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Proteomic effects of different Hg concentration in Dictyostelium discoideum amoebae
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Abstracts / Comparative Biochemistry and Physiology, Part A 151 (2008) S37–S43
9. Proteomic effects of different Hg concentration in Dictyostelium discoideum amoebae F. Marsano, L. Boatti, A. Negri, M. Cigar, F. Dondero, A. Viarengo (DiSAV, Università del Piemonte Orientale “A. Avogadro”, Alessandria, Italy) We used bidimensional electrophoresis with immunological and mass spectrometry (MALDI and nanoLC-ESI-Q-TOF) techniques to identify proteomic changes evoked in Dictyostelium amoebae by different Hg concentrations. Indeed, it was used a lower dose-2 µMdetermining physiological changes(e.g., lysosomal membrane stability), and an higher concentration-10 µM-eliciting also effects on mortality (few%) and cell replication. High-resolution 2DE proteomic maps of soluble proteins unveiled two distinct signatures. Peptide identification showed a response to oxidative stress at low Hg dose:13 proteins were up-regulated and among these GST-catalase and thioredoxin were involved. Such molecular fingerprinting disappeared at higher concentration, where12 proteins were downregulated and only 2 were overexpressed. A battery of antibodies was used to get clues on protein responses. Westernblot with anti-GST antibodies revealed the overexpression in both conditions, but immunofluorescence staining showed a compartimentalization at the higher concentration, which is consistent with 2DE results. Antiubiquitin staining revealed augmented protein ubiquitination at high molecular weights and also an increase of ubiquitin availability only at the high dose. Data were supported by DNA-microarrays indicating a dose dependent overexpression of the proteasome complex genes, which – together with dramatic lysosomal destabilization – suggests the occurrence of non-physiological catabolic processes. Protein carbonylation was assayed by spectrophotometric and 2DE westernblot analyses. Results demonstrated an increase in protein oxidation after the treatment with the higher Hg dose (14 spots). Severe oxidative stress injuries were also confirmed by the lysosomal accumulation of lipofuscin. In conclusion, the observed proteomic changes may be related to two different physiological status of Hg exposed amoebae: (i) a stress syndrome during which cells adapt, propagate and survive; (ii) a toxic syndrome affecting several cellular, physiological functions and finally cell survival.
doi:10.1016/j.cbpa.2008.05.147 10. Monitoring of environmental soil contamination through transcriptomics B. Nota, N. van Straalen, D. Roelofs (Vrije Universiteit Amsterdam, The Netherlands) Increasing concern about pollution of our environment calls for advanced and rapid methods to estimate ecological toxicity. The use of gene expression microarrays in environmental studies, can potentially meet this challenge. We present a novel method to examine soil toxicity. We exposed Folsomia candida to soil containing an ecological relevant cadmium concentration, and found a cumulative total of 1586 differentially expressed genes across three exposure durations, including genes involved in stress response, detoxification, and hypoxia. Additional enrichment analysis of Gene Ontology (GO) terms revealed that antibiotic biosynthesis is important in all time points. This included genes encoding isopenicillin N synthases, which have never been identified in other animals. Digoxygenin in situ hybridization confirms that the genes are expressed in tissue of F. candida, and not in possible commensal microorganisms. Production of antibiotics suggests increased cadmium-induced susceptibility to
invading pathogens, which might be caused by repression of genes involved in the immune-system (C-type lectins and Toll receptor). This study presents a first global view on the environmental stress response of arthropods exposed to contaminated soil, and provides a mechanistic basis for the development of a gene expression soil quality test. doi:10.1016/j.cbpa.2008.05.148 11. Cloning and expression of cancer related genes in turbot (Scophthalmus maximus) exposed to styrene and heavy fuel oil # 2 P. Ruiz, A. Orbea, I. Cancio, M.P. Cajaraville (Laboratory of Cell Biology and Histology, University of the Basque Country, Spain) Several accidental spills in European coastal areas have resulted in the release of different toxic compounds into the marine environment. In recent oil spills such as those of the tankers “Erika” (1999) and “Prestige” (2002) heavy fuel oil type # 2 was released, heavily contaminating coastal areas of Brittany and Galicia and the Bay of Biscay. In 2000 the loss of the tanker “Levoli Sun” in the English Channel resulted in the release of several tonnes of the highly toxic and corrosive hydrocarbon styrene. Both heavy fuel oil and styrene are classified as potentially genotoxic and carcinogenic. The aim of the present study was to clone several cancer related genes and to investigate the expression of some of these genes in liver of juvenile turbots (Scophthalmus maximus) exposed to styrene and to heavy fuel oil # 2 for 1 and 2 weeks, respectively, followed by 1 and 2 weeks of recovery. PCR-based screening of turbot cDNA with degenerate primers resulted in the amplification, cloning and sequencing of cDNA fragments coding for tumor suppressor protein p53 (171 bp), retinoblastoma (786 bp), rasrelated nuclear protein (307 bp), ras protein (256 bp), cyclin G1 (367 bp), cyclin H (114 bp), growth arrest- and DNA damageinducible GADD45 (362 bp) and proliferating cell nuclear antigen (486 bp). Quantitative real time PCR was used to characterize the expression of some of these genes employing 18S-rRNA, β-actin or elongation-factor α (EF126038, AY008305 and AF467776, respectively) as housekeeping genes. doi:10.1016/j.cbpa.2008.05.149 12. The Power of the transcriptome: Prognostic tool for populations of free-ranging bottlenose dolphins, Tursiops truncatus A. Mancia (Medical University of South Carolina, USA; Università degli studi di Bologna, Italy); G.W. Warr (Medical University of South Carolina, USA); J. Almeida (MD Anderson Cancer Center, USA); R. Wells (Chicago Zoological Society, USA); A. Veloso (College of Charleston, USA); R.W. Chapman (South Carolina Department of Natural Resources, USA) Peripheral blood leukocyte (PBL) samples were collected from 151 bottlenose dolphins (Tursiops truncatus) in the course of capture/ release health evaluation studies at 4 different sites: Charleston Harbor SC, Indian River Lagoon FL, Sarasota Bay, FL and St Joseph Bay FL, USA. RNA was extracted and hybridized to a first-generation dolphin microarray. We tested the hypothesis that individual dolphins could be assigned (by using artificial neural networks, ANNs, a machine-learning approach) to their home regions using only their transcriptomic signatures as classifiers. ANNs could correctly classify
Abstracts / Comparative Biochemistry and Physiology, Part A 151 (2008) S37–S43
9. Proteomic effects of different Hg concentration in Dictyostelium discoideum amoebae F. Marsano, L. Boatti, A. Negri, M. Cigar, F. Dondero, A. Viarengo (DiSAV, Università del Piemonte Orientale “A. Avogadro”, Alessandria, Italy) We used bidimensional electrophoresis with immunological and mass spectrometry (MALDI and nanoLC-ESI-Q-TOF) techniques to identify proteomic changes evoked in Dictyostelium amoebae by different Hg concentrations. Indeed, it was used a lower dose-2 µMdetermining physiological changes(e.g., lysosomal membrane stability), and an higher concentration-10 µM-eliciting also effects on mortality (few%) and cell replication. High-resolution 2DE proteomic maps of soluble proteins unveiled two distinct signatures. Peptide identification showed a response to oxidative stress at low Hg dose:13 proteins were up-regulated and among these GST-catalase and thioredoxin were involved. Such molecular fingerprinting disappeared at higher concentration, where12 proteins were downregulated and only 2 were overexpressed. A battery of antibodies was used to get clues on protein responses. Westernblot with anti-GST antibodies revealed the overexpression in both conditions, but immunofluorescence staining showed a compartimentalization at the higher concentration, which is consistent with 2DE results. Antiubiquitin staining revealed augmented protein ubiquitination at high molecular weights and also an increase of ubiquitin availability only at the high dose. Data were supported by DNA-microarrays indicating a dose dependent overexpression of the proteasome complex genes, which – together with dramatic lysosomal destabilization – suggests the occurrence of non-physiological catabolic processes. Protein carbonylation was assayed by spectrophotometric and 2DE westernblot analyses. Results demonstrated an increase in protein oxidation after the treatment with the higher Hg dose (14 spots). Severe oxidative stress injuries were also confirmed by the lysosomal accumulation of lipofuscin. In conclusion, the observed proteomic changes may be related to two different physiological status of Hg exposed amoebae: (i) a stress syndrome during which cells adapt, propagate and survive; (ii) a toxic syndrome affecting several cellular, physiological functions and finally cell survival.
doi:10.1016/j.cbpa.2008.05.147 10. Monitoring of environmental soil contamination through transcriptomics B. Nota, N. van Straalen, D. Roelofs (Vrije Universiteit Amsterdam, The Netherlands) Increasing concern about pollution of our environment calls for advanced and rapid methods to estimate ecological toxicity. The use of gene expression microarrays in environmental studies, can potentially meet this challenge. We present a novel method to examine soil toxicity. We exposed Folsomia candida to soil containing an ecological relevant cadmium concentration, and found a cumulative total of 1586 differentially expressed genes across three exposure durations, including genes involved in stress response, detoxification, and hypoxia. Additional enrichment analysis of Gene Ontology (GO) terms revealed that antibiotic biosynthesis is important in all time points. This included genes encoding isopenicillin N synthases, which have never been identified in other animals. Digoxygenin in situ hybridization confirms that the genes are expressed in tissue of F. candida, and not in possible commensal microorganisms. Production of antibiotics suggests increased cadmium-induced susceptibility to
invading pathogens, which might be caused by repression of genes involved in the immune-system (C-type lectins and Toll receptor). This study presents a first global view on the environmental stress response of arthropods exposed to contaminated soil, and provides a mechanistic basis for the development of a gene expression soil quality test. doi:10.1016/j.cbpa.2008.05.148 11. Cloning and expression of cancer related genes in turbot (Scophthalmus maximus) exposed to styrene and heavy fuel oil # 2 P. Ruiz, A. Orbea, I. Cancio, M.P. Cajaraville (Laboratory of Cell Biology and Histology, University of the Basque Country, Spain) Several accidental spills in European coastal areas have resulted in the release of different toxic compounds into the marine environment. In recent oil spills such as those of the tankers “Erika” (1999) and “Prestige” (2002) heavy fuel oil type # 2 was released, heavily contaminating coastal areas of Brittany and Galicia and the Bay of Biscay. In 2000 the loss of the tanker “Levoli Sun” in the English Channel resulted in the release of several tonnes of the highly toxic and corrosive hydrocarbon styrene. Both heavy fuel oil and styrene are classified as potentially genotoxic and carcinogenic. The aim of the present study was to clone several cancer related genes and to investigate the expression of some of these genes in liver of juvenile turbots (Scophthalmus maximus) exposed to styrene and to heavy fuel oil # 2 for 1 and 2 weeks, respectively, followed by 1 and 2 weeks of recovery. PCR-based screening of turbot cDNA with degenerate primers resulted in the amplification, cloning and sequencing of cDNA fragments coding for tumor suppressor protein p53 (171 bp), retinoblastoma (786 bp), rasrelated nuclear protein (307 bp), ras protein (256 bp), cyclin G1 (367 bp), cyclin H (114 bp), growth arrest- and DNA damageinducible GADD45 (362 bp) and proliferating cell nuclear antigen (486 bp). Quantitative real time PCR was used to characterize the expression of some of these genes employing 18S-rRNA, β-actin or elongation-factor α (EF126038, AY008305 and AF467776, respectively) as housekeeping genes. doi:10.1016/j.cbpa.2008.05.149 12. The Power of the transcriptome: Prognostic tool for populations of free-ranging bottlenose dolphins, Tursiops truncatus A. Mancia (Medical University of South Carolina, USA; Università degli studi di Bologna, Italy); G.W. Warr (Medical University of South Carolina, USA); J. Almeida (MD Anderson Cancer Center, USA); R. Wells (Chicago Zoological Society, USA); A. Veloso (College of Charleston, USA); R.W. Chapman (South Carolina Department of Natural Resources, USA) Peripheral blood leukocyte (PBL) samples were collected from 151 bottlenose dolphins (Tursiops truncatus) in the course of capture/ release health evaluation studies at 4 different sites: Charleston Harbor SC, Indian River Lagoon FL, Sarasota Bay, FL and St Joseph Bay FL, USA. RNA was extracted and hybridized to a first-generation dolphin microarray. We tested the hypothesis that individual dolphins could be assigned (by using artificial neural networks, ANNs, a machine-learning approach) to their home regions using only their transcriptomic signatures as classifiers. ANNs could correctly classify