.
Abstract: The carbapenem-hydrolyzing B-lactamase SFC-1 from Serratia fonticola UTAD54 was overexpressed in
Escherichia coli, purified, and characterized. The enzyme exhibited an apparent molecular mass of 30.5
kDa, determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. SFC-1 hydrolyzes penicillins,
cephalosporins, aztreonam, and carbapenems and is inhibited by clavulanic acid, sulbactam, and
tazobactam
Abstract: The kinetics of the structural changes affecting cardosin A, a plant aspartic proteinase (AP) from Cynara cardunculus L., in the presence of a mixture of acetonitrile (AN) in water (W) was studied. Incubation of cardosin A with 10% (v/v) AN resulted in a gradual increase in protein helicity, accompanied by changes in the tertiary structure, seen by changes in the intrinsic fluorescence of tryptophan. Differential scanning calorimetry (DSC) revealed that the temperature of denaturation of cardosin A decreased upon the addition of AN. With longer incubation times, the small chain of cardosin A denatured completely, consequent exposure of the single tryptophan residue accounting well for the observed spectral shift intrinsic fluorescence of the protein. Enzymatic activity assays demonstrated that the kinetically determined unfolding of the small chain of cardosin A does not result in loss of the activity of this enzyme.
Abstract: Cardosin A, a plant aspartic proteinase, capable of synthesising peptides, was investigated through synthesis of five methyl esters amino acid substrates as amino donors and nine benzyloxycarbonyl amino acid and peptide carboxyl donors. It was found that cardosin A is able to catalyse the synthesis of several peptide bonds, being the preference order for the carboxyl components the following: CBz.Phe >CBz.Trp. Unpredictably, Tyr could not be accepted in P1. Results were compared and discussed according to the known specificity of pepsin, the most studied aspartic proteinase.
Abstract: A Bacillus licheniformis strain, 189, isolated from a hot spring environment in the Azores, Portugal, strongly inhibited growth of Gram-positive bacteria. It produced a peptide antibiotic at 50 degrees C. The antibiotic was purified and biochemically characterized. It was highly resistant to several proteolytic enzymes. Additionally, it retained its antimicrobial activity after incubation at pH values between 3.5 and 8; it was thermostable, retaining about 85% and 20% of its activity after 6 h at 50 degrees C and 100 degrees C, respectively. Its molecular mass determined by mass spectrometry was 3249.7 Da.
Abstract: This investigation showed that cardosin A not only is active in media with organic solvents, cleaving the -chain of oxidised insulin at three susceptible peptide bonds, but also maintains its specificity in all media tested. Additionally, the presence of organic solvents in the reaction media led to modifications of enzyme selectivity, which enabled the detection of intermediate products. While solvents like ethyl acetate induced a decrease in enzymatic activity, both by reducing the amount of active enzyme and presumably due to an inhibiting effect of ethyl acetate (which might compete with the substrate for the active site of the enzyme), n-hexane caused an increase in the hydrolysis velocity of one peptide bond. In view of the activity and specificity of cardosin A (which shows high preference for hydrophobic residues), it is proposed as a reliable probe for limited proteolysis in the presence of organic solvents. This may become particularly useful for structural characterisation of membrane proteins.
