Triterpenoids are a class of natural products of great commercial value that are widely used in pharmaceutical, health care and cosmetic industries. The biosynthesis of triterpenoids relies on the efficient synthesis of squalene epoxide, which is synthesized from the NADPH dependent oxidation of squalene catalyzed by squalene epoxidase. We screened squalene epoxidases derived from different species, and found the truncated squalene epoxidase from Rattus norvegicus (RnSETC) showed the highest activity in engineered Escherichia coli. Further examination of the effect of endogenous cytochrome P450 reductase like (CPRL) proteins showed that overexpression of NADH: quinone oxidoreductase (WrbA) under Lac promoter in a medium-copy number plasmid increased the production of squalene epoxide by nearly 2.5 folds. These results demonstrated that the constructed pathway led to the production of squalene epoxide, an important precursor for the biosynthesis of triterpenoids.
Animals ; Escherichia coli/genetics* ; NADPH-Ferrihemoprotein Reductase ; Protein Engineering ; Rats ; Repressor Proteins ; Squalene ; Squalene Monooxygenase/genetics*

OBJECTIVE: To compare the accuracy of five warfarin-dosing algorithms and warfarin stable dose model (2.5 mg/day) for Shandong population. METHODS: One hundred and twenty five patients who achieved stable warfarin dose were enrolled. Clinical and genetic data were used to evaluate the value of each algorithm by calculating the percentage of patients whose predicted warfarin dose was within 20% of the actual stable therapeutic dose and mean absolute error (MAE). RESULTS: The frequency of patients with CYP2C9*1/*1, CYP2C9*1/*3 and CYP2C9*1/*2 genotype was 92.00%, 7.20%, 0.80%, respectively. That of VKORC1-1639 AA, AG and GG genotype was 82.40%, 15.20%, 2.40%, respectively. CYP4F2*1/*1, *1/*3, *3/*3 genotype was 50.40%, 39.20%, 10.40%, respectively. With the same genotypes for other loci, patients who carried at least one VKORC1-16398G mutant allele had increased warfarin stable daily dose compared with VKORC1-1639AA. Compared with CYP4F2*1/*1, those carrying at least one CYP4F2*3 mutant allele had warfarin stable daily dose increased by 5.9%-13.00%. The percentage of ideal prediction calculated from IWPC model (59.20%), Huang model (57.60%) and Ohno model (52.80%) were higher than others. The MAE were 0.35 (95%CI: 0.11-0.49), 0.15 (95%CI: 0.10-0.32), 0.39 (95%CI: 0.12-0.51), respectively. CONCLUSION The polymorphisms of CYP2C9, VKORC1 and CYP4F2 genes can influence the stable dose of warfarin in Shandong population. IWPC algorithm is suitable for guiding the use of warfarin in this population.
Anticoagulants ; administration & dosage ; Aryl Hydrocarbon Hydroxylases ; Cytochrome P-450 CYP2C9 ; genetics ; Cytochrome P450 Family 4 ; genetics ; Dose-Response Relationship, Drug ; Genotype ; Humans ; Models, Theoretical ; Polymorphism, Genetic ; Vitamin K Epoxide Reductases ; genetics ; Warfarin ; administration & dosage

Andrographolide is a main active ingredient in traditional Chinese medicine Andrographis paniculata，with a variety of pharmacological activity，widely used in clinical practice. However its biosynthetic pathway has not been resolved. Cytochrome P450 reductase provides electrons for CYP450 and plays an important role in the CYP450 catalytic process. In this study，the coding sequence of A. paniculata CPR was screened and cloned by homologous alignment，named ApCPR4. The ApCPR4 protein was obtained by prokaryotic expression. After isolation and purification，the enzyme activity was identified . The results showed that ApCPR4 could reduce the cytochrome c and ferricyanide in NADPH-dependent manner. In order to verify its function，ApCPR4 and CYP76AH1 were co-transformed into yeast engineering bacteria. The results showed that ApCPR4 could help CYP76AH1 catalyze the formation of rustols in yeast. Real-time quantitative PCR results showed that the expression of ApCPR4 increased gradually in leaves treated with methyl jasmonate (MeJA). The expression pattern was consistent with the trend of induction and accumulation of andrographolide by MeJA，suggesting that ApCPR4 was associated with biosynthesis of andrographolide.
Acetates ; Andrographis ; enzymology ; genetics ; Biosynthetic Pathways ; Cloning, Molecular ; Cyclopentanes ; Diterpenes ; metabolism ; NADPH-Ferrihemoprotein Reductase ; genetics ; Oxylipins ; Plant Leaves ; enzymology ; Plant Proteins ; genetics

Pharmacogenetic testing for clinical applications is steadily increasing. Correct and adequate use of pharmacogenetic tests is important to reduce unnecessary medical costs and adverse patient outcomes. This document contains recommended pharmacogenetic testing guidelines for clinical application, interpretation, and result reporting through a literature review and evidence-based expert opinions for the clinical pharmacogenetic testing covered by public medical insurance in Korea. This document aims to improve the utility of pharmacogenetic testing in routine clinical settings.
Anticoagulants/therapeutic use ; Antidepressive Agents/therapeutic use ; Antimetabolites, Antineoplastic/therapeutic use ; Antitubercular Agents/therapeutic use ; Arylamine N-Acetyltransferase/genetics ; Coronary Artery Disease/drug therapy/genetics ; Cytochrome P-450 CYP2C19/genetics ; Cytochrome P-450 CYP2C9/genetics ; Cytochrome P-450 CYP2D6/genetics ; Depressive Disorder/drug therapy/genetics ; Genotype ; Isoniazid/therapeutic use ; Laboratories, Hospital/standards ; Methyltransferases/genetics ; Pharmacogenomic Testing/*methods/standards ; Platelet Aggregation Inhibitors/therapeutic use ; Pulmonary Embolism/drug therapy/genetics ; Ticlopidine/analogs & derivatives/therapeutic use ; Tuberculosis/drug therapy/genetics ; Vitamin K Epoxide Reductases/genetics ; Warfarin/therapeutic use

PURPOSE: The genes for cytochrome P450 2C9 (CYP2C9) and vitamin K epoxide reductase complex subunit 1 (VKORC1) have been identified as important genetic determinants of warfarin dosing and have been studied. We developed warfarin algorithm for Korean patients with stroke and compared the accuracy of warfarin dose prediction algorithms based on the pharmacogenetics. MATERIALS AND METHODS: A total of 101 patients on stable maintenance dose of warfarin were enrolled. Warfarin dosing algorithm was developed using multiple linear regression analysis. The performance of all the algorithms was characterized with coefficient of determination, determined by linear regression, and the mean of percent deviation was used to predict doses from the actual dose. In addition, we compared the performance of the algorithms using percentage of predicted dose falling within ±20% of clinically observed doses and dividing the patients into a low-dose group (≤3 mg/day), an intermediate-dose group (3-7 mg/day), and high-dose group (≥7 mg/day). RESULTS: A new developed algorithms including the variables of age, body weight, and CYP2C9 and VKORC1 genotype. Our algorithm accounted for 51% of variation in the warfarin stable dose, and performed best in predicting dose within 20% of actual dose and intermediate-dose group. CONCLUSION: Our warfarin dosing algorithm may be useful for Korean patients with stroke. Further studies to elucidate clinical utility of genotype-guided dosing and find the additional genetic association are necessary.
Aged ; *Algorithms ; Anticoagulants/*administration & dosage/therapeutic use ; Cytochrome P-450 CYP2C9/*genetics ; Dose-Response Relationship, Drug ; Female ; Genotype ; Humans ; International Normalized Ratio ; Linear Models ; Male ; Middle Aged ; Multivariate Analysis ; Pharmacogenetics ; Regression Analysis ; Republic of Korea ; Stroke/*drug therapy/ethnology ; Vitamin K Epoxide Reductases/*genetics ; Warfarin/*administration & dosage/therapeutic use

<b>BACKGROUNDb>Cytochrome b5 reductase 2 (CYB5R2) is a potential tumor suppressor that inhibits cell proliferation and motility in nasopharyngeal carcinoma (NPC). Inactivation of CYB5R2 is associated with lymph node metastasis in NPC. This study aimed to explore the mechanisms contributing to the anti-neoplastic effects of CYB5R2.

<b>METHODSb>Polymerase chain reaction (PCR) assays were used to analyze the transcription of 84 genes known to be involved in representative cancer pathways in the NPC cell line HONE1. NPC cell lines CNE2 and HONE1 were transiently transfected with CYB5R2, and data was validated by real-time PCR. A chick chorioallantoic membrane (CAM) embryo model was implanted with CYB5R2-expressing CNE2 and HONE1 cells to evaluate the effect of CYB5R2 on angiogenesis. An immunohistochemical assay of the CAM model was used to analyze the protein expression of vascular endothelial growth factor (VEGF).

<b>RESULTSb>In CYB5R2-transfected NPC cells, PCR assays revealed up-regulated mRNA levels of Fas cell surface death receptor (FAS), FBJ murine osteosarcoma viral oncogene homolog (FOS), phosphoinositide-3-kinase regulatory subunit 1 (PIK3R1), integrin beta 3 (ITGB3), metastasis suppressor 1 (MTSS1), interferon beta 1 (IFNB1), and cyclin-dependent kinase inhibitor 2A (CDKN2A) and down-regulated levels of integrin beta 5 (ITGB5), insulin-like growth factor 1 (IGF1), TEK tyrosine kinase (TEK), transforming growth factor beta receptor 1 (TGFBR1), and VEGF. The angiogenesis in the CAM model implanted with CYB5R2-transfected NPC cells was inhibited. Down-regulation of VEGF by CYB5R2 in NPC cells was confirmed by immunohistochemical staining in the CAM model.

<b>CONCLUSIONb>CYB5R2 up-regulates the expression of genes that negatively modulate angiogenesis in NPC cells and down-regulates the expression of VEGF to reduce angiogenesis, thereby suppressing tumor formation.

Animals ; Carcinoma ; Cell Line, Tumor ; Cell Movement ; Cell Proliferation ; Chickens ; Cytochrome-B(5) Reductase ; Down-Regulation ; Gene Regulatory Networks ; Genes, Tumor Suppressor ; Humans ; Nasopharyngeal Neoplasms ; Neovascularization, Pathologic ; Oxidoreductases ; Real-Time Polymerase Chain Reaction ; Transfection ; Up-Regulation ; Vascular Endothelial Growth Factor A

OBJECTIVE To assess the influence of genetic polymorphisms and non-genetic factors on warfarin maintenance dose variations in order to provide guidance for personalized use of warfarin. METHODS Two hundred patients from outpatient and inpatient with stable international normalized ratio(INR) were recruited. Clinical data and blood samples were collected. Genotypes of 4 genes involved in warfarin metabolic pathways were determined with Sanger sequencing. Based on statistical analysis of warfarin maintenance dosage, a mathematical model was established. RESULTS Among non-genetic factors, the age and height have significant influence in warfarin dosage. The dosage is negatively correlated with age but positively correlated with height. The difference in dosage for between the 20-year-old group and 60-year-old group has reached 1.81 mg/day, and that for between the 140 cm in height and 180 cm in height groups has reached 1.06 mg/day. VKORC1 -1639G/A, CYP2C9 430C/T, CYP2C9 1075A/C and CYP4F2 V433M polymorphisms have significant influence on stable warfarin dosage. The dosage for patients with wild type and mutant genotypes has varied from 0.35 mg/day to 0.84 mg/day. CONCLUSION Non-genetic factors and genetic polymorphisms play important roles in personalized variations of warfarin maintenance dose. The establishment of mathematical models considering multiple factors is helpful in evaluating the safety and effectiveness of warfarin dosage.
Adult ; Aged ; Anticoagulants ; administration & dosage ; Cytochrome P-450 CYP2C9 ; genetics ; Cytochrome P-450 Enzyme System ; genetics ; Cytochrome P450 Family 4 ; Female ; Humans ; Male ; Middle Aged ; Polymorphism, Genetic ; Vitamin K Epoxide Reductases ; genetics ; Warfarin ; administration & dosage

The study aimed to establish a population pharmacokinetic/pharmacodynamic (PPK/PD) model of warfarin. PCR-RFLP technique was used to genotype the CYP2C9 and VKORC1 polymorphisms of 73 patients. RP-HPLC-UV method was used to determine the 190 plasma concentrations of warfarin. Application of NONMEM, the clinical information and 263 international normalized ratio (INR) monitoring data were used to investigate the effect of genetic, physiological, pathological factors, other medication on clearance and anticoagulant response. The final model of warfarin PPK/PD was described as follows: CL = θCL · (WT/60)θWT · θCYP · eηCL (if CYP2C9*1/*1, θCYP = 1; if *1/*3, θCYP = 0.708); EC50 = θEC50 · θVKOR · eηEC50 (if VKORC1- 1639AA, θVKOR = 1; if GA, θVKOR = 2.01; V = θV; K(E0) = θK(E0); Emax = θEmax; E0 = θE0 · eηE0. Among them, the body weight (WT), CYP2C9 and VKORC1 genotype had conspicuous effect on warfarin PK/PD parameters. The goodness diagnosis, Bootstrap, NPDE verification showed that the final model was stable, effective and predictable. It may provide a reference for opitimizing the dose regimen of warfarin.
Anticoagulants ; pharmacology ; Body Weight ; Cytochrome P-450 CYP2C9 ; genetics ; Genotype ; Humans ; International Normalized Ratio ; Nonlinear Dynamics ; Polymorphism, Genetic ; Vitamin K Epoxide Reductases ; genetics ; Warfarin ; pharmacokinetics

A 73-yr-old Korean man with permanent atrial fibrillation visited outpatient clinic with severely increased International Normalized Ratio (INR) values after taking a usual starting dosage of warfarin to prevent thromboembolism. We found out later from his blood tests that he had hyperthyroidism at the time of treatment initiation. His genetic analysis showed CYP2C9*1/*3 and VKORC1+1173TT genotypes. We suspect that both hyperthyroidism and genetic variant would have contributed to his extremely increased INR at the beginning of warfarin therapy. From this case, we learned that pharmacogenetic and thyroid function test might be useful when deciding the starting dosage of warfarin in patients with atrial fibrillation.
Aged ; Anticoagulants/blood/metabolism/therapeutic use ; Aspirin/therapeutic use ; Atrial Fibrillation/*diagnosis ; Chromatography, High Pressure Liquid ; Cytochrome P-450 CYP2C9/*genetics ; Genotype ; Humans ; Male ; Polymorphism, Single Nucleotide ; Tandem Mass Spectrometry ; Thromboembolism/prevention & control ; Thyrotoxicosis/*diagnosis ; Vitamin K Epoxide Reductases/genetics ; Warfarin/*blood/metabolism/therapeutic use

Acute-On-Chronic Liver Failure ; blood ; pathology ; Case-Control Studies ; Cytochrome Reductases ; metabolism ; Humans ; Prospective Studies ; alpha-Fetoproteins ; metabolism