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1.
Chinese Journal of Biotechnology ; (12): 1109-1116, 2019.
Article in Chinese | WPRIM | ID: wpr-771817

ABSTRACT

The discovery of hydroxylases in the anticancer drug taxol biosynthesis pathway is a hotspot and difficulty in current research. In this study, a new hydroxylase gene TcCYP725A22 (GenBank accession number: MF448646.1) was used to construct a sub-cellular localization vector pCAMIBA1303-TcCYP725A22-EGFP to get the transient expression in onion epidermal cells. Laser confocal microscopy revealed that the protein encoded by this gene was localized in the cell membrane. Furthermore, the recombinant plant expression plasmid pBI121-TcCYP725A22 was constructed. After transient transformation to the Taxus chinensis mediated by Agrobacterium tumefaciens LBA4404, qRT-PCR and LC-MS were utilized to analyze the effects of TcCYP725A22 overexpression on the synthesis of taxol. The results showed that, in the TcCYP725A22 overexpressed cell line, expression levels of most defined hydroxylase genes for taxol biosynthesis were increased, and the yield of taxanes were also increased. It was concluded that the hydroxylase gene TcCYP725A22 is likely involved in the biosynthetic pathway of taxol.


Subject(s)
Biosynthetic Pathways , Mixed Function Oxygenases , Paclitaxel , Taxoids , Taxus
2.
Chinese Journal of Biotechnology ; (12): 351-362, 2019.
Article in Chinese | WPRIM | ID: wpr-771371

ABSTRACT

Baeyer-Villiger monooxygenases, a well-studied class of flavin-dependent enzymes, catalyze the conversion of ketones to lactones or esters and the oxygenation of heteroatoms, which possesses great practical prospect in synthetic chemistry and biocatalysis. In this review, we focus on Baeyer-Villiger oxidations involved in biosynthesis of microbial secondary metabolites and discuss the characteristics of these Baeyer-Villiger oxidations and Baeyer-Villiger monooxygenases, to provide reference for the protein engineering of Baeyer-Villiger monooxygenases.


Subject(s)
Biocatalysis , Catalysis , Mixed Function Oxygenases , Oxidation-Reduction , Protein Engineering
3.
Journal of Stroke ; : 151-159, 2019.
Article in English | WPRIM | ID: wpr-766251

ABSTRACT

Trimethylamine N-oxide (TMAO) is produced when trimethylamine, a waste product of gut microbes, is converted via hepatic flavin monooxygenases. As TMAO is a potential causative factor in various cardiovascular diseases (CVDs) considerable research interest has arisen on its use as a biomarker. Higher TMAO levels are associated with future risk of both incident CVD in the general population and established CVD, including stroke. The addition of TMAO into models with traditional risk factors significantly improved the prediction of future CVD risk. TMAO promotes atherosclerosis and is associated with platelet hyperreactivity and inflammation, which are in turn associated with the development of stroke and its secondary consequences. Additionally, TMAO may play a key mediator role in the relationship between the diet, gut microbiota, and CVD development. Compelling evidence suggesting that TMAO is both a risk factor and prognostic marker of stroke and CVD. Potential therapeutic strategy of diet and drugs in reducing TMAO levels have emerged. Thus, TMAO is a novel biomarker and target in stroke and CVD prevention.


Subject(s)
Atherosclerosis , Blood Platelets , Cardiovascular Diseases , Diet , Gastrointestinal Microbiome , Inflammation , Mixed Function Oxygenases , Prognosis , Risk Factors , Stroke , Waste Products
4.
Article in Chinese | WPRIM | ID: wpr-776421

ABSTRACT

Hyoscyamine and scopolamine are two main alkaloids in Atropa belladonna with great medicinal value. In this paper, the contents of hyoscyamine and scopolamine, the upstream products in alkaloid synthesis, and the expression levels of key enzyme genes PMT, TRⅠ and H6H in secondary metabolism of A. belladonna seedlings were measured to clarify the mechanism of nitrogen forms regulating alkaloids synthesis.The results showed that the 50/50 (NH⁺₄/NO⁻₃) treatment was more favorable for the accumulation of alkaloids and the conversion of hyoscyamine to scopolamine. The content of putrescine was almost consistent with the change of key enzymes activities in the synthesis of putrescine, they both increased with the rise of ammonium ratio, reaching the highest at 75/25 (NH⁺₄/NO⁻₃). The detection of signaling molecule nitric oxide (NO) showed that the NO concentration decreased with the decrease of nitrate proportion. Further detection of gene expression levels of PMT, TRⅠ and H6H in TAs synthesis pathway showed that a certain amount of ammonium promoted the expression of PMT and H6H in roots. When the ratio of ammonium to nitrate was 50/50, PMT, TRⅠ and H6H in leaves and roots had higher expression levels. It can be speculated that the regulation of the formation of hyoscyamine to scopolamine by nitrogen forms mainly through affecting the expression of key enzyme genes. 50/50 (NH⁺₄/NO⁻₃) treatment increased the gene expression of TRⅠ in both leaves and roots as well as PMT and H6H in roots, promoting the synthesis of putrescine to hyoscyamine and the conversion of hyoscyamine to scopolamine.


Subject(s)
Atropa belladonna , Genetics , Gene Expression Regulation, Plant , Hyoscyamine , Mixed Function Oxygenases , Nitrogen , Metabolism , Scopolamine , Metabolism
5.
Rev. ecuat. pediatr ; 18(1): 23-25, 201706.
Article in Spanish | LILACS | ID: biblio-996607

ABSTRACT

La hiperplasia suprarrenal congénita (HSC), es un desorden endócrino genético debido a alteraciones en la esteroidogénesis adrenal por déficit de una de las enzimas participantes en este ciclo, caracterizado por diferentes grados de alteración en la síntesis de glucocorticoides y mineralocorticoides más una sobreestimulación de la vía androgénica provocando durante el desarrollo embrionario la virilización de los genitales externos. En el 90 a 95 % de los casos la enzima más afectada es la 21 hidroxilasa y de éstas el 75% tiene déficit de aldosterona con pérdida de sal que da lugar a crisis adrenal con riesgo de vida. Se clasifica en su forma clásica y no clásica. El diagnóstico prenatal es fundamental para delinear estrategias de tratamiento perinatal. El déficit de 21-hidroxilasa es el primer diagnóstico que se plantea ante un recién nacido con genitales ambiguos y se debería iniciar terapia de reemplazo hormonal. Presentamos un caso clínico de un neonato con hiperplasia suprarrenal congénita por déficit de 21-hidroxilasa en su forma clásica perdedor de sal.


Congenital adrenal hyperplasia (CAH) is an endocrine genetic disorder due to alterations in adrenal steroidogenesis due to a deficiency of one of the enzymes participating in this cycle, characterized by different degrees of alteration in the synthesis of glucocorticoids and mineralocorticoids plus an overstimulation of the androgenic pathway causing during the embryonic development the virilization of the external genitalia. In 90 to 95% of the cases the most affected enzyme is 21 hydroxylase and of these 75% have aldosterone deficit with salt loss that leads to adrenal crisis with life risk. It is classified in classical and non-classical form. Prenatal diagnosis is essential to delineate perinatal treatment strategies. Deficiency of 21-hydroxylase is the first diagnosis that is presented to a newborn with ambiguous genitalia and should initiate hormone replacement therapy. We present a clinical case of a neonate with congenital adrenal hyperplasia due to a deficiency of 21-hydroxylase in its classical salt-losing form.


Subject(s)
Humans , Male , Infant, Newborn , Infant, Newborn , Adrenal Hyperplasia, Congenital , Mixed Function Oxygenases
6.
Braz. j. microbiol ; 47(4): 846-852, Oct.-Dec. 2016. tab, graf
Article in English | LILACS | ID: biblio-828191

ABSTRACT

Abstract Copper mine drainages are restricted environments that have been overlooked as sources of new biocatalysts for bioremediation and organic syntheses. Therefore, this study aimed to determine the enzymatic activities (esterase, epoxide hydrolase and monooxygenase) of 56 heterotrophic bacteria isolated from a neutral copper mine drainage (Sossego Mine, Canaã dos Carajás, Brazil). Hydrolase and monooxygenase activities were detected in 75% and 20% of the evaluated bacteria, respectively. Bacterial strains with good oxidative performance were also evaluated for biotransformation of organic sulfides. Fourteen strains with good enzymatic activity were identified by 16S rRNA gene sequencing, revealing the presence of three genera: Bacillus, Pseudomonas and Stenotrophomonas. The bacterial strains B. megaterium (SO5-4 and SO6-2) and Pseudomonas sp. (SO5-9) efficiently oxidized three different organic sulfides to their corresponding sulfoxides. In conclusion, this study revealed that neutral copper mine drainages are a promising source of biocatalysts for ester hydrolysis and sulfide oxidation/bioremediation. Furthermore, this is a novel biotechnological overview of the heterotrophic bacteria from a copper mine drainage, and this report may support further microbiological monitoring of this type of mine environment.


Subject(s)
Bacteria/classification , Bacteria/enzymology , Copper , Environmental Microbiology , Oxidation-Reduction , Phylogeny , Sulfides/metabolism , Bacteria/isolation & purification , Bacteria/genetics , RNA, Ribosomal, 16S/genetics , Sequence Analysis, DNA , Enzymes , Esterases/genetics , Esterases/metabolism , Mixed Function Oxygenases/genetics , Mixed Function Oxygenases/metabolism , Mining
7.
Chinese Journal of Biotechnology ; (12): 554-564, 2016.
Article in Chinese | WPRIM | ID: wpr-337442

ABSTRACT

Taxol is a secondary metabolite with prominent anti-tumor activity, but the yield cannot meet the growing clinical demand due to lower content in yew. Now, most enzyme genes involved in taxol biosynthesis have been cloned and identified, so that obtaining this drug by using synthetic biology method has become a hotspot in recent years. However, most hydroxylases involved in taxol biosynthetic pathway have not been explored. Here, we reviewed the progress on the biosynthesis pathway of taxol, especially concerning hydroxylase. The future research areas of taxol biosynthesis through synthetic biology were also discussed to provide basis for the discovery of uncharacterized hydroxylase genes and the mass taxol production by synthetic biology technology.


Subject(s)
Biosynthetic Pathways , Mixed Function Oxygenases , Metabolism , Paclitaxel , Synthetic Biology , Taxus
8.
Chinese Medical Journal ; (24): 1744-1751, 2016.
Article in English | WPRIM | ID: wpr-251312

ABSTRACT

<p><b>OBJECTIVE</b>Aberrant expression of ten-eleven translocation 1 (TET1) plays a critical role in tumor development and progression. We systematically summarized the latest research progress on the role and mechanisms of TET1 in cancer biology.</p><p><b>DATA SOURCES</b>Relevant articles published in English from 1980 to April 2016 were selected from the PubMed database. The terms "ten-eleven translocation 1," "5mC," "5hmC," "microRNA," "hypoxia," and "embryonic stem cell" were used for the search.</p><p><b>STUDY SELECTION</b>Articles focusing on the role and mechanism of TET1 in tumor were reviewed, including clinical and basic research articles.</p><p><b>RESULTS</b>TET proteins, the key enzymes converting 5-methylcytosine to 5-hydroxymethylcytosine, play vital roles in DNA demethylation regulation. Recent studies have shown that loss of TET1 is associated with tumorigenesis and can be used as a potential biomarker for cancer therapy, which indicates that TET1 serves as tumor suppressor gene. Moreover, besides its dioxygenase activity, TET1 could induce epithelial-mesenchymal transition and act as a coactivator to regulate gene transcription, such as developmental regulator in embryonic stem cells (ESCs) and hypoxia-responsive gene in cancer. The regulation of TET1 is also correlated with microRNA in a posttranscriptional modification process. Hence, it is complex but critical to comprehend the mechanisms of TET1 in the biology of ESCs and cancer.</p><p><b>CONCLUSIONS</b>TET1 not only serves as a demethylation enzyme but also plays multiple roles during tumorigenesis and progression. More studies should be carried out to elucidate the exact mechanisms of TET1 and its associations with cancer before considering it as a therapeutic tool.</p>


Subject(s)
Animals , Biomarkers , Metabolism , Carcinogenesis , Genetics , Metabolism , Pathology , Humans , MicroRNAs , Genetics , Mixed Function Oxygenases , Genetics , Metabolism , Proto-Oncogene Proteins , Genetics , Metabolism
9.
Acta Pharmaceutica Sinica ; (12): 1346-1355, 2015.
Article in Chinese | WPRIM | ID: wpr-320078

ABSTRACT

Hyoscyamine 6 beta-hydroxylase (H6H) is the last rate-limiting enzyme directly catalyzing the formation of scopolamine in tropane alkaloids (TAs) biosynthesis pathway. It is the primary target gene in the genetic modification of TAs metabolic pathway. Full-length cDNA and gDNA sequences of a novel H6H gene were cloned from Datura arborea (DaH6H, GenBank accession numbers for cDNA and gDNA are KR006981 and KR006983, respectively). Nucleotide sequence analysis reveals an open reading frame of 1375 bp encoding 347 amino acids in the cDNA of DaH6H, while the gDNA of DaH6H contains four exons and three introns, with the highest similarity to the gDNA of H6H from D. stramonium. DaH6H also exhibited the most identity of 90.5% with DsH6H in amino acids and harbored conserved 2-oxoglutarate binding motif and two iron binding motifs. The expression level of DaH6H was highest in the mature leaf, followed by the secondary root, and with no expression in the primary root based on qPCR analysis. Its expression was inhibited by MeJA. DaH6H was expressed in E. coli and a 39 kD recombinant protein was detected in SDS-PAGE. Comparison of the contents of scopolamine and hyoscyamine in various TAs-producing plants revealed that D. arborea was one of the rare scopolamine predominant plants. Cloning of DaH6H gene will allow more research in the molecular regulatory mechanism of TAs biosynthesis in distinct plants and provide a new candidate gene for scopolamine metabolic engineering.


Subject(s)
Cloning, Molecular , DNA, Complementary , Datura , Genetics , Escherichia coli , Hyoscyamine , Chemistry , Mixed Function Oxygenases , Genetics , Plant Leaves , Plant Roots , Recombinant Proteins , Genetics , Scopolamine , Chemistry
10.
Chinese Journal of Biotechnology ; (12): 361-374, 2015.
Article in Chinese | WPRIM | ID: wpr-240637

ABSTRACT

Baeyer-Villiger monooxygenases (BVMOs) are important biocatalysts to synthesize a series of valuable esters and lactones. Based on protein sequence alignment and crystal structure analysis, a nonconserved hinge which linked NADPH domain and FAD domain was speculated to play an important role in substrate recognition and catalytic oxidation process. Cyclohexanone monooxygenase (CHMO) was selected as a model. Mutants obtained by homologous replacement of the whole hinge almost completely lost its original catalytic activity, demonstrating that the overall hinge structure was of great importance. Some significant sites were identified to greatly affect the catalytic activity and stereoselectivity by alanine scanning mutagenesis, accompanied by enzyme activity assessments and chiral kinetic resolutions. Altering K153 decreased the activity of the enzyme but enhanced the stereoselectivity. Changing L143 site reduced stereoselectivity but had little effect on enzyme activity. Mutation at L144 site dramatically weakened both activity and stereoselectivity. Subsequently, these corresponding sites in phenylacetone monooxygenase were also illustrated to follow a similar rule, revealing a universal importance of these sites in the BVMO family. These results expanded our understanding of the structure-activity relationship of these enzymes and provided more proofs for future directed evolution of BVMOs.


Subject(s)
Amino Acid Sequence , Catalysis , Mixed Function Oxygenases , Chemistry , Models, Molecular , NADP , Oxidation-Reduction , Oxygenases , Chemistry , Protein Conformation , Sequence Alignment , Structure-Activity Relationship , Substrate Specificity
11.
Chinese Journal of Biotechnology ; (12): 523-533, 2015.
Article in Chinese | WPRIM | ID: wpr-240622

ABSTRACT

9α-hydroxy-4-androstene-3,17-dione (9-OH-AD) is an important intermediate in the steroidal drugs production. 3-ketosteroid-9α-hydroxylase (KSH), a two protein system of KshA and KshB, is a key-enzyme in the microbial steroid ring B-opening pathway. KSH catalyzes the transformation of 4-androstene-3,17-dione (AD) into 9-OH-AD specifically. In the present study, the putative KshA and KshB genes were cloned from Mycobacterium smegmatis mc(2)155 and Gordonia neofelifaecis NRRL B-59395 respectively, and were inserted into the expression vector pNIT, the co-expression plasmids of kshA-kshB were obtained and electroporated into Mycobacterium sp. NRRL B-3805 cells. The recombinants were used to transform steroids, the main product was characterized as 9α-hydroxy-4-androstene-3,17-dione (9-OH-AD), showing that kshA and kshB were expressed successfully. Different from the original strain Mycobacterium sp. NRRL B-3805 that accumulates 4-androstene-3,17-dione, the recombinants accumulates 9α-hydroxy-4-androstene-3,17-dione as the main product. This results indicates that the putative genes kshA, kshB encode active KshA and KshB, respectively. The process of biotransformation was investigated and the results show that phytosterol is the most suitable substrate for biotransformation, kshA and kshB from M. smegmatis mc(2)155 seemed to exhibit high activity, because the resultant recombinant of them catalyzed the biotransformation of phytosterol to 9-OH-AD in a percent conversion of 90%, which was much higher than that of G. neofelifaecis NRRL B-59395. This study on the manipulation of the ksh genes in Mycobacterium sp. NRRL B-3805 provides a new pathway for producing steroid medicines.


Subject(s)
Androstenedione , Bacterial Proteins , Genetics , Metabolism , Biotransformation , Ketosteroids , Mixed Function Oxygenases , Genetics , Metabolism , Mycobacterium , Metabolism , Mycobacterium smegmatis , Plasmids
12.
Acta Pharmaceutica Sinica ; (12): 1346-1352, 2014.
Article in Chinese | WPRIM | ID: wpr-299128

ABSTRACT

Artemisnin is a novel sesquiterpene lactone with an internal peroxide bridge structure, which is extracted from traditional Chinese herb Artemisia annua L. (Qinghao). Recommended by World Health Organization, artemisinin is the first-line drug in the treatment of encephalic and chloroquine-resistant malaria. In the present study, transgenic A. annua plants were developed by overexpressing the key enzymes involved in the biosynthetic pathway of artemisinin. Based on Agrobacterium-mediated transformation methods, transgenic plants of A. annua with overexpression of both HDR and ADS were obtained through hygromycin screening. The genomic PCR analysis confirmed six transgenic lines in which both HDR and ADS were integrated into genome. The gene expression analysis given by real-time quantitative PCR showed that all the transgenic lines had higher expression levels of HDR and ADS than the non-transgenic control (except ah3 in which the expression level of ADS showed no significant difference compared with control); and the HPLC analysis of artemisinin demonstrated that transgenic A. annua plants produced artemisinin at significantly higher level than non-transgenic plants. Especially, the highest content of artemisinin was found in transgenic line ah70, in which the artemisinin content was 3.48 times compared with that in non-transgenic lines. In summary, overexpression of HDR and ADS facilitated artemisinin biosynthesis and this method could be applied to develop transgenic plants of A. annua with higher yield of artemisinin.


Subject(s)
Artemisia annua , Genetics , Metabolism , Artemisinins , Metabolism , Biosynthetic Pathways , Drugs, Chinese Herbal , Mixed Function Oxygenases , Genetics , Oxidoreductases , Genetics , Plant Proteins , Genetics , Plants, Genetically Modified , Genetics , Metabolism , Plants, Medicinal , Genetics , Metabolism
13.
Acta Pharmaceutica Sinica ; (12): 243-249, 2013.
Article in Chinese | WPRIM | ID: wpr-235677

ABSTRACT

Atropa belladonna L. is the officially medicinal plant species and the main commercial source of scopolamine and hyoscyamine in China. In this study, we reported the simultaneous overexpression of two functional genes involved in biosynthesis of scopolamine, which respectively encoded the upstream key enzyme putrescine N-methyltransferase (PMT; EC 2.1.1.53) and the downstream key enzyme hyoscyamine 6beta-hydroxylase (H6H; EC 1.14.11.11) in transgenic hair root cultures of Atropa belladonna L. HPLC results suggested that four transgenic hair root lines produced higher content of scopolamine at different levels compared with nontransgenic hair root cultures. And scopolamine content increased to 8.2 fold in transgenic line PH2 compared with that of control line; and the other four transgenic lines showed an increase of scopolamine compared with the control. Two of the transgenic hair root lines produced higher levels of tropane alkaloids, and the content increased to 2.7 fold in transgenic line PH2 compared with the control. The gene expression profile indicated that both PMT and H6H expressed at a different levels in different transgenic hair root lines, which would be helpful for biosynthesis of scopolamine. Our studies suggested that overexpression of A. belladonna endogenous genes PMT and H6H could enhance tropane alkaloid biosynthesis.


Subject(s)
Atropa belladonna , Genetics , Gene Expression Regulation, Enzymologic , Gene Expression Regulation, Plant , Hyoscyamine , Metabolism , Methyltransferases , Genetics , Metabolism , Mixed Function Oxygenases , Genetics , Metabolism , Plant Roots , Genetics , Plants, Genetically Modified , Genetics , Plants, Medicinal , Genetics , Scopolamine , Metabolism , Synthetic Biology , Tropanes , Metabolism
14.
Article in Chinese | WPRIM | ID: wpr-294035

ABSTRACT

Transgenic Atropa belladonna with high levels of scopolamine was developed by metabolic engineering. A functional gene involved in the rate limiting enzyme of h6h involved in the biosynthetic pathway of scopolamine was over expressed in A. belladonna via Agrobacterium-mediation. The transgenic plants were culturing till fruiting through micropropogating and acclimating. The integration of the h6h genes into the genomic DNA of transgenic plants were confirmed by genomic polymerase chain reaction (PCR) analysis. Analysis of the difference of plant height, crown width, stem diameter, leaf length, leaf width, branch number and fresh weight was carried out using SPSS software. The content of hyoscyamine and scopolamine in roots, stems, leaves and fruits was determined by HPLC. The investigation of the expression levels of Hnh6h by qPCR. Both Kan(r) and Hnh6h genes were detected in five transgenic lines of A. belladonna plants (A8, A11, A12, C8 and C19), but were not detected in the controls. The plant height, crown width, stem diameter, leaf length, leaf width, branch number and fresh weight of transgenic plants did not decrease by comparison with the non-transgenic ones, and furthermore some agronomic characters of transgenic plants were better than those of the controls. The highest level of scopolamine was found in leaves of transgenic A. belladonna, and the content of scopolamine was also higher than that of hyoscyamine in leaves. The contents of scopolamine of leaves in different transgenic lines were listed in order: C8 > A12 > C19 > A11 > A8, especially, the content of scopolamine in transgenic line C8 was 2.17 mg x g(-1) DW that was 4.2 folds of the non-transgenic ones (0.42 mg x g(-1) DW). The expression of transgenic Hnh6h was detected in all the transgenic plants but not in the control. The highest level of Hnh6h expression was found in transgenic leaves. Overexpression of Hnh6h is able to break the rate limiting steps involved in the downstream pathway of scopolamine biosynthesis, and thus promotes the metabolic flux flowing toward biosynthesis of scopolamine to improve the capacity of scopolamine biosynthesis in transgenic plants. As a result, transgenic plants of A. belladonna with higher level of scopolamine were developed.


Subject(s)
Atropa belladonna , Genetics , Metabolism , Atropine , Metabolism , Gene Expression , Mixed Function Oxygenases , Genetics , Metabolism , Plant Proteins , Genetics , Metabolism , Plants, Genetically Modified , Genetics , Metabolism , Scopolamine , Metabolism , Solanaceae , Genetics
15.
Article in English | WPRIM | ID: wpr-727482

ABSTRACT

Prolyl 4 hydroxylases (P4H) are iron- and 2-oxoglutamate-dependent dioxygenase enzymes and hypoxia-inducible transcription factor (HIF)-P4Hs play a critical role in the regulating oxygen homeostasis in the local tissues as well in the systemic circulation. Over a period of time, a number of prolyl hydroxylase inhibitors and activators have been developed. By employing the pharmacological tools and transgenic knock out animals, the critical role of these enzymes has been established in the pathophysiology of number of diseases including myocardial infarction, congestive heart failure, stroke, neurodegeneration, inflammatory disease, respiratory diseases, retinopathy and others. The present review discusses the different aspects of these enzymes including their pathophysiological role in disease development.


Subject(s)
Animals , Heart Failure , Homeostasis , Inflammation , Ischemia , Mixed Function Oxygenases , Myocardial Infarction , Oxygen , Procollagen-Proline Dioxygenase , Stroke , Transcription Factors
16.
Chinese Journal of Biotechnology ; (12): 950-958, 2012.
Article in Chinese | WPRIM | ID: wpr-342426

ABSTRACT

JadH is a bifunctional hydoxylase/dehydrase involved in jadomycin biosynthesis; it catalyzes a post-PKS modification reaction to convert 2,3-dehydro-UWM6 to dehydrorabelomycin. To identify the key residues involved in substrate-binding and catalysis, structural modeling and multiple sequence alignments of JadH homologs were performed to predict nine residues at the proximity of substrate. Site-directed mutagenesis of the corresponding residues and in vitro evaluation of the activities of the mutant enzymes, indicate these mutations severely reduced JadH activity. Our results indicate these residues are specifically involved in substrate-binding or catalysis in JadH.


Subject(s)
Amino Acid Sequence , Catalysis , Hydro-Lyases , Genetics , Metabolism , Isoquinolines , Metabolism , Mixed Function Oxygenases , Genetics , Metabolism , Molecular Sequence Data , Mutagenesis, Site-Directed , Mutant Proteins , Metabolism , Naphthoquinones , Metabolism , Streptomyces , Metabolism , Substrate Specificity
17.
Article in Chinese | WPRIM | ID: wpr-329900

ABSTRACT

<p><b>OBJECTIVE</b>To observe the expression of hypoxia-inducible factor-lalpha subunit (HIF-1alpha), HIF prolyl hydroxylase domain-containing protein(PHDs) and factor inhibiting HIF-1(FIH) in pulmonary arteries of patient with chronic obstructive pulmonary disease (COPD).</p><p><b>METHODS</b>Pulmonary specimens were obtained from patients undergoing lobectomy for lung cancer, 12 had concurrent COPD (COPD group) and 14 without COPD (control group). The ratio of vascular wall area to total vascular area (WA%) and pulmonary artery media thickness (PAMT) was observed, and HIF-1alpha and its hydroxylases(PHD1, PHD2, PHD3, FIH) mRNA and protein were detected by in situ hybridization and immunohistochemistry respectively.</p><p><b>RESULTS</b>WA% and PAMT of COPD patients(50 microm +/- 9 microm, 40% +/- 5%, were statistically different from those of the control subjects (39 microm +/- 6 microm, 31% +/- 4%, P < 0.01). Relative quantification of mRNA and protein levels (absorbance, A) showed that HIF-lalpha mRNA and protein levels in COPD group (0.230 +/- 0.036,0.275 +/- 0.039) were statistically higher than those of the control subjects (0.174 +/- 0.029, 0.102 +/- 0.015, P < 0.01 ), and that the protein level increased more markedly. PHD1 mRNA in COPD subjects (0.180 +/- 0.030) was comparable to that in control group (0.191 +/- 0.029, P > 0.05); PHD2 and PHD3 mRNA levels in COPD (0.245 +/- 0.044, 0.252 +/- 0.023) were significantly higher than those in control group(0.182 +/- 0.028, 0.127 +/- 0.017, P < 0.01). On the other hand, in COPD subjects PHD1 protein (0.104 +/- 0.015) was significantly lower(P < 0.01), whereas PHD2 protein (0.274 +/- 0.044) was significantly higher(P < 0.01) than those in control group(0.209 +/- 0.023, 0.219+/- 0.043). As for PHD3 protein, no significant changes were observed between the two groups (0.161+/- 0.023 in COPD, 0.146 +/- 0.021 in control, P > 0.05). FIH mRNA and protein both showed no differences between the two groups. Linear correlation analysis showed that HIF1alpha protein was positively correlated with WA%, PAMT, PHD2 mRNA and protein, PHD3 mRNA, and that HIF1alpha protein was negatively correlated with PHD1 protein.</p><p><b>CONCLUSION</b>PHDs may be involved in the process of hypoxic pulmonary vascular remodeling in COPD via regulation of HIF-1alpha gene expression</p>


Subject(s)
Aged , Case-Control Studies , Female , Humans , Hypoxia-Inducible Factor 1, alpha Subunit , Metabolism , Lung , Metabolism , Male , Middle Aged , Mixed Function Oxygenases , Metabolism , Procollagen-Proline Dioxygenase , Metabolism , Pulmonary Artery , Metabolism , Pulmonary Disease, Chronic Obstructive , Metabolism , RNA, Messenger , Genetics , Repressor Proteins , Metabolism
18.
Article in English | WPRIM | ID: wpr-184663

ABSTRACT

Warfarin is a frequently prescribed anticoagulant in rehabilitation patients. Adverse drug reactions of warfarin were reported as bleeding and cutaneous microvascular thrombosis. Major bleeding, such as intracranial hemorrhage and psoas hematoma, in patients receiving anticoagulation therapy is a rare condition, but sometimes very serious complication that can even be fatal. Patient-specific factors (eg, age, body size, race, concurrent diseases, and medications) explain some of the individual variability in warfarin dose, but genetic factors, which influence warfarin response, explain a significantly higher proportion of the variability in the dose. There are two identified genes that are responsible for the main proportion of the genetic effect: CYP2C9, which codes for the enzyme cytochrome P450 2C9 that metabolizes S-warfarin, and VKORC1, which codes for warfarin's target, vitamin K epoxide reductase. We report a case of intolerance to warfarin dosing, due to impaired drug metabolism in a patient with CYP2C9*1/*3 and VKORC 1173TT. Fortunately, there are no severe complications.


Subject(s)
Body Size , Continental Population Groups , Cytochrome P-450 Enzyme System , Drug-Related Side Effects and Adverse Reactions , Hematoma , Hemorrhage , Humans , Intracranial Hemorrhages , Mixed Function Oxygenases , Oxidoreductases , Thrombosis , Vitamin K , Warfarin
19.
Chinese Journal of Cardiology ; (12): 477-481, 2012.
Article in Chinese | WPRIM | ID: wpr-275021

ABSTRACT

<p><b>OBJECTIVE</b>To establish the high-resolution melting curve (HRM) approach for genotyping CYP2C9*3 (1075A/C, rs1057910) and VKORC1 (-1639A/G, rs9923231) and explore its value on estimation of the Warfarin initial dose in comparison with various traditional genotyping methods.</p><p><b>METHODS</b>CYP2C9*3 (1075A/C, rs1057910) and VKORC1 (-1639A/G, rs9923231) genotyping was detected in 100 patients receiving Warfarin therapy by the newly developed HRM method and traditional genotyping methods including PCR-restriction fragment length polymorphism (PCR-RFLP), TaqMan probe and DNA sequencing.</p><p><b>RESULTS</b>The results of the genotypes obtained from above mentioned methods to detect CYP2C9*3 (1075A/C, rs1057910) and VKORC1 (-1639A/G, rs9923231) were similar and consistent. The HRM method is simpler, more economical, and faster compared to the traditional methods. The frequencies of the VKORC1-1639 AA, AG, GG genotypes from these 100 clinical samples were 73 (73%), 23 (23%), 4 (4%), respectively; Frequencies of the CYP2C9 1075 AA, AC, CC genotypes were 94(94%), 6 cases (6%), 0, respectively.</p><p><b>CONCLUSIONS</b>HRM approach can effectively detect CYP2C9*3 (1075A/C, rs1057910) and VKORC1 (-1639A/G, rs9923231) polymorphisms and this method is simpler, more economical, and faster compared to the traditional methods for detecting CYP2C9*3 (1075A/C, rs1057910) and VKORC1 (-1639A/G, rs9923231) polymorphisms.</p>


Subject(s)
Adult , Aged , Aged, 80 and over , Aryl Hydrocarbon Hydroxylases , Genetics , Cytochrome P-450 CYP2C9 , Dose-Response Relationship, Drug , Female , Freezing , Genotype , Genotyping Techniques , Humans , Male , Middle Aged , Mixed Function Oxygenases , Genetics , Polymorphism, Restriction Fragment Length , Vitamin K Epoxide Reductases , Warfarin , Therapeutic Uses , Young Adult
20.
Diagnóstico (Perú) ; 50(4): 192-195, oct.-dic. 2011. tab
Article in Spanish | LILACS, LIPECS | ID: lil-646579

ABSTRACT

Antecedentes: La deficiencia de 21-hidroxilasa es la causa de cerca del 95% de casos de hiperplasia suprarrenal congénita y en nuestro medio es responsable de 1 de cada 3000 consultas en el Instituto Nacional de Salud del Niño. El gen que codifica la producción de enzima 21OH es el CYP21B está localizado en el brazo corto del cromosoma 6. Objetivos: Conocer la frecuencia de las principales mutaciones en pacientes peruanos con las formas clásica y no clásica de HSC con deficiencia de 21-hidroxilasa, así como las deleciones del gen CYP21B. Materiales y pacientes: La técnica utilizada para el estudio genético fue la de la polymerase chain reaction (PCR) y se analizaron las mutaciones y delecciones más frecuentes encontradas tanto en Japón como en los EE.UU. de NA. Se han estudiado 20 pacientes que fueron diagnosticados y seguidos en el Servicio de Endocrinología del Instituto de Salud del Niño, 13 padres, 13 madres y 5 hermanos para el estudio genético. Cuando el padre estaba ausente se estudió un familiar de la línea paterna de sexo masculino abuelo o tío, en nuestro estudio se hizo con un abuelo paterno. Resultados: De los 20 pacientes estudiados, 11 fueron de sexo femenino y 9 de sexo masculino; de otro lado, 16 pacientes eran portadores de la variedad perdedora de sal y 4 de la forma virilizante. Tres hermanos de los pacientes no estuvieron afectados y 2 presentaron alteraciones genéticas y cuadro clínico (nacieron cuando se realizaba este estudio). Los pacientes portadores de HSC en el Perú presentan en la variedad perdedora de sal como mutaciones más frecuentes la I172N (7,42%), la P30L (7,42%) y la R356W (7,42%), 7,42% corresponden a deleciones y 21,4% a alteraciones genéticas no detectadas con las técnicas utilizadas; en el caso de la variedad virilizante simple 14,28% de mutaciones corresponden al alelo V28IL, 52,14% a deleciones y 28,57 a alteraciones genéticas no detectadas con la técnica empleada...


Background: Congenital adrenal hyperplasia (CAH) in 95% of cases is due to 21-hydroxylase deficiency and of one of every 3000 consultations in the Instituto Nacional de Salud del Niño in Lima, Peru. Deficiency of 21OH is related to mutations in gene CYP21B, located in chromosome 6p. Objectives: To assess the frequency of the main disorders of the CYP 21B gene in peruvian patients with classic and non classic CAH due to 21-hydroxylase deficiency. Methods: 20 patients with CAH of the Endocrinology unit of the IESN were studied. Also, 13 fathers, 13 mothers and 5 brothers where studied. In one case, the paternal grandfather was studied in absence of the father. Polymerase chain reaction (PCR) technique was used in order to determine the mutations and deletions of the samples. The results were compared with those of similar studies in Japan and USA. Results: The frequency by sex in the 20 patients was: 11 females and 9 males. 16 patients had salt loss- CAH and 4 had simple virilizing CAH. Gene disorders and clinical characteristics of CAH were detected in 2 siblings (both were born during the study). The most frequent allele mutations found in salt loss-CAH were I172N (7,42%), P30L (7,42%) and R356W (7,42%). Deletions were detected in 7,42% of cases and unknown gene disorders in 21,4%. In the virilizing CAH cases, the most frequent allele mutation was V281L (14,28), deletions were present in 52,14% and were detected in 28,57% of cases the gene anomaly could not be detected. Conclusions: The frequency of allele mutations in CAH was similar to those found in Japan and USA. The unknown gene disorders may be mutations the gene anomaly could not be detected.


Subject(s)
Humans , Male , Female , Family , Adrenal Hyperplasia, Congenital/genetics , Mixed Function Oxygenases , Patients , Polymerase Chain Reaction
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