To study the effect of Fuzheng Huayu recipe (FZHY) on five types of isozymes of cytochrome P450 (CYP450) of normal and liver fibrosis rats by using the cocktail probe method. Dimethylnitrosamine ( DMN) was injected to induce the liver fibrosis model. After the tail vein injection with Cocktail probe solutions prepared with five CYP450s probe substrates (phenacetin-CYP1A2, omeprazole-CYP2C9, tolbutamide-CYP2C19, dextromethorphan-CYP2D6, midazolam-CYP3A4), the plasma concentrations of the five probe substrates were determined by LC-MS/MS, and the pharmacokinetic parameters were calculated by PK solutions 2. After the oral administration with FZHY, normal rats given phenacetin, omeprazole, tolbutamide and dextromethorphan showed increase in AUC(0-t) and decrease in CL to varying degrees, indicating that FZHY obviously inhibited the activities of CYP1A2, CYP2C9, CYP2C19 and CYP2D6 in normal rats, but with no obvious effect on the activity of CYP3A4. After the oral administration with FZHY, liver fibrosis rats treated with CYP2C9 showed the significant increase in AUC(0-t) and significant decrease in Vd, hut with no obvious changes in the pharmacokinetic parameters of other four types of prove substances, suggesting that FZHY could significantly inhibit the activity of CYP2C9 in rats but had no effect on the activities of CYP1A2, CYP2C19, CYP2D6 and CYP3A4. The changes in the activity of CYP450 isozymes in liver fibrosis rats may be the reason for FZHY's different effects on CYP450 isozymes in normal and liver fibrosis rats.
Animals ; Cytochrome P-450 Enzyme System ; genetics ; metabolism ; Disease Models, Animal ; Drugs, Chinese Herbal ; administration & dosage ; chemistry ; pharmacokinetics ; Humans ; Isoenzymes ; genetics ; metabolism ; Liver Cirrhosis ; drug therapy ; enzymology ; genetics ; Male ; Mass Spectrometry ; Rats ; Rats, Wistar

OBJECTIVETo clone and sequence the cDNA of squalene epoxidase gene in Eleutherococcus senticosus.

METHODTotal RNA of E. senticosus was extracted by the improved isothiocyanate method and reverse transcripted into cDNA. The primers were designed depending on the reported SE cDNA sequences of Panax ginseng. The SE cDNAs in E. senticosus was amplified using RT-PCR strategy.

RESULTSequencing results showed two different cDNA fragments (SE1, SE2) with 1665, 1629 bp each ORF which encoded 554,542 amino acids, respectively. The identities of nucleotides and amino acids between SE1, SE2 were 91.49%, 92.55%. SE1, SE2 had the highest amino acids similarity to the SE1 of P. notoginseng, 93.45%, 94.87% respectively. SE1, SE2 both had a FAD binding domain. The deduced speculated amino acids of SE1, SE2 each had 2,4 membrane-spanning helices.

CONCLUSIONThe two SE sequences in E. senticosus were firstly separated and reported, which has made foundation for E. senticosus secondary metabolite engineering researches.

Amino Acid Sequence ; Cloning, Molecular ; DNA, Complementary ; chemistry ; genetics ; Eleutherococcus ; enzymology ; genetics ; Isoenzymes ; classification ; genetics ; Molecular Sequence Data ; Phylogeny ; Plant Proteins ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Sequence Analysis, DNA ; Sequence Homology, Amino Acid ; Squalene Monooxygenase ; classification ; genetics

OBJECTIVETo reveal the 3-hydroxy-3-methylglutary-coenzyme A reductase (HMGR) gene polymorphism of Glycyrrhiza uralensis, and the correlation between HMGR gene polymorphism and the content of glycyrrhizic acid.

METHODLiquorice plants containing different content of glycyrrhizic acid were used as materials. RT-PCR was used to amplify their HMGR gene sequences, which were connected with vector pMD19-T for clone sequencing. Multiple alignments were performed to analyse HMGR gene polymorphism of G. uralensis. Then the correlation between HMGR gene polymorphism and the content of glycyrrhizic acid was revealed.

RESULTHMGR gene sequences polymorphism included codon mutation, base substitution mutation, copy number polymorphism and allele heterozygosity. There were 4 types of mutations in HMGR gene coding amino acid sequences, namely -HSL, -HSV, GALLV, GALSV. Among them, -HSV type was common in liquorice plants, -HSL type only existed in liquorice plants with low content of glycyrrhizic acid, and GALSV type only existed in liquorice plants with high content of glycyrrhizic acid.

CONCLUSIONHMGR gene sequences of G. uralensis are highly polymorphic and related to the content of glycyrrhizic acid.

Cloning, Molecular ; DNA, Complementary ; chemistry ; classification ; genetics ; Glycyrrhiza uralensis ; enzymology ; genetics ; metabolism ; Glycyrrhizic Acid ; metabolism ; Hydroxymethylglutaryl CoA Reductases ; genetics ; metabolism ; Isoenzymes ; genetics ; metabolism ; Mutation ; Phylogeny ; Plant Proteins ; genetics ; metabolism ; Polymorphism, Genetic ; Sequence Analysis, DNA

OBJECTIVETo analyse the polymorphism of squalene synthase gene and reveal the influence of squalene synthase (SQS) gene polymorphism on the catalytic efficiency of its encode enzyme in Glycyrrhiza uralensi.

METHODThe total RNA was extracted. PCR was used to amplify the coding sequences of squalene synthase gene, which were sequenced and analysed. The expression vectors containing different SQS gene sequences, including SQS1C, SQS1F, SQS2A, SQS2B, were constructed and transformed into Escherichia coli BL21. The fusion protein was induced to express by IPTG, then was isolated, purified and used to carry out the enzymatic reaction in vitro. GC-MS was used to analyse the production.

RESULTThere were three kinds of gene polymorphism existing in SQS1 gene of G. uralensis, including single nucleotide polymorphism (SNPs), insertion/deletion length polymorphism (InDels) and level of amino acid, the proportion of conservative replace of SQS1 was 53.94%, and there were 2 mutational sites in structural domains. The proportion of conservative replace of SQS2 was 60%, and there was 1 mutational site in structural domains. The production squalene could be detected by GC-MS in all the 4 kinds of enzymatic reactions. The capacity of accumulating squalene of SQS1F was higher than other SQS genes.

CONCLUSIONThe polymorphism of SQS gene was quite abundant in G. uralensis, which maybe the molecular foundation of the formation of high-quality liquorice.

Amino Acid Substitution ; Biocatalysis ; Cloning, Molecular ; DNA, Complementary ; chemistry ; genetics ; Electrophoresis, Polyacrylamide Gel ; Escherichia coli ; genetics ; Farnesyl-Diphosphate Farnesyltransferase ; genetics ; metabolism ; Gas Chromatography-Mass Spectrometry ; Glycyrrhiza uralensis ; enzymology ; genetics ; INDEL Mutation ; Isoenzymes ; genetics ; metabolism ; Molecular Sequence Data ; Plant Proteins ; genetics ; metabolism ; Polymorphism, Genetic ; Polymorphism, Single Nucleotide ; Recombinant Proteins ; metabolism ; Sequence Analysis, DNA ; Squalene ; metabolism

During severe acute respiratory syndrome coronavirus (SARS-CoV) infection, the activity of the replication/transcription complexes (RTC) quickly peaks at 6 hours post infection (h.p.i) and then diminishes significantly in the late post-infection stages. This "down-up-down" regulation of RNA synthesis distinguishes different viral stages: primary translation, genome replication, and finally viron assembly. Regarding the nsp8 as the primase in RNA synthesis, we confirmed that the proteolysis product of the primase (nsp8) contains the globular domain (nsp8C), and indentified the resectioning site that is notably conserved in all the three groups of coronavirus. We subsequently crystallized the complex of SARS-CoV nsp8C and nsp7, and the 3-D structure of this domain revealed its capability to interfuse into the hexadecamer super-complex. This specific proteolysis may indicate one possible mechanism by which coronaviruses to switch from viral infection to genome replication and viral assembly stages.
Amino Acid Sequence ; Crystallography, X-Ray ; DNA Primase ; chemistry ; genetics ; physiology ; Humans ; Isoenzymes ; chemistry ; genetics ; physiology ; Molecular Sequence Data ; Protein Structure, Secondary ; RNA, Viral ; biosynthesis ; SARS Virus ; chemistry ; genetics ; physiology ; Sequence Alignment ; Severe Acute Respiratory Syndrome ; virology ; Virus Replication

Acetohydroxyacid synthase (AHAS) catalyses the first reaction in the pathway for synthesis of the branched-chain amino acids. AHAS is the target for sulfonylurea, imidazolinone and other AHAS-inhibitor herbicides. Herbicides-resistant AHAS genes have potential application in plant transgenetic engineering and development of new generation herbicide. The AHAS isozyme genes ilvBN, ilvGM and ilvIH were cloned from metsulfuron-methyl resistant strain Klebsiella sp. HR11 and metsulfuron-methyl sensitive strain Klebsiella pneumoniae MGH 78578. Homologous sequences comparison indicated that the differences in AHAS isozyme genes at amino acid levels between strain HR11 and strain MGH 78578 were mainly on the large subunits of ilvBN and ilvGM. The three AHAS isozyme genes from HR11 and MGH 78578 were ligated into the expression vector pET29a(+) and expressed in Escherichia coli BL21, respectively. The results of enzyme inhibition assay showed that only ilvBN and ilvGM from strain HR11 showed strong resistance to AHAS-inhibitor herbicides, while ilvIH from strain HR11 and ilvBN, ilvGM and ilvIH from strain MGH78578 were sensitive to AHAS-inhibitor herbicides.
Acetolactate Synthase ; chemistry ; genetics ; Escherichia coli ; genetics ; metabolism ; Gene Expression ; Genes, Bacterial ; drug effects ; Herbicide Resistance ; genetics ; Herbicides ; pharmacology ; Imidazolines ; pharmacology ; Isoenzymes ; genetics ; Klebsiella ; genetics ; Sulfonylurea Compounds ; pharmacology

OBJECTIVETo identify adenine phosphoribosyltransferases in Schistosoma japonicum and analyze their structural features.

METHODSBased on the accessible transcriptome and proteomic data, the S. japonicum adenine phosphoribosyl transferases were identified using bioinformatics approaches including bi-directional homology comparison, domain search and phylogenetic analysis. Homology modeling was also performed to describe the structural features of the proteins.

RESULTS AND CONCLUSIONTwo homologue sequences of adenine phosphoribosyltransferase were obtained from S. japonicum, and the EST abundance, physico-chemical properties and three-dimensional structures of them were also acquired.

Adenine Phosphoribosyltransferase ; chemistry ; genetics ; Animals ; Computational Biology ; methods ; Helminth Proteins ; chemistry ; genetics ; Isoenzymes ; chemistry ; genetics ; Models, Molecular ; Phylogeny ; Protein Conformation ; Schistosoma japonicum ; enzymology

When treated with protopine and alkalized extracts of the tuber of Corydalis ternata for one year, significant decrease in glutamate level and increase in glutamate dehydrogenase (GDH) activity was observed in rat brains. The expression of GDH between the two groups remained unchanged as determined by Western and Northern blot analysis, suggesting a post-translational regulation of GDH activity in alkalized extracts treated rat brains. The stimulatory effects of alkalized extracts and protopine on the GDH activity was further examined in vitro with two types of human GDH isozymes, hGDH1 (house-keeping GDH) and hGDH2 (nerve-specific GDH). Alkalized extracts and protopine activated the human GDH isozymes up to 4.8-fold. hGDH2 (nervespecific GDH) was more sensitively affected by 1 mM ADP than hGDH1 (house-keeping GDH) on the activation by alkalized extracts. Studies with cassette mutagenesis at ADP-binding site showed that hGDH2 was more sensitively regulated by ADP than hGDH1 on the activation by Corydalis ternata. Our results suggest that prolonged exposure to Corydalis ternata may be one of the ways to regulate glutamate concentration in brain through the activation of GDH.
Animals ; Berberine Alkaloids/pharmacology ; Brain/*drug effects/enzymology ; Corydalis/*chemistry ; Enzyme Activation/drug effects ; Glutamate Dehydrogenase/genetics/*metabolism ; Glutamic Acid/*metabolism ; Isoenzymes/genetics/metabolism ; Plant Extracts/pharmacology ; RNA, Messenger/analysis/metabolism ; Rats ; Research Support, Non-U.S. Gov't

This study describes the cDNA sequencing and the bioinformatic analysis of a novel NADP(H)-dependent retinol dehydrogenase/reductases isoform (NRDRiso). Based upon the concensus sequences of human and mouse NRDR coding region, we have identified a short 377 bp RT-PCR product from human liver tissue. The cDNA sequence of a NRDR isoform was then isolated using RACE approach and its sequence was analysed. The full-length cDNA is 1,003bp in length and was submitted to GenBank as NADP-dependent retinol dehydrogenase/reductase short isoform (NRDRiso). The open reading frames of NRDRiso cDNA is 525 bp.
Alcohol Oxidoreductases ; chemistry ; genetics ; Amino Acid Sequence ; Animals ; Base Sequence ; Chromosome Mapping ; Computational Biology ; DNA, Complementary ; chemistry ; Humans ; Isoenzymes ; genetics ; Liver ; enzymology ; Mice ; Molecular Sequence Data ; NADP ; metabolism ; Open Reading Frames ; Sequence Analysis, DNA

OBJECTIVETo study the modulatory effect of Panax gingseng and coadministration with Veratrum nigrum on the activity and mRNA expression of cytochrome P450 isoenzymes in rat liver.

METHODRat liver microsomal cytochrome P450, b5, aminopyrine N-demethylase(APND), p-nitrophenol-hydroxylase(pNPH)activities were quantitated by UV chromatography. The mRNA expression level of five CYP isoenzymes CYP1A1, CYP2B1/2, CYP2C11, CYP2E1 and CYP3A1 were detected by semi-quantitative reverse transcriptase-polymerase chain reaction(RT-PCR).

RESULTP. gingseng coadministrated with V. nigrum obviously decreased the P450 contents of liver microsomes, and the b5 contents. Both single and combined used inhibited the activities of aminopyrine N-demethylase. At the mRNA level, the expression of CYP2C11 markedly induced exposure to V. nigrum, but combinative groups decreased the expression of CYP2C11. The combination of P. gingseng and V. nigrum induced the expression of CYP1A1. P. gingseng has inhibitory effect on CYP2B1/2 and inductive effect used with V. nigrum. The combination of P. gingseng with V. nigrum also induced the expression of CYP3A1.

CONCLUSIONP. gingseng used singly has some different modulation effects compared with combinative used, which may occur because of drug-drug interaction based on cytochrome P450. To elucidate the drug-drug interaction, it needs further analysis and metabolism research.

Aminopyrine N-Demethylase ; metabolism ; Animals ; Cytochrome P-450 Enzyme System ; biosynthesis ; genetics ; Cytochromes b5 ; metabolism ; Drug Incompatibility ; Drugs, Chinese Herbal ; isolation & purification ; pharmacology ; Female ; In Vitro Techniques ; Isoenzymes ; biosynthesis ; genetics ; Male ; Microsomes, Liver ; metabolism ; Panax ; chemistry ; RNA, Messenger ; biosynthesis ; genetics ; Rats ; Rats, Wistar ; Veratrum ; chemistry