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Expression profiling of Escherichia coli small RNAs using custom microarrays
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Abstracts / Comparative Biochemistry and Physiology, Part A 163 (2012) S43–S47
control of the zebra mussel invasion requires previous knowledge about the main colonization strategies and dispersal ability, as well as a comprehensive study of the mechanisms involved in coping with environmental factors. The study included six populations sampled at diverse locations from the Ebro and Júcar basins, differing in their water quality. We have used osmoregulatory and ionoregulatory characters, defenses against metal toxicity, and a general health status index for the ecophysiological characterization of zebra mussel populations. Furthermore, proteomics (DIGE and iTRAQ methodologies) allowed us to find some of the proteins involved in the physiological adaptation of Dreissena polymorpha to the environmental conditions of the studied sites. This research was supported by the project CEBRAPOP (reference 042/RN08/03.4) funded by the Ministry of the Environment and Rural and Marine Affairs. doi:10.1016/j.cbpa.2012.05.136
10. Effect of heavy metal exposure on blood hemoglobin in Lumbricus terrestris A. Leomanni, A. Calisi, M.G. Lionetto, T. Schettino (Department of Biological and Environmental Science and Technologies, University of Salento, Italy) Earthworms, very important organisms for organic matter breakdown in terrestrial environments and for their particular interactions with the soil, are significantly affected by pollutants reaching the soil system. Earthworms have a closed circulatory system. The blood contains hemoglobin which is a large extracellular hemoprotein flowing in a closed circulatory system. In spite of the fundamental role of this pigment in earthworm physiology, little is known about its sensitivity to environmental pollutants. The aim of this research was to investigate the metal-induced alterations in Hemoglobin (Hb) and Methemoglobin (MetHb) blood concentrations in the earthworm Lumbricus terrestris. The animals were exposed to increasing concentrations of Cd and Cu utilizing the standard acute toxicity test, “Filter paper test” for 48 h. The blood Hb concentration and MetHb percentage were colorimetrically determined. A comparative electrophoretic analysis of blood hemoglobin between control and exposed animals was also performed. Results showed that metal exposure caused a dose-dependent increase of blood Hb concentration. In addition a dramatic dose-dependent increase of MetHb % was observed in exposed organisms, suggesting a strong oxidation effect on the respiratory pigment. Quantitative electrophoretic analysis showed the occurrence of Hb structural alterations, in terms of changes of the molecular weight of the single Hb subunits. The results obtained in the present work indicated multiple effects of Cd and Cu exposures on L. terrestris Hb: an increase of blood Hb concentration, an oxidative effect in terms of marked increase of MetHb percentage, and alteration of the molecular weight of single Hb subunits. These results provide evidences of the responses of Hb to metal exposure in earthworms and suggest a possible role of this respiratory pigment in the monitoring of soil chemical contamination. doi:10.1016/j.cbpa.2012.05.137
11. Expression profiling of Escherichia coli small RNAs using custom microarrays V.R. Kaberdin, O. Ruiz Larrabeiti, E. Sevillano Peña, L. Gallego Andrés (Immunology, Microbiology and Parasitology, University of the Basque Country, Spain)
The ubiquitous presence of microorganisms is largely associated with their unique abilities to adapt and strive in adverse environments. Recent studies revealed a new class of small antisense RNAs (sRNAs) that play important roles in regulatory mechanisms enabling Escherichia coli (E. coli) and other bacteria (including essential genera of pathogenic enterobacteria) to adapt to environmental stresses. Many stress-related sRNAs of E. coli exert their regulatory functions by base pairing with specific mRNA targets, thereby affecting mRNA translation and stability (reviewed in Kaberdin and Bläsi, 2006). Although approximately 120 stress-related antisense RNAs have been discovered in E. coli and other enterobacteria (Repoila and Darfeuille, 2009) and many other candidate sRNAs were predicted in silico (reviewed in Hershberg et al., 2003), only a small fraction of them have been characterized. As many sRNAs are normally transcribed from intergenic regions and are barely detected by many commercial microarrays, we designed our own microarray with improved capacity for detection of these riboregulators. Apart from the standard set of probes developed by Agilent Technologies for hybridization with E. coli transcripts, our custom microarray additionally contains a group of probes selected for detection of known and putative sRNAs. RNA profiling experiments performed with total RNA extracted from E. coli cells grown on rich (Luria-Bertani) and minimal (M9) media demonstrated the suitability of our custom microarray for efficient profiling of sRNAs. Moreover, our microarray data also revealed new sRNAs with potential roles in bacterial stress responses. Their presence in E. coli cells was further validated by northern blot analysis. References: Kaberdin V.R., Bläsi U. (2006) FEMS Microbiol Reviews 30: 967–979. Repoila F., Darfeuille F, (2009) Biol Cell 101: 117–131. Hershberg R., Altuvia S., Margalit H. (2003) Nucleic Acids Res 31: 1813– 1820.
doi:10.1016/j.cbpa.2012.05.138
12. Isolation and functional characterisation of a stearoyl-CoA desaturase from the marine invertebrate Octopus vulgaris O. Monroig, I. Varó (Instituto de Acuicultura de Torre de la Sal, CSIC, Spain); D.R. Tocher (Institute of Aquaculture, University of Stirling, UK); J.C. Navarro (Institute of Aquaculture, University of Stirling, UK) Culture of the common octopus (Octopus vulgaris) is hindered by massive mortalities encountered during early stages (paralarvae). A variety of stressors have been hypothesised to account for paralarvae mortalities, with suboptimal diets emerging as one of the most likely determining factors. Our overarching objective is to establish the essential fatty acid (FA) requirements of paralarval stages of the common octopus through characterisation of the enzymes of endogenous biosynthetic pathways. Here we report on the molecular and functional characterisations of a stearoyl-CoA desaturase (Scd) from the common octopus, Scd being a key enzyme controlling the desaturation of FA, a mechanism by which cells regulate membrane fluidity and thus physiological homeostasis. Using PCR-based methodologies, we isolated an Scd-like cDNA whose coding sequence comprised 981 bp encoding a putative protein of 326 amino acids. Phylogenetic analysis grouped the newly cloned O. vulgaris desaturase into a cluster with greatest similarity to orthologues from both vertebrates and invertebrates, and separated from the group of fatty acyl desaturases (Fad) including the octopus Δ5like Fad recently identified by our group. Consistent with the function described in other organisms, heterologous expression of the octopus desaturase cDNA in yeast Saccharomyces cerevisiae confirmed Δ9desaturation specificity, and established the enzyme as an Scd
Abstracts / Comparative Biochemistry and Physiology, Part A 163 (2012) S43–S47
control of the zebra mussel invasion requires previous knowledge about the main colonization strategies and dispersal ability, as well as a comprehensive study of the mechanisms involved in coping with environmental factors. The study included six populations sampled at diverse locations from the Ebro and Júcar basins, differing in their water quality. We have used osmoregulatory and ionoregulatory characters, defenses against metal toxicity, and a general health status index for the ecophysiological characterization of zebra mussel populations. Furthermore, proteomics (DIGE and iTRAQ methodologies) allowed us to find some of the proteins involved in the physiological adaptation of Dreissena polymorpha to the environmental conditions of the studied sites. This research was supported by the project CEBRAPOP (reference 042/RN08/03.4) funded by the Ministry of the Environment and Rural and Marine Affairs. doi:10.1016/j.cbpa.2012.05.136
10. Effect of heavy metal exposure on blood hemoglobin in Lumbricus terrestris A. Leomanni, A. Calisi, M.G. Lionetto, T. Schettino (Department of Biological and Environmental Science and Technologies, University of Salento, Italy) Earthworms, very important organisms for organic matter breakdown in terrestrial environments and for their particular interactions with the soil, are significantly affected by pollutants reaching the soil system. Earthworms have a closed circulatory system. The blood contains hemoglobin which is a large extracellular hemoprotein flowing in a closed circulatory system. In spite of the fundamental role of this pigment in earthworm physiology, little is known about its sensitivity to environmental pollutants. The aim of this research was to investigate the metal-induced alterations in Hemoglobin (Hb) and Methemoglobin (MetHb) blood concentrations in the earthworm Lumbricus terrestris. The animals were exposed to increasing concentrations of Cd and Cu utilizing the standard acute toxicity test, “Filter paper test” for 48 h. The blood Hb concentration and MetHb percentage were colorimetrically determined. A comparative electrophoretic analysis of blood hemoglobin between control and exposed animals was also performed. Results showed that metal exposure caused a dose-dependent increase of blood Hb concentration. In addition a dramatic dose-dependent increase of MetHb % was observed in exposed organisms, suggesting a strong oxidation effect on the respiratory pigment. Quantitative electrophoretic analysis showed the occurrence of Hb structural alterations, in terms of changes of the molecular weight of the single Hb subunits. The results obtained in the present work indicated multiple effects of Cd and Cu exposures on L. terrestris Hb: an increase of blood Hb concentration, an oxidative effect in terms of marked increase of MetHb percentage, and alteration of the molecular weight of single Hb subunits. These results provide evidences of the responses of Hb to metal exposure in earthworms and suggest a possible role of this respiratory pigment in the monitoring of soil chemical contamination. doi:10.1016/j.cbpa.2012.05.137
11. Expression profiling of Escherichia coli small RNAs using custom microarrays V.R. Kaberdin, O. Ruiz Larrabeiti, E. Sevillano Peña, L. Gallego Andrés (Immunology, Microbiology and Parasitology, University of the Basque Country, Spain)
The ubiquitous presence of microorganisms is largely associated with their unique abilities to adapt and strive in adverse environments. Recent studies revealed a new class of small antisense RNAs (sRNAs) that play important roles in regulatory mechanisms enabling Escherichia coli (E. coli) and other bacteria (including essential genera of pathogenic enterobacteria) to adapt to environmental stresses. Many stress-related sRNAs of E. coli exert their regulatory functions by base pairing with specific mRNA targets, thereby affecting mRNA translation and stability (reviewed in Kaberdin and Bläsi, 2006). Although approximately 120 stress-related antisense RNAs have been discovered in E. coli and other enterobacteria (Repoila and Darfeuille, 2009) and many other candidate sRNAs were predicted in silico (reviewed in Hershberg et al., 2003), only a small fraction of them have been characterized. As many sRNAs are normally transcribed from intergenic regions and are barely detected by many commercial microarrays, we designed our own microarray with improved capacity for detection of these riboregulators. Apart from the standard set of probes developed by Agilent Technologies for hybridization with E. coli transcripts, our custom microarray additionally contains a group of probes selected for detection of known and putative sRNAs. RNA profiling experiments performed with total RNA extracted from E. coli cells grown on rich (Luria-Bertani) and minimal (M9) media demonstrated the suitability of our custom microarray for efficient profiling of sRNAs. Moreover, our microarray data also revealed new sRNAs with potential roles in bacterial stress responses. Their presence in E. coli cells was further validated by northern blot analysis. References: Kaberdin V.R., Bläsi U. (2006) FEMS Microbiol Reviews 30: 967–979. Repoila F., Darfeuille F, (2009) Biol Cell 101: 117–131. Hershberg R., Altuvia S., Margalit H. (2003) Nucleic Acids Res 31: 1813– 1820.
doi:10.1016/j.cbpa.2012.05.138
12. Isolation and functional characterisation of a stearoyl-CoA desaturase from the marine invertebrate Octopus vulgaris O. Monroig, I. Varó (Instituto de Acuicultura de Torre de la Sal, CSIC, Spain); D.R. Tocher (Institute of Aquaculture, University of Stirling, UK); J.C. Navarro (Institute of Aquaculture, University of Stirling, UK) Culture of the common octopus (Octopus vulgaris) is hindered by massive mortalities encountered during early stages (paralarvae). A variety of stressors have been hypothesised to account for paralarvae mortalities, with suboptimal diets emerging as one of the most likely determining factors. Our overarching objective is to establish the essential fatty acid (FA) requirements of paralarval stages of the common octopus through characterisation of the enzymes of endogenous biosynthetic pathways. Here we report on the molecular and functional characterisations of a stearoyl-CoA desaturase (Scd) from the common octopus, Scd being a key enzyme controlling the desaturation of FA, a mechanism by which cells regulate membrane fluidity and thus physiological homeostasis. Using PCR-based methodologies, we isolated an Scd-like cDNA whose coding sequence comprised 981 bp encoding a putative protein of 326 amino acids. Phylogenetic analysis grouped the newly cloned O. vulgaris desaturase into a cluster with greatest similarity to orthologues from both vertebrates and invertebrates, and separated from the group of fatty acyl desaturases (Fad) including the octopus Δ5like Fad recently identified by our group. Consistent with the function described in other organisms, heterologous expression of the octopus desaturase cDNA in yeast Saccharomyces cerevisiae confirmed Δ9desaturation specificity, and established the enzyme as an Scd