Identification of amine receptors from a swallowtail butterfly, Papilio xuthus L.: cloning and mRNA localization in foreleg chemosensory organ for recognition of host plants

The swallowtail butterfly, Papilio xuthus L., feeds exclusively on members of the plant family, Rutaceae. Female butterflies lay eggs in response to specific chemicals contained in their host plants. They perceive a variety of polar compounds as oviposition stimulants through the tarsal chemosensilla of the foreleg by drumming upon the leaf surface. Some biogenic amine analogs have been characterized as oviposition stimulants. We have cloned three amine receptors, serotonin, tyramine, and dopamine, from cDNA derived from foreleg tarsus of P. xuthus, and determined structures of both cDNA and genomic genes. The phenylethylamine (tyramine and dopamine) receptors were expressed preferentially in brain and chemosensory organs. Moreover, we observed the localized expression of dopamine receptors at the base of tarsal chemosensilla by in situ hybridization. These results suggest that amine receptors in tarsal chemosensilla have a functional role in chemoreception for host plant recognition.     

Identification and genomic structure of chemosensory proteins (CSP) and odorant binding proteins (OBP) genes expressed in foreleg tarsi of the swallowtail butterfly Papilio xuthus

Chemoreception is a key feature for selection of host plants by phytophagous insects. Female swallowtail butterflies recognize their host plants using chemosensilla present on foreleg tarsi. We constructed a cDNA library of female tarsi and a genome library of Papilio xuthus. We identified 11 chemosensory protein (CSP) genes and three odorant binding proteins (OBP) genes from the cDNA library and eight additional CSP genes from the genome library using the ESTs as probes. A sequence similarity tree of insect CSPs showed that lepidopteran CSPs constructed big branches of the order. Small numbers of CSPs have been identified from the whole genomes of several insect orders which belong to branches separated from those of Lepidoptera. The CSP gene family of Lepidoptera may have diverged in at least two steps, the first on a small scale and the second on a large scale before and after the diversification of insect orders, respectively. Seventeen of 19 CSP genes of P. xuthus clustered in a specific region of the genome, suggesting that they were diversified by gene duplication from a common ancestral gene.     

灰飞虱唾液腺三大解毒酶家族的转录组分析

灰飞虱Laodelphax striatellus （Fallén）是危害多种禾本科经济作物的重要刺吸式害虫。唾液腺对刺吸式口器昆虫取食植物尤其重要, 其分泌的唾液可以帮助刺吸式口器昆虫刺穿植物、 消化食物、 解毒植物的次生物质。细胞色素P450单加氧酶（cytochrome P450 monooxygenase, P450）、 谷胱甘肽S 转移酶（glutathione S transferase, GST）和羧酸酯酶（carboxylesterase, CarE）是昆虫主要的解毒酶系。为了分析解毒酶基因在灰飞虱唾液腺中的表达谱, 本研究对灰飞虱成虫唾液腺进行转录组测序、 重头组装和注释, 并与豌豆蚜Acyrthosiphon pisum和西方蜜蜂Apis mellifera的同源蛋白进行系统发育分析。发现有9个谷胱甘肽S 转移酶（glutathione S transferase, GST）基因、 22个羧酸酯酶（carboxylesterase, CarE）基因和39个细胞色素P450单加氧酶（cytochrome P450 monooxygenase, P450）基因在灰飞虱唾液腺中表达。通过对同源蛋白进行系统发育分析, 发现灰飞虱唾液腺大部分的CarE是参与消化/解毒和激素/信息素的加工, 而参与神经/发育的CarE很少; 灰飞虱唾液腺表达的P450基因远远少于豌豆蚜和西方蜜蜂基因组的P450基因数, 且只有CYP6和CYP4家族的成员; GST家族在3种昆虫的保守性最高。研究结果为灰飞虱对寄主植物和杀虫剂的适应性研究奠定了基础。     

Induction of cytochrome P450 1A1 and conjugating enzymes in rainbow trout (Oncorhynchus mykiss) liver: A time course study

1. The effects of i.p. injections of isosafrole (ISF) or β-naphthoflavone (β-NF) on the cytochrome P450 (CYP) 1A1 system and conjugating enzymes were investigated in livers from juvenile rainbow trout in a time course study employing catalytic, immunochemical and cDNA probes.2. β-NF treatment resulted in a rapid rise in CYP1A1 mRNA followed by accumulation of P450 1A1 protein and P450 1A1 mediated enzyme activity measured as ethoxyresorufin-O-deethylase (EROD) activity.3. ISF treatment resulted in a comparatively weak induction of CYP1A1 mRNA and P450 1A1 protein levels whilst EROD activity was markedly induced; thus when expressed on the basis of immunoquantified P450 1A1 protein, the specific EROD activity was signficantly higher in ISF than β-NF treated fish.4. In vitro inhibition studies revealed that ISF inhibited EROD activity to a far lesser extent than β-NF.5. Conjugation enzymes represented by phenol UDP-glucuronosyltransferase and glutathione S-transferase (GST) activities, were induced by β-NF, whereas ISF treatment had no effect on these enzyme activities.6. Immunoblotting using antibodies raised against rat GST7-7 showed that a Pi class trout GST enzyme was induced by β-NF treatment.     

Intron-exon organization and phylogeny in a large superfamily, the paralogous cytochrome P450 genes of Arabidopsis thaliana

The cytochrome P450 gene superfamily is represented by 80 genes in animal genomes and perhaps more than 300 genes in plant genomes. We analyzed about half of all Arabidopsis P450 genes, a very large dataset of truly paralogous genes. Sequence alignments were used to draw phylogenetic trees, and this information was compared with the intron-exon organization of each P450 gene. We found 60 unique intron positions, of which 37 were phase 0 introns. Our results confirm the polyphyletic origin of plant P450 genes. One group of these genes, the A-type P450s, are plant specific and characterized by a simple organization, with one highly conserved intron. Closely related A-type P450 genes are often clustered in the genome with as many as a dozen genes (e.g., of the CYP71 subfamily) on a short stretch of chromosome. The other P450 genes (non-A-type) form several distinct clades and are characterized by numerous introns. One such clade contains the two CYP51 genes, which are thought to encode obtusifoliol 14a demethylase. The two CYP51 genes have a single intron that is not shared with CYP51 genes from vertebrates or fungi, or with any other Arabidopsis P450 gene. Only a few of the Arabidopsis P450 genes are intronless (e.g., the CYP710A and CYP96A subfamilies). There was a relatively good correlation between intron conservation and phylogenetic relationships between members of the P450 subfamilies. Gene organization appears to be a useful tool in establishing the evolutionary relatedness of P450 genes, which may help in predictions of P450 function.     

A tissue specific cytochrome P450 required for the structure and function of Drosophila sensory organs

Cytochrome P450s have generally been acknowledged as broadly tuned detoxifying enzymes. However, emerging evidence argues P450s have an integral role in cell signaling and developmental processes, via their metabolism of retinoic acid, arachidonic acid, steroids, and other cellular ligands. To study the morphogenesis of Drosophila sensory organs, we examined mutants with impaired mechanosensation and discovered one, nompH, encodes the cytochrome P450 CYP303a1. We now report the characterization of nompH, a mutant defective in the function of peripheral chemo- and mechanoreceptor cells, and demonstrate CYP303a1 is essential for the development and structure of external sensory organs which mediate the reception of vital mechanosensory and chemosensory stimuli. Notably this P450 is expressed only in sensory bristles, localizing in the apical region of the socket cell. The wide diversity of the P450 family and the growing number of P450s with developmental phenotypes suggests the exquisite tissue and subcellular specificity of CYP303a1 illustrates an important aspect of P450 function; namely, a strategy to process critical developmental signals in a tissue- and cell-specific manner.     

Identification of beta-amyrin and sophoradiol 24-hydroxylase by expressed sequence tag mining and functional expression assay

Triterpenes exhibit a wide range of structural diversity produced by a sequence of biosynthetic reactions. Cyclization of oxidosqualene is the initial origin of structural diversity of skeletons in their biosynthesis, and subsequent regio- and stereospecific hydroxylation of the triterpene skeleton produces further structural diversity. The enzymes responsible for this hydroxylation were thought to be cytochrome P450-dependent monooxygenase, although their cloning has not been reported. To mine these hydroxylases from cytochrome P450 genes, five genes (CYP71D8, CYP82A2, CYP82A3, CYP82A4 and CYP93E1) reported to be elicitor-inducible genes in Glycine max expressed sequence tags (EST), were amplified by PCR, and screened for their ability to hydroxylate triterpenes (beta-amyrin or sophoradiol) by heterologous expression in the yeast Saccharomyces cerevisiae. Among them, CYP93E1 transformant showed hydroxylating activity on both substrates. The products were identified as olean-12-ene-3beta,24-diol and soyasapogenol B, respectively, by GC-MS. Co-expression of CYP93E1 and beta-amyrin synthase in S. cerevisiae yielded olean-12-ene-3beta,24-diol. This is the first identification of triterpene hydroxylase cDNA from any plant species. Successful identification of a beta-amyrin and sophoradiol 24-hydroxylase from the inducible family of cytochrome P450 genes suggests that other triterpene hydroxylases belong to this family. In addition, substrate specificity with the obtained P450 hydroxylase indicates the two possible biosynthetic routes from triterpene-monool to triterpene-triol.     

A comparison of Drosophila melanogaster detoxification gene induction responses for six insecticides, caffeine and phenobarbital

Modifications of metabolic pathways are important in insecticide resistance evolution. Mutations leading to changes in expression levels or substrate specificities of cytochrome P450 (P450), glutathione-S-transferase (GST) and esterase genes have been linked to many cases of resistance with the responsible enzyme shown to utilize the insecticide as a substrate. Many studies show that the substrates of enzymes are capable of inducing the expression of those enzymes. We investigated if this was the case for insecticides and the enzymes responsible for their metabolism. The induction responses for P450s, GSTs and esterases to six different insecticides were investigated using a custom designed microarray in Drosophila melanogaster. Even though these gene families can all contribute to insecticide resistance, their induction responses when exposed to insecticides are minimal. The insecticides spinosad, diazinon, nitenpyram, lufenuron and dicyclanil did not induce any P450, GST or esterase gene expression after a short exposure to high lethal concentrations of insecticide. DDT elicited the low-level induction of one GST and one P450. These results are in contrast to induction responses we observed for the natural plant compound caffeine and the barbituate drug phenobarbital, both of which highly induced a number of P450 and GST genes under the same short exposure regime. Our results indicate that, under the insecticide exposure conditions we used, constitutive over-expression of metabolic genes play more of a role in insect survival than induction of members of these gene families.     

Characterization of biotransformation enzyme activities in primary rat proximal tubular cells

The proximal tubule is a frequent target for nephrotoxic compounds due to it's ability to transport and accumulate xenobiotics and their metabolites, as well as by the presence of an organ-selective set of biotransformation enzymes. The aim of the present study was to characterize the activities of different biotransformation enzymes during primary culturing of rat proximal tubular cells (PT cells). Specific marker substrates for determining cytochrome P450 (CYP450) activity of primary cultured PT cells include 7-ethoxyresorufin (CYP1A1), caffeine (CYP1A), testosterone (CY2B/C, CYP3A), tolbutamide (CYP2C) and dextromethorphan (CYP2D1). Activities of the CYP450 isoenzymes decreased considerably during culture with the greatest loss in activity within 24 h of culture. In addition, expression of CYP450 apoprotein, including CYP1A, CYP2C, CYP2D, CYP2E and CYP4A, was detected in microsomes from freshly isolated PT cells by immunoblotting using specific antibodies. CYP2B and CYP3A apoprotein could not be detected. Activity of the phase II biotransformation enzymes GST, GGT, beta-lyase and UGT was determined with 1-chloro-2,4-dinitrobenzene, L-glutamic acid gamma-(7-amido-4-methyl-coumarin), S-(1,1,2,2-tetrafluoroethyl)-L-cysteine and 1-naphthol, respectively, as marker substrates. Activity of the phase II enzymes remained more stable and, in contrast to CYP450 activity, significant activity was still expressed after 1 week of PT cell culture. Thus, despite the obvious advantages of PT cells as an in-vitro model for studies of biotransformation mediated toxicity, the strong time dependency of especially phase I and, to a lesser extent, phase II biotransformation activities confers limitations to their application.     

Expression of active human P450 3A4 on the cell surface of Escherichia coli by Autodisplay

The cytochrome P450 enzyme system comprises a large group of enzymes catalyzing a broad diversity of reactions and an extensive substrate specificity, which makes them the most versatile known catalysts. CYP3A4 is one of the important human P450 enzymes and involved in the oxidation of a large range of substrates including toxins and pharmaceuticals. Bottlenecks in studying this enzyme include the difficulty in expressing it in a bacterial host, its need for membrane surroundings and the limited substrate accessibility of enzymes expressed within the cell. To circumvent these difficulties, human CYP3A4 was expressed on the outer membrane of Escherichia coli using Autodisplay. Transport of CYP3A4 to the cell surface was monitored by SDS-PAGE and Western blot analysis of outer membrane proteins. Localization on the cell envelope was determined by flow cytometry after immunolabeling, a whole cell ELISA and a protease accessibility assay. A HPLC assay confirmed the catalytic activity of displayed CYP3A4, using testosterone as a substrate. This activity required the external addition of electron supplying enzymes, however surprisingly, we found that the external addition of a heme group was not necessary. Our results indicate that human CYP3A4 can be recombinantly expressed by surface display in a gram-negative bacterium.     

Host suitability and diet mixing influence activities of detoxification enzymes in adult Japanese beetles

Induction of cytochrome P450, glutathione S transferase (GST), and carboxylesterase (CoE) activity was measured in guts of the scarab Popillia japonica Newman, after consumption of single or mixed plant diets of previously ranked preferred (rose, Virginia creeper, crape myrtle and sassafras) or non-preferred hosts (boxelder, riverbirch and red oak). The goal of this study was to quantify activities of P450, GST and CoE enzymes in the midgut of adult P. japonica using multiple substrates in response to host plant suitability (preferred host vs non-preferred hosts), and single and mixed diets. Non-preferred hosts were only sparingly fed upon, and as a group induced higher activities of P450, GST and CoE than did preferred hosts. However, enzyme activities for some individual plant species were similar across categories of host suitability. Similarly, beetles tended to have greater enzyme activities after feeding on a mixture of plants compared to a single plant type, but mixing per se does not seem as important as the species represented in the mix. Induction of detoxification enzymes on non-preferred hosts, or when switching between hosts, may explain, in part, the perceived feeding preferences of this polyphagous insect. The potential consequences of induced enzyme activities on the ecology of adult Japanese beetles are discussed.     

梨小食心虫幼虫中肠中高表达消化酶和解毒酶基因的筛选

【目的】挖掘梨小食心虫Grapholita molesta幼虫中肠中高表达消化酶和解毒酶基因,为今后研究以肠道为靶标的新型农药和转基因作物提供理论依据。【方法】基于梨小食心虫4龄幼虫中肠转录组高通量测序数据的FPKM值,筛选高表达基因,进行GO功能注释和KEGG通路富集分析,并使用BLAST软件进行比对筛选高表达的消化酶和解毒酶基因,利用MEGA对这些高表达的消化酶和解毒酶及其他鳞翅目昆虫的同源蛋白进行系统发育分析。利用qRT-PCR技术对梨小食心虫幼虫不同龄期中肠中的高表达代表性消化酶和解毒酶基因表达量进行定量分析和验证。【结果】在GO数据库中注释了103 677个在梨小食心虫4龄幼虫中肠中高表达基因,包括细胞组分、分子功能和生物学进程三大类功能共41个分支。KEGG通路分析表明,10 846个高表达基因参与了5类生化代谢通路。筛选到具有完整开放阅读框的消化酶基因17个［5个胰蛋白酶(trypsin, TRY)基因、3个氨肽酶(aminopeptidase, APN)基因和9个羧肽酶(carboxypeptidase, CP)基因］和解毒酶基因32个［11个谷胱甘肽S-转移酶(glutathione S-transferase, GST)基因、13个细胞色素P450(cytochrome P450, CYP450)基因和8个羧酸脂酶(carboxylesterase, CarE)基因］。系统发育分析结果表明,梨小食心虫的消化酶同源聚类分支较为分散,GSTs和CYP450s分支聚类较为集中,但都至少与1个鳞翅目昆虫同源蛋白聚在一支。qRT-PCR验证结果表明,消化酶和解毒酶基因在不同龄期梨小食心虫幼虫中肠中的表达量差异显著,表达量均在4龄幼虫期最高。【结论】本研究成功筛选和验证部分梨小食心虫幼虫中肠中高表达的消化酶和解毒酶基因,明确其与鳞翅目其他昆虫同源蛋白的进化关系。研究结果为鳞翅目其他近缘昆虫的转录组分析和以肠道为靶标的害虫防治提供了参考。     

Indole alkaloid biosynthesis in Catharanthus roseus: new enzyme activities and identification of cytochrome P450 CYP72A1 as secologanin synthase

The molecular characterization of CYP72A1 from Catharanthus roseus (Madagascar periwinkle) was described nearly a decade ago, but the enzyme function remained unknown. We now show by in situ hybridization and immunohistochemistry that the expression in immature leaves is epidermis-specific. It thus follows the pattern previously established for early enzymes in the pathway to indole alkaloids, suggesting that CYP72A1 may be involved in their biosynthesis. The early reactions in that pathway, i.e. from geraniol to strictosidine, contain several candidates for P450 activities. We investigated in this work two reactions, the conversion of 7-deoxyloganin to loganin (deoxyloganin 7-hydroxylase, DL7H) and the oxidative ring cleavage converting loganin into secologanin (secologanin synthase, SLS). The action of DL7H has not been demonstrated in vitro previously, and SLS has only recently been identified as P450 activity in one other plant. We show for the first time that both enzyme activities are present in microsomes from C. roseus cell cultures. We then tested whether CYP72A1 expressed in E. coli as a translational fusion with the C. roseus P450 reductase (P450Red) has one or both of these activities. The results show that CYP72A1 converts loganin into secologanin.     

Garlic Oil Attenuated Nitrosodiethylamine-Induced Hepatocarcinogenesis by Modulating the Metabolic Activation and Detoxification Enzymes

Nitrosodiethylamine (NDEA) is a potent carcinogen widely existing in the environment. Our previous study has demonstrated that garlic oil (GO) could prevent NDEA-induced hepatocarcinogenesis in rats, but the underlying mechanisms are not fully understood. It has been well documented that the metabolic activation may play important roles in NDEA-induced hepatocarcinogenesis. Therefore, we designed the current study to explore the potential mechanisms by investigating the changes of hepatic phase Ⅰ enzymes (including cytochrome P450 enzyme (CYP) 2E1, CYP1A2 and CYP1A1) and phase Ⅱ enzymes (including glutathione S transferases (GSTs) and UDP- Glucuronosyltransferases (UGTs)) by using enzymatic methods, real-time PCR, and western blotting analysis. We found that NDEA treatment resulted in significant decreases of the activities of CYP2E1, CYP1A2, GST alpha, GST mu, UGTs and increases of the activities of CYP1A1 and GST pi. Furthermore, the mRNA and protein levels of CYP2E1, CYP1A2, GST alpha, GST mu and UGT1A6 in the liver of NDEA-treated rats were significantly decreased compared with those of the control group rats, while the mRNA and protein levels of CYP1A1 and GST pi were dramatically increased. Interestingly, all these adverse effects induced by NDEA were simultaneously and significantly suppressed by GO co-treatment. These data suggest that the protective effects of GO against NDEA-induced hepatocarcinogenesis might be, at least partially, attributed to the modulation of phase I and phase II enzymes.