Pil Gue Jo

Cloning and mRNA expression of antioxidant enzymes in the Pacific oyster, Crassostrea gigas in response to cadmium exposure

Cadmium (Cd) is one of the most toxic heavy metal pollutants in the aquatic environment and can induce the formation of reactive oxygen species (ROS) that cause oxidative stress. In present study, we cloned catalase (CAT) and glutathione peroxidase (GPX) cDNA, and investigated its time- and dose-related effects of three Cd concentrations (0.01, 0.05 or 0.1 ppm) on mRNA levels of antioxidant enzymes (superoxide dismutase (SOD), CAT, GPX) in the gill and changes enzyme levels in the hemolymph of the Pacific oyster, Crassostrea gigas. The cDNA indentified encoded proteins of 516 and 244 amino acids corresponding to CAT and GPX, respectively. BLAST analysis from other species indicated that the residues essential to the enzymatic function of CAT and GPX proteins of C. gigas are highly conserved. Cd treatment significantly increased antioxidant enzyme mRNA expression in the gill in a time- and dose-dependent manner. The mRNA expression at 0.1 ppm Cd concentration increased up to 3 days (CAT, GPX) or 7 days (SOD) and then decreased by 7 days (CAT, GPX) or 11 days (SOD). Aspartate aminotransferase, alanine amintransferase and hydrogen peroxide (H(2)O(2)) concentrations levels increased significantly with exposure to 0.05 or 0.1 ppm Cd for 7 days. These results suggest that antioxidant enzymes play important roles in the physiological changes related to metabolism and cell protection that occur in Pacific oysters exposed to Cd.

Cadmium affects the expression of heat shock protein 90 and metallothionein mRNA in the Pacific oyster, Crassostrea gigas

Cadmium (Cd) is a widespread nonessential heavy metal that enters the aquatic environment as a result of natural processes and human activities such as wastewater production, agriculture, and mining. To determine the effects of Cd on organisms, we investigated its time- and dose-related effects on mRNA levels of heat shock protein 90 (HSP90) and metallothionein (MT) in the gill and digestive gland and changes enzyme levels in the hemolymph of the Pacific oyster Crassostrea gigas. Full-length HSP90 cDNA was isolated from C. gigas by rapid amplification of cDNA end (RACE) techniques and found to contain 2154 nucleotides, including an open reading frame, and was predicted to encode a protein of 717 amino acids. BLAST analysis indicated that the HSP90 gene of C. gigas shared high homology with known HSP90 genes of other mollusks. The expression of HSP90 mRNA increased significantly with exposure to 0.01 ppm Cd for 11 days or 0.05 or 0.1 ppm Cd for 7 days. The expression of MT mRNA increased significantly with exposure to 0.01, 0.05, or 0.1 ppm Cd for 11 days. Glutamate oxaloacetate and glutamate pyruvate levels increased significantly with exposure to 0.05 or 0.1 ppm Cd for 7 days. These results indicate that HSP90 and MT play important roles in the physiological changes related to metabolism and cell protection that occur in Pacific oysters exposed to Cd.

Characterization and mRNA expression of Mn-SOD and physiological responses to stresses in the Pacific oyster Crassostrea gigas.

Abstract Superoxide dismutases (SODs) are metalloenzymes that play an important role in mollusc immune defence systems by eliminating oxidative stress to reactive oxygen species. We investigated physiological changes in the Pacific oyster Crassostrea gigas caused by exposure to pollutants (cadmium, tributyltin) and acute water temperature change. We analysed mRNA expression of Mn-SOD in gills using a quantitative polymerase chain reaction (QPCR), and measured the glutamate oxaloacetate transaminase (GOT), glutamate pyruvate transaminase (GPT) and hydrogen peroxide (H2O2) levels in haemolymph based on time- and dose-related effects of pollutants and acute water temperature treatments. We cloned a cgMn-SOD full-length cDNA that included an open reading frame of 675 nucleotides that was predicted to encode proteins of 225 amino acids. BLAST analysis of other species indicated that residues essential to the enzymatic functions of Mn-SOD proteins are highly conserved. Levels of Mn-SOD mRNA expression, GOT and GPT were gradually increased and then decreased during the experimental periods. On the other hand, H2O2 levels increased continuously during the exposure periods. These results suggest that Mn-SOD plays an important role in the physiological changes related to metabolism and cell protection that occur in C. gigas when exposed to oxidative stress by pollutants and fluctuations in water temperature.

Expression of warm temperature acclimation‐related protein 65‐kDa (Wap65) mRNA, and physiological changes with increasing water temperature in black porgy, Acanthopagrus schlegeli

We isolated the warm temperature acclimation-related protein 65-kDa (Wap65) cDNA from the liver of black porgy and investigated the expression by increasing water temperature in black porgy, Acanthopagrus schlegeli. Black porgy Wap65 full-length cDNA consists of 1,338 nucleotides, including an open reading frame, predicted to encode a protein of 425 amino acids and showed high homology to pufferfish (79%), Medaka (73%), carp (70%), and goldfish (68%) Wap65. Increase in water temperature (20 degrees C --> 30 degrees C; 1 degrees C/day) induced the rise of Wap65 mRNA expression in liver of black porgy. Also, the levels of cortisol and glucose in plasma were significantly higher at 30 degrees C than at 20 degrees C. To determine the high water temperature stressor specificity of the induction of Wap65, black porgy were transferred from seawater (SW) to freshwater (FW) for 24 hr. Wap65 expression was not detected when the fish were transferred from SW to FW (in fish transferred from SW to FW), although the levels of cortisol and glucose in plasma were increased. These results suggest that increase in Wap65 gene is related to high water temperature stress and play important roles in high water temperature environment of black porgy.

Characterization of estrogen receptor β2 and expression of the estrogen receptor subtypes α, β1, and β2 in the protandrous black porgy (Acanthopagrus schlegeli) during the sex change process

Estrogens play an important role in many physiological processes in both female and male vertebrates, mediated by specific nuclear receptor, estrogen receptors (ERs). We have isolated a third ER (ERbeta2), which was found to contain 2004 nucleotides including an open reading frame that encodes 667 amino acids. We have also cloned ERalpha and ERbeta1 from the published information (GenBank accession nos. AY074780 and AY074779) and investigated the expression pattern of these ER subtypes in the gonads during gonad sex change of black porgy by quantitative polymerase chain reaction. Maturity stages can be divided into five stages during the sex change process from immature male to female (immature male, mature male, male of mostly testis, male of mostly ovary and mature female). The expression of ERalpha mRNA was highest in the ovary of mature female, followed by the testis of mature male and testicular portion of mostly testis. ERbeta1 expression was higher in the mature testis and ovary than in the gonads of other maturity stages. In contrast to that, ERbeta2 was highest in the ovary of mature female, and significantly lower levels of ERbeta2 expression were observed in the gonads of the other maturity stages. The present study describes the molecular characterization of ERbeta2, and documents the expression changes of three ER subtypes during sex change process of the protandrous black porgy.

Physiological responses and expression of arginine vasotocin receptor, prolactin and prolactin receptor mRNA in olive flounder Paralichthys olivaceus during osmotic stress

We cloned complementary DNA (cDNA) encoding the arginine vasotocin receptor (AVT-R) from the kidney of olive flounder (Paralichthys olivaceus). Olive flounder AVT-R cDNA consists of 1155 bp, and encodes a protein of 384 amino acids. To investigate the stress responses and osmoregulatory abilities of olive flounder in changing salinities (35, 17.5, 8.75, 4, and 0 psu), we examined the expression of AVT-R, prolactin (PRL), and PRL receptor (PRL-R) messenger RNA (mRNA) in osmoregulatory organs using quantitative real time PCR (QPCR). AVT-R and PRL-R were expressed in the gill, kidney, and intestine, whereas PRL mRNA was expressed only in the pituitary gland. In addition, we measured the levels of plasma glucose, cortisol, aspartate aminotransferase (AST), and alanine aminotransferase (ALT). The mRNA expression and plasma parameters increased in hypoosmotic environments. Furthermore, osmolality decreased significantly according to salinity concentration decreased. These results suggest that AVT-R, PRL, and PRL-R mRNA play important roles in the hormonal regulation of osmoregulatory organs, thereby improving the osmoregulatory ability of olive flounder in hypoosmotic environments.

Changes of cytochrome P4501A mRNA expression and physiology responses in the olive flounder, Paralichthys olivaceus, exposed to benzo[a]pyrene

Abstract Benzo[a]pyrene (BaP) is generated by the incomplete combustion of organic substances such as oil and coal, and is a widespread organic environmental contaminant in terrestrial and aquatic ecosystems. To determine the effects of BaP on organisms, we investigated its time- and dose-related effects on the levels of cytochrome P4501A (P4501A) mRNA in the liver and gills of the olive flounder (Paralichthys olivaceus) using quantitative polymerase chain reaction (QPCR) and measured the plasma glucose, cortisol, aspartate aminotransferase (AST), and alanine aminotransferase (ALT) levels. The full-length olive flounder P4501A cDNA consists of 1566 nucleotides and encodes a 521-amino-acid protein. In the liver and gills, the expression of P4501A mRNA was highest 6 h after exposure to both 10 and 30 µg l−1 BaP, and then decreased. In addition, the plasma parameters increased with exposure. These results suggest that P4501A plays an important role in the detoxification of BaP, which stressed the olive flounder. Therefore, these physiological parameters may be indicators of BaP-induced stress responses.