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Immunohistochemistry of Stanniocalcin isoforms in fish tissues
Abstracts / Comparative Biochemistry and Physiology, Part A 151 (2008) S19–S30
28. Size and site differences in both phase I and II cytochrome P450 in Mullus barbatus from Southern Tyrrhenian Sea L. Pirrera (IAMC-CNR of Messina, Italy); I. Corsi, A.M. Pastore, C. Della Torre, S. Focardi (Department of Environmental Science, University of Siena, Italy); F. Perdichizzi, A. Profeta, B. Busalacchi, A. Perdichizzi, V. Micale, P. Rinelli (IAMC-CNR of Messina, Italy) In the last years the use of biological markers in the environmental monitoring became an effective approach to assess the environmental pollution in coastal areas. The aim of the present study was to evaluate the environmental quality of a coastal marine area with high anthropic impact investigating cytochrome P450 responses in a sentinel species, the red mullet Mullus barbatus (Linnaeus, 1758). The species is widely used in environmental monitoring programs as bioindicator, due to its physiological and trophic characteristics and also due to sensitivity of its cytochrome P450 in pollution studies. Male specimens of two sizes (b17 and 17– 20 cm TL) were collected in three different sites along east coast of the Southern Tyrrhenian Sea, in two different period (first campaign, June 2007; second January 2008): site 3 in front of an oil refinery, site 2 an agricultural area inside of Patti gulf and site 1 as reference site. Phase I EROD activities resulted significant higher in samples from site 3 as well as phase II GST and UDPGT. Season seems not to affect EROD activity in all specimens from the three sites. No differences were observed between the other two sites. Size of the fish seems to influence EROD enzymes with greater activities in longer size fish. Gonad histology was also evaluated in order to detect possible relationships with the reproductive status of fish. doi:10.1016/j.cbpa.2008.05.101
29. Physiological antioxidant responses against high environmental oxygen concentration: Glutathione peroxidase from the Antarctic teleost Trematomus eulepidotus
S27
This research was supported by the Italian National Program for Antarctic Research (PNRA). doi:10.1016/j.cbpa.2008.05.102
30. Immunohistochemistry of Stanniocalcin isoforms in fish tissues V. Schein, N. Silva, D.M. Power, A.V.M. Canario (Centre of Marine Sciences, University of Algarve, Portugal) Stanniocalcin 1 (STC1) is a homodimeric glycoprotein synthesized and secreted by the Corpuscles of Stannius in response to a rise in serum Ca2+. Although STC1 has received most attention, at least one other isoform, STC2, encoded by a separate gene has been identified in all vertebrates. Taking into consideration the duplication events in teleost genomes we decided to investigate the presence of STC paralogues in teleost fish genomes. Our search revealed the presence of duplicated STC1 and STC2 paralogue genes. We have produced polyclonal antibodies against synthetic peptides specific to amino acids in the C-terminal regions of STC1, STC1B and STC2 proteins and analysed their tissue distribution by immunohistochemistry in rainbow trout (Oncorhynchus mykiss), sea bream (Sparus auratus) and sea bass (Dicentrarchus labrax). STC1 was immunoreactive in CS gland cells and in kidney cells. STC1B was more prominent in gills, but staining was also present in kidney, skin and mesentery fat cells. STC2 was present in a variety of tissues, especially in gonad, kidney, pituitary, teeth cells, gill racker, olfactory epithelium and olfactory bulb. These findings indicate differential distribution of the expression of STC duplicated genes in a variety of tissues from several teleost species, suggesting subfunctional specialization of this family of proteins during evolution. Acknowledgments: Research funded by the European Social Fund and the Portuguese National Science Foundation: project POCTI/CVT/ 55683/2004 and fellowship SFRH/BPD/25353/2005 to VS. doi:10.1016/j.cbpa.2008.05.103
G. Sattin, G. Santovito, A. Cassini (University of Padua, Italy) Antarctic marine environment is characterised by increased oxygen concentration, correlated to low temperature of waters (−1.9 °C). In these conditions, reactive oxygen specie (ROS) formation is favoured. The organisms living in this environment perform strong cellular antioxidant homeostatic control, mainly inducing antioxidant enzymes. Among these, glutathione peroxidase (GPX) is a ubiquitous cytosolic enzyme which catalyzes the reduction of hydrogen peroxide and other organic peroxides. In the present study we have characterised the cDNA sequence of GPX from the Antarctic fish Trematomus eulepidotus. Specimens of this teleost were sampled in the Ross Sea (Terra Nova Bay, 74°42′S, 164°7′E) during the XXI Italian Antarctic Expedition. cDNA sequence of GPX has been obtained from hepatic tissue by a combination of RT-PCR, 3′RACE and 5′RACE techniques. The obtained nucleotide and amino acid sequences were compared to those of other teleost GPXs available in GenBank, SwissProt and published literature. The results shown high identity and similarity levels. The knowledge of cDNA sequence allowed to design specific oligonucleotide primers for the quantification of mRNA levels in different tissues by semiquantitative RT-PCR analyses. The obtained results are the first data about GPX gene induction in Antarctic teleost species and act as startup for further studies on physiological antioxidant responses to the high oxygen concentration Antarctic environment.
31. Cellular adaptation and recovery in response to heat stress in Mediterranean snails (Xeropicta derbentina, Theba pisana and Cernuella virgata) R. Triebskorn (Steinbeis Transfer Center, Rottenburg, Germany / Animal Physiological Ecology, University of Tuebingen, Germany); C. Mazzia, Y. Capowiez (UMR INRA, Avignon, France); N. Dittbrenner, A. Zuern, H.-R. Koehler (Animal Physiological Ecology, University of Tuebingen, Germany) In order to cope with heat stress in hot and sun-exposed environments, different terrestrial snail species favour to climb up vertical objects, as e.g. blades of grass, pales, power poles or walls during daytime. Up to now, it is still unknown which physiological and biochemical mechanisms of adaptation in these snail species are responsible for their extreme heat tolerance. In the present study, we investigated cellular adaptations to heat stress and cellular recovery processes after termination of heat stress in Xeropicta derbentina, Cernuella virgata and Theba pisana. Prior to histopathological investigations, snails were exposed to different
28. Size and site differences in both phase I and II cytochrome P450 in Mullus barbatus from Southern Tyrrhenian Sea L. Pirrera (IAMC-CNR of Messina, Italy); I. Corsi, A.M. Pastore, C. Della Torre, S. Focardi (Department of Environmental Science, University of Siena, Italy); F. Perdichizzi, A. Profeta, B. Busalacchi, A. Perdichizzi, V. Micale, P. Rinelli (IAMC-CNR of Messina, Italy) In the last years the use of biological markers in the environmental monitoring became an effective approach to assess the environmental pollution in coastal areas. The aim of the present study was to evaluate the environmental quality of a coastal marine area with high anthropic impact investigating cytochrome P450 responses in a sentinel species, the red mullet Mullus barbatus (Linnaeus, 1758). The species is widely used in environmental monitoring programs as bioindicator, due to its physiological and trophic characteristics and also due to sensitivity of its cytochrome P450 in pollution studies. Male specimens of two sizes (b17 and 17– 20 cm TL) were collected in three different sites along east coast of the Southern Tyrrhenian Sea, in two different period (first campaign, June 2007; second January 2008): site 3 in front of an oil refinery, site 2 an agricultural area inside of Patti gulf and site 1 as reference site. Phase I EROD activities resulted significant higher in samples from site 3 as well as phase II GST and UDPGT. Season seems not to affect EROD activity in all specimens from the three sites. No differences were observed between the other two sites. Size of the fish seems to influence EROD enzymes with greater activities in longer size fish. Gonad histology was also evaluated in order to detect possible relationships with the reproductive status of fish. doi:10.1016/j.cbpa.2008.05.101
29. Physiological antioxidant responses against high environmental oxygen concentration: Glutathione peroxidase from the Antarctic teleost Trematomus eulepidotus
S27
This research was supported by the Italian National Program for Antarctic Research (PNRA). doi:10.1016/j.cbpa.2008.05.102
30. Immunohistochemistry of Stanniocalcin isoforms in fish tissues V. Schein, N. Silva, D.M. Power, A.V.M. Canario (Centre of Marine Sciences, University of Algarve, Portugal) Stanniocalcin 1 (STC1) is a homodimeric glycoprotein synthesized and secreted by the Corpuscles of Stannius in response to a rise in serum Ca2+. Although STC1 has received most attention, at least one other isoform, STC2, encoded by a separate gene has been identified in all vertebrates. Taking into consideration the duplication events in teleost genomes we decided to investigate the presence of STC paralogues in teleost fish genomes. Our search revealed the presence of duplicated STC1 and STC2 paralogue genes. We have produced polyclonal antibodies against synthetic peptides specific to amino acids in the C-terminal regions of STC1, STC1B and STC2 proteins and analysed their tissue distribution by immunohistochemistry in rainbow trout (Oncorhynchus mykiss), sea bream (Sparus auratus) and sea bass (Dicentrarchus labrax). STC1 was immunoreactive in CS gland cells and in kidney cells. STC1B was more prominent in gills, but staining was also present in kidney, skin and mesentery fat cells. STC2 was present in a variety of tissues, especially in gonad, kidney, pituitary, teeth cells, gill racker, olfactory epithelium and olfactory bulb. These findings indicate differential distribution of the expression of STC duplicated genes in a variety of tissues from several teleost species, suggesting subfunctional specialization of this family of proteins during evolution. Acknowledgments: Research funded by the European Social Fund and the Portuguese National Science Foundation: project POCTI/CVT/ 55683/2004 and fellowship SFRH/BPD/25353/2005 to VS. doi:10.1016/j.cbpa.2008.05.103
G. Sattin, G. Santovito, A. Cassini (University of Padua, Italy) Antarctic marine environment is characterised by increased oxygen concentration, correlated to low temperature of waters (−1.9 °C). In these conditions, reactive oxygen specie (ROS) formation is favoured. The organisms living in this environment perform strong cellular antioxidant homeostatic control, mainly inducing antioxidant enzymes. Among these, glutathione peroxidase (GPX) is a ubiquitous cytosolic enzyme which catalyzes the reduction of hydrogen peroxide and other organic peroxides. In the present study we have characterised the cDNA sequence of GPX from the Antarctic fish Trematomus eulepidotus. Specimens of this teleost were sampled in the Ross Sea (Terra Nova Bay, 74°42′S, 164°7′E) during the XXI Italian Antarctic Expedition. cDNA sequence of GPX has been obtained from hepatic tissue by a combination of RT-PCR, 3′RACE and 5′RACE techniques. The obtained nucleotide and amino acid sequences were compared to those of other teleost GPXs available in GenBank, SwissProt and published literature. The results shown high identity and similarity levels. The knowledge of cDNA sequence allowed to design specific oligonucleotide primers for the quantification of mRNA levels in different tissues by semiquantitative RT-PCR analyses. The obtained results are the first data about GPX gene induction in Antarctic teleost species and act as startup for further studies on physiological antioxidant responses to the high oxygen concentration Antarctic environment.
31. Cellular adaptation and recovery in response to heat stress in Mediterranean snails (Xeropicta derbentina, Theba pisana and Cernuella virgata) R. Triebskorn (Steinbeis Transfer Center, Rottenburg, Germany / Animal Physiological Ecology, University of Tuebingen, Germany); C. Mazzia, Y. Capowiez (UMR INRA, Avignon, France); N. Dittbrenner, A. Zuern, H.-R. Koehler (Animal Physiological Ecology, University of Tuebingen, Germany) In order to cope with heat stress in hot and sun-exposed environments, different terrestrial snail species favour to climb up vertical objects, as e.g. blades of grass, pales, power poles or walls during daytime. Up to now, it is still unknown which physiological and biochemical mechanisms of adaptation in these snail species are responsible for their extreme heat tolerance. In the present study, we investigated cellular adaptations to heat stress and cellular recovery processes after termination of heat stress in Xeropicta derbentina, Cernuella virgata and Theba pisana. Prior to histopathological investigations, snails were exposed to different