Cytochrome P450 1 enzymes in black rockfish, Sebastes schlegelii: Molecular characterization and expression patterns after exposure to benzo[a]pyrene.

Cytochrome P450 (CYP) enzymes play important roles in the detoxification and bioactivation of environmental contaminants and are involved in the responses to pollution in fish. In this study, we cloned four new CYP1 genes, CYP1A, CYP1B, CYP1C1, and CYP1C2, from black rockfish (Sebastes schlegelii), a dominant and economically important fish species in Korea. This species is at a significant risk of exposure to petrohydrocarbons, such as benzo[a]pyrene(B[a]P), due to frequent oil spills along the Korean coast.

Effects of trichlorfon on oxidative stress, neurotoxicity, and cortisol levels in common carp, Cyprinus carpio L., at different temperatures.

Trichlorfon (TCF) is an organophosphate compound used extensively as an anti-parasitic in aquaculture. In this study, we investigated the effects of TCF on the antioxidant defense system, acetylcholinesterase (AChE) activity, and stress responses in various tissues of common carp (Cyprinus carpio L.).

Toxicological effects of trichlorfon on hematological and biochemical parameters in Cyprinus carpio L. following thermal stress.

Trichlorfon is a moderately toxic organophosphate pesticide that is widely used in aquaculture. This study investigated the effects of trichlorfon on hematological parameters, biochemical factors, and stress reaction in Cyprinus carpio L. The fish were exposed to acute concentrations of trichlorfon (0, 0.

Molecular characterization and expression analysis of olive flounder (Paralichthys olivaceus) phospholipase C gamma 1 and gamma 2.

Phospholipase C gamma 1 and gamma 2 (PLCG1 and PLCG2) are influential in modulating Ca and diacylglycerol, second messengers involved in tyrosine kinase-dependent signaling, including growth factor activation. Here, we used RACE (rapid amplification of cDNA ends) to clone cDNA encoding PLCG1 (PoPLCG1) and PLCG2 (PoPLCG2) in the olive flounder (Paralichthys olivaceus). The respective 1313 and 1249 amino acid sequences share high identity with human PLCG1 and PLCG2, and contain the following domains: pleckstrin homology (PH), EF-hand, catalytic X and Y, Src homology 2 (SH2), Src homology 3 (SH3), and C2.

Functional analysis of the extended N-terminal region in PLC-δ1 (MlPLC-δ1) from the mud loach, Misgurnus mizolepis.

Mud loach phospholipase C-δ1 (MlPLC-δ1) contains all the characteristic domains found in mammalian PLC-δ isozymes (pleckstrin homology domain, EF-hands, X–Y catalytic region, and C2 domain) as well as an extended 26-amino acid (aa)-long N-terminal region that is an alternative splice form of PLC-δ1 and is novel to vertebrate PLC-δ. In the present structure-function analysis, deletion of the extended N-terminal region caused complete loss of phosphatidylinositol (PI)- and phosphatidylinositol 4,5-bisphosphate (PIP2)-hydrolyzing activity in MlPLC-δ1. Additionally, recombinant full-length MlPLC-δ1 PLC activity was reduced in a dose-dependent manner by coincubation with the 26-aa protein fragment.

Expression analysis and enzymatic characterization of phospholipase Cδ4 from olive flounder (Paralichthys olivaceus).

Phospholipase Cδ4 (PLCδ4) plays a significant role in cell proliferation, tumorigenesis, and in an early stage of fertilization. Despite the characterization of the mammalian PLCδ4, extensive study in aquatic organisms has not been carried out so far. Here, we performed the molecular and biochemical characterization of flatfish Paralichthys olivaceus PLCδ4 (PoPLCδ4) to understand its enzymatic properties and physiological functions.

PLC-δ1-Lf, a novel N-terminal extended phospholipase C-δ1.

Phospholipase C-δ (PLC-δ), a key enzyme in phosphoinositide turnover, is involved in a variety of physiological functions. The widely expressed PLC-δ1 isoform is the best characterized and the most well understood phospholipase family member. However, the functional and molecular mechanisms of PLC-δ1 remain obscure.

Olive flounder (Paralichthys olivaceus) cystatin C: cloning, mRNA expression, and enzymatic characterization of olive flounder cystatin C.

Cystatins are endogenous inhibitors of mammalian lysosomal cysteine proteinases, such as cathepsins B, L, H, and S. Cystatin C belongs to the type 2 cystatin family. In this study, the 751-bp cystatin C cDNA (PoCystatin C) of olive flounder (Paralichthys olivaceus) was cloned by screening from the olive flounder cDNA library.

Olive flounder (Paralichthys olivaceus) cystatin B: cloning, tissue distribution, expression and inhibitory profile of piscine cystatin B.

Among the cystatin superfamily, cystatin B, also known as stefin B, is an intracellular inhibitor that regulates the activities of cysteine proteases, such as papain and cathepsins. In this study, the 536 bp cystatin B cDNA (referred to hereafter as PoCystatin B) was cloned from olive flounder (Paralichthys olivaceus) using a combination of the rapid amplification of cDNA ends (RACE) approach and olive flounder cDNA library screening. To determine the tissue distribution of PoCystatin B mRNA, the expression of PoCystatin B in normal and lipopolysaccharide (LPS)-stimulated flounder tissues were compared with that of the inflammatory cytokines interleukin (IL)-1β, IL-6, and IL-8 by reverse transcription (RT)-polymerase chain reaction (PCR).

Functional analysis of duplicated genes and N-terminal splice variant of phospholipase C-δ1 in Paralichthys olivaceus.

Phosphoinositide-specific phospholipase C δ (PLC δ) plays an important role in many cellular responses and is involved in the production of second messenger. Here, we describe the presence of novel N-terminal extended alternative splice form of PLC-δ1B in Paralichthys olivaceus, which differs from the reported mammalian PLC-δ1 isoform. The two variants PoPLC-δ1B-Lf and PoPLC-δ1B-Sf share exon 3 (including the PH domain) to exon 16, but differ at the exon 1 (Short form: Sf) and novel exon 2 (Long form: Lf) of the transcript.

Molecular cloning, expression, and enzymatic analysis of cathepsin X from starfish (Asterina pectinifera).

Cathepsin X, also known as cathepsin Z, is referred to as a "lysosomal proteolytic enzyme" and a member of the peptidase C1 family, which is involved in various biological processes such as immune response, cell adhesion, and proliferation. In the present study, the cDNA of starfish (Asterina pectinifera), which is known to cause serious damage to commercial shellfish mariculture, cathepsin X (ApCtX) was isolated through the combination of homology molecular cloning and rapid amplification of cDNA ends (RACE) methods for the application to find a way to reduce/control starfish densities. The full-length of ApCtX gene was determined to consist of the 2,240 bp nucleotide sequence, which encoded for a preproprotein of 296 amino acids with a molecular mass of about 32.

Cloning, heterologous expression, and enzymatic characterization of cathepsin L from starfish (Asterina pectinifera).

To determine the role of cathepsin L in Echinoderms, starfish (Asterina pectinifera) cathepsin L (ApCtL) was cloned. The results of RT-PCR analysis indicated that the expression of ApCtL in all of the tissues. The pro-mature enzyme of ApCtL, proApCtL, was expressed in Escherichia coli, and cathepsin activity was detected by cleaving of synthetic fluorogenic peptide substrates and gelatin zymography.

Cloning, expression analysis and enzymatic characterization of cathepsin S from olive flounder (Paralichthys olivaceus).

Cathepsin S is a critical protease for the regulation of MHC class II immune responses, and thus is a potential target for developing immunosuppressive drugs in the pathogenesis of degenerative and autoimmune diseases. In this study, we cloned a cDNA encoding for cathepsin S (PoCtS) from the olive flounder, Paralichthys olivaceus. The 1170 bp PoCtS cDNA contained an open reading frame of 1014 bp, which consisted of a 25-residue putative signal peptide, a 96-residue propeptide and the 216-residue mature enzyme.

Molecular cloning, expression, and characterization of cathepsin L from mud loach (Misgurnus mizolepis).

Cathepsin L is an important protease in the initiation of protein degradation and one of the most powerful endopeptidases. In this study, we cloned mud loach (Misgurnus mizolepis) cathepsin L (MlCtL) cDNA, and the pro-mature enzyme of MlCtL (proMlCtL) was expressed in Escherichia coli as a fusion protein with glutathione S-transferase in a pGEX-4 T-1 vector. The recombinant proMlCtL was overexpressed in E.

Purification, molecular cloning, and biochemical characterization of subtilisin JB1 from a newly isolated Bacillus subtilis JB1.

An extracellular gelatinolytic enzyme obtained from the newly isolated Bacillus subtilis JB1, a thermophilic microorganism relevant to the aerobic biodegradation process of fish-meal production, was purified via ammonium sulfate precipitation, Sephadex G-200 Gel filtration chromatography, and one-dimensional gel electrophoresis separation and subsequently identified via peptide mass fingerprinting and chemically assisted fragmentation matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The subtilisin JB1 gene was sequenced and its recombinant protein prosubtilisin JB1 was expressed in Escherichia coli, and the purified prosubtilisin JB1 (62 kDa) protein was digested with gelatin, bovine serum albumin, azocasein, fibrinogen, and the fluorogenic peptide substrate Ala-Ala-Phe-7-amido-4-methylcoumarin hydrochloride, whereas the serine protease inhibitors phenylmethylsulfonyl fluoride and chymostatin completely inhibited its enzyme activity at an optimal pH of 7.5.

Molecular cloning, expression analysis and enzymatic characterization of cathepsin K from olive flounder (Paralichthys olivaceus).

We assessed the putative physiological roles of cathepsin K from a flatfish, olive flounder. We cloned a cDNA encoding for cathepsin K (PoCtK), a cysteine protease of the papain family from olive flounder, Paralichthys olivaceus. The tissue-specific expression pattern of PoCtK, determined via real-time PCR analysis, revealed ubiquitous expression in normal tissues with high levels of expression in the spleen and bone marrow.

Molecular cloning, mRNA expression and enzymatic characterization of cathepsin F from olive flounder (Paralichthys olivaceus).

Cathepsin F is a recently described papain-like cysteine protease of unknown function, and unique among cathepsins due to an elongated N-terminal pro-region, which contains a cystatin domain. In the present study, the cDNA of olive flounder (Paralichthys olivaceus) cathepsin F (PoCtF) was cloned by the combination of homology molecular cloning and rapid amplification of cDNA ends (RACE) approaches. The PoCtF gene was determined to consist of the 1844 bp nucleotide sequence which encodes for a 475-amino acid polypeptide.

Cloning and characterization of muscarinic receptor genes from the nile tilapia (Oreochromis niloticus).

To investigate the regulatory mechanism underlying the contractile response in the intestinal smooth muscle of the nile tilapia (Orechromis niloticus), we used pharmacologic and molecular approaches to identify the muscarinic subreceptors and the intracellular signaling pathways involved in this motility. Myography assays revealed that an M1- and M3-subtype selective antagonist, but not a M2-subtype selective antagonist, inhibited carbachol HCI (CCH)-induced intestinal smooth muscle contraction. In addition, a phospholipase C inhibitor, but not an adenylate cyclase inhibitor, blocked the contractile response to CCH.

Transmission of iridovirus from freshwater ornamental fish (pearl gourami) to marine fish (rock bream).

Freshwater pearl gourami Trichogaster leeri and seawater rock bream Oplegnathus fasciatus infected by the iridoviruses PGIV-SP and IVS-1 were carrying similar numbers of viral particles (2.52 x 10(8) and 2.46 x 10(8) viral genome copies mg(-1) spleen tissue, respectively).

Duplication of phospholipase C-delta gene family in fish genomes.

Fishes possess more genes than other vertebrates, possibly because of a genome duplication event during the evolution of the teleost (ray-finned) fish lineage. To further explore this idea, we cloned five genes encoding phosphoinositide-specific phospholipase C-delta (PLC-delta), designated respectively PoPLC-deltas, from olive flounder (Paralichthys olivaceus), and we performed phylogenetic analysis and sequence comparison to compare our putative gene products (PoPLC-deltas) with the sequences of known human PLC isoforms. The deduced amino acid sequences shared high sequence identity with human PLC-delta1, -delta3, and -delta4 isozymes and exhibited similar primary structures.

Molecular cloning, tissue distribution and enzymatic characterization of cathepsin X from olive flounder (Paralichthys olivaceus).

In this study, we have cloned a cDNA encoding for cathepsin X (PoCtX) from the olive flounder, Paralichthys olivaceus. The presence of an HIP motif, which is conserved in the unique cathepsin X family, PoCtX, clearly shows its relation to the cathepsin X group, apart from the cathepsin L or B subfamily. The results of RT-PCR and real-time PCR analyses revealed ubiquitous PoCtX expression in normal and LPS-stimulated tissues.

Cloning and characterization of phospholipase D from olive flounder (Paralichthys olivaceus).

The phospholipase D1 (PLD1) cDNA, designated PoPLD, encoding a predicted protein of 1053 amino acids in olive flounder (Paralichthys olivaceus) has been cloned. The deduced amino acid sequence shares high identity with that of PLD1s and PLD2 in human, rat and mouse. The phylogenic analysis and sequence comparison of PoPLD with other PLD isozymes were found to be closely related to the PLD1 isozyme in primary structure.

Cloning, site-directed mutagenesis and expression of cathepsin L-like cysteine protease from Uronema marinum (Ciliophora: Scuticociliatida).

A cysteine protease gene (ScCtL) homologous to the cathepsin L genes was isolated from a cDNA library of the scuticociliate parasite (Uronema marinum). To express the ScCtL recombinant protein in heterologous system, 17 codons were redesigned to conform to the standard eukaryotic genetic code using PCR-based site-directed mutagenesis. The synthetic U.

Prevalence of tet(B) and tet(M) genes among tetracycline-resistant Vibrio spp. in the aquatic environments of Korea.

Of 24 tetracycline(Tc)-resistant Vibrio spp. isolated from different marine sources in Korea between 1993 and 2003, 23 were identified as carrying both tet(B) and tet(M), while 1 strain carried tet(B) only. In conjugation experiments, 3 strains appeared to be able to transfer both tet(B) and tet(M) to the recipient.

Molecular cDNA cloning and analysis of the organization and expression of the IL-1beta gene in the Nile tilapia, Oreochromis niloticus.

The full-length cDNA sequence of interleukin-1beta (IL-1beta) from the Nile tilapia, Oreochromis niloticus, was determined by using PCR with primers designed from known fish IL-1beta sequences followed by elongation of the 5' and 3' ends using Rapid Amplification of cDNA Ends (RACE). The cDNA contains a 92-bp 5' untranslated region (UTR), a single open reading frame (ORF) of 732 bp that translates into a 243-amino acid molecule, a 341-bp 3' UTR with four cytokine RNA instability motifs (ATTTA), and a polyadenylation signal (AATAAA) at 15 nucleotides upstream of the poly(A) tail. The organization of the genomic IL-1beta based on the cDNA sequence appeared to be 4 introns and 5 exons.