Can the Herbicide Dicamba Produce Oxidative Stress in the Native South American fish Jenynsia lineata at Environmentally Relevant Concentrations?

Dicamba (DIC) is one of the most applied auxin herbicides worldwide. Sublethal effects in the South American native fish Jenynsia lineata exposed to DIC concentrations close to environmental concentrations (0.03-30 µg/L) during 48 h were analysed thorough the evaluation of catalase (CAT), glutathione S-transferase (GST), superoxide dismutase (SOD) activities and malondialdehyde (MDA) and H2O2 levels for detecting potential oxidative stress. In gills MDA increased showing oxidative damage probably because of an inefficient antioxidant defense. This response evidenced the important role of gills as an organ of direct contact with waterborne contaminants. In addition, other changes in the biomarkers of oxidative stress were observed such as the inhibition of SOD activities in brain and the inhibition of GST in liver. These results show that short- term exposures to environmentally relevant concentrations of DIC could induce sublethal effects in native fish.

Wheat germin-like protein: Studies on chitin/chitosan matrix for tissue engineering applications.

Advances in tissue engineering require the development of new biomaterials with adequate properties of cell attachment and growth. The properties of biomaterials can be improved by incorporation of bioactive molecules to enhance in vitro and/or in vivo functions. In this work, we study the role of a wheat germin-like protease inhibitor (GLPI), free or immobilized in biocompatible matrices to improve cell-attachment ability on different mammalian cell lines. The phylogenetic relationships and functional diversity of the GLPI were analyzed among diverse genera to get insights into sequence motif conservations. The cytocompatibility effect of free GLPI on C2C12 premyoblastic cells and B16 cells as tumoral model has been tested. GLPI promoted proliferation and metabolic activity of both cell types on in vitro models, not showing cytotoxic effects. Furthermore, GLPI was immobilized in chitin microparticles and in chitosan films; we demonstrated an accelerated cell adhesion process in both biomaterials.

Liver damage and caspase-dependent apoptosis is related to protein malnutrition in mice: effect of methionine.

This study aimed to determine whether the effects on the mouse liver caused by three periods of feeding a protein-free diet for 5 days followed by a normal complete diet for 5 days (3PFD-CD) are prevented by a constant methionine supply (3PFD+Met-CD). The expressions of carbonic anhydrase III (CAIII), fatty acid synthase (FAS), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and glutathione S-transferase P1 (GSTP1) were assessed by proteomics and reverse transcriptase-polymerase chain reactions. The liver redox status was examined by measuring the activities of superoxide dismutase (SOD) and catalase (CAT), as well as protein carbonylation. Because oxidative stress can result in apoptosis, the activity and content of caspase-3, as well as the x-linked inhibitor of the apoptosis protein (XIAP) and mitochondrial caspase-independent apoptosis inducing factor (AIF) contents were assessed. In addition, the liver histomorphology was examined. Compared to the controls fed a normal complete diet throughout, feeding with 3PFD-CD increased the FAS content, decreased the CAIII content, decreased both the SOD and CAT activities, and increased protein carbonylation. It also activated caspase-3, decreased the XIAP content, decreased the AIF content, increased the number of GSTP1-positive foci and caspase-3-positive cells, and caused fatty livers. Conversely, the changes were lessened to varying degrees in mice fed 3PFD+Met-CD. The present results indicate that a regular Met supply lessens the biochemical changes, damage, and caspase-dependent apoptosis provoked by recurrent dietary amino acid deprivation in the mouse liver.

Alternation between dietary protein depletion and normal feeding cause liver damage in mouse

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The effect of frequent protein malnutrition on liver function has not been intensively examined. Thus, the effects of alternating 5 days of a protein and amino acid-free diet followed by 5 days of a complete diet repeated three times (3 PFD-CD) on female mouse liver were examined. The expression of carbonic anhydrase III (CAIII), fatty acid synthase (FAS), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and glutathione S-transferase P1 (GSTP1) in liver were assessed by proteomics, reverse transcriptase-polymerase chain reaction and Northern blotting. The activities of liver GSTs, glutathione reductase (GR) and catalase (CAT), as well as serum glutamic-oxaloacetic transaminase (SGOT) and glutamic-pyruvic transaminase (SGPT) were also tested. Additionally, oxidative damage was examined by measuring of protein carbonylation and lipid peroxidation. Liver histology was examined by light and electron microscopy. Compared with control mice, 3 PFD-CD increased the content of FAS protein (+90%) and FAS mRNA (+30%), while the levels of CAIII and CAIII mRNAs were decreased (−48% and −64%, respectively). In addition, 3 PFD-CD did not significantly change the content of GSTP1 but produced an increase in its mRNA level (+20%), while it decreased the activities of both CAT (−66%) and GSTs (−26%). After 3 PFD-CD, liver protein carbonylation and lipid peroxidation were increased by +55% and +95%, respectively. In serum, 3 PFD-CD increased the activities of both SGOT (+30%) and SGPT (+61%). In addition, 3 PFD-CD showed a histological pattern characteristic of hepatic damage. All together, these data suggest that frequent dietary amino acid deprivation causes hepatic metabolic and ultrastructural changes in a fashion similar to precancerous or cancerous conditions (AU)

Alternation between dietary protein depletion and normal feeding cause liver damage in mouse.

The effect of frequent protein malnutrition on liver function has not been intensively examined. Thus, the effects of alternating 5 days of a protein and amino acid-free diet followed by 5 days of a complete diet repeated three times (3 PFD-CD) on female mouse liver were examined. The expression of carbonic anhydrase III (CAIII), fatty acid synthase (FAS), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and glutathione S-transferase P1 (GSTP1) in liver were assessed by proteomics, reverse transcriptase-polymerase chain reaction and Northern blotting. The activities of liver GSTs, glutathione reductase (GR) and catalase (CAT), as well as serum glutamic-oxaloacetic transaminase (SGOT) and glutamic-pyruvic transaminase (SGPT) were also tested. Additionally, oxidative damage was examined by measuring of protein carbonylation and lipid peroxidation. Liver histology was examined by light and electron microscopy. Compared with control mice, 3 PFD-CD increased the content of FAS protein (+90%) and FAS mRNA (+30%), while the levels of CAIII and CAIII mRNAs were decreased (-48% and -64%, respectively). In addition, 3 PFD-CD did not significantly change the content of GSTP1 but produced an increase in its mRNA level (+20%), while it decreased the activities of both CAT (-66%) and GSTs (-26%). After 3 PFD-CD, liver protein carbonylation and lipid peroxidation were increased by +55% and +95%, respectively. In serum, 3 PFD-CD increased the activities of both SGOT (+30%) and SGPT (+61%). In addition, 3 PFD-CD showed a histological pattern characteristic of hepatic damage. All together, these data suggest that frequent dietary amino acid deprivation causes hepatic metabolic and ultrastructural changes in a fashion similar to precancerous or cancerous conditions.

Oxidative stress in mouse liver caused by dietary amino acid deprivation: protective effect of methionine.

The aim of this work was to evaluate the effects of a diet depleted of amino acids (protein-free diet, or PFD), as well as the supplementation with methionine (PFD+Met), on the antioxidant status of the female mouse liver. With this purpose, cytosolic protein spots from two-dimensional non-equilibrium pH gel electrophoresis were identified by several procedures, such as mass spectrometry, Western blot, gel matching and enzymatic activity. PFD decreased the contents of catalase (CAT), peroxiredoxin I (Prx-I), and glutathione peroxidase (GPx) by 67%, 37% and 45%, respectively. Gene expression analyses showed that PFD caused a decrease in CAT (-20%) and GPx (-30%) mRNA levels but did not change that of Prx-I. It was also found that, when compared to a normal diet, PFD increased the liver contents of both reactive oxygen species (+50%) and oxidized protein (+88%) and decreased that of glutathione (-45%). Supplementation of PFD with Met prevented these latter effects to varying degrees, whereas CAT, Prx-I and GPx mRNA levels resulted unmodified. Present results suggest that dietary amino acid deprivation deranges the liver antioxidant defences, and this can be, in part, overcome by supplementation with Met.

Oxidative stress in mouse liver caused by dietary amino acid deprivation: protective effect of methionine

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The aim of this work was to evaluate the effects of a diet depleted of amino acids (protein-free diet, or PFD), as well as the supplementation with methionine (PFD+Met), on the antioxidant status of the female mouse liver. With this purpose, cytosolic protein spots from two-dimensional non-equilibrium pH gel electrophoresis were identified by several procedures, such as mass spectrometry, Western blot, gel matching and enzymatic activity. PFD decreased the contents of catalase (CAT),peroxiredoxin I (Prx-I), and glutathione peroxidase (GPx) by 67%, 37% and 45%, respectively. Gene expression analyses showed that PFD caused a decrease in CAT (−20%) and GPx (−30%) mRNAlevels but did not change that of Prx-I. It was also found that, when compared to a normal diet, PFD increased the liver contents of both reactive oxygen species (+50%) and oxidized protein (+88%) and decreased that of glutathione (−45%). Supplementation of PFD with Met prevented these latter effects to varying degrees, whereas CAT, Prx-I and GPx mRNA levels resulted unmodified. Present results suggest that dietary amino acid deprivation deranges the liver antioxidant defences, and this can be, in part, overcome by supplementation with Met (AU)

The swaposin-like domain of potato aspartic protease (StAsp-PSI) exerts antimicrobial activity on plant and human pathogens.

Plant-specific insert domain (PSI) is a region of approximately 100 amino acid residues present in most plant aspartic protease (AP) precursors. PSI is not a true saposin domain; it is the exchange of the N- and C-terminal portions of the saposin like domain. Hence, PSI is called a swaposin domain. Here, we report the cloned, heterologous expression and purification of PSI from StAsp 1 (Solanum tuberosum aspartic protease 1), called StAsp-PSI. Results obtained here show that StAsp-PSI is able to kill spores of two potato pathogens in a dose-dependent manner without any deleterious effect on plant cells. As reported for StAPs (S. tuberosum aspartic proteases), the StAsp-PSI ability to kill microbial pathogens is dependent on the direct interaction of the protein with the microbial cell wall/or membrane, leading to increased permeability and lysis. Additionally, we demonstrated that, like proteins of the SAPLIP family, StAsp-PSI and StAPs are cytotoxic to Gram-negative and Gram-positive bacteria in a dose dependent manner. The amino acid residues conserved in SP_B (pulmonary surfactant protein B) and StAsp-PSI could explain the cytotoxic activity exerted by StAsp-PSI and StAPs against Gram-positive bacteria. These results and data previously reported suggest that the presence of the PSI domain in mature StAPs could be related to their antimicrobial activity.

Cytotoxic effect of potato aspartic proteases (StAPs) on Jurkat T cells.

StAPs are potato aspartic proteases with cytotoxic activity against plant pathogens and spermatozoa. StAPs cytotoxic activity is selective, since these proteins do not exert toxic effect on plant cells and erythrocytes. In this work, we investigated the capacity of StAPs to exert cytotoxicity on human leukaemia cells. Obtained results show that StAPs induce apoptosis on Jurkat T cells after a short time of incubation in a dose-dependent manner. However, no significative effect on the T lymphocytes viability was observed at all StAPs incubation times and concentrations tested. These results suggest that StAPs can be conceptually promising leads for cancer therapy.

Roles of glycosylation on the antifungal activity and apoplast accumulation of StAPs (Solanum tuberosum aspartic proteases).

Specific roles of glycosylation appear to be protein-dependent. Plant aspartic proteases (APs) contain two or more consensus N-glycosylation sites; however, the importance of them is not well understood. StAPs (Solanum tuberosum aspartic proteases) are bifunctional proteins with both proteolytic and antimicrobial activities. These proteins are accumulated into the intercellular washing fluid of potato tubers and leaves after wounding or infection. In this paper we investigated the importance of glycosylation on the StAPs apoplast accumulation, biochemical parameters, and fungicidal activity. Assays to evaluate the importance of StAPs glycosylation groups by using glycosylation inhibitors demonstrate that carbohydrate portions are essential to StAPs accumulation into the apoplast of tubers and leaves after wounding or detachment, respectively. Bifunctional activity of StAPs is differentially affected by this post-translational modification. Results obtained show that not significant changes were produced in the physicochemical properties after StAPs deglycosylation (pH and thermal-optimum activity and index of protein surface hydrophobicity). Otherwise, StAPs antifungal activity is affected by deglycosylation. Deglycosylated StAPs (dgStAPs) fungicidal activity is lower than native StAPs at all concentrations and times assayed. In summary, glycosylation has not a significant role on the StAPs conformational structure. However, it is involved in the StAPs subcellular accumulation and antifungal activity suggesting that it could be necessary for StAPs membrane and/or protein interactions and subsequently its biological function(s).

Potato aspartic proteases (StAPs) exert cytotoxic activity on bovine and human spermatozoa.

OBJECTIVE: To evaluate the in vitro spermicidal activity of Solanum tuberosum aspartic proteinases (StAPs) on bovine and human sperm. DESIGN: Controlled laboratory study. SETTING: Three research laboratories at a university of biologic science. ANIMAL(S) AND DONOR(S): Frozen semen from five Aberdeen Angus bulls and six proven fertile men volunteers. INTERVENTION(S): The effect of StAPs on sperm motility was studied in vitro by incubation of different concentrations of StAPs with sperm suspensions, and motility was assessed by direct microscopic observation. Membrane integrity was analyzed by SYTOX Green uptake after incubation with different StAP concentrations. The effect of StAPs was evaluated by human erythrocyte lysis, as a control in somatic cells. The StAPs binding was monitored by fluorescence. MAIN OUTCOME MEASURE(S): Total and progressive sperm motility; hypoosmotic swelling test and SYTOX Green uptake as a measure of membrane damage; fluorescein isothiocyanate-labeled StAP binding by an optical microscopy. RESULT(S): The StAPs reduced sperm motility in a dose-dependent manner, and 25 microM of StAP1 and 35 microM of StAP3 completely abolished the progressive motility. The StAPs were able to bind in the postacrosomal and midpiece region only in bovine sperm. Also, StAPs caused spermatozoa agglutination. In vitro cell toxicity was observed by a dose-dependent increase in hypoosmotic swelling negative sperm and SYTOX Green uptake in both human and bovine spermatozoa; however, no toxic effect was observed on erythrocytes. CONCLUSION(S): The spermicidal effect of StAPs involves plasma membrane permeabilization.

Antimicrobial activity of potato aspartic proteases (StAPs) involves membrane permeabilization.

Solanum tuberosum aspartic proteases (StAPs) with antimicrobial activity are induced after abiotic and biotic stress. In this study the ability of StAPs to produce a direct antimicrobial effect was investigated. Viability assays demonstrated that StAPs are able to kill spores of Fusarium solani and Phytophthora infestans in a dose-dependent manner. Localization experiments with FITC-labelled StAPs proved that the proteins interact directly with the surface of spores and hyphae of F. solani and P. infestans. Moreover, incubation of spores and hyphae with StAPs resulted in membrane permeabilization, as shown by the uptake of the fluorescent dye SYTOX Green. It is concluded that the antimicrobial effect of StAPs against F. solani and P. infestans is caused by a direct interaction with the microbial surfaces followed by membrane permeabilization.

Molecular cloning of a potato leaf cDNA encoding an aspartic protease (StAsp) and its expression after P. infestans infection.

Aspartic proteinases (EC 3.4.23) are widely distributed in the plant kingdom, and a number of cDNAs have been isolated from different plants. Here we report the isolation an expression analysis of a cDNA from Solanum tuberosum L. (cv. Pampeana) named StAsp. The StAsp cDNA clone was obtained using a reverse transcriptase-polymerase chain reaction (RT-PCR) and degenerated primers encoding to plant aspartic proteinases conserved domains. The coding region of the gene is 1494 bp long encoding 497 amino acids of a predicted 54 kDa molecular mass and with a pI of 5.5. The gene shares a high homology with an aspartic proteinase cDNA of tomato, 97% and 94% homology on the level of DNA and protein, respectively. The deduced amino acid sequence contains the conserved features of plant aspartic proteinases, including the plant specific insert. Northern blot analysis indicated that StAps transcripts are differentially accumulated in potato leaves after Phytophthora infestans infection in two potato cultivars with different degree of field resistance to this pathogen. In the resistant cultivar (Pampeana), induction was higher and more durable than in the susceptible cultivar (Bintje), suggesting that the StAsp level expression are associated with the resistance degree of potato cultivars to P. infestans. Results obtained previously about the induction of StAP proteins in stress conditions and these results suggest that potato aspartic proteinases are components of the plant defense response.