In vivo immunostimulation of the flat oyster Ostrea edulis and tissue targeting by microspheres uptake (original) (raw)
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Chemosphere, 2007
Glutathione S-transferases (GSTs) play a major role in detoxification of xenobiotics and antioxidant defense. Here we report fulllength cDNA sequence of a novel Sigma-class of GST (GST-S) from the intertidal copepod Tigriopus japonicus. The full sequence was of 1136 bp in length containing an open reading frame (ORF) of 651 bp that encoded 217 amino acid residues. The recombinant Tigriopus GST-S was highly expressed in transformed Escherichia coli. Kinetic properties and effects of pH, temperature and chemical inhibitors on Tigriopus GST-S were also studied. The expression of GST-S was studied using real-time RT-PCR in response to exposure to two oxidative stresses-inducing agents, viz., hydrogen peroxide (H 2 O 2 ) and heavy metals (copper, manganese). It was observed that H 2 O 2 (2 mM) exposure down-regulated its expression at the initial stage but there was recovery and up-regulation shortly afterwards. In case of heavy metal exposure there was concentration-dependent increase in Tigriopus GST-S gene expression up to 24 h. These results suggest that Tigriopus GST-S expression is modulated by prooxidant chemicals and it may play a role against oxidative stress. A majority of other GST isoforms is known to play an important role in antioxidant defense. This study provides a preliminary insight into the possible antioxidant role for Sigma-class of GST in T. japonicus.
Glutathione S-transferases play an important role in cellular detoxification and may have evolved to protect cells against reactive oxygen metabolites. In this study, we report the molecular characterization of glutathione s-transferase-theta (GST-θ) from freshwater prawn Macrobrachium rosenbergii. A full length cDNA of GSTT (1417 base pairs) was isolated and characterized bioinformatically. Exposure to virus (white spot syndrome baculovirus or M. rosenbergii nodovirus), bacteria (Aeromonas hydrophila or Vibrio harveyi) or heavy metals (cadmium or lead) significantly increased the expression of GSTT (P b 0.05) in hepatopancreas. Recombinant GST-θ with monochlorobimane substrate had an optimum activity at pH 7.5 and 35 °C. Furthermore recombinant GST-θ activity was abolished by the denaturants triton X-100, Gua-HCl, Gua-thiocyanate, SDS and urea in a dose-dependent manner. Overall, the results suggest a potential role for M. rosenbergii GST-θ in detoxification and possibly conferring immune protection.
Glutathione S-transferases (GSTs) are versatile enzymes, act as primary intracellular detoxifiers and contribute to a broad range of physiological processes including cellular defense. In this study, a full-length cDNA representing a novel sigma-like GST was identified from Manila clam, Ruditapes philippinarum (RpGSTσ). RpGSTσ (884 bp) was found to possess an open reading frame of 609 bp. The encoded polypeptide (203 amino acids) had a predicted molecular mass of 23.21 kDa and an isoelectric point of 7.64. Sequence analysis revealed two conserved GST domain profiles in N-and C-termini. Alignment studies revealed that the identity between deduced peptides of RpGSTσ and known GSTσ members was relatively low (b 35%), except a previously identified Manila clam GSTσ isoform (87.2%). Phylogenetic analysis indicated that RpGSTσ clustered together with molluscan GSTσ homologs, which were closely related to insect GSTσs. The RpGSTσ was subsequently cloned and expressed as recombinant protein, in order to characterize its biological activity. The recombinant RpGSTσ exhibited characteristic glutathione conjugating catalytic activity toward 1-chloro-2,4-dinitrobenzene, 3,4-dichloronitrobenzene and ethacrynic acid. It had an optimal pH and temperature of 8.0 and 35 °C, respectively. Expression profiles under normal conditions and in response to lipopolysaccharide-, poly I:C-and Vibrio tapetis-challenges were also investigated. RpGSTσ demonstrated a differential tissue distribution with robust transcription in gills of normal animals. We explored potential association of GSTσ in cellular defense during bacterial infection and found that in challenged clams, RpGSTσ gene was significantly induced in internal and external tissues, in conjunction with manganese-as well as copper–zinc superoxide dismutase (MnSOD and CuZnSOD) genes. Moreover, the induction was remarkably higher in hemocytes than in gill. Collectively, our findings suggested that RpGSTσ could play a significant role in cellular defense against oxidative stress caused by bacteria, in conjunction with other antioxidant enzymes, such as SODs.
Biochemical and Biophysical Research Communications, 2006
We cloned and sequenced full-length cDNA of a h-class-like glutathione S-transferase (GST-T) from liver tissue of the self-fertilizing fish Rivulus marmoratus. The full-length cDNA of rm-GST-T was 907 bp in length containing an open reading frame of 666 bp that encoded a 221-amino acid putative protein. Its derived amino acid sequence was clustered with other vertebrate h-class GSTs in a phylogenetic tree. The deduced amino acid sequence of h-like rm-GST (rm-GST-T) was compared with both classes (a and h) of GST and a-class rm-GST (rm-GST-A). Tissue-specific expression of two rm-GST mRNAs was investigated using real-time RT-PCR. To further characterize the catalytic properties of this enzyme along with rm-GST-A, we constructed the recombinant h-like rm-GST plasmid with a 6·His-Tag at the N-terminal of rm-GST-T cDNA. Recombinant rm-GST-T was highly expressed in transformed Escherichia coli, and its soluble fraction was purified by His-Tag affinity column chromatography. The kinetic properties and effects of pH and temperature on rm-GST-T were further studied, along with enzyme activity and inhibition effects, and compared with recombinant rm-GST-A. These results suggest that recombinant rm-GSTs such as rm-GST-A and rm-GST-T play a conserved functional role in R. marmoratus.
Journal of Biochemical and Molecular Toxicology, 2010
The expression of glutathione S-transferase (GST) is a crucial factor in determining the sensitivity of cells and organs in response to a variety of toxicants. In this study, we cloned the core nucleotide of alpha, kappa, mu, mGST, pi, rho, and theta-like GST genes from bighead carp (Aristichthys nobilis). Their derived amino acid sequences were clustered with other vertebrate GSTs in a phylogenetic tree, and the bighead carp GST sequences have the highest similarity with those from common carp and zebrafish. We quantified the constitutive mRNA transcription of GST isoforms in eight different tissues (liver, kidney, spleen, intestine, muscle, heart, brain, and gill). The information obtained from the present study could be distilled into a few generalized principles: multiple GST isoenzymes were ubiquitously expressed in all tissues; majority of GSTs had high constitutive expression in intestine, liver, and kidney. These findings are in agreement with the roles of these tissues in xenobiotic metabolism. At the same time, some unique findings were detected in the current experiment: (1) higher expression of most GSTs was observed in spleen; (2) the expression of GST pi was highest in almost all the studied tissues except muscle; the other two isoforms, GST alpha and rho, were also highly expressed in liver, kidney, intestine, spleen, heart, and brain of bighead carp. All these results strongly imply an important role of these GST isoforms in detoxification of ingested xenobiotics. © 2010 Wiley Periodicals, Inc. J Biochem Mol Toxicol 24:250–259, 2010; View this article online at wileyonlinelibrary.com. DOI 10.1002/jbt.20333
A Mu-class glutathione S-transferase (GSTM) from the rock shell Thais clavigera
Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology, 2008
The rock shell (Thais clavigera) has attracted interest due to high frequency of imposex induced by endocrine disrupting chemicals (EDCs) in its natural populations. Oxidative stress is one of the mechanisms of action of EDCs. Glutathione S-transferases (GSTs) play an important role in antioxidant defense protecting the cells from oxidative stress. So far, there is no information on antioxidant defense or detoxification genes from T. clavigera. We cloned the full length cDNA sequence for a Mu-class of GST gene from T. clavigera (Tc-GSTM) and purified recombinant Tc-GSTM protein by bacterial expression. The deduced amino acid sequence of Tc-GSTM exhibited 45 to 66% identity with other Mu-class GSTs. Real-time RT-PCR analysis showed highest expression of Tc-GSTM in gill, while reproductive organs showed low expression. The biochemical characteristics of purified recombinant Tc-GSTM were typical, and thus Tc-GSTM showed highest specific activity for the universal GST substrate, 1-chloro-2,4-dinitrobenzene (CDNB). After exposure to prooxidant H 2 O 2 , transformed Escherichia coli containing Tc-GSTM showed higher survival rate compared to control bacteria without expressed Tc-GSTM. The present study reveals a conserved antioxidant role for GSTM in rock shells, and the tissue-specific differences in Tc-GSTM transcripts would partly reflect vulnerability of reproductive organs to chemically induced oxidative stress.
Marine Environmental Research, 2004
We have previously shown that largemouth bass (Micropterus salmoides) has a remarkable ability to conjugate 4-hydroxy-2-nonenal (4HNE), a mutagenic and cytotoxic a; b-unsaturated aldehyde produced during the peroxidation of lipids. In addition, we have isolated a glutathione S-transferase cDNA (bass GSTA) that encodes a recombinant protein which is highly active in 4HNE conjugation and structurally similar to plaice (Pleuronectes platessa) GSTA. In the present study, HPLC-GST subunit analysis revealed the presence of at least two major GST isoforms in bass liver, with one peak constituting 80% of the total bass liver GST protein.
Environmental Toxicology, 2005
Cyanobacterial toxins have been shown to have a far-reaching impact-from aquatic organisms to human health. Aquatic organisms are typically exposed in their natural environment to toxic cyanobacteria, and exposure can occur via ingestion of cyanobacterial cells or by bioaccumulation of water-borne toxin. The aquaculture and fisheries of crustaceans are among the most important seafood industries. Concomitant with the growth of this industry, the importance of the health of crustaceans increased. The black tiger prawn is the major cultivated prawn in Australia. The aquaculture of these prawns takes place in shallow ponds, where blooms, often of cyanobacteria, develop. Cyanobacterial toxins were hypothesized to contribute to the mortality of prawns. Many aquatic organisms have the possibility of detoxifying cyanobacterial toxins via conjugation to glutathione. The presence of several classes of the cytosolic glutathione S-transferase system in black tiger prawns-mu, pi, theta, alpha, and tau-was shown using different substrates for measurement. Injection experiments with microcystin-LR and feeding experiments with nodularin revealed elevation of GST activity in different types of prawn tissue in parallel with reduction in the GST classes. Correlation analyses of toxin content of the prawns with GST activity showed that low toxin content was correlated with high elevation of enzymes and high toxin content with low elevation of enzymes. Wiley Periodicals, Inc. Environ Toxicol 20: 301-307, 2005