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1.
Artigo em Inglês | MEDLINE | ID: mdl-31235362

RESUMO

LC-MS quantification of drug metabolites is sometimes impeded by the availability of internal standards that often requires customized synthesis and/or extensive purification. Although isotopically labeled internal standards are considered ideal for LC-MS/MS based quantification, de novo synthesis using costly isotope-enriched starting materials makes it impractical for early stage of drug discovery. Therefore, quick access to these isotope-enriched compounds without chemical derivatization and purification will greatly facilitate LC-MS/MS based quantification. Herein, we report a novel 18O-labeling technique using metabolizing enzyme carboxylesterase (CES) and its potential application in metabolites quantification study. Substrates of CES typically undergo a two-step oxygen exchange with H218O in the presence of the enzyme, generating singly- and doubly-18O-labeled carboxylic acids; however, unexpected hydrolytic behavior was observed for three of the test compounds - indomethacin, piperacillin and clopidogrel. These unusual observations led to the discovery of several novel hydrolytic mechanisms. Finally, when used as internal standard for LC-MS/MS based quantification, these in situ labeled compounds generated accurate quantitation comparable to the conventional standard curve method. The preliminary results suggest that this method has potential to eliminate laborious chemical synthesis of isotope-labeled internal standards for carboxylic acid-containing compounds, and can be developed to facilitate quantitative analysis in early-stage drug discovery.

2.
J Pharm Sci ; 107(7): 1973-1986, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29548977

RESUMO

Methodology for analysis of a microdosing drug cocktail designed to evaluate the contribution of drug transporters and drug metabolizing enzymes to disposition was developed using liquid chromatography-mass spectrometry-based detection. Fast and sensitive methods were developed and qualified for the quantification of statins (pitavastatin, pitavastain lactone, rosuvastatin, atorvastatin, 2-hydroxy, and 4-hydroxy atorvastatin), midazolam, and dabigatran in human plasma. Chromatographic separation was accomplished using reversed-phase liquid chromatography or hydrophilic interaction liquid chromatography with gradient elution and detection by tandem mass spectrometry in the positive ionization mode using electrospray ionization. The lower limit of quantitation (LLOQ) for the statins assay was 1 pg/mL for the 6 analytes with a linear range from 1 to 1000 pg/mL processing 250 µL plasma sample. The midazolam assay LLOQ was 0.5 pg/mL with a linear range of 0.5 to 1000 pg/mL. For the dabigatran assay, the LLOQ was 10 pg/mL with a linear range of 10 to 5000 pg/mL processing 100 µL plasma sample. The intraday and interday precision and accuracy of the assays were within acceptable ranges, and the assays were successfully applied to support a study where a microdose cocktail was dosed to healthy human subjects for simultaneous assessment of clinical drug-drug interactions mediated by major drug transporters and CYP3A.

3.
Drug Metab Dispos ; 44(3): 428-34, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26712818

RESUMO

The cholesteryl ester transfer protein (CETP) inhibitor anacetrapib exhibits a long terminal half-life (t½) in humans; however, the dispositional mechanisms that lead to this long t½ are still being elucidated. As it is hypothesized that disposition into adipose tissue and binding to CETP might play a role, we sought to delineate the relative importance of these factors using a preclinical animal model. A multiple-dose pharmacokinetic study was conducted in C57BL6 wild-type (WT) lean, WT diet-induced obese (DIO), natural flanking region (NFR) CETP-transgenic lean, and NFR-DIO mice. Mice were dosed orally with 10 mg/kg anacetrapib daily for 42 days. Drug concentrations in blood, brown and white adipose tissue, liver, and brain were measured up to 35 weeks postdose. During dosing, a 3- to 9-fold accumulation in 72-hour postdose blood concentrations of anacetrapib was observed. Drug concentrations in white adipose tissue accumulated ∼20- to 40-fold, whereas 10- to 17-fold accumulation occurred in brown adipose and approximately 4-fold in liver. Brain levels were very low (<0.1 µM), and a trend of accumulation was not seen. The presence of CETP as well as adiposity seems to play a role in determining the blood concentrations of anacetrapib. The highest blood concentrations were observed in NFR DIO mice, whereas the lowest concentrations were seen in WT lean mice. In adipose and liver tissue, higher concentrations were seen in DIO mice, irrespective of the presence of CETP. This finding suggests that white adipose tissue serves as a potential depot and that disposition into adipose tissue governs the long-term kinetics of anacetrapib in vivo.


Assuntos
Tecido Adiposo/metabolismo , Proteínas de Transferência de Ésteres de Colesterol/antagonistas & inibidores , Proteínas de Transferência de Ésteres de Colesterol/metabolismo , Oxazolidinonas/metabolismo , Animais , Dieta , Cinética , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Obesidade/metabolismo
4.
AAPS J ; 15(2): 498-504, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23355301

RESUMO

Rolofylline is a potent, selective adenosine A1 receptor antagonist that was under development for the treatment of patients with acute congestive heart failure and renal impairment. Rolofylline is metabolized primarily to the pharmacologically active M1-trans and M1-cis metabolites (metabolites) by cytochrome P450 (CYP) 3A4. The aim of this investigation was to provide a pharmacokinetic (PK) model for rolofylline and metabolites following intravenous administration to healthy volunteers. Data included for this investigation came from a randomized, double-blind, dose-escalation trial in four groups of healthy volunteers (N=36) where single doses of rolofylline, spanning 1 to 60 mg ,were infused over 1-2 h. The rolofylline and metabolite data were analyzed simultaneously using NONMEM. The simultaneous PK model comprised, in part, a two-compartment linear PK model for rolofylline, with estimates of clearance and volume of distribution at steady-state of 24.4 L/h and 239 L, respectively. In addition, the final PK model contained provisions for both conversion of rolofylline to metabolites and stereochemical conversion of M1-trans to M1-cis. Accordingly, the final model captured known aspects of rolofylline metabolism and was capable of simultaneously describing the PK of rolofylline and metabolites in healthy volunteers.


Assuntos
Antagonistas do Receptor A1 de Adenosina/farmacocinética , Modelos Biológicos , Xantinas/farmacocinética , Antagonistas do Receptor A1 de Adenosina/administração & dosagem , Antagonistas do Receptor A1 de Adenosina/sangue , Adolescente , Adulto , Biotransformação , Citocromo P-450 CYP3A/metabolismo , Método Duplo-Cego , Humanos , Infusões Intravenosas , Modelos Lineares , Masculino , Taxa de Depuração Metabólica , Xantinas/administração & dosagem , Xantinas/sangue , Adulto Jovem
5.
Intensive Care Med ; 38(4): 642-8, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22231174

RESUMO

PURPOSE: To assess coagulation status and factor Xa inhibition in surgical intensive care unit (ICU) patients administered prophylactic unfractionated heparin for venous thromboembolism (VTE) prophylaxis. METHODS: We conducted a randomized, single-blind study at a tertiary academic medical center. Included were patients 18 years and older admitted to the surgical ICU directly after major abdominal surgery. Exclusion criteria included significant bleeding risk, preoperative anticoagulation, or history of heparin-induced thrombocytopenia. Patients were randomized to two regimens for VTE prophylaxis: standard of care unfractionated heparin, 5,000 units subcutaneously three times daily (SQH) versus unfractionated heparin via intravenous infusion, titrated to an activated partial thromboplastin time of 40-45 s (IVH). Blood samples were taken prior to surgical incision on day 0 and daily for 5 days after surgery. Samples were analyzed for factor Xa inhibition and viscoelastic whole blood clotting parameters (Sonoclot analyzer). RESULTS: A total of 50 patients were randomized to either SQH or IVH. The majority of patients had cancer. Patients in the SQH group had no detectable peak anti-factor Xa (aFXa) activity for 5 days after surgery, while patients in the IVH group had statistically elevated levels compared to the SQH group on days 3-5. SQH patients demonstrated a hypercoagulable profile on Sonoclot, while IVH patients displayed a normal profile. CONCLUSIONS: Standard of care subcutaneous dosing of unfractionated heparin for VTE prophylaxis in surgical ICU patients leads to subtherapeutic levels of factor Xa inhibition.


Assuntos
Anticoagulantes/administração & dosagem , Cuidados Críticos , Heparina/administração & dosagem , Tromboembolia Venosa/prevenção & controle , APACHE , Análise de Variância , Testes de Coagulação Sanguínea , Inibidores do Fator Xa , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Complicações Pós-Operatórias/prevenção & controle , Estudos Prospectivos , Método Simples-Cego , Estatísticas não Paramétricas , Resultado do Tratamento
6.
Am J Ther ; 17(1): 8-16, 2010 Jan-Feb.
Artigo em Inglês | MEDLINE | ID: mdl-20027105

RESUMO

Rolofylline is a potent, selective adenosine A1 receptor antagonist that was under development for the treatment of patients with acute decompensated heart failure and renal function impairment. The 30-mg dose of rolofylline administered by intravenous infusion over 4 hours for 3 days represented the anticipated recommended clinical regimen of rolofylline. This was a randomized, double-blind, double-dummy, placebo-controlled, three-period crossover study performed with a single 2-hour intravenous infusion of 60 mg rolofylline, placebo, or oral moxifloxacin in healthy subjects. Plasma samples were collected for determination of rolofylline, M1-trans, and M1-cis pharmacokinetic parameters. The upper limit of the two-sided 90% confidence interval for the placebo-adjusted least squares mean change from baseline in QTcF interval for rolofylline was less than 5 msec at every time point. Moxifloxacin demonstrated an increase in QTcF of greater than 10 msec at 2, 2.5, and 3 hours postdose, thus establishing the sensitivity of the assay to detect modest increases in QTcF interval. Mean Cmax values of 1947.4, 739.2, and 54.8 nM were attained for rolofylline and its metabolites M1-trans and M1-cis, respectively, which were 2.2- to 3.1-fold higher than historic Cmax values seen at the anticipated clinical dose and regimen. Adenosine A1 receptor antagonism from a single supratherapeutic intravenous dose of 60 mg rolofylline over 2 hours was generally well tolerated and did not prolong the QTcF interval relative to placebo.


Assuntos
Antagonistas do Receptor A1 de Adenosina , Diuréticos/efeitos adversos , Xantinas/efeitos adversos , Administração Oral , Adulto , Compostos Aza/efeitos adversos , Estudos Cross-Over , Diuréticos/administração & dosagem , Diuréticos/farmacocinética , Relação Dose-Resposta a Droga , Método Duplo-Cego , Eletrocardiografia , Feminino , Fluoroquinolonas , Humanos , Infusões Intravenosas , Síndrome do QT Longo/induzido quimicamente , Masculino , Pessoa de Meia-Idade , Moxifloxacina , Quinolinas/efeitos adversos , Xantinas/administração & dosagem , Xantinas/farmacocinética , Adulto Jovem
7.
Am J Ther ; 17(1): 53-60, 2010 Jan-Feb.
Artigo em Inglês | MEDLINE | ID: mdl-20027108

RESUMO

Rolofylline is a potent, selective adenosine A1 receptor antagonist that was under development for the treatment of patients with acute decompensated heart failure and renal function impairment. This was a phase I, randomized, open-label, 2-period, fixed-sequence study in 19 healthy adult volunteers to examine the effect of multiple intravenous rolofylline doses on the single-dose pharmacokinetics of midazolam, a sensitive CYP3A4 substrate. In period 1, subjects received a single oral dose of midazolam 7.5 mg on day 1. In period 2, subjects received 30 mg, 4-hour infusions of rolofylline (intended clinical dose and duration) once daily for 4 consecutive days; midazolam 7.5 mg was coadministered on day 4. The geometric mean ratios and 90% confidence intervals for AUC0-infinity and Cmax of midazolam in the presence/absence of rolofylline were 1.20 (1.12-1.29) and 1.17 (1.03-1.32), respectively. The apparent terminal half-life (t1/2) for midazolam was similar in the presence/absence of rolofylline (4.31 and 4.27 hours, respectively). The geometric mean ratios (90% confidence intervals) for AUC0-infinity and Cmax of 1'-hydroxymidazolam in the presence/absence of rolofylline were 1.04 (0.96-1.13) and 0.98 (0.84-1.14), respectively. The t1/2 for 1'-hydroxymidazolam was slightly higher in the presence relative to absence of rolofylline (4.24 and 3.17 hours, respectively). Multiple doses of intravenous rolofylline 30 mg for 4 days were generally well tolerated and did not result in clinically important inhibition of CYP3A4 as indicated by little or no change in the pharmacokinetics of midazolam.


Assuntos
Citocromo P-450 CYP3A/efeitos dos fármacos , Diuréticos/farmacologia , Midazolam/farmacocinética , Xantinas/farmacologia , Antagonistas do Receptor A1 de Adenosina , Adolescente , Adulto , Área Sob a Curva , Citocromo P-450 CYP3A/metabolismo , Diuréticos/administração & dosagem , Diuréticos/efeitos adversos , Esquema de Medicação , Interações de Medicamentos , Feminino , Meia-Vida , Humanos , Infusões Intravenosas , Masculino , Midazolam/análogos & derivados , Pessoa de Meia-Idade , Xantinas/administração & dosagem , Xantinas/efeitos adversos , Adulto Jovem
8.
Br J Clin Pharmacol ; 68(4): 535-45, 2009 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-19843057

RESUMO

AIMS: Anacetrapib is an orally active and potent inhibitor of CETP in development for the treatment of dyslipidaemia. These studies endeavoured to establish the safety, tolerability, pharmacokinetics and pharmacodynamics of rising single doses of anacetrapib, administered in fasted or fed conditions, and to preliminarily assess the effect of food, age, gender and obesity on the single-dose pharmacokinetics and pharmacodynamics of anacetrapib. METHODS: Safety, tolerability, anacetrapib concentrations and CETP activity were evaluated. RESULTS: Anacetrapib was rapidly absorbed, with peak concentrations occurring at approximately 4 h post-dose and an apparent terminal half-life ranging from approximately 9 to 62 h in the fasted state and from approximately 42 to approximately 83 h in the fed state. Plasma AUC and C(max) appeared to increase in a less than approximately dose-dependent manner in the fasted state, with an apparent plateau in absorption at higher doses. Single doses of anacetrapib markedly and dose-dependently inhibited serum CETP activity with peak effects of approximately 90% inhibition at t(max) and approximately 58% inhibition at 24 h post-dose. An E(max) model best described the plasma anacetrapib concentration vs CETP activity relationship with an EC(50) of approximately 22 nm. Food increased exposure to anacetrapib; up to approximately two-three-fold with a low-fat meal and by up to approximately six-eight fold with a high-fat meal. Anacetrapib pharmacokinetics and pharmacodynamics were similar in elderly vs young adults, women vs men, and obese vs non-obese young adults. Anacetrapib was well tolerated and was not associated with any meaningful increase in blood pressure. CONCLUSIONS: Whereas food increased exposure to anacetrapib significantly, age, gender and obese status did not meaningfully influence anacetrapib pharmacokinetics and pharmacodynamics.


Assuntos
Proteínas de Transferência de Ésteres de Colesterol/antagonistas & inibidores , Dislipidemias/tratamento farmacológico , Oxazolidinonas/farmacocinética , Adolescente , Adulto , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Estudos Cross-Over , Método Duplo-Cego , Jejum , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Obesidade , Oxazolidinonas/administração & dosagem , Oxazolidinonas/farmacologia , Fatores Sexuais , Resultado do Tratamento , Adulto Jovem
9.
J Mol Biol ; 392(2): 452-64, 2009 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-19616560

RESUMO

Pax5 (paired box binding factor 5) is a critical regulator of transcription and lineage commitment in B lymphocytes. In B cells, mb-1 (Ig-alpha/immunoglobulin-associated alpha) promoter transcription is activated by Pax5 through its recruitment of E74-like transforming sequence (Ets) family proteins to a composite site, the P5-EBS (Pax5-Ets binding site). Previously, X-ray crystallographic analysis revealed a network of contacts between the DNA-binding domains of Pax5 and Ets-1 while bound to the P5-EBS. Here, we report that Pax5 assembles these ternary complexes via highly cooperative interactions that overcome the autoinhibition of Ets-1. Using recombinant proteins, we calculated K(d(app)) values for the binding of Pax5, Ets-1, and GA-binding proteins, separately or together, to the P5-EBS. By itself, Pax5 binds the P5-EBS with high affinity (K(d) approximately equal 2 nM). Ets-1(331-440) bound the P5-EBS by itself with low affinity (K(d)=136 nM). However, autoinhibited Ets-1(280-440) alone does not bind detectably to the suboptimal sequences of the P5-EBS. Recruitment of Ets-1(331-440) or Ets-1(280-440) resulted in highly efficient ternary complex assembly with Pax5. Pax5 counteracts autoinhibition and increases binding of Ets-1 of the mb-1 promoter by >1000-fold. Mutation of Pax5 Gln22 to alanine (Q22A) enhances promoter binding by Pax5; however, Q22A greatly reduces recruitment of Ets-1(331-440) and Ets-1(280-440) by Pax5 (8.9- or >300-fold, respectively). Thus, Gln22 of Pax5 is essential for overcoming Ets-1 autoinhibition. Pax5 wild type and Q22A each recruited GA-binding protein alpha/beta1 to the mb-1 promoter with similar affinities, but recruitment was less efficient than that of Ets-1 (reduced by approximately 8-fold). Our results suggest a mechanism that allows Pax5 to overcome autoinhibition of Ets-1 DNA binding. In summary, these data illustrate requirements for partnerships between Ets proteins and Pax5.


Assuntos
DNA/metabolismo , Fator de Transcrição PAX5/metabolismo , Proteína Proto-Oncogênica c-ets-1/metabolismo , Substituição de Aminoácidos/genética , Ensaio de Desvio de Mobilidade Eletroforética , Humanos , Cinética , Mutagênese Sítio-Dirigida , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Fator de Transcrição PAX5/genética , Regiões Promotoras Genéticas , Ligação Proteica
10.
Tuberculosis (Edinb) ; 89(4): 294-303, 2009 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-19556165

RESUMO

In mice, and possibly in humans, nitric oxide (NO) is an important host-defense molecule against Mycobacterium tuberculosis. Inducible nitric oxide synthase (iNOS) and NO are upregulated in murine macrophages stimulated with interferon-gamma (IFNgamma) and mannose-capped lipoarabinomannan (ManLAM), a major lipoglycan in the cell wall of M. tuberculosis. Interleukin-4 (IL-4) can inhibit NO expression and may impair host immune response to M. tuberculosis. Therefore, we sought to determine the mechanism by which IL-4 inhibits IFNgamma+ManLAM-induced NO production. Since l-arginine is the substrate for both iNOS and arginase, and IL-4 increases arginase activity by inducing its production, a plausible mechanism of IL-4 inhibition of NO expression is via depletion of l-arginine through increased arginase activity. Herein, we show that IL-4 inhibited iNOS gene expression at the transcriptional level, suggesting an inhibitory mechanism that is independent of the competition for l-arginine between iNOS and arginase. Furthermore, pharmacologic inhibition of IL-4-induced arginase activity did not abrogate IL-4 inhibition of IFNgamma+ManLAM-induced NO expression. Instead, inhibition by IL-4 was mediated principally by the ability of IL-4 to inhibit the production of IFNgamma-induced interferon-gamma response factor-1 (IRF-1) protein, a critically important transcriptional element that enhances expression of IFNgamma-inducible genes such as iNOS.


Assuntos
Interferon gama/farmacologia , Interleucina-4/farmacologia , Lipopolissacarídeos/farmacologia , Óxido Nítrico/biossíntese , Animais , Arginase/metabolismo , Arginina/metabolismo , Western Blotting , Células Cultivadas , Ensaio de Imunoadsorção Enzimática , Expressão Gênica/efeitos dos fármacos , Humanos , Fator Regulador 1 de Interferon/metabolismo , Interferon gama/metabolismo , Interleucina-18/metabolismo , Lipopolissacarídeos/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Mycobacterium tuberculosis/imunologia , Óxido Nítrico/genética , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Regiões Promotoras Genéticas/fisiologia , RNA Mensageiro/metabolismo , Fator de Transcrição STAT6/metabolismo
11.
Lancet ; 370(9603): 1907-14, 2007 Dec 08.
Artigo em Inglês | MEDLINE | ID: mdl-18068514

RESUMO

BACKGROUND: The inhibition of cholesteryl ester transfer protein (CETP) is considered a potential new mechanism for treatment of dyslipidaemia. Anacetrapib (MK-0859) is a CETP inhibitor currently under development. We aimed to assess anacetrapib's effects as monotherapy on low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C) and on 24-h ambulatory blood pressure. METHODS: We did two double-blind, randomised, placebo-controlled phase I studies. In the first study, 50 patients with dyslipidaemia (LDL-C 100-190 mg/dL; 40 active, 10 placebo) aged 18-75 years received anacetrapib doses of 0, 10, 40, 150, or 300 mg orally once a day with a meal for 28 days. Standard lipid and lipoprotein monitoring, safety monitoring, and anacetrapib concentrations for pharmacokinetics were done. In the second study, 22 healthy participants aged 45-75 years received either 150 mg of anacetrapib once a day or matching placebo with a meal for 10 days in each crossover period, in a randomised sequence, with at least a 14-day washout between the treatment periods. Continuous 24-h ambulatory blood pressure monitoring was done on day -1 and day 10 of each treatment period in this study. The primary or secondary endpoints of safety and tolerability were assessed in both studies by monitoring clinical adverse experiences, physical examinations, vital signs, 12-lead electrocardiogram, and laboratory safety. Analysis was per protocol. These trials are registered with ClinicalTrials.gov, number NCT00565292 and NCT00565006. FINDINGS: In the dyslipidaemia study, one patient withdrew consent and one was excluded from the data analysis for HDL-C and LDL-C because complete pre-dose measurements were not available. Anacetrapib produced dose-dependent lipid-altering effects with peak lipid-altering effects of 129% (mean 51.1 [SD 3.8]-114.9 [7.9] mg/dL) increase in HDL-C and a 38% (138.2 [11.4]-77.6 [7.9] mg/dL) decrease in LDL-C in patients with dyslipidaemia. In the 24-h ambulatory blood pressure study in healthy individuals, least squares difference between anacetrapib and placebo groups on day 10 were 0.60 (90% CI -1.54 to 2.74; p=0.634) mm Hg for systolic blood pressure and 0.47 (90% CI -0.90 to 1.84; p=0.561) mm Hg for diastolic blood pressure. INTERPRETATION: Anacetrapib seems to exhibit HDL-C increases greater than those seen with other investigational drugs in this class and LDL-C lowering effects similar to statins. Despite greater lipid-altering effects relative to other members of this class, anacetrapib seems not to increase blood pressure, suggesting that potent CETP inhibition by itself might not lead to increased blood pressure.


Assuntos
Pressão Sanguínea/efeitos dos fármacos , Proteínas de Transferência de Ésteres de Colesterol/antagonistas & inibidores , Dislipidemias/tratamento farmacológico , Oxazolidinonas/uso terapêutico , Adolescente , Adulto , Idoso , Relação Dose-Resposta a Droga , Método Duplo-Cego , Dislipidemias/sangue , Eletrocardiografia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade
12.
Mol Immunol ; 41(12): 1145-53, 2004 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-15482850

RESUMO

Antigen-stimulated B lymphocytes undergo genetic and phenotypic changes in germinal centers (GCs), including affinity maturation of immunoglobulin (Ig) genes and Ig heavy chain isotype switching. Expression of the Germinal Center Expressed Transcript (GCET) gene is up-regulated in murine GC B cells. The human homolog of GCET, HGAL/GCET2, is an important prognostic marker for staging lymphomas derived from GCs. To identify mechanisms that control cell type-specific transcription of GCET, we localized promoter sequences using S1 nuclease protection and functional assays. Sequences comprising a TATA-less promoter were localized to a short region upstream of multiple mRNA start sites. In functional assays, the promoter is active in cells irrespectively of endogenous GCET gene expression. In vitro binding assays identified a non-consensus binding site for Sp factors near sites of transcriptional initiation. The site binds Spl and Sp3 in nuclear extracts and recombinant Spl in vitro, and is required for full promoter function in transient promoter assays. Activation of the promoter by Spl or Sp3 in Spl/3-deficient cells was largely dependent on the Sp site. Together, these data provide the first analysis of regulatory modules necessary for GCET expression, a model for GC B cell-specific transcription.


Assuntos
Linfócitos B/metabolismo , Centro Germinativo/citologia , Regiões Promotoras Genéticas/genética , Fator de Transcrição Sp1/fisiologia , Fatores de Transcrição/genética , Animais , Sítios de Ligação/genética , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/fisiologia , Camundongos , RNA Mensageiro/análise , Fator de Transcrição Sp3 , Fatores de Transcrição/fisiologia , Sítio de Iniciação de Transcrição
14.
Infect Immun ; 71(3): 1442-52, 2003 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-12595462

RESUMO

Nitric oxide (NO(.)) produced by inducible nitric oxide synthase (iNOS) is an important host defense molecule against Mycobacterium tuberculosis in mononuclear phagocytes. The objective of this study was to determine the role of the IkappaBalpha kinase-nuclear factor kappaB (IKK-NF-kappaB) signaling pathway in the induction of iNOS and NO(.) by a mycobacterial cell wall lipoglycan known as mannose-capped lipoarabinomannan (ManLAM) in mouse macrophages costimulated with gamma interferon (IFN-gamma). NF-kappaB was activated by ManLAM as shown by electrophoretic mobility shift assay, by immunofluorescence of translocated NF-kappaB in intact cells, and by a reporter gene driven by four NF-kappaB-binding elements. Transduction of an IkappaBalpha mutant (Ser32/36Ala) significantly inhibited NO(.) expression induced by IFN-gamma plus ManLAM. An activated SCF complex, a heterotetramer (Skp1, Cul-1, beta-TrCP [F-box protein], and ROC1) involved with ubiquitination, is also required for iNOS-NO(.) induction. Two NF-kappaB-binding sites (kappaBI and kappaBII) present on the 5'-flanking region of the iNOS promoter bound ManLAM-induced NF-kappaB similarly. By use of reporter constructs in which one or both sites are mutated, both NF-kappaB-binding positions were essential in iNOS induction by IFN-gamma plus ManLAM. IFN-gamma-induced activation of the IRF-1 transcriptional complex is a necessary component in host defense against tuberculosis. Although the 5'-flanking region of the IRF-1 promoter contains an NF-kappaB-binding site and ManLAM-induced NF-kappaB also binds to this site, ManLAM was unable to induce IRF-1 expression. The influence of mitogen-activated protein kinases on IFN-gamma plus ManLAM induction of iNOS-NO(.) is not due to any effects on ManLAM induction of NF-kappaB.


Assuntos
Proteínas de Ligação a DNA/genética , Lipopolissacarídeos/farmacologia , NF-kappa B/fisiologia , Óxido Nítrico Sintase/genética , Óxido Nítrico/biossíntese , Fosfoproteínas/genética , Regiões Promotoras Genéticas , Região 5'-Flanqueadora , Animais , Ensaio de Desvio de Mobilidade Eletroforética , Proteínas I-kappa B/fisiologia , Fator Regulador 1 de Interferon , Interferon gama/farmacologia , Camundongos , Proteínas Quinases Ativadas por Mitógeno/fisiologia , Inibidor de NF-kappaB alfa , Óxido Nítrico Sintase Tipo II , Transdução de Sinais
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