Exposure to environmental pollutants such as polychlorinated biphenyls (PCBs) is now taken into a... more Exposure to environmental pollutants such as polychlorinated biphenyls (PCBs) is now taken into account to partly explain the worldwide decline of amphibians. PCBs induce deleterious effects on developing amphibians including deformities and delays in metamorphosis. However, the molecular mechanisms by which they express their toxicity during the development of tadpoles are still largely unknown. A proteomics analysis was performed on developing Xenopus laevis tadpoles exposed from 2 to 5 days postfertilization to either 0.1 or 1 ppm Aroclor 1254, a PCB mixture. Two-dimensional DIGE with a minimal labeling method coupled to nanoflow liquid chromatography-tandem mass spectrometry was used to detect and identify proteins differentially expressed under PCBs conditions. Results showed that 59 spots from the 0.1 ppm Aroclor 1254 condition and 57 spots from the 1 ppm Aroclor 1254 condition displayed a significant increase or decrease of abundance compared with the control. In total, 28 pr...
The identification of the regulatory proteins that control DNA transcription as well as RNA stabi... more The identification of the regulatory proteins that control DNA transcription as well as RNA stability and translation represents a key step in the comprehension of gene expression regulation. Those proteins can be purified by DNA- or RNA-affinity chromatography, followed by identification by mass spectrometry. Although very simple in the concept, this represents a real technological challenge due to the low abundance of regulatory proteins compared to the highly abundant proteins binding to nucleic acids in a nonsequence-specific manner. Here we review the different strategies that have been set up to reach this purpose, discussing the key parameters that should be considered to increase the chances of success. Typically, two categories of biological questions can be distinguished: the identification of proteins that specifically interact with a precisely defined binding site, mostly addressed by quantitative mass spectrometry, and the identification in a non-comparative manner of the protein complexes recruited by a poorly characterized long regulatory region of nucleic acids. Finally, beside the numerous studies devoted to in vitro-assembled nucleic acid-protein complexes, the scarce data reported on proteomic analyses of in vivo-assembled complexes are described, with a special emphasis on the associated challenges.
Previous investigations of the potential of metal-organic compounds as inhibitors of human immuno... more Previous investigations of the potential of metal-organic compounds as inhibitors of human immunodeficiency virus type I protease (HIV-1 PR) showed that the copper(II) complex diaqua [bis(2-pyridylcarbonyl)amido] copper(II) nitrate dihydrate and the complex bis[N2-(2,3,6-trimethoxybenzyl)-4-2-pyridinecarboxamide] copper(II) behaved as inhibitors of HIV-1 PR. In a search for similar readily accessible ligands, we synthesised and studied the structural properties of N2-(2-pyridylmethyl)-2-pyridinecarboxamide (L) copper(II) complexes. Three different crystal structures were obtained. Two were found to contain ligand L simultaneously in a tridentate and bidentate conformation [Cu(L L )]. The other contained two symmetry-related ligands, coordinated through the pyridine nitrogen and tri bi the amide oxygen atoms [Cu(L ) ]. A search of the Cambridge Structural Database indicated that L resulting from nitrogen bound bi 2 tri amide hydrogen metal substitution is favoured over chelation through the amide oxygen atom. In our case, we calculated that the conformation of L is 11 kcal / mol more favourable than that of L . ESI-MS experiments showed that the Cu(L ) structure could not tri bi bi 2 be observed in solution, while Cu(L L )-related complexes were indeed present. The lack of protease inhibition of the pyridine tri bi carboxamide copper(II) complexes was explained by the fact that the Cu(L L ) complex could not fit into the HIV-1 active site.
Abstraet. The arachidonic acid cascade in interleukin-1-stimulated chondrocytes was investigated ... more Abstraet. The arachidonic acid cascade in interleukin-1-stimulated chondrocytes was investigated by studying the activation of phospholipase A2 and the regulation of cyclooxygenase. ILl stimulated the [3H]-arachidonic acid release and analyses of the [3Hl-arachidonic acidderived metabolites stressed that most of the released radioactivity after IL 1 activation was associated with prostaglandin E2. Human IL 1-stimulated chondrocytes also synthesized prostaglandin Fz~ and 6-keto-prostaglandin F1,. Using transcription and translation inhibitors it was shown that protein synthesis was required for PG synthesis. Aspirin, a specific inhibitor of cyclooxygenase, was also used and confirmed that the production of PG induced by IL 1 is imputable to a de novo synthesis of cyclooxygenase. In human chondrocytes, cyclooxygenase is thus a key enzyme in the AA cascade in addition of PLA2.
Comparative Biochemistry and Physiology Part D: Genomics and Proteomics, 2010
The intensification of shrimp farming has been related to the increasing use of chemotherapeutics... more The intensification of shrimp farming has been related to the increasing use of chemotherapeutics and potentially suboptimal rearing conditions. For the purpose of assessing the stress level of cultured giant tiger shrimp Penaeus monodon, a proteomic analysis (2D-DIGE) was performed on hemolymph. On the one hand, shrimp were exposed for 7 days to the antibiotics enrofloxacin or furazolidone via feed (4 g kg(-1)) under laboratory conditions. On the other hand, shrimp were submitted to enrofloxacin directly in field conditions in Vietnam, for which two different culture systems were distinguished (intensive and improved extensive). No significant different protein abundance pattern was induced by antibiotics under laboratory conditions, while only one protein spot displayed a 1.53-fold reduction in intensity after exposure to enrofloxacin in improved extensive ponds. When we compared the proteome of shrimp bred either in intensive or in improved extensive system, we observed 9 protein spots displaying significant difference in abundance. Among them, 3 spots of hemocyanin were under-expressed in shrimp from improved extensive ponds. At the opposite 2 spots corresponding to Sarcoplasmic Calcium-binding Protein (SCP) were less abundant in hemolymph of shrimp from intensive ponds. These results demonstrate that the very subtle effects of tested antibiotics on patterns of hemolymph protein expression are overwhelmed by the effects of conditions encountered in different production management systems, such as different oxygen and nitric concentrations.
Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology, 2014
Domestication might be a possible way to reduce the physiological response to long-term stressors... more Domestication might be a possible way to reduce the physiological response to long-term stressors and deleterious effects on immunity. The present study aimed to evaluate the chronic immune response induced by repeated emersions and the possible impact of domestication by comparing farmed Eurasian perch with short (F1) and long (F4) captive-life history. In the first experiment, fish were exposed to a single emersion and physiological stress response was measured in the short term to characterize fish sensitivity to the tested stressor. Serum cortisol and glucose elevated within 6 h post-stress and splenosomatic index (SSI) decreased within 48 h, indicating that the species was affected by emersion stressor. In the second experiment, F1 and F4 generations were submitted to repeated water emersions (3 times/week during 44 days). On day 9, 18 and 44, samplings were performed 48 h post-stressor to highlight any sustained disruption of immune system. Serum cortisol, glucose, SSI and lysozyme activity were evaluated and serum proteome was analyzed using 2D-DIGE. Any of the tested variables were affected by repeated emersions and proteomic analysis only revealed that alpha-2 macroglobulins (a2Ms) were up-regulated in the serum of stressed individuals. Domestication also resulted in the up-regulation of five a2M isoforms and down-regulation of complement C3 and Ig light chain proteins, independently of any stressor exposure. In conclusion, the results suggested that repeated emersions are not severe stressors for Eurasian perch, probably explaining why domestication had no influence on fish responses. Changes associated with domestication are highly complex and certainly need further investigations.
Background: Repeated exposures to UVB of human keratinocytes lacking functional p16 and able to d... more Background: Repeated exposures to UVB of human keratinocytes lacking functional p16 and able to differentiate induce an alternative state of differentiation rather than stress-induced premature senescence.
Exposure to environmental pollutants such as polychlorinated biphenyls (PCBs) is now taken into a... more Exposure to environmental pollutants such as polychlorinated biphenyls (PCBs) is now taken into account to partly explain the worldwide decline of amphibians. PCBs induce deleterious effects on developing amphibians including deformities and delays in metamorphosis. However, the molecular mechanisms by which they express their toxicity during the development of tadpoles are still largely unknown. A proteomics analysis was performed on developing Xenopus laevis tadpoles exposed from 2 to 5 days postfertilization to either 0.1 or 1 ppm Aroclor 1254, a PCB mixture. Two-dimensional DIGE with a minimal labeling method coupled to nanoflow liquid chromatography-tandem mass spectrometry was used to detect and identify proteins differentially expressed under PCBs conditions. Results showed that 59 spots from the 0.1 ppm Aroclor 1254 condition and 57 spots from the 1 ppm Aroclor 1254 condition displayed a significant increase or decrease of abundance compared with the control. In total, 28 pr...
The identification of the regulatory proteins that control DNA transcription as well as RNA stabi... more The identification of the regulatory proteins that control DNA transcription as well as RNA stability and translation represents a key step in the comprehension of gene expression regulation. Those proteins can be purified by DNA- or RNA-affinity chromatography, followed by identification by mass spectrometry. Although very simple in the concept, this represents a real technological challenge due to the low abundance of regulatory proteins compared to the highly abundant proteins binding to nucleic acids in a nonsequence-specific manner. Here we review the different strategies that have been set up to reach this purpose, discussing the key parameters that should be considered to increase the chances of success. Typically, two categories of biological questions can be distinguished: the identification of proteins that specifically interact with a precisely defined binding site, mostly addressed by quantitative mass spectrometry, and the identification in a non-comparative manner of the protein complexes recruited by a poorly characterized long regulatory region of nucleic acids. Finally, beside the numerous studies devoted to in vitro-assembled nucleic acid-protein complexes, the scarce data reported on proteomic analyses of in vivo-assembled complexes are described, with a special emphasis on the associated challenges.
Previous investigations of the potential of metal-organic compounds as inhibitors of human immuno... more Previous investigations of the potential of metal-organic compounds as inhibitors of human immunodeficiency virus type I protease (HIV-1 PR) showed that the copper(II) complex diaqua [bis(2-pyridylcarbonyl)amido] copper(II) nitrate dihydrate and the complex bis[N2-(2,3,6-trimethoxybenzyl)-4-2-pyridinecarboxamide] copper(II) behaved as inhibitors of HIV-1 PR. In a search for similar readily accessible ligands, we synthesised and studied the structural properties of N2-(2-pyridylmethyl)-2-pyridinecarboxamide (L) copper(II) complexes. Three different crystal structures were obtained. Two were found to contain ligand L simultaneously in a tridentate and bidentate conformation [Cu(L L )]. The other contained two symmetry-related ligands, coordinated through the pyridine nitrogen and tri bi the amide oxygen atoms [Cu(L ) ]. A search of the Cambridge Structural Database indicated that L resulting from nitrogen bound bi 2 tri amide hydrogen metal substitution is favoured over chelation through the amide oxygen atom. In our case, we calculated that the conformation of L is 11 kcal / mol more favourable than that of L . ESI-MS experiments showed that the Cu(L ) structure could not tri bi bi 2 be observed in solution, while Cu(L L )-related complexes were indeed present. The lack of protease inhibition of the pyridine tri bi carboxamide copper(II) complexes was explained by the fact that the Cu(L L ) complex could not fit into the HIV-1 active site.
Abstraet. The arachidonic acid cascade in interleukin-1-stimulated chondrocytes was investigated ... more Abstraet. The arachidonic acid cascade in interleukin-1-stimulated chondrocytes was investigated by studying the activation of phospholipase A2 and the regulation of cyclooxygenase. ILl stimulated the [3H]-arachidonic acid release and analyses of the [3Hl-arachidonic acidderived metabolites stressed that most of the released radioactivity after IL 1 activation was associated with prostaglandin E2. Human IL 1-stimulated chondrocytes also synthesized prostaglandin Fz~ and 6-keto-prostaglandin F1,. Using transcription and translation inhibitors it was shown that protein synthesis was required for PG synthesis. Aspirin, a specific inhibitor of cyclooxygenase, was also used and confirmed that the production of PG induced by IL 1 is imputable to a de novo synthesis of cyclooxygenase. In human chondrocytes, cyclooxygenase is thus a key enzyme in the AA cascade in addition of PLA2.
Comparative Biochemistry and Physiology Part D: Genomics and Proteomics, 2010
The intensification of shrimp farming has been related to the increasing use of chemotherapeutics... more The intensification of shrimp farming has been related to the increasing use of chemotherapeutics and potentially suboptimal rearing conditions. For the purpose of assessing the stress level of cultured giant tiger shrimp Penaeus monodon, a proteomic analysis (2D-DIGE) was performed on hemolymph. On the one hand, shrimp were exposed for 7 days to the antibiotics enrofloxacin or furazolidone via feed (4 g kg(-1)) under laboratory conditions. On the other hand, shrimp were submitted to enrofloxacin directly in field conditions in Vietnam, for which two different culture systems were distinguished (intensive and improved extensive). No significant different protein abundance pattern was induced by antibiotics under laboratory conditions, while only one protein spot displayed a 1.53-fold reduction in intensity after exposure to enrofloxacin in improved extensive ponds. When we compared the proteome of shrimp bred either in intensive or in improved extensive system, we observed 9 protein spots displaying significant difference in abundance. Among them, 3 spots of hemocyanin were under-expressed in shrimp from improved extensive ponds. At the opposite 2 spots corresponding to Sarcoplasmic Calcium-binding Protein (SCP) were less abundant in hemolymph of shrimp from intensive ponds. These results demonstrate that the very subtle effects of tested antibiotics on patterns of hemolymph protein expression are overwhelmed by the effects of conditions encountered in different production management systems, such as different oxygen and nitric concentrations.
Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology, 2014
Domestication might be a possible way to reduce the physiological response to long-term stressors... more Domestication might be a possible way to reduce the physiological response to long-term stressors and deleterious effects on immunity. The present study aimed to evaluate the chronic immune response induced by repeated emersions and the possible impact of domestication by comparing farmed Eurasian perch with short (F1) and long (F4) captive-life history. In the first experiment, fish were exposed to a single emersion and physiological stress response was measured in the short term to characterize fish sensitivity to the tested stressor. Serum cortisol and glucose elevated within 6 h post-stress and splenosomatic index (SSI) decreased within 48 h, indicating that the species was affected by emersion stressor. In the second experiment, F1 and F4 generations were submitted to repeated water emersions (3 times/week during 44 days). On day 9, 18 and 44, samplings were performed 48 h post-stressor to highlight any sustained disruption of immune system. Serum cortisol, glucose, SSI and lysozyme activity were evaluated and serum proteome was analyzed using 2D-DIGE. Any of the tested variables were affected by repeated emersions and proteomic analysis only revealed that alpha-2 macroglobulins (a2Ms) were up-regulated in the serum of stressed individuals. Domestication also resulted in the up-regulation of five a2M isoforms and down-regulation of complement C3 and Ig light chain proteins, independently of any stressor exposure. In conclusion, the results suggested that repeated emersions are not severe stressors for Eurasian perch, probably explaining why domestication had no influence on fish responses. Changes associated with domestication are highly complex and certainly need further investigations.
Background: Repeated exposures to UVB of human keratinocytes lacking functional p16 and able to d... more Background: Repeated exposures to UVB of human keratinocytes lacking functional p16 and able to differentiate induce an alternative state of differentiation rather than stress-induced premature senescence.
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