Cytochrome P450 Gene Superfamily Research Articles

Acceleration of infectious disease drug discovery and development using a humanized model of drug metabolism

A key step in drug discovery, common to many disease areas, is preclinical demonstration of efficacy in a mouse model of disease. However, this demonstration and its translation to the clinic can be impeded by mouse-specific pathways of drug metabolism. Here, we show that a mouse line extensively humanized for the cytochrome P450 gene superfamily ("8HUM") can circumvent these problems. The pharmacokinetics, metabolite profiles, and magnitude of drug-drug interactions of a test set of approved medicines were in much closer alignment with clinical observations than in wild-type mice. Infection with Mycobacterium tuberculosis, Leishmania donovani, and Trypanosoma cruzi was well tolerated in 8HUM, permitting efficacy assessment. During such assessments, mouse-specific metabolic liabilities were bypassed while the impact of clinically relevant active metabolites and DDI on efficacy were well captured. Removal of species differences in metabolism by replacement of wild-type mice with 8HUM therefore reduces compound attrition while improving clinical translation, accelerating drug discovery.

Genome wide and comprehensive analysis of the cytochrome P450 (CYPs) gene family in Pyrus bretschneideri: Expression patterns during Sporidiobolus pararoseus Y16 enhanced with ascorbic acid (VC) treatment

Cytochrome P450s (CYPs) constitute the largest group of enzymes in plants and are involved in a variety of processes related to growth and protection. However, the CYP gene superfamily in pear (Pyrus bretschneideri) and their characteristics is unclear. Through a comprehensive genome-wide analysis, this article identified a total of 74 CYP genes in the P. bretschneideri genome, which were categorized into fourteen families. Motif analysis reveals that most of the ten motifs predicted were with the p450 conserved domain. The majority of the CYP genes have exon arrangements. Furthermore, promoter analysis unveiled a multitude of cis-acting elements associated with diverse responsiveness including hormones, light responsive, anoxic specific inducibility and anaerobic induction. Analysis of the transcriptome data reveal that about 80% of the pear CYPs genes were upregulated and they were positively correlated with the antioxidant's parameters such as total flavonoids and total phenol content as well as ABTS and DPPH radicals. RT-qPCR analysis confirmed that the CYP genes could be regulated in pear. Collectively, our results reveal comprehensive insights into the CYP superfamily in pear and make a valuable contribution to the ongoing process of functional validation.

Phylogenomic Analysis of Cytochrome P450 Gene Superfamily and Their Association with Flavonoids Biosynthesis in Peanut (Arachis hypogaea L.).

Cytochrome P450s (CYPs) constitute extensive enzyme superfamilies in the plants, playing pivotal roles in a multitude of biosynthetic and detoxification pathways essential for growth and development, such as the flavonoid biosynthesis pathway. However, CYPs have not yet been systematically studied in the cultivated peanuts (Arachis hypogaea L.), a globally significant cash crop. This study addresses this knowledge deficit through a comprehensive genome-wide analysis, leading to the identification of 589 AhCYP genes in peanuts. Through phylogenetic analysis, all AhCYPs were systematically classified into 9 clans, 43 gene families. The variability in the number of gene family members suggests specialization in biological functions. Intriguingly, both tandem duplication and fragment duplication events have emerged as pivotal drivers in the evolutionary expansion of the AhCYP superfamily. Ka/Ks analysis underscored the substantial influence of strong purifying selection on the evolution of AhCYPs. Furthermore, we selected 21 genes encoding 8 enzymes associated with the flavonoid pathway. The results of quantitative real-time PCR (qRT-PCR) experiments unveiled stage-specific expression patterns during the development of peanut testa, with discernible variations between pink and red testa. Importantly, we identified a direct correlation between gene expression levels and the accumulation of metabolites. These findings offer valuable insights into elucidating the comprehensive functions of AhCYPs and the underlying mechanisms governing the divergent accumulation of flavonoids in testa of different colors.

Dual roles of CYP302A1 in regulating ovarian maturation and molting in Macrobrachium nipponense

CYP302A1 is a member of the Halloween genes in the cytochrome P450 supergene family, which play an important regulatory role in the synthesis of 20-hydroxyecdysone (20E) in crustaceans and insects. In this study, we found that the Mn-CYP302A1sequence included typical CYP450 conserved domains. Phylogenic showed that it is closely related to crustaceans and insects. q-PCR analysis indicated that Mn-CYP302A1 was highly expressed in the ovaries and peaked before ovarian maturation. Mn-CYP302A1 expression was higher at the post-larval stage of day 15 than at other stages of embryogenesis. In situ hybridization indicated that Mn-CYP302A1 was mainly distributed in the nucleus, yolk granules, cell membrane and cytoplasm To further establish the function of CYP302A1, a 21-day RNA interference experiment was conducted. On day 16, the Gonad Somatic Index of the control group and the experimental group showed significant differences, with GSI of 11.72% in the control group and 3.21% in the experimental group. The cumulative proportion of the second entry into stage O-Ⅲ was 100% in the control group, while it was 41.67% in the experimental group on day 21. The ecdysone content was 8.91nmol/L in the control group and 6.11nmol/L in the experimental group on day 9. A significant difference in the molting proportion between the control group and the experimental group was also observed (49% in the control group and 34% in the experimental group) on day 16. Statistical results showed that the average molting cycle of the control group was 14.5 days, while that of the experimental group was 16.5 days. However, the morphological structure of ovarian tissue did not abnormal change. Therefore, the results of this study suggest that Mn-CYP302A1 can promote ovarian maturation and molting in female M. nipponense.

Transcriptome Analysis of CYP450 Family Members in Fritillaria cirrhosa D. Don and Profiling of Key CYP450s Related to Isosteroidal Alkaloid Biosynthesis.

Fritillaria cirrhosa D. Don (known as Chuan-Bei-Mu in Chinese) can synthesize isosteroidal alkaloids (ISA) with excellent medicinal value, and its bulb has become an indispensable ingredient in many patented drugs. Members of the cytochrome P450 (CYP450) gene superfamily have been shown to play essential roles in regulating steroidal alkaloids biosynthesis. However, little information is available on the P450s in F. cirrhosa. Here, we performed full-length transcriptome analysis and discovered 48 CYP450 genes belonging to 10 clans, 25 families, and 46 subfamilies. By combining phylogenetic trees, gene expression, and key F. cirrhosa ISA content analysis, we presumably identify seven FcCYP candidate genes, which may be hydroxylases active at the C-22, C-23, or C-26 positions in the late stages of ISA biosynthesis. The transcript expression levels of seven FcCYP candidate genes were positively correlated with the accumulation of three major alkaloids in bulbs of different ages. These data suggest that the candidate genes are most likely to be associated with ISA biosynthesis. Finally, the subcellular localization prediction of FcCYPs and transient expression analysis within Nicotiana benthamiana showed that the FcCYPs were mainly localized in the chloroplast. This study presents a systematic analysis of the CYP450 gene family in F. cirrhosa and provides a foundation for further functional characterization of the CYPs involved in ISA biosynthesis.

Characterization, expression and functional analysis of CYP306a1 in the oriental river prawn, Macrobrachium nipponense

The CYP306a1 gene is a member of the Halloween genes in the cytochrome P450 supergene family, which play an important regulatory role in the synthesis of 20-hydroxyecdysone (20E) in arthropods. In this study, we cloned the full-length DNA sequence of the CYP306a1 (Mn-CYP306a1) gene in the oriental river prawn Macrobrachium nipponense. The sequence encoded 600 amino acids, including typical CYP450 conserved domains. We detected the expression of Mn-CYP306a1 in different tissues of male and female M. nipponense by qRT-PCR. The results showed that the gene was highly expressed in its respective gonadal tissues (ovary and testis). Mn-CYP306a1 was expressed in embryonic and metamorphic developmental stages, and the expression trend appeared in a wave pattern, firstly rising gradually, with peaks appearing in the L1 gastrulation stage and later at the PL10 and PL25 metamorphic stages. RNA interference (RNAi) experiments have found that Mn-CYP306a1 dsRNA can effectively reduce the content of 20E and the molting frequency of M. nipponense. The molting frequency of the experimental group was almost half that of the control group. Moreover, the body weight of M. nipponense was also significantly negatively affected in the late stage of the RNAi experiment. This study showed that Mn-CYP306a1 played an important role in promoting 20E synthesis in M. nipponense.

Genome-wide identification and transcriptomic data exploring of the cytochrome P450 family in Chinese cabbage (Brassica rapa L. ssp. pekinensis)

ABSTRACT Cytochrome P450 genes are involved in the catalysis of various reactions, including growth, development and secondary metabolite biosynthetic pathways. However, little is known about the characteristics and functions of the P450 gene family in Chinese cabbage (Brassica rapa L. ssp. pekinensis). In this study, we identified 258 non-redundant members of the cytochrome P450 gene superfamily in Brassica. rapa from Brassica database (http://www.brassicadb.cn). We also did transcriptomic data mining derived from abiotic/biotic stresses and developmental related RNA-seq in Chinese cabbage. These differentially expressed genes (DEGs) were involved in cadmium (Cd) detoxification, salinity tolerance, calcium signaling transduction and homeostasis, pathogen triggered immunity and leafy head formation in Chinese cabbage. These P450 genes are essential candidates for a better understanding of the mechanism of P450 genes, which help Chinese cabbage adapt to environmental changes.

Highly homologous mouse Cyp2a4 and Cyp2a5 genes are differentially expressed in the liver and both express long non-coding antisense RNAs.

The mammalian Cytochrome P450 (Cyp) gene superfamily encodes enzymes involved in numerous metabolic pathways and are frequently expressed in the liver. Despite the remarkably high sequence similarity of Cyp2a4 and Cyp2a5 genes and their surrounding genomic regions, they exhibit differences in expression in the adult mouse liver. For example, Cyp2a4 is highly female-biased whereas Cyp2a5 is only moderately female-biased and Cyp2a4, but not Cyp2a5, is activated in liver cancer. We hypothesized that the limited sequence differences may help us identify the basis for this differential expression. An antisense expressed sequence tag had been uniquely annotated to the Cyp2a4 gene which led us to investigate this transcript as a possible regulator of this gene. We characterized the full-length antisense transcript and also discovered a similar transcript in the Cyp2a5 gene. These transcripts are nuclear long noncoding RNAs that are expressed similarly to their sense mRNA counterparts. This includes the sex-biased and liver tumor differences seen between the Cyp2a4 and Cyp2a5 genes, but we also find that these two genes and their antisense transcripts are expressed within different zones of the liver structure. Interestingly, while the differences in sex-biased expression of the mRNAs are established 1-2months after birth, the antisense transcripts exhibit these expression differences earlier, at 3-4weeks after birth. By analyzing published genomic data, we have identified candidate transcription factor binding sites that could account for differences in Cyp2a4/Cyp2a5 expression. Taken together, these studies characterize the first antisense RNAs within the Cyp supergene family and identify potential transcriptional and post-transcriptional mechanisms governing different Cyp2a4 and Cyp2a5 expression patterns in mouse liver.

Identification and analysis of CYP450 and UGT supergene family members from the transcriptome of Aralia elata (Miq.) seem reveal candidate genes for triterpenoid saponin biosynthesis

BackgroundMembers of the cytochrome P450 (CYP450) and UDP-glycosyltransferase (UGT) gene superfamily have been shown to play essential roles in regulating secondary metabolite biosynthesis. However, the systematic identification of CYP450s and UGTs has not been reported in Aralia elata (Miq.) Seem, a highly valued medicinal plant.ResultsIn the present study, we conducted the RNA-sequencing (RNA-seq) analysis of the leaves, stems, and roots of A. elata, yielding 66,713 total unigenes. Following annotation and KEGG pathway analysis, we were able to identify 64 unigenes related to triterpenoid skeleton biosynthesis, 254 CYP450s and 122 UGTs, respectively. A total of 150 CYP450s and 92 UGTs encoding > 300 amino acid proteins were utilized for phylogenetic and tissue-specific expression analyses. This allowed us to cluster 150 CYP450s into 9 clans and 40 families, and then these CYP450 proteins were further grouped into two primary branches: A-type (53%) and non-A-type (47%). A phylogenetic analysis of 92 UGTs and other plant UGTs led to clustering into 16 groups (A-P). We further assessed the expression patterns of these CYP450 and UGT genes across A. elata tissues, with 23 CYP450 and 16 UGT members being selected for qRT-PCR validation, respectively. From these data, we identified CYP716A295 and CYP716A296 as the candidate genes most likely to be associated with oleanolic acid synthesis, while CYP72A763 and CYP72A776 were identified as being the most likely to play roles in hederagenin biosynthesis. We also selected five unigenes as the best candidates for oleanolic acid 3-O-glucosyltransferase. Finally, we assessed the subcellular localization of three CYP450 proteins within Arabidopsis protoplasts, highlighting the fact that they localize to the endoplasmic reticulum.ConclusionsThis study presents a systematic analysis of the CYP450 and UGT gene family in A. elata and provides a foundation for further functional characterization of these two multigene families.

Comparative functional genomics analysis of cytochrome P450 gene superfamily in wheat and maize

BackgroundThe cytochrome P450s (CYP450s) as the largest enzyme family of plant metabolism participate in various physiological processes, whereas no study has demonstrated interest in comprehensive comparison of the genes in wheat and maize. Genome-wide survey, characterization and comparison of wheat and maize CYP450 gene superfamily are useful for genetic manipulation of the Gramineae crops.ResultsIn total, 1285 and 263 full-length CYP450s were identified in wheat and maize, respectively. According to standard nomenclature, wheat CYP450s (TaCYP450s) were categorized into 45 families, while maize CYP450s (ZmCYP450s) into 43 families. A comprehensive analysis of wheat and maize CYP450s, involved in functional domains, conserved motifs, phylogeny, gene structures, chromosome locations and duplicated events was performed. The result showed that each family/subfamily in both species exhibited characteristic features, suggesting their phylogenetic relationship and the potential divergence in their functions. Functional divergence analysis at the amino acid level of representative clans CYP51, CYP74 and CYP97 in wheat, maize and rice identified some critical amino acid sites that are responsible for functional divergence of a gene family. Expression profiles of Ta-, ZmCYP450s were investigated using RNA-seq data, which contribute to infer the potential functions of the genes during development and stress responses. We found in both species CYP450s had preferential expression in specific tissues, and many tissue-specific genes were identified. Under water-deficit condition, 82 and 39 significantly differentially expressed CYP450s were respectively detected in wheat and maize. These genes may have some roles in protecting plants against drought damage. Thereinto, fourteen CYP450s were selected to validate their expression level through qRT-PCR. To further elucidating molecular mechanisms of CYP450 action, gene co-expression network was constructed. In total, 477 TaCYP450s were distributed in 22 co-expression modules, and some co-expressed genes that likely take part in the same biochemical pathway were identified. For instance, the expression of TaCYP74A98_4D was highly correlated with TaLOX9, TaLOX36, TaLOX39, TaLOX44 and TaOPR8, and all of them may be involved in jasmonate (JA) biosynthesis. TaCYP73A201_3A showed coexpression with TaPAL1.25, TaCCoAOMT1.2, TaCOMT.1, TaCCR1.6 and TaLAC5, which probably act in the wheat stem and/or root lignin synthesis pathway.ConclusionOur study first established systematic information about evolutionary relationship, expression pattern and function characterization of CYP450s in wheat and maize.

T-2 toxin in the diet suppresses growth and induces immunotoxicity in juvenile Chinese mitten crab (Eriocheir sinensis)

The T-2 toxin is a trichothecene mycotoxin and is highly toxic to aquatic animals, but little is known on its toxic effect in crustaceans. In the present study, the crab juveniles were fed with diets containing four levels of T-2 toxin: 0 (control), 0.6 (T1), 2.5 (T2) and 5.0 (T3) mg/kg diet for 56 days to evaluate its impact on the juvenile of Chinese mitten crab (Eriocheir sinensis). The crabs fed the T-2 toxin diets had significantly lower weight gain and specific growth rate than those fed the control diet. Moreover, crab survival in T3 group was obviously lower than that in the control. Oxidative stress occurred in all the treatment groups as indicated by higher activities of total superoxide dismutase, glutathione peroxidase, and total antioxidant capacity than those in the control. The total hemocyte count, respiratory burst, phenoloxidase in the hemolymph, and phenoloxidase, acid phosphatase and alkaline phosphatase in the hepatopancreas of crabs fed T-2 toxin were significantly lower than those in the control. The transcriptional expressions of lipopolysaccharide-induced TNF-alpha factor, relish, and the apoptosis genes in the hepatopancreas were induced by dietary T-2 toxin. The genes related to detoxication including cytochrome P450 gene superfamily and glutathione S transferase were induced in low concentration, then decreased in high concentration. Dietary T-2 toxin damaged the hepatopancreas structure, especially as seen in the detached basal membrane of hepatopancreatic tubules. This study indicates that dietary T-2 toxin can reduce growth performance, deteriorate health status and cause hepatopancreas dysfunction in crabs.

Development of organoid-based drug metabolism model

Cytochrome P450 (CYP) gene superfamily catalyzes oxidative metabolism of a wide variety of drugs, carcinogens, and endogenous biomolecules in the liver and intestinal organs. In vitro assay platforms such as primary hepatocyte and immortalized liver-derived cell lines have been developed to evaluate drug effects. However, several limitations have been suggested regarding discrepancies between in vitro and in vivo assays. In this study, we aimed to investigate drug metabolism and toxicity based on mouse small intestinal and liver organoids derived from resident stem cells. At first, expressions and activities of CYP subfamilies (CYPs) in intestinal and liver organoids were investigated. Organoids treated with three CYPs-inducers dexamethasone (Dex), β-naphthoflavone (BNF), and 1,4-bis-2-(3, 5-dichloropyridyloxy)-benzene (TCPOBOP) were evaluated for CYPs activities. The CYPs-induced intestinal and liver organoids were confirmed to digest more docetaxel, as colon cancer cell-line survived more in CYPs-induced organoid's medium than in non-induced organoid's medium. Then, the activity of docetaxel in a co-culture platform of mouse liver organoids and human pancreatic tumoroids was measured. We obtained significant statistical values on CYPs-induced metabolic activities: cell survival rates of pancreatic tumoroids co-cultured with docetaxel-treated undifferentiated, differentiated, and CYPs-induced differentiated organoids were 66.05 ± 2.14%, 89.20 ± 2.67%, and 101.90 ± 0.94%, respectively. To sum up, gene expression modification and drug metabolism evaluation were able to be done with organoids as done with tissues. In vivo-like in vitro investigation on drug toxicity may potentially be done with organoids as a stepping bridge to the clinical trial.

Overexpression of a Novel Cytochrome P450 Promotes Flavonoid Biosynthesis and Osmotic Stress Tolerance in Transgenic Arabidopsis.

Flavonoids are mainly associated with growth, development, and responses to diverse abiotic stresses in plants. A growing amount of data have demonstrated the biosynthesis of flavonoids through multienzyme complexes of which the membrane-bounded cytochrome P450 supergene family shares a crucial part. However, the explicit regulation mechanism of Cytochrome P450s related to flavonoid biosynthesis largely remains elusive. In the present study, we reported the identification of a stress-tolerant flavonoid biosynthetic CtCYP82G24 gene from Carthamus tinctorius. The transient transformation of CtCYP82G24 determined the subcellular localization to the cytosol. Heterologously expressed CtCYP82G24 was effective to catalyze the substrate-specific conversion, promoting the de novo biosynthesis of flavonoids in vitro. Furthermore, a qRT-PCR assay and the accumulation of metabolites demonstrated that the expression of CtCYP82G24 was effectively induced by Polyethylene glycol stress in transgenic Arabidopsis. In addition, the overexpression of CtCYP82G24 could also trigger expression levels of several other flavonoid biosynthetic genes in transgenic plants. Taken together, our findings suggest that CtCYP82G24 overexpression plays a decisive regulatory role in PEG-induced osmotic stress tolerance and alleviates flavonoid accumulation in transgenic Arabidopsis.

Transcriptome analysis identifies strong candidate genes for ginsenoside biosynthesis and reveals its underlying molecular mechanism in Panax ginseng C.A. Meyer

Ginseng, Panax ginseng C.A. Meyer, is one of the most important medicinal herbs for human health and medicine in which ginsenosides are known to play critical roles. The genes from the cytochrome P450 (CYP) gene superfamily have been shown to play important roles in ginsenoside biosynthesis. Here we report genome-wide identification of the candidate PgCYP genes for ginsenoside biosynthesis, development of functional SNP markers for its manipulation and systems analysis of its underlying molecular mechanism. Correlation analysis identified 100 PgCYP genes, including all three published ginsenoside biosynthesis PgCYP genes, whose expressions were significantly correlated with the ginsenoside contents. Mutation association analysis identified that six of these 100 PgCYP genes contained SNPs/InDels that were significantly associated with ginsenosides biosynthesis (P ≤ 1.0e-04). These six PgCYP genes, along with all ten published ginsenoside biosynthesis genes from the PgCYP and other gene families, formed a strong co-expression network, even though they varied greatly in spatio-temporal expressions. Therefore, this study has identified six new ginsenoside biosynthesis candidate genes, provided a genome-wide insight into how they are involved in ginsenoside biosynthesis and developed a set of functional SNP markers useful for enhanced ginsenoside biosynthesis research and breeding in ginseng and related species.

Structural Variation, Functional Differentiation, and Activity Correlation of the Cytochrome P450 Gene Superfamily Revealed in Ginseng.

Ginseng ( C.A. Mey.) is one of the most important medicinal herbs for human health and medicine, for which ginsenosides are the major bioactive components. The cytochrome P450 genes, , form a large gene superfamily; however, only three genes have been identified from ginseng and shown to be involved in ginsenoside biosynthesis, indicating the importance of the gene superfamily in the process. Here we report genome-wide identification and systems analysis of the genes in ginseng, defined as genes. We identified 414 genes, including the three published genes. These genes formed a superfamily consisting of 41 gene families, with a substantial diversity in phylogeny and dramatic variation in spatiotemporal expression. Gene ontology (GO) analysis categorized the gene superfamily into 12 functional subcategories distributing among all three primary functional categories, suggesting its functional differentiation. Nevertheless, the majority of its gene members expressed correlatively and tended to form a coexpression network and some of them were commonly regulated in expression across tissues and developmental stages. These results have led to genome-wide identification of genes useful for genome-wide identification of the genes involved in ginsenoside biosynthesis in ginseng and provided the first insight into how a gene superfamily functionally differentiates and acts correlatively in plants.

MIPhy: identify and quantify rapidly evolving members of large gene families.

After transitioning to a new environment, species often exhibit rapid phenotypic innovation. One of the fastest mechanisms for this is duplication followed by specialization of existing genes. When this happens to a member of a gene family, it tends to leave a detectable phylogenetic signature of lineage-specific expansions and contractions. These can be identified by analyzing the gene family across several species and identifying patterns of gene duplication and loss that do not correlate with the known relationships between those species. This signature, termed phylogenetic instability, has been previously linked to adaptations that change the way an organism samples and responds to its environment; conversely, low phylogenetic instability has been previously linked to proteins with endogenous functions. With the increase in genome-level data, there is a need to identify and quantify phylogenetic instability. Here, we present Minimizing Instability in Phylogenetics (MIPhy), a tool that solves this problem by quantifying the incongruence of a gene’s evolutionary history. The motivation behind MIPhy was to produce a tool to aid in interpreting phylogenetic trees. It can predict which members of a gene family are under adaptive evolution, working only from a gene tree and the relationship between the species under consideration. While it does not conduct any estimation of positive selection—which is the typical indication of adaptive evolution—the results tend to agree. We demonstrate the usefulness of MIPhy by accurately predicting which members of the mammalian cytochrome P450 gene superfamily metabolize xenobiotics and which metabolize endogenous compounds. Our predictions correlate very well with known substrate specificities of the human enzymes. We also analyze the Caenorhabditis collagen gene family and use MIPhy to predict genes that produce an observable phenotype when knocked down in C. elegans, and show that our predictions correlate well with existing knowledge. The software can be downloaded and installed from https://github.com/dave-the-scientist/miphy and is also available as an online web tool at http://www.miphy.wasmuthlab.org.

Molecular characterization, expression and cellular localization of CYP17 gene during geese (Anser cygnoides) follicular development

As a key member of the cytochrome P450 gene superfamily, CYP17 gene encodes 17α-hydroxylase/C17,20-lyase that is critical for directing androgen synthesis. The CYP17 gene has been identified in several species, yet little is known about its distribution and expression profile during goose follicular development. In the present study, we obtained the full-length coding sequence of goose CYP17 (gCYP17) gene for the first time using RACE method. Its sequence alignment and phylogenetic analysis suggested that gCYP17 was highly conserved with those of other birds and consisted of four main functional domains like other species. Results from immunohistochemistry, quantitative real-time PCR and Western blotting suggested that gCYP17 was predominantly located in theca interna throughout follicular development. Furthermore, levels of gCYP17 reached the maximum in theca layer of the 6–8 mm follicles which were significantly higher than in those of other follicles (P < 0.05). In addition, gCYP17 was expressed at much higher levels in the F4 theca layer than the F1 follicle (P < 0.05). Therefore, these results indicated that the fluctuating expression pattern and specific cellular localization of gCYP17 during follicular development might be closely related to androgen secretion, and thereby follicular maturation.

Evolutionary history and functional divergence of the cytochrome P450 gene superfamily between Arabidopsis thaliana and Brassica species uncover effects of whole genome and tandem duplications

BackgroundThe cytochrome P450 monooxygenase (P450) superfamily is involved in the biosynthesis of various primary and secondary metabolites. However, little is known about the effects of whole genome duplication (WGD) and tandem duplication (TD) events on the evolutionary history and functional divergence of P450s in Brassica after splitting from a common ancestor with Arabidopsis thaliana.ResultsUsing Hidden Markov Model search and manual curation, we detected that Brassica species have nearly 1.4-fold as many P450 members as A. thaliana. Most P450s in A. thaliana and Brassica species were located on pseudo-chromosomes. The inferred phylogeny indicated that all P450s were clustered into two different subgroups. Analysis of WGD event revealed that different P450 gene families had appeared after evolutionary events of species. For the TD event analyses, the P450s from TD events in Brassica species can be divided into ancient and recent parts. Our comparison of influence of WGD and TD events on the P450 gene superfamily between A. thaliana and Brassica species indicated that the family-specific evolution in the Brassica lineage can be attributed to both WGD and TD, whereas WGD was recognized as the major mechanism for the recent evolution of the P450 super gene family. Expression analysis of P450s from A. thaliana and Brassica species indicated that WGD-type P450s showed the same expression pattern but completely different expression with TD-type P450s across different tissues in Brassica species. Selection force analysis suggested that P450 orthologous gene pairs between A. thaliana and Brassica species underwent negative selection, but no significant differences were found between P450 orthologous gene pairs in A. thaliana–B. rapa and A. thaliana–B. oleracea lineages, as well as in different subgenomes in B. rapa or B. oleracea compared with A. thaliana.ConclusionsThis study is the first to investigate the effects of WGD and TD on the evolutionary history and functional divergence of P450 gene families in A. thaliana and Brassica species. This study provides a biology model to study the mechanism of gene family formation, particularly in the context of the evolutionary history of angiosperms, and offers novel insights for the study of angiosperm genomes.

Characterization of the Tissue Distribution of the Mouse Cyp2c Subfamily by Quantitative PCR Analysis.

The CYP2C subfamily of the cytochrome P450 gene superfamily encodes heme-thiolate proteins that have a myriad of biologic functions. CYP2C proteins detoxify xenobiotics and metabolize endogenous lipids such as arachidonic acid to bioactive eicosanoids. We report new methods and results for the quantitative polymerase reaction (qPCR) analysis for the 15 members of the mouse Cyp2c subfamily (Cyp2c29, Cyp2c37, Cyp2c38, Cyp2c39, Cyp2c40, Cyp2c44, Cyp2c50, Cyp2c54, Cyp2c55, Cyp2c65, Cyp2c66, Cyp2c67, Cyp2c68, Cyp2c69, and Cyp2c70). Commercially available TaqMan primer/probe assays were compared with developed SYBR Green primer sets for specificity toward the mouse Cyp2c cDNAs and analysis of their tissue distribution. TaqMan primer/probe assays for 10 of the mouse Cyp2c isoforms were shown to be specific for their intended mouse Cyp2c cDNA; however, there were no TaqMan primer/probe assays specific for the mouse Cyp2c29, Cyp2c40, Cyp2c67, Cyp2c68, or Cyp2c69 transcripts. Each of the SYBR Green primer sets was specific for its intended mouse Cyp2c cDNA. The two qPCR methods confirmed similar patterns of Cyp2c tissue expression: Cyp2c37, Cyp2c38, Cyp2c39, Cyp2c44, Cyp2c50, Cyp2c54, and Cyp2c70 were most highly expressed in liver; Cyp2c55 was highly expressed in large intestine; Cyp2c65 was highly expressed in stomach, duodenum, and large intestine; and Cyp2c66 was highly expressed in both duodenum and jejunum. For isoforms without specific TaqMan primer/probe assays, the SYBR Green primer sets detected high level expression of Cyp2c29, Cyp2c40, Cyp2c67, Cyp2c68, and Cyp2c69 in the liver. Lower expression levels of the mouse Cyp2cs were also detected in other tissues.

Genetics of tailored medicine: Focus on CNS drugs

The genetics of tailored medicine (TM), a medicine in which drug types and dosages are tailored to the clinical needs of each patient, greatly overlap cytochrome P450 (CYP) genetics, probably the most active area of multidisciplinary research interrelating the interest of medicine, biology, biochemistry, and pharmacology, and potentially crossing all medical disciplines. The great genetics variability showed by the CYP gene superfamily permitted on one side to encode a series of enzyme capable to metabolize almost all drugs, and on the other side to have specific genetic profiles for each individual, resulting in inter-individual difference in drug efficacy also responsible of severe clinical consequences such as therapeutic failures (TFs) and adverse drug reactions (ADRs), worldwide primary causes of morbidity and mortality in Western countries. Among medical disciplines, neurology, psychiatry and geriatrics resulted the most interesting for TM since the administration of concomitant treatment is a common practice, raising the prevalence of TFs and ADRs in the treatment of neurological and psychiatric diseases. i.e. in older patients with in treatment with acetylcholinesterase inhibitors, antidepressants, and antipsychotics, all substrate of CYPs. Thus, in these clinical settings the genetic analysis of CYPs may be pivotal for the identification of patients to be addressed toward alternative treatments, and tailor drug types and dosages to the individual patient's clinical needs.