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1.
Orphanet J Rare Dis ; 14(1): 297, 2019 12 26.
Artigo em Inglês | MEDLINE | ID: mdl-31878983

RESUMO

BACKGROUND: Primary hypertrophic osteoarthropathy (PHO) is a rare disease related to HPGD and SLCO2A1 gene mutation. Gastrointestinal involvement of PHO is even rarer with unknown pathogenesis. Clinical features of GI complication in PHO mimics other auto-immune based bowel entities, such as inflammatory bowel diseases and cryptogenic multifocal ulcerous stenosing enteritis (CMUSE). We aimed to analyze the clinical, genetic, radiological and pathological features of Chinese patients with PHO and determine the difference between PHO patients presenting with and without GI involvement. METHODS: We reported two PHO cases with gastrointestinal involvement and reviewed all the studies of PHO in Chinese population published from January 1, 2000, to April 30, 2018. Clinical and genetic presentations of PHO in Chinese patients were analyzed. We compared the characteristics of those patients with gastrointestinal involvement against those without. RESULTS: The two patients were both males with complete-form PHO for more than 10 years. GI related symptoms included diarrhea, chronic gastrointestinal hemorrhage, incomplete intestinal obstruction, anemia, and edema, which were unresponsive to etoricoxib treatment. Radiological examinations revealed segmental intestinal stenosis and thickened intestinal wall. Endoscopic findings included multiple ulcers and mucosal inflammation. Both patients had mutations of SLCO2A1 according to sequence analysis. The surgical pathology revealed chronic inflammation involving the intestinal mucosa and submucosa, similar to histological changes in CMUSE. According to the systemic review of 158 Chinese patients with PHO, 17.2% had gastrointestinal involvement, including peptic ulcer, gastric polyps, hypertrophic gastritis, and segmental intestinal stenosis. Patients with gastrointestinal involvement were more likely to have anemia (40.0% vs. 4.5%, P < 0.001), hypoalbuminemia (16.7% vs. 0.9%, P = 0.003), and myelofibrosis (19.0% vs. 0.9%, P = 0.002) than those without. Most patients with gastrointestinal complication had SLCO2A1 mutation (86.7%, 13 /15). CONCLUSIONS: Digestive tract involvement is uncommon in patients with PHO and often presents with anemia, and hypoalbuminemia resulted from intestinal inflammation. The intestinal pathologic characteristics are distinct from Crohn's disease but similar to CMUSE. Mutations in SLCO2A1 might be the pathogenic cause of GI involvement of PHO. NSAIDs may not be effective for PHO patients with gastrointestinal complications.

2.
Sensors (Basel) ; 19(1)2019 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-30626144

RESUMO

Doppler parameter estimation and compensation (DPEC) is an important technique for airborne SAR imaging due to the unpredictable disturbance of real aircraft trajectory. Traditional DPEC methods can be only applied for broadside, small- or medium-squint geometries, as they at most consider the spatial variance of the second-order Doppler phase. To implement the DPEC in very-high-squint geometries, we propose an extended multiple aperture mapdrift (EMAM) method in this paper for better accuracy. This advantage is achieved by further estimating and compensating the spatial variation of the third-order Doppler phase, i.e., the derivative of the Doppler rate. The main procedures of the EMAM, including the steps of sub-view image generation, sliding-window-based cross-correlation, and image-offset-based Doppler parameter estimation, are derived in detail, followed by the analyses for the EMAM performance. The presented approach is evaluated by both computer simulations and real airborne data.

3.
Sci China Life Sci ; 61(11): 1333-1351, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-29797182

RESUMO

Multi-walled carbon nanotubes (MWCNTs) have wide application prospects but also exhibit notable biotoxicity that is tightly associated with macrophages. Macrophages simultaneously act as initiators and defenders in MWCNT-induced organ lesions, and targeting macrophages with MWCNTs may be a potential immunotherapy and oncotherapy approach. This review focuses on the impacts of MWCNTs on macrophages and further discusses the influence of MWCNT characteristics on their bioactivity. Based on existing studies, MWCNTs stimulate macrophage migration, induce secretion of various cytokines and activate inflammatory pathways in macrophages, especially NLRP3-mediated IL-1ß production. This inflammatory state, together with the oxidative stress and cell membrane lesions induced by MWCNTs, contributes to decreased phagocytic ability and cell viability, which finally results in cell apoptosis and necrosis. A series of intracellular and systemic components, such as toll-like receptor, high-mobility group box 1, Rho-associated kinases, scavenger receptor and complement components, may be involved in the above-mentioned cell-MWCNT interactions. The characteristics of MWCNTs can influence their bioactivity in macrophages both mechanically and chemically. The size (length and/or diameter), functionalization, purification and even the experimental method can affect the influence of MWCNTs on macrophages, and a better understanding of these MWCNT characteristics may benefit utilization of this nanomaterial in associated nanomedical applications.


Assuntos
Macrófagos/efeitos dos fármacos , Nanotubos de Carbono/química , Nanotubos de Carbono/toxicidade , Animais , Apoptose/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Citocinas/metabolismo , Humanos , Inflamação/metabolismo , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/patologia , Modelos Biológicos , Estresse Oxidativo/efeitos dos fármacos
4.
J Chromatogr B Analyt Technol Biomed Life Sci ; 1087-1088: 29-35, 2018 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-29704798

RESUMO

A sensitive, selective, and reliable LC-MS/MS method was developed and validated for simultaneous quantification of venlafaxine (VEN) and its 5 metabolites (ODV, NDV, NNDDV, OHV and NODDV) in rat plasma. The calibration ranges are 15.0 to 6000 ng/mL for VEN, 1.00 to 400 ng/mL for ODV, 5.00 to 2000 ng/mL for NDV, 1.00 to 400 ng/mL for NNDDV, 10.0 to 4000 ng/mL for OHV, and 0.200 to 20.0 ng/mL for NODDV. Briefly, 50 µL of rat plasma was extracted using liquid-liquid extraction (LLE) with methyl tert-butyl ether (MTBE). The analytes were separated on an Agilent SB-Phenyl (50 mm × 4.6 mm, 3.5 µm) column using a binary gradient of 0.1% formic acid in water versus 0.1% formic acid in acetonitrile at a flow rate of 0.8 mL/min. The method was validated following FDA guidance for bioanalytical method validation. Validated method was successfully applied to a pharmacokinetic study of VEN orally administered to rats.


Assuntos
Cromatografia Líquida/métodos , Espectrometria de Massas em Tandem/métodos , Cloridrato de Venlafaxina/sangue , Cloridrato de Venlafaxina/farmacocinética , Animais , Estabilidade de Medicamentos , Feminino , Modelos Lineares , Ratos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Cloridrato de Venlafaxina/química
5.
Bioanalysis ; 9(7): 505-516, 2017 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-28339299

RESUMO

The 10th Global CRO Council (GCC) Closed Forum was held in Orlando, FL, USA on 18 April 2016. In attendance were decision makers from international CRO member companies offering bioanalytical services. The objective of this meeting was for GCC members to meet and discuss scientific and regulatory issues specific to bioanalysis. The issues discussed at this closed forum included reporting data from failed method validation runs, GCP for clinical sample bioanalysis, extracted sample stability, biomarker assay validation, processed batch acceptance criteria, electronic laboratory notebooks and data integrity, Health Canada's Notice regarding replicates in matrix stability evaluations, critical reagents and regulatory approaches to counteract fraud. In order to obtain the pharma perspectives on some of these topics, the first joint CRO-Pharma Scientific Interchange Meeting was held on 12 November 2016, in Denver, Colorado, USA. The five topics discussed at this Interchange meeting were reporting data from failed method validation runs, GCP for clinical sample bioanalysis, extracted sample stability, processed batch acceptance criteria and electronic laboratory notebooks and data integrity. The conclusions from the discussions of these topics at both meetings are included in this report.


Assuntos
Biomarcadores/análise , Técnicas de Química Analítica/normas , Coleta de Dados/normas , Guias como Assunto , Preparações Farmacêuticas/análise , Estabilidade de Medicamentos , Regulamentação Governamental , Humanos , Relatório de Pesquisa
6.
Artigo em Inglês | MEDLINE | ID: mdl-26218770

RESUMO

N-acetylneuraminic acid (Neu5Ac or NANA) is the most predominant sialic acid in mammals. As a terminal component in many glycoproteins and glycolipids, sialic acid is believed to be an important biomarker related to various diseases. Its precursor, N-acetylmannosamine (ManNAc), is being investigated as a potential treatment for GNE myopathy. In this work, we developed two highly sensitive and selective liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods for the quantitation of ManNAc and free Neu5Ac in human plasma. A fit-for-purpose approach was adopted during method validation and sample analysis. To measure the endogenous compounds and overcome the interference from plasma samples, a surrogate matrix that contained 5% bovine serum albumin (BSA) was used for the preparation of calibration standards and certain levels of quality control (QC) samples. QC samples at higher concentrations were prepared in the authentic matrix (human plasma) to best mimic incurred samples. For both methods, an Ostro 96-well phospholipid removal plate was used for sample extraction, which efficiently removed the phospholipids from the plasma samples prior to LC injection, eliminated matrix effect, and improved sensitivity. Chromatographic separation was achieved using hydrophilic interaction chromatography (HILIC) and gradient elution in order to retain the two polar compounds. The lower limit of quantitation (LLOQ) for ManNAc and Neu5Ac was 10.0 and 25.0ng/mL, respectively. The overall accuracy of the two assays was within 100%±8.3% based on three levels of QC samples. Inter- and intra-run precision (coefficient of variation (%CV)) across three analytical runs was less than 6.7% for ManNAc and less than 10.8% for Neu5Ac. These methods have been validated to support clinical studies.


Assuntos
Cromatografia Líquida/métodos , Hexosaminas/sangue , Ácido N-Acetilneuramínico/sangue , Espectrometria de Massas em Tandem/métodos , Estabilidade de Medicamentos , Humanos , Interações Hidrofóbicas e Hidrofílicas , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
7.
J Chromatogr B Analyt Technol Biomed Life Sci ; 877(8-9): 799-806, 2009 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-19237324

RESUMO

Dried blood spots (DBSs) technology was evaluated in an assay for the quantitation of dextromethorphan (DM) and its metabolite, dextrorphan (DT), in human whole blood using high performance liquid chromatography with tandem mass spectrometry method (LC-MS/MS). Both the parent drug and metabolite were spiked in the blood matrix and subsequently allowed to dry on a specimen collection card. The dried blood spots were removed using a manual punch and then extracted into methyl tert-butyl ether (MTBE). The organic supernatant was transferred and evaporated and the residue was reconstituted in 20% acetonitrile. The overall method recovery of DM and DT was 87.8% and 95.4%, respectively. The assay was linear over the concentration range of 0.2-200ng/mL for both analytes. Several factors that potentially affect DBS assay quantitation were investigated, such as punch size, DBS sample punch-out location, and the volume of the blood sample pipetted on the specimen collection cards. The study determined that punch size does not affect assay quantitation accuracy. Indeed, a larger punch size increases the sensitivity due to the larger sampled blood spots. Sampling from different location on the specimen collection cards shows no significant variation for both drugs. The study also shows that acceptable results can be achieved with some variation of the sample volume, which allows a simple blood sampling procedure at the test sites. To achieve the similar lower limit of quantitation (LLOQ) as the plasma assay, several blood spots at the same concentration level were stacked together and extracted. Bioanalytical assays using the DBS technique are promising given the advantages of the method over the plasma assay.


Assuntos
Coleta de Amostras Sanguíneas/métodos , Dextrometorfano/sangue , Dextrorfano/sangue , Cromatografia Líquida de Alta Pressão , Humanos , Espectrometria de Massas em Tandem
8.
J Chromatogr A ; 1192(2): 230-8, 2008 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-18395729

RESUMO

A sensitive and selective bioanalytical assay was developed and validated for the determination of enantiomeric molindone in human plasma using high-performance liquid chromatography-tandem mass spectrometry along with supported liquid extraction procedures. The chiral separation was evaluated and optimized on macrocyclic antibiotic type chiral stationary phases (CSPs) based on teicoplanin aglycone (Chirobiotic TAG) in polar organic, polar ionic, and reversed-phase mode chromatography, respectively. Complete baseline separation was achieved on a Chirobiotic TAG column under isocratic condition in reversed-phase chromatography. The method validation was conducted using a Chirobiotic TAG column (100 mm x 2.1 mm) over the curve range 0.100-100 ng/ml for each molindone enantiomer using 0.0500 ml of plasma sample. The flow rate was 0.8 ml/min and the total run time was 9 min. Supported liquid extraction in a 96-well plate format was used for sample preparation. Parameters including recovery, matrix effect, linearity, sensitivity, specificity, carryover, precision, accuracy, dilution integrity, and stability were evaluated. The intra- and inter-day precision and accuracy of the quality control samples at low, medium, and high concentration levels were RSD

Assuntos
Antipsicóticos/sangue , Compostos Macrocíclicos/química , Molindona/sangue , Antibacterianos/química , Antipsicóticos/química , Calibragem , Cromatografia Líquida de Alta Pressão , Interpretação Estatística de Dados , Congelamento , Humanos , Molindona/química , Controle de Qualidade , Padrões de Referência , Reprodutibilidade dos Testes , Estereoisomerismo , Espectrometria de Massas em Tandem
9.
J Pharm Biomed Anal ; 43(1): 277-84, 2007 Jan 04.
Artigo em Inglês | MEDLINE | ID: mdl-16887315

RESUMO

A new analytical method is described here for the quantitation of anti-inflammatory drug cyclosporin A (CyA) in monkey and rat plasma. The method used tetrahydrofuran (THF)-water mobile phases to elute the analyte and internal standard, cyclosporin C (CyC). The gradient mobile phase program successfully eluted CyA into a sharp peak and therefore improved resolution between the analyte and possible interfering materials compared with previously reported analytical approaches, where CyA was eluted as a broad peak due to the rapid conversion between different conformers. The sharp peak resulted from this method facilitated the quantitative calculation as multiple smoothing and large number of bunching factors were not necessary. The chromatography in the new method was performed at 30 degrees C instead of 65-70 degrees C as reported previously. Other advantages of the method included simple and fast sample extraction-protein precipitation, direct injection of the extraction supernatant to column for analysis, and elimination of evaporation and reconstitution steps, which were needed in solid phase extraction or liquid-liquid extraction reported before. This method is amenable to high-throughput analysis with a total chromatographic run time of 3 min. This approach has been verified as sensitive, linear (0.977-4000 ng/mL), accurate and precise for the quantitation of CyA in monkey and rat plasma. However, compared with the usage of conventional mobile phases, the only drawback of this approach was the reduced detection response from the mass spectrometer that was possibly caused by poor desolvation in the ionization source. This is the first report to demonstrate the advantages of using THF-water mobile phases to elute CyA in liquid chromatography.


Assuntos
Ciclosporina/sangue , Imunossupressores/sangue , Animais , Área Sob a Curva , Calibragem , Cromatografia Líquida , Ciclosporina/farmacocinética , Relação Dose-Resposta a Droga , Furanos , Imunossupressores/farmacocinética , Macaca fascicularis , Masculino , Espectrometria de Massas , Controle de Qualidade , Ratos , Padrões de Referência , Reprodutibilidade dos Testes , Água
10.
Biomed Chromatogr ; 20(6-7): 597-604, 2006.
Artigo em Inglês | MEDLINE | ID: mdl-16779771

RESUMO

S-phenylmercapturic acid is widely accepted as a specific biomarker for the evaluation of benzene exposure. Here, we describe a fast, specific and sensitive high-performance liquid achromatography coupled with tandem mass spectrometry (LC-MS/MS) method that has been developed and validated for the determination of S-phenylmercapturic acid in human urine. Isotope-labeled S-phenylmercapturic acid-d5 was used as internal standard to improve the method ruggedness. The fully automated solid-phase extraction method on a 96-well Oasis MAX (mix-mode anion exchange) plate was employed to clean up the urine samples before analysis. The rapid LC-MS/MS analysis of extracted samples was achieved on a Genesis C18 column with a run time of only 3 min. Negative electrospray ionization with multiple reaction monitoring (ESI-MRM) mode was used to detect S-phenylmercapturic acid (m/z 238 --> 109) and S-phenylmercapturic acid -d5 (m/z 243 --> 114). The method fulfils all the standard requirements of method validation. The calibration curve was linear within the concentration range 0.400-200 ng/mL. The method performed accurately and precisely in validation with <7.5% relative error and <6.5% relative standard deviation of quality control samples. The method efficacy was also verified by the analysis of urine samples from 12 smokers and 12 non-smokers. With the fully automated sample cleanup procedure and the fast LC-MS/MS analysis, a sample analysis throughput of 384 samples per day could be achieved.


Assuntos
Acetilcisteína/análogos & derivados , Cromatografia Líquida de Alta Pressão/métodos , Espectrometria de Massas/métodos , Acetilcisteína/urina , Automação , Feminino , Humanos , Padrões de Referência , Sensibilidade e Especificidade
11.
Artigo em Inglês | MEDLINE | ID: mdl-16213451

RESUMO

In the present work, for the first time, a liquid chromatographic method with tandem mass spectrometric detection (LC-MS/MS) for the simultaneous analysis of norethindrone, and ethinyl estradiol, was developed and validated over the concentration range of 50-10000pg/ml and 2.5-500pg/ml, respectively, using 0.5 ml of plasma sample. Norethindrone, ethinyl estradiol, and their internal standards norethindrone-(13)C2, and ethinyl estradiol-d4, were extracted from human plasma matrix with n-butyl chloride. After evaporation of the organic solvent, the extract was derivatized with dansyl chloride and the mixture was injected onto the LC-MS/MS system. The gradient chromatographic elution was achieved on a Genesis RP-18 (50 mm x 4.6 mm, 3 microm) column with mobile phase consisted of acetonitrile, water and formic acid. The flow rate was 1.0 ml/min and the total run time was 5.0 min. Important parameters such as sensitivity, linearity, matrix effect, reproducibility, stability, carry-over and recovery were investigated during the validation. The inter-day precision and accuracy of the quality control samples at low, medium and high concentration levels were <6.8% relative standard deviation (RSD) and 4.4% relative error (RE) for norethindrone, and 4.2% RSD and 5.9% RE for ethinyl estradiol, respectively. Chromatographic conditions were optimized to separate analytes of interest from the potential interference peaks, arising from the derivatization. This method could be used for pharmacokinetic and drug-drug interaction studies in human subjects.


Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Etinilestradiol/sangue , Espectrometria de Massas/métodos , Noretindrona/sangue , Compostos de Dansil/química , Estabilidade de Medicamentos , Etinilestradiol/química , Humanos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
12.
Rapid Commun Mass Spectrom ; 19(22): 3331-8, 2005.
Artigo em Inglês | MEDLINE | ID: mdl-16235235

RESUMO

This article presents an analytical approach that used chemical derivatization to enhance mass spectrometric (MS) response in electrospray ionization (ESI) mode of 1-hydroxypyrene (1-OHP), a commonly used biomarker to monitor human exposure to polycyclic aromatic hydrocarbons (PAHs). The enhancement successfully enabled the desired detection of 50 pg/mL in human urine. The introduction of an MS-friendly dansyl group to 1-OHP enhanced both ionization efficiency in the ESI source and collision-activated dissociation (CAD) in the collision cell. The response increase was estimated to be at least 200-fold, and enabled the reduction of sample size to only 100 microL. The selective MS detection also facilitated a fast (run time 3 min) liquid chromatography (LC) method which successfully resolved the analyte and interferences. The sample processing procedure included enzymatic hydrolysis of glucuronide and sulfate conjugates, liquid-liquid extraction, derivatization with dansyl chloride and a final liquid-liquid extraction to generate clean extracts for LC/MS/MS analysis. This approach has been validated as sensitive, linear (50-1000 pg/mL), accurate and precise for the quantitation of 1-OHP in human urine. This is the first report of using chemical derivatization to enhance MS/MS detection with fast chromatography in the determination of 1-OHP in human urine.


Assuntos
Biomarcadores/urina , Cromatografia Líquida/métodos , Hidrocarbonetos Policíclicos Aromáticos/urina , Pirenos/análise , Espectrometria de Massas por Ionização por Electrospray/métodos , Calibragem , Humanos , Estrutura Molecular
13.
Biomed Chromatogr ; 19(5): 385-93, 2005 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-15651086

RESUMO

A hydrophilic interaction liquid chromatographic method with tandem mass spectrometry for the determination of atenolol, a beta-blocking agent, in human plasma has been developed and validated over the curve range of 10--2000 ng/mL. The assay was based on protein precipitation followed by evaporation of the extraction solvent, reconstitution with acetonitrile, and chromatography on an Hypersil silica column (50 x 4.6 mm) using a low aqueous--high organic mobile phase. The mobile phase consists of 85% acetonitrile, 15% water, 0.5% acetic acid and 0.04% trifluoroacetic acid and runs isocratically at a flow rate of 2.0 mL/min. The column ef fluent was split so that 50% of it was transferred into the LC-MS/MS interface operated in positive electrospray ionization mode. The chromatographic run time was 2.0 min per injection. Atenolol and the internal standard, atenolol-d(7), showed a retention time of 1.0 min. The inter-day and intra-day precision and accuracy of the quality control samples were <5.3% relative standard deviation and <8.0% relative error, respectively. To explore the application of the current method for the analysis of other beta-blocking agents, propranolol and metoprolol were tested under the same chromatographic conditions with retention times of 0.68 and 0.75 min, respectively. The present method could be used for therapeutic drug monitoring, pharmacokinetic and drug--drug interaction studies of beta-blocking agents.


Assuntos
Antagonistas Adrenérgicos beta/sangue , Atenolol/sangue , Cromatografia Líquida de Alta Pressão/métodos , Estabilidade de Medicamentos , Humanos , Espectrometria de Massas , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
14.
Drug Metab Dispos ; 31(9): 1161-9, 2003 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-12920172

RESUMO

Seven dog cytochromes p450 (p450s) were heterologously expressed in baculovirus-Sf21 insect cells. Of all enzymes examined, CYP1A1 exhibited high 7-ethoxyresorufin O-deethylase activity (low Km enzyme, 1 microM). CYP2B11 and CYP3A12 effectively catalyzed the N1-demethylation and C3-hydroxylation of diazepam (and its derivatives), whereas CYP3A12 and CYP2D15 catalyzed exclusively the N- and O-demethylation, respectively, of dextromethorphan. However, no saturation velocity curves for the N-demethylation of dextromethorphan (up to 500 microM) were achieved, suggesting a high Km for CYP3A12. In contrast to CYP3A12, the CYP2D15-dependent O-demethylation of dextromethorphan was a low Km process (Km = 0.7 microM), similar to that in dog liver microsomes (Km = 2.3 microM). CYP2D15 was also capable of metabolizing bufuralol (1'-hydroxylation), with a Km of 3.9 microM, consistent with that obtained with dog liver microsomes. CYP3A12 was shown to primarily oxidize testosterone at 16alpha-, 2alpha/2beta-, and 6beta-positions. Selectivity of CYP3A12 was observed toward testosterone 6beta-(Km = 83 microM) and 2alpha/2beta-hydroxylations (Km = 154 microM). However, the 16alpha-hydroxylation of testosterone was catalyzed by CYP2C21 also (Km = 6.4 microM for CYP2C21). Therefore, the 6beta- and 16alpha-hydroxylation of testosterone can potentially be employed as markers of CYP3A12 and CYP2C21 (at low concentration), respectively. CYP2C21 was also capable of catalyzing diclofenac 4'-hydroxylation, although some activity was detected with CYP2B11. Surprisingly, none of the p450s selectively metabolized (S)-mephenytoin 4'-hydroxylation. The results described herein are a first step toward the systematic evaluation of a panel of dog p450s and the development of dog p450 isoenzyme-selective marker substrates, as well as providing useful information on prediction and extrapolation of the results from in vitro to in vivo and from dog to human.


Assuntos
Sistema Enzimático do Citocromo P-450/biossíntese , Preparações Farmacêuticas/metabolismo , Animais , Baculoviridae/genética , Linhagem Celular , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Cães , Insetos/citologia , Isoenzimas/biossíntese , Isoenzimas/genética , Isoenzimas/metabolismo , Cinética , Masculino , Microssomos/enzimologia , Microssomos/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/metabolismo , Especificidade por Substrato
15.
Sheng Li Xue Bao ; 54(6): 519-24, 2002 Dec 25.
Artigo em Chinês | MEDLINE | ID: mdl-12506326

RESUMO

This study was intended to evaluate the effects of hypoxic exposure on gene expression and coordination of cytochrome oxidase (COX) subunits I (COX I) and IV (COX IV) encoded by mtDNA and nDNA respectively in rat cerebral cortex. Male Wistar rats were exposed to hypoxia in a hypobaric chamber simulating high altitude at 5000 m for 2, 5, 15 and 30 d. Control rats were fed outside the hypobaric chamber (the height was 300 m above sea level). Rats were sacrificed and mitochondria from cerebral cortex were isolated by differential centrifugation at each time point. COX I and COX IV proteins in isolated rat cerebral cortex mitochondria were detected by Western blot analysis and mRNA in the cerebral cortex by RT-PCR. The ratios of protein and mRNA were used to estimate the coordinative expression of two subunits. The results showed that COX I mRNA increased significantly at 2 and 5 d, and decreased to the control level at 15 and 30 d; COX IV mRNA remarkably increased at 2, 5 and 15 d, and dropped below the control level at 30 d. The mRNA ratio of COX IV to COX I reached a peak at 15 d, but showed no differences between other time points. The Western blot analysis of COX I and COX IV in isolated rat cerebral cortex mitochondria showed no obvious changes during hypoxic exposure. Our findings demonstrate that hypoxia can affect mRNA expression of COX I and COX IV and their coordination, while protein expression of both subunits are stable and coordinative. This study suggests that the expression of COX I and COX IV proteins during hypoxic exposure is coordinately regulated by post-transcriptional mechanisms.


Assuntos
Córtex Cerebral/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Hipóxia/metabolismo , Animais , Regulação Enzimológica da Expressão Gênica , Masculino , Mitocôndrias/metabolismo , Ratos , Ratos Wistar
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