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This study planned to isolation and characterization of AKR1A1 cDNA from Bap injected nile tilapia (Oreochromis niloticus), comparison of its characteristic structures with those of other species, characterization of AKR1A1 gene and promoter, and investigation of AKR1A1 mRNA expression in various organs of Bap injected tilapia. The cDNA was 1172 bp long which includes an open reading frame of 975 bp encoding a 324 amino acids protein and a stop codon. The sequence showed 3' and 5' non-coding regions of 179 and 18 bp. The amino acid sequence ofO. niloticusAKR1A1 shows similarities of 60, 60, 60.6, 61.2 62.2, and 57.8% with mouse AKR1A1, Norway rat AKR1A1, zebrafish AKR1A1, African clawed frog AKR1A1, human, and yellow perch AKR1A1, respectively. Nucleotide sequence investigation ofAKR1A1gene and 5′-flanking region showed that the structural gene and the 5′-flanking region were approximately 2975 bp and 4006 bp in length, respectively. The protein-coding region contained eight exons, and one additional upstream exon. Real-time polymerase chain reaction (PCR) results showed that the highest level of AKR1A1 expression was found in bile (108.7), followed by kidney (77.9), muscles (37.3), and liver (24.7). mRNA levels of AKR1A1 were almost negligible in gills (0.6) while no detectable (ND) constitutive expression was detected in gut. In conclusion, our results concluded that tilapia AKR1A1 is inducible by BaP and have a significant function in the metabolism of xenobiotics and, therefore, may used as biomarker in fish [ABSTRACT FROM PUBLISHER]

polipoproteins such as apolipoprotein (apo) A-I, apoA-IV, and apoE are lipid binding proteins synthesized mainly in the liver and the intestine and play an important role in the transfer of exogenous or endogenous lipids through the circulatory system. To investigate the mechanism of lipid transport in fish, we have isolated some novel genes of the apoA-I family, apoIA-I (apoA-I isoform) 1-11, from Japanese eel by PCR amplification. Some of the isolated genes of apoIA-I corresponded to 28kDa-1 cDNAs which had already been deposited into the database and encoded an apolipoprotein with molecular weight of 28 kDa in the LDL, whereas others seemed to be novel genes. The structural organization of all apoIA-Is consisted of four exons separated by three introns. ApoIA-I10 had a total length of 3232 bp, whereas other genes except for apoIA-I9 ranged from 1280 to 1441 bp. The sequences of apoIA-Is at the exon-intron junctions were mostly consistent with the consensus sequence (GT/AG) at exon-intron boundaries, whereas the sequences of 3′ splice acceptor in intron 1 of apoIA-I1-7 were (AC) but not (AG). The deduced amino acid sequences of all apoIA-Is contained a putative signal peptide and a propeptide of 17 and 5 amino acid residues, respectively. The mature proteins of apoIA-I1-3, 7, and 8 consisted of 237 amino acids, whereas those of apoIA-I4-6 consisted of 239 amino acids. The mature apoIA-I10 sequence showed 65% identity to amino acid sequence of apoIA-I11 which was associated with an apolipoprotein with molecular weight of 23 kDa in the VLDL. All these mature apoIA-I sequences satisfied the common structural features depicted for the exchangeable apolipoproteins such as apoA-I, apoA-IV, and apoE but apoIA-I11 lacked internal repeats 7, 8, and 9 when compared with other members of apoA-I family. Phylogenetic analysis showed that these novel apoIA-Is isolated from Japanese eel were much closer to apoA-I than apoA-IV and apoE, suggesting new members of the apoA-I family. [ABSTRACT FROM AUTHOR]

Porcine heart adenylate kinase can be separated into acidic and basic components by electrofocusing, and the latter is identical with the skeletal muscle adenylate kinase. The acidic adenylate kinase was purified approximately 3600-fold from porcine heart extract in an overall yield of 59yo by DEAE-cellulose chromatography, substrate elution from a column of phosphocellulose, affinity chromatography on a column of blue dextran-Sepharose 4B, and gel filtration. The purified acidic enzyme had a specific activity of 1100 units per mg of enzyme, and was found to be a homogeneous protein in all the procedures used, i.e. disc and sodium dodecylsulfate electrophoresis, and gel filtration. However, electrophoretic variants were observed on isoelectrofocusing and this variance may depend on the formation of protein-ampholine complexes. The purified acidic enzyme had a molecular weight of 26900 and an optimum pH of 5.8 in the forward reaction (ATP + AMP → 2ADP). The amino acid composition of the acidic enzyme was found to be 22 aspartic acid or asparagine, 13 threonine. 14 serine, 29 glutamic acid or glutamine, 17 proline, 17 glycine; 22 alanine, 4 half-cystine, 13 valine, 8 methionine, 13 isoieucine, 24 leucine, 4 tyrosine, 7 phenylalanine, 5 histidine, 19 lysine, 14 arginine and no tryptophan, totaling 245 residues. The enzyme had two -SS- bonds but no free -SH, and was not inhibited at all by 5,5'-dithio-bis(2-nitrobenzoic acid). From these results, it may be concluded that the acidic adenylate kinase of porcine heart is similar to liver mitochondrial adenylate kinase. [ABSTRACT FROM AUTHOR]

Cytochrome P450 (CYP) genes, which make up a large gene superfamily, are known to play an important role in drug metabolism. The CYP1 family, one of the gene families of the CYP superfamily, has three subfamilies of genes whose sequences have been deposited in the GenBank/EMBL thus far: CYP1A, CYP1B, and CYP1C. Mammals as well as fish confront numerous foreign chemicals in the environment that may accumulate to toxic levels unless they are metabolized and eliminated by processes largely mediated by CYP enzymes. A new complementary DNA of the CYP1C subfamily encoding CYP1C2 was isolated from the carp liver after a single intraperitoneal injection of beta-napthoflavone (BNF). The full-length cDNA obtained contained a 5' noncoding region of 198 bp, an open reading frame of 1575 bp coding for 524 amino acids and a stop codon, and a 3' noncoding region of 531 bp. The predicted molecular weight of the protein was approximately 59.3 kDa. The amino acid sequence deduced from the carp CYP1C2 sequence showed a similarity of 76.6% with that deduced from our previously reported carp CYP1C1. It exhibited similarities of 77.3, 73.7, and 76.4% with those deduced from scup CYP1C2, scup CYP1C1, and Japanese eel CYP1C1 sequences, respectively. Carp CYP1C2 cDNA showed similarities with reported CYP1Bs of teleosts and mammals, namely, 47.6, 45.3, 45.7, 44.0, and 44.6% for carp, plaice, human, rat, and mouse CYP1B1s, respectively, while it exhibited a similarity of 49.0% with carp CYP1B2. The carp CYP1C2 sequence was aligned with the CYP1 sequences and has been deposited in the GenBank/EMBL data bank with the accession number AY437777. The phylogenetic tree constructed using fish and mammalian CYP1 sequences suggested a closer relationship of CYP1C with CYP1B than with CYP1A. The tree showed possibile existence of CYP1C subfamily genes in mammalian species. Northern blot analysis of the liver, intestines, kidneys, and gills revealed a distinct induced expression only in the kidneys, with no detectable constitutive expression in the other organs studied.