Cloning and characterization of the trout perforin

The pore-forming protein, perforin is one of the effectors of cell-mediated killing. A perforin cDNA clone was isolated from rainbow trout (Oncorhynchus mykiss) after screening of a spleen cDNA library. The full-length cDNA is 2070 bp in size, encoding for a polypeptide of 589 amino acids. The predicted amino acid sequence of the trout perforin is 64, 58 and 40% identical to those of Japanese flounder, zebrafish and human perforins, respectively. Although its membrane attack complex/perforin (MACPF) domain is conserved, trout perforin shows low homology to human and trout terminal complement components (C6, C7, C8 and C9), ranging from 19 to 26% identity. Expression analysis reveals that the trout perforin gene is expressed in the blood, brain, heart, kidney, intestine and spleen. Phylogenetic analysis of proteins which belong to the MACPF superfamily clusters the trout perforin in the same group with other known perforins.     

Comparative binding and sequence interaction specificities of aflatoxin B1, aflatoxicol, aflatoxin M1, and aflatoxicol M1 with purified DNA

The covalent binding of the activated forms of several aflatoxins to N-7 of guanine residues on purified DNA has been studied. The aflatoxins include aflatoxin B1 (AFB1) and two human metabolites, aflatoxicol and aflatoxin M1, along with aflatoxicol M1, a rabbit and trout metabolite. DNA binding studies using tritiated [3H]aflatoxins indicate that equimolar solutions of each aflatoxin upon activation with chloroperoxybenzoic acid readily react to produce covalently bound adducts. These reactions produce alkali-labile sites which can be identified using a simple variation of the Maxam-Gilbert sequencing procedure. Two DNA fragments were exposed to each aflatoxin, and the reaction intensities at 33 guanine residues were determined. As much as 10-fold variation in reaction intensities was observed for various guanyl sites. Data indicate that none of the aflatoxins had identical reaction profiles, although AFB1 and aflatoxicol M1 were similar, as were aflatoxicol and aflatoxin M1. Hence, the frequency with which the various aflatoxin epoxides might damage specific sites critical for tumor initiation in vivo would not be predictable from total covalent binding indices. The frequency of occurrence of modifications at particular sites for AFB1 was also compared with the empirical "rules" established for AFB1 by Misra et al. (Misra, R. P., Muench, K. F., and Humayun, M. Z. (1983) Biochemistry 22, 3351-3359). Identical sites within fragments were compared for each aflatoxin, and the data showed that the attacking frequency for some such sites varied significantly. These results indicate that binding intensity rules based on nearest neighbor nucleotides do not reliably predict guanyl-AFB1 binding frequencies.     

Cloning, Sequencing, and Phylogenetic Analysis of Complementary DNA of Novel Cytochrome P-450 CYP1A in Japanese Eel (Anguilla japonica)

Complementary DNA of cytochrome P-450 CYP1A, in addition to CYP1A1, has been isolated from Japanese eel (Anguilla japonica) liver treated with 3-methylcholanthrene. The cDNA contained a 5′ untranslated region of 66 bp, an open reading frame of 1554 bp coding for 517 amino acids and a stop codon, and a 3′ untranslated region of 1166 bp. The predicted molecular weight of the Japanese eel CYP1A was approximately 58.5 kDa. The nucleotide sequence exhibited identities with the reported CYP1A1 sequences of 77% for Japanese eel, 75% for rainbow trout, 72% for scup, plaice, and butterfly fish, and 71% for toadfish. The deduced amino acid sequence exhibited identities with the reported CYP1A1 sequences of 78% for Japanese eel, 77% for rainbow trout, 75% for scup, 74% for toadfish, 73% for plaice, and 72% for butterfly fish. The novel eel CYP1A obtained had less similarity to the other teleost CYP1A1 proteins (72%–78%) than that of the eel CYP1A1 (74%–80%). When compared with mammalian CYP proteins, the novel eel CYP1A was more similar to the CYP1A1 proteins (54%–56%) than to the CYP1A2 proteins (50%–53%). The phylogenetic tree of the teleost CYP1A genes constructed using the maximum likelihood method suggested that the novel eel CYP1A is ubiquitous among the Anguilliformes. Received August 25, 2000; accepted November 30, 2000     

Molecular cloning and characterization of cDNAs coding for apopolysialoglycoproteins in cherry salmon (Oncorhynchus masou) eggs

Recently we have cloned the cDNAs and genomic DNAs for apopolysialoglycoproteins (apoPSGPs) of Salmo gairdneri (rainbow trout) [Sorimachi, H., Emori, Y., Kawasaki, H., Kitajima, K., Inoue, S., Suzuki, K., & Inoue, Y. (1988) J. Biol. Chem. 262, 17678-17684], and the sequence analyses have indicated that the mRNAs for apoPSGPs vary in length and contain different numbers of identical 39-bp repeating units encoding the tridecapeptide (Asp-Asp-Ala-Thr-Ser-Glu-Ala-Ala-Thr-Gly-Pro-Ser-Gly) as well as highly conserved sequences encoding pre-, pro-, and telo-peptide regions. In this study we isolated cDNA clones for yamame (cherry salmon, river resident form; Oncorhynchus masou ishikawai) apoPSGP using a genomic DNA fragment for rainbow trout apoPSGP as a probe. The nucleotide sequence analyses revealed that the structures of mRNAs for yamame apoPSGP including the noncoding regions are essentially identical to those for rainbow trout, showing 90% sequence identity. Within the repeating region, 4 bp out of the 39 were replaced, producing a different tridecapeptide, Asp-Asp-Ala-Thr-Ser-Glu-Ala-Ala-Thr-Gly-Pro-Ser-Ser. This tridecapeptide is unique to yamame and common among all cDNAs obtained from yamame. Genomic Southern blot analysis showed that the yamame apoPSGP genes constituted a multiple gene family with a similar gene organization to that of rainbow trout. Oligodeoxynucleotide probes (18 bases) synthesized based on specific sequences for the yamame repeating unit hybridized only to the yamame DNA and not to the rainbow trout DNA, and vice versa.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cloning,pharmacology, and distribution of the neuropeptide Y-receptor Yb in rainbow trout

This work describes the isolation and pharmacological characterization of a neuropeptide Y (NPY) receptor from rainbow trout (Oncorhynchus mykiss). The receptor exhibits approximately 45% amino acid sequence identity to mammalian Y1-subfamily receptors, Y1, Y4 and y6, a similar degree of identity as these subtypes display to one another. Because it displays highest sequence identity to zebrafish Yb (75%), we named it the trout Yb receptor. The receptor exhibits high binding affinity for zebrafish and human NPY and peptide YY (PYY) but not truncated forms of the peptides. Human pancreatic polypeptide (PP) also binds with high affinity. Y1 selective antagonists exhibit poor binding as is the case for Y2 and Y5 selective ligands. This binding profile supports membership in the Y1 subfamily. Sequence data also support this relationship suggesting that Yb is a fourth and separate member of the Y1 subfamily. NPY has a number of important physiological functions such as regulating food intake and reproduction. The expression of the receptor in the hypothalamus and telencephalon suggests a possible role in these processes. This and other receptors from this species have potential for improving aquaculture.     

黄颡鱼PGRP-L基因克隆及其对爱德华氏菌的反应

为了研究肽聚糖识别蛋白家族(Peptidoglycan recognition proteins, PGRPs)在黄颡鱼(Pelteobagrusfulvidraco)先天免疫应答中发挥的作用, 根据NCBI中斑马鱼(Danio rerio) 和虹鳟(Oncorhynchus mykiss) PGRP-L的基因信息, 采用简并引物和RACE方法从黄颡鱼肝脏中克隆得到了一个长型PGRP (PfPGRP-L)基因. PfPGRP-L基因的全长cDNA序列大小为1617 bp, 其中5'和3'非翻译区的长度分别为135和72 bp, 开放阅读框为1410 bp, 编码469个氨基酸. 同源性和系统进化分析表明, 黄颡鱼PGRP-L与虹鳟的同源性为60%, 与脊椎动物的PGLYRP2 或PGRP-L聚在一起. 半定量RT-PCR分析发现PfPGRP-L基因在黄颡鱼鳃、胸腺、肝脏、脾脏、肠道、肾脏、头肾、心脏、血液和肌肉组织中均有分布, 但在肠道和脾脏中的表达量较为丰富, 而在肌肉和血液中表达则很少. 用爱德华氏菌刺激后, PfPGRP-L在肝脏、脾脏、肠道及头肾中的表达明显上调. 结果表明, PfPGRP-L在黄颡鱼抵抗病原菌中具有重要作用.     

Purification and characterization of hepatic steroid hydroxylases from untreated rainbow trout

Purification of cytochrome P450 from liver microsomes of untreated juvenile male rainbow trout yielded five fractions designated LMC1 to LMC5. All fractions, except LMC4 and LMC5, appeared homogeneous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and showed minimum molecular weights of 50,000 (LMC1), 54,000 (LMC2), 56,000 (LMC3), 58,000 (LMC4), and 59,000 (LMC5). Specific contents ranged from 2.8 (LMC3) to 14.9 (LMC5) nmol heme/mg protein. The catalytic activity of LMC1, LMC2, and LMC5 toward various substrates was examined. LMC2 exhibited the highest estradiol 2-hydroxylase activity and progesterone 16 alpha-hydroxylase activity. LMC2 also was most active in the metabolic activation of aflatoxin B1 (AFB1). In contrast, LMC5 was most active in catalyzing the 6 beta- and 16 beta-hydroxylation of testosterone and the 6 beta-hydroxylation of progesterone. LMC1 showed the highest lauric acid hydroxylase activity. The three isozymes tested had low activity (for LMC2 and LMC5) or no activity (for LMC1) toward benzphetamine or benzo[a]pyrene. Polyclonal antibodies to all five isozymes were raised in rabbits and the antibodies were used to examine the contribution of the P450s to microsomal enzyme activities. The results of microsomal enzyme inhibition studies with polyclonal antibodies showed that anti-LMC2 IgG significantly inhibited the oxidative metabolism of testosterone, lauric acid, AFB1, and benzphetamine. Anti-LMC5 IgG inhibited the oxidation of progesterone, estradiol, benzo[a]pyrene, and benzphetamine. Anti-LMC1 IgG slightly inhibited the microsomal hydroxylation of lauric acid. Anti-LMC3 and anti-LMC4 IgG did not inhibit any of the measured microsomal enzyme activities. These findings suggest that individual constitutive isozymes of trout cytochrome P450 have well-defined contributions to the microsomal metabolism of steroids, fatty acids, and xenobiotics.     

Inhibition of in vitro aflatoxin B1-DNA binding in rainbow trout by CYP1A inhibitors: α-naphthoflavone, β-naphthoflavone and trout CYP1A1 peptide antibody

Rainbow trout cytochrome P450 (CYP)1A detoxifies aflatoxin B₁ (AFB₁) to aflatoxin M₁ (AFM₁), whereas CYP2K1 activates AFB₁ to AFB₁-8,9-epoxide. We report that α-naphthoflavone (ANF) and β-naphthoflavone (BNF) both strongly inhibit CYP1A-mediated ethoxyresorufin O-deethylase (EROD) activity (K_i = 9.1 ± 0.8 and 7.6 ± 1.1 nM, respectively). These inhibitors (selective for mammalian CYP1A at low concentrations), as well as rabbit polyclonal antibody to a trout CYP1A1 peptide (residues 277–294), also strongly inhibited trout microsome-catalyzed AFB₁-DNA binding and lauric acid (ω-1) hydroxylation in vitro, reactions previously established to be CYP2K1-dependent. ANF at 0.5, 5, 50 and 500 μM inhibited liver microsome-catalyzed AFB₁-DNA binding by 22, 58, 84 and 91%, respectively, whereas BNF at the same concentrations inhibited 22, 74, 78 and 81%, respectively. The CYP1A1 peptide and CYP2K1 polyclonal antibodies (10 mg IgG/mg microsomal protein) inhibited AFB₁-DNA binding by 84 and 66%, respectively, compared with pre-immune IgG. Lauric acid (ω-1) hydroxylation was inhibited 61% by 5 μM ANF, 69% by 5 μM BNF and 100% by either antibody at 12 mg IgG/mg microsomal protein. These results demonstrate that mammalian CYP1A inhibitors also inhibit trout microsomal AFB₁-DNA binding and lauric acid (ω-1) hydroxylation, catalyzed primarily by CYP2K1. In the absence of evidence that trout CYP1A can catalyze AFB₁-DNA binding, the results suggest configuration similarities at, or near, the active sites for these two fish enzymes that result in antibody crossreaction and loss of the inhibitor specificity observed with mammalian CYP1A.     

Inhibition by green tea catechins of metabolic activation of procarcinogens by human cytochrome P450

Catechins, major polyphenol constituents of green tea, are potent chemopreventive agents against cancers caused by chemical carcinogens in rodents. The effects of four epicatechin derivatives, epigallocatechin gallate (EGCG), epicatechin gallate (ECG), epigallocatechin (EGC) and epicatechin (EC), on the metabolic activation of benzo[a]pyrene (B[a]P), 2-amino-1-methyl-6-phenylimidazo-[4,5-b]pyridine (PhIP) and aflatoxin B(1) (AFB(1)) by human cytochrome P450 (CYP) were examined. B[a]P, PhIP and AFB(1) were activated by respective human CYP1A1, CYP1A2 and CYP3A4 expressed in the membrane fraction of genetically engineered Salmonella typhimurium (S. typhimurium) TA1538 cells harboring the human CYP and human NADPH-CYP reductase (OR), when the membrane fraction was added to S. typhimurium TA98. Galloylated catechins, ECG and EGCG inhibited the mutagenic activation potently, while EGC and EC showed relatively weak inhibitory effects. Catechins also inhibited the oxidations of typical substrates catalyzed by human CYPs, namely ethoxycoumarin O-deethylation by CYP1A1, ethoxyresorufin O-deethylation by CYP1A2 and midazolam 1'-hydroxylation by CYP3A4. The IC(50) values of catechins for the inhibition of human CYP were roughly the same as those seen in the mutagenic activation. EGCG inhibited other forms of human CYP such as CYP2A6, CYP2C19 and CYP2E1, indicating the non-specific inhibitory effects of EGCG toward human CYPs. Furthermore, EGCG inhibited human NADPH-cytochrome CYP reductase (OR) with a K(i) value of 2.5 microM. These results suggest that the inhibition of the enzyme activity of CYP is accounted for partially by the inhibition of OR.     

Cloning and Sequencing of Cytochrome P450 1A Complementary DNA in Eel (Anguilla japonica)

Cytochrome P450 1A (CYP1A) complementary DNA was isolated from eel (Anguilla japonica) liver treated with 3-methylcholanthrene. The cDNA contained a 5′ untranslated region of 163 bp, an open reading flame of 1560 bp coding for 519 amino acids and a stop codon, and a 3′ untranslated region of 1730 bp. The predicted molecular weight was approximately 58.4 kDa. The deduced amino acid sequence exhibited identities with reported CYP1A sequences of 80% for rainbow trout, 79% for scup, 76% for plaice and butterfly fish, and 74% for toadfish. When compared with mammalian CYP proteins, the eel CYP1A was more similar to CYP1A1 (54%–56%) than to CYP1A2 (49%–52%). Northern and Southern blot analyses showed two distinct bands, suggesting the existence of another 3-methylcholanthrene-inducible CYP1A gene in eel. Received December 19, 1998; accepted February 18, 1999     

Two inducible, functional cyclooxygenase-2 genes are present in the rainbow trout genome

The cyclooxygenases (Cox) catalyze the initial reactions in prostanoid biosynthesis, and produce the common prostanoids precursor, PGH(2). Mammalian species have two Cox isoforms; constitutively expressed cyclooxygenase-1 (Cox-1) and inducible cyclooxygenase-2 (Cox-2). Database searches suggest three Cox genes are present in many fish species. In this study, we cloned and characterized a second Cox-2 cDNA, Cox-2b, from the rainbow trout. Rainbow trout Cox-2b protein contains all the functionally important conserved amino acids for Cox enzyme activity. Moreover, the Cox-2b message contains AU-rich elements (AREs) in the 3' untranslated region (3'UTR) characteristic of inducible Cox-2 mRNAs. We took advantage of the existence of a rainbow trout cell line to demonstrate that expression from both the originally reported Cox-2 (Cox-2a) and Cox-2b genes is inducible. However, differential induction responses to alternative inducers are observed for rainbow trout Cox-2a and Cox-2b. Both Cox-2a and Cox-2b proteins expressed in COS cells are enzymatically active. Thus the rainbow trout has two functional, inducible Cox-2 genes. The zebrafish also contains two Cox-2 genes. However, genome structure analysis suggests diversion of the Cox-2a gene between zebrafish and rainbow trout.     

Cloning, sequencing, and characterization of CYP1A1 cDNA from leaping mullet (Liza Saliens) liver and implications for the potential functions of its conserved amino acids

A 2,037 bp CYP1A1 cDNA (GenBank AF072899) was cloned through screening of a lambdaZipLox cDNA library constructed from the liver of a leaping mullet (Liza saliens) fish captured from Izmir Bay on the Aegean coast of Turkey using rainbow trout CYP1A1 cDNA as a probe. This clone has a 130 bp 5'-flanking region, a 1,563 bp open reading frame (ORF) encoding a 521-amino acid protein (58,972 Da), and a 344 bp 3'-untranslated region without a poly (A) tail. Alignment of the deduced amino acids of CYP1A1 cDNAs showed 58% and 69-96% identities with human and 12 other fish species, respectively. Southern blot analysis suggested that this CYP1A1 cDNA was from a single-copy gene. Based on the comparison with CYP1A1 genes reported for fish and mammals, the leaping mullet CYP1A1 gene is probably split into 7 exons. The intron insertion sites were predicted. Alignment of the CYP1A1 cDNA encoded amino acids from 13 fish and 7 mammalian species disclosed differences in highly conserved amino acids between aquatic and land vertebrates. The possible associated secondary structure; conserved motifs and substrate-binding sites were discussed. The phylogenetic relationships of CYP1A1s among 13 fish species were analyzed by a distance method.