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1.
Biochim Biophys Acta ; 1848(10 Pt A): 1974-80, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26074010

RESUMO

Integral membrane proteins (IMPs) are of therapeutic interest and are targeted by a majority of approved drugs. It's difficult to express, purify, and maintain the functional conformation of IMPs. Nanodisc presents a reliable method to solubilize and stabilize IMPs in detergent-free condition. In this study, we demonstrate the assembly and purification of a chimeric ion channel, KcsA-Kv1.3 Nanodisc. We further detail biophysical analysis of the assembled Nanodisc using analytical ultracentrifugation (AUC), surface plasmon resonance (SPR), and back scattering interferometry (BSI). AUC is employed to determine the molecular composition of the empty and KcsA-Kv1.3 Nanodisc. Combination of SPR and BSI overcomes each other's limitation and provides insight of equilibrium binding properties of peptide and small molecule ligands to KcsA-Kv1.3.


Assuntos
Proteínas de Bactérias/química , Canal de Potássio Kv1.3/química , Nanopartículas/química , Nanopartículas/ultraestrutura , Bloqueadores dos Canais de Potássio/química , Canais de Potássio/química , Sequência de Aminoácidos , Proteínas de Bactérias/antagonistas & inibidores , Sítios de Ligação , Canal de Potássio Kv1.3/antagonistas & inibidores , Dados de Sequência Molecular , Complexos Multiproteicos/síntese química , Complexos Multiproteicos/ultraestrutura , Ligação Proteica
2.
PLoS One ; 10(12): e0145849, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26720709

RESUMO

Type 2 diabetes (T2D) occurs when there is insufficient insulin release to control blood glucose, due to insulin resistance and impaired ß-cell function. The GPR39 receptor is expressed in metabolic tissues including pancreatic ß-cells and has been proposed as a T2D target. Specifically, GPR39 agonists might improve ß-cell function leading to more adequate and sustained insulin release and glucose control. The present study aimed to test the hypothesis that GPR39 agonism would improve glucose stimulated insulin secretion in vivo. A high throughput screen, followed by a medicinal chemistry program, identified three novel potent Zn2+ modulated GPR39 agonists. These agonists were evaluated in acute rodent glucose tolerance tests. The results showed a lack of glucose lowering and insulinotropic effects not only in lean mice, but also in diet-induced obese (DIO) mice and Zucker fatty rats. It is concluded that Zn2+ modulated GPR39 agonists do not acutely stimulate insulin release in rodents.


Assuntos
Descoberta de Drogas , Insulina/metabolismo , Receptores Acoplados a Proteínas G/agonistas , Animais , Glicemia/efeitos dos fármacos , Relação Dose-Resposta a Droga , Teste de Tolerância a Glucose , Ensaios de Triagem em Larga Escala , Humanos , Secreção de Insulina , Ilhotas Pancreáticas/metabolismo , Masculino , Camundongos , Ratos , Ratos Zucker , Receptores Acoplados a Proteínas G/genética , Bibliotecas de Moléculas Pequenas , Zinco/metabolismo , Zinco/farmacologia
3.
Chest ; 147(6): 1494-1502, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25474571

RESUMO

BACKGROUND: Ventilator-associated pneumonia (VAP) remains a common complication in critically ill surgical patients, and its diagnosis remains problematic. Exhaled breath contains aerosolized droplets that reflect the lung microbiota. We hypothesized that exhaled breath condensate fluid (EBCF) in hygroscopic condenser humidifier/heat and moisture exchanger (HCH/HME) filters would contain bacterial DNA that qualitatively and quantitatively correlate with pathogens isolated from quantitative BAL samples obtained for clinical suspicion of pneumonia. METHODS: Forty-eight adult patients who were mechanically ventilated and undergoing quantitative BAL (n = 51) for suspected pneumonia in the surgical ICU were enrolled. Per protocol, patients fulfilling VAP clinical criteria undergo quantitative BAL bacterial culture. Immediately prior to BAL, time-matched HCH/HME filters were collected for study of EBCF by real-time polymerase chain reaction. Additionally, convenience samples of serially collected filters in patients with BAL-diagnosed VAP were analyzed. RESULTS: Forty-nine of 51 time-matched EBCF/BAL fluid samples were fully concordant (concordance > 95% by κ statistic) relative to identified pathogens and strongly correlated with clinical cultures. Regression analysis of quantitative bacterial DNA in paired samples revealed a statistically significant positive correlation (r = 0.85). In a convenience sample, qualitative and quantitative polymerase chain reaction analysis of serial HCH/HME samples for bacterial DNA demonstrated an increase in load that preceded the suspicion of pneumonia. CONCLUSIONS: Bacterial DNA within EBCF demonstrates a high correlation with BAL fluid and clinical cultures. Bacterial DNA within EBCF increases prior to the suspicion of pneumonia. Further study of this novel approach may allow development of a noninvasive tool for the early diagnosis of VAP.


Assuntos
Testes Diagnósticos de Rotina/métodos , Expiração , Pulmão/microbiologia , Técnicas Microbiológicas/métodos , Microbiota/genética , Pneumonia Associada à Ventilação Mecânica/diagnóstico , Pneumonia Associada à Ventilação Mecânica/microbiologia , Lavagem Broncoalveolar/instrumentação , Lavagem Broncoalveolar/métodos , Estado Terminal , DNA Bacteriano/genética , Testes Diagnósticos de Rotina/instrumentação , Humanos , Unidades de Terapia Intensiva , Técnicas Microbiológicas/instrumentação , Projetos Piloto , Reação em Cadeia da Polimerase em Tempo Real , Análise de Regressão , Sensibilidade e Especificidade
4.
Front Oncol ; 3: 333, 2014 Jan 13.
Artigo em Inglês | MEDLINE | ID: mdl-24455489

RESUMO

Previous whole-exome sequencing has demonstrated that melanoma tumors harbor mutations in the GRIN2A gene. GRIN2A encodes the regulatory GluN2A subunit of the glutamate-gated N-methyl-d-aspartate receptor (NMDAR), involvement of which in melanoma remains undefined. Here, we sequenced coding exons of GRIN2A in 19 low-passage melanoma cell lines derived from patients with metastatic melanoma. Potential mutation impact was evaluated in silico, including within the GluN2A crystal structure, and clinical correlations were sought. We found that of 19 metastatic melanoma tumors, four (21%) carried five missense mutations in the evolutionarily conserved domains of GRIN2A; two were previously reported. Melanoma cells that carried these mutations were treatment-naïve. Sorting intolerant from tolerant analysis predicted that S349F, G762E, and P1132L would disrupt protein function. When modeled into the crystal structure of GluN2A, G762E was seen to potentially alter GluN1-GluN2A interactions and ligand binding, implying disruption to NMDAR functionality. Patients whose tumors carried non-synonymous GRIN2A mutations had faster disease progression and shorter overall survival (P < 0.05). This was in contrast to the BRAF V600E mutation, found in 58% of tumors but showing no correlation with clinical outcome (P = 0.963). Although numbers of patients in this study are small, and firm conclusions about the association between GRIN2A mutations and poor clinical outcome cannot be drawn, our results highlight the high prevalence of GRIN2A mutations in metastatic melanoma and suggest for the first time that mutated NMDARs impact melanoma progression.

5.
Anticancer Drugs ; 23(6): 627-37, 2012 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-22293659

RESUMO

Adjuvant therapies can incorporate a number of different drugs to minimize the cardiotoxicity of cancer chemotherapy, decrease the development of drug resistance and increase the overall efficacy of the treatment regime. Topoisomerase IIα is a major target of many commonly used anticancer drugs, where cell death is brought about by an accumulation of double-strand DNA breaks. Poly (ADP-ribose) polymerase (PARP)-1 has been extensively studied for its role in the repair of double-strand DNA breaks, but its ability to add highly negative biopolymers (ribosylation) to target proteins provides a vast number of pathways where it can also be important in mediating cell death. In this study, we combine the classical topoisomerase IIα poison doxorubicin with the PARP inhibitor PJ34 to investigate the potentiation of chemotherapeutic efficiency in HeLa cells. We demonstrate that PJ34 treatment has the capacity to increase endogenous topoisomerase IIα protein by about 20%, and by combining doxorubicin treatment with PJ34, we observed a 50% improvement in doxorubicin-mediated cell death in HeLa cells. These results were correlated with the ribosylation of transcription factor specificity factor 1 after doxorubicin treatment, thereby altering its affinity for binding to known regulatory elements within the human topoisomerase IIα promoter. Taken together, these results highlight the synergistic potential of combining PARP inhibitors with classical topoisomerase IIα-targeting drugs.


Assuntos
Doxorrubicina/farmacologia , Inibidores Enzimáticos/farmacologia , Fenantrenos/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Morte Celular/efeitos dos fármacos , DNA/metabolismo , DNA Topoisomerases Tipo II/genética , DNA Topoisomerases Tipo II/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Sinergismo Farmacológico , Regulação da Expressão Gênica/efeitos dos fármacos , Células HeLa/efeitos dos fármacos , Humanos , Poli(ADP-Ribose) Polimerase-1 , Poli(ADP-Ribose) Polimerases/metabolismo , Regiões Promotoras Genéticas , Fator de Transcrição Sp1/metabolismo
6.
Am J Transl Res ; 4(1): 72-82, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22347523

RESUMO

Ventilator associated pneumonia is a common and costly complication in critically ill and injured surgical patients. The diagnosis of pneumonia remains problematic and non-specific. Using clinical criteria, a diagnosis of pneumonia is typically not made until an infection is well established. Semi-quantitative cultures of endotracheal aspirate and broncho-alveolar lavage are employed to improve the accuracy of diagnosis but are invasive and require time for culture results to become available. We report data that show that an inexpensive, rapid and non-invasive alternative may exist. In particular we show that: 1). Bio-aerosols evolved in the breath of ventilated patients and captured in the hygroscopic condenser humidifier filter of the ventilator circuit contain pathogenic micro-organisms. 2). The number (CFU/ml) and identity (Genus, species) of the pathogens in the aerosol samples can rapidly and inexpensively be determined by PCR. 3). Data from a convenience sample of filters correlate with clinical findings from standard microbiological methods such as broncho-alveolar lavage. The evaluation of the bacterial load evolved in exhaled breath by PCR is amenable to repeated sampling. Since increasing bacterial burden is believed to correlate with the establishment of infection, the use of quantitative PCR may provide a method to rapidly, inexpensively, and effectively detect and diagnose the early onset of pneumonia and identify pathogens involved.

7.
Anal Chem ; 84(4): 2017-24, 2012 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-22229858

RESUMO

Through integration of a MOSFET-based microfluidic Coulter counter with a dc-dielectrophoretic cell sorter, we demonstrate simultaneous on-chip cell separation and sizing with three different samples including 1) binary mixtures of polystyrene beads, 2) yeast cells of continuous size distribution, and 3) mixtures of 4T1 tumor cells and murine bone marrow cells. For cells with continuous size distribution, it is found that the receiver operator characteristic analysis is an ideal method to characterize the separation performance. The characterization results indicate that dc-DEP separation performance degrades as the sorting throughput (cell sorting rate) increases, which provides insights into the design and operation of size-based microfluidic cell separation.


Assuntos
Células da Medula Óssea/citologia , Separação Celular/instrumentação , Eletroforese em Microchip/instrumentação , Neoplasias Mamárias Experimentais/química , Técnicas Analíticas Microfluídicas , Saccharomyces cerevisiae/química , Animais , Células Cultivadas , Feminino , Citometria de Fluxo , Camundongos , Microesferas , Análise de Sequência com Séries de Oligonucleotídeos , Poliestirenos/química
8.
Expert Opin Drug Discov ; 3(6): 671-676, 2008 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-19750206

RESUMO

BACKGROUND: Antagonizing the production of infectious virus inside cells requires drugs that can cross the cell membrane without harming host cells. OBJECTIVE: It is therefore advantageous to establish intracellular potency of anti-viral drug candidates early in the drug-discovery pipeline. METHODS: To this end, cell-based assays are being developed and employed in high-throughput drug screening, ranging from assays that monitor replication of intact viruses to those that monitor activity of specific viral proteins. While numerous cell-based assays have been developed and investigated, rapid counter screens are also needed to define the specific viral targets of identified inhibitors and to eliminate nonspecific screening hits. RESULTS/CONCLUSIONS: Here, we describe the types of cell-based assays being used in antiviral drug screens and evaluate the equally important counter screens that are being employed to reach the full potential of cell-based high-throughput screening.

10.
BMC Mol Biol ; 8: 36, 2007 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-17511886

RESUMO

BACKGROUND: Topoisomerase IIalpha has been shown to be down-regulated in doxorubicin-resistant cell lines. The specificity proteins Sp1 and Sp3 have been implicated in regulation of topoisomerase IIalpha transcription, although the mechanism by which they regulate expression is not fully understood. Sp1 has been shown to bind specifically to both proximal and distal GC elements of the human topoisomerase IIalpha promoter in vitro, while Sp3 binds only to the distal GC element unless additional flanking sequences are included. While Sp1 is thought to be an activator of human topoisomerase IIalpha, the functional significance of Sp3 binding is not known. Therefore, we sought to determine the functional relationship between Sp1 and Sp3 binding to the topoisomerase IIalpha promoter in vivo. We investigated endogenous levels of Sp1, Sp3 and topoisomerase IIalpha as well as binding of both Sp1 and Sp3 to the GC boxes of the topoisomerase IIalpha promoter in breast cancer cell lines in vivo after short term doxorubicin exposure. RESULTS: Functional effects of Sp1 and Sp3 were studied using transient cotransfection assays using a topoisomerase IIalpha promoter reporter construct. The in vivo interactions of Sp1 and Sp3 with the GC elements of the topoisomerase IIalpha promoter were studied in doxorubicin-treated breast cancer cell lines using chromatin immunoprecipitation assays. Relative amounts of endogenous proteins were measured using immunoblotting. In vivo DNA looping mediated by proteins bound at the GC1 and GC2 elements was studied using the chromatin conformation capture assay. Both Sp1 and Sp3 bound to the GC1 and GC2 regions. Sp1 and Sp3 were transcriptional activators and repressors respectively, with Sp3 repression being dominant over Sp1-mediated activation. The GC1 and GC2 elements are linked in vivo to form a loop, thus bringing distal regulatory elements and their cognate transcription factors into close proximity with the transcription start site. CONCLUSION: These observations provide a mechanistic explanation for the modulation of topoisomerase IIalpha and concomitant down-regulation that can be mediated by topoisomerase II poisons. Competition between Sp1 and Sp3 for the same cognate DNA would result in activation or repression depending on absolute amounts of each transcription factor in cells treated with doxorubicin.


Assuntos
Antígenos de Neoplasias/metabolismo , DNA Topoisomerases Tipo II/metabolismo , Proteínas de Ligação a DNA/metabolismo , Regulação Enzimológica da Expressão Gênica/genética , Regiões Promotoras Genéticas/genética , Fator de Transcrição Sp1/metabolismo , Fator de Transcrição Sp3/metabolismo , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Doxorrubicina , Humanos , Immunoblotting
11.
Anticancer Drugs ; 18(4): 419-25, 2007 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-17351394

RESUMO

Radiotherapy and chemotherapy often induce DNA double-strand breaks in both normal and malignant cells. The proteins involved in the repair of such lesions are central to cancer prognosis and treatment, as they can be overexpressed in many cancers, accelerating malignant transformation and increasing repair capacity, potentially leading to cellular resistance. If malignant cells can be selectively targeted repair proteins could also be candidates for targeted therapy. In this study, two keyplayers in eukaryotic DNA double-strand break repair, Rad51 and DNA-dependent protein kinase catalytic subunit, were analysed in noncancerous human breast cells (MCF12A) and the breast cancer cell lines (MDA MB 231 and MCF7) in response to treatment with doxorubicin. A cell cycle-independent increase in Rad51 protein levels (a recombinase involved in homologous recombination repair) was observed 24 and 48 h after treatment in MDA MB 231 and MCF12A when exposed to low levels of doxorubicin, whereas MCF7 cells displayed a continuous decrease in Rad51 protein with increasing drug concentration. DNA-dependent protein kinase catalytic subunit, which is involved in nonhomologous end joining of DNA lesions, remained unaltered under all conditions tested. Topoisomerase II-alpha protein, the primary target of doxorubicin, was upregulated at low concentrations of doxorubicin in all cell lines tested. Here we show that Rad51 protein levels can be differentially regulated in normal and malignant breast cell lines in response to doxorubicin, independent of cell cycle state. These observations have direct relevance to chemosensitivity and add an additional prognostic factor that could be taken into account when designing targeted therapeutic regimes.


Assuntos
Antibióticos Antineoplásicos/farmacologia , Reparo do DNA/fisiologia , Doxorrubicina/farmacologia , Rad51 Recombinase/biossíntese , Rad51 Recombinase/genética , Western Blotting , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Dano ao DNA , Relação Dose-Resposta a Droga , Feminino , Citometria de Fluxo , Genes p53/genética , Humanos , Receptores de Estrogênio/genética , Receptores de Estrogênio/fisiologia , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/fisiologia
12.
Oncol Res ; 14(6): 297-304, 2004.
Artigo em Inglês | MEDLINE | ID: mdl-15206492

RESUMO

A short-term assay method able to estimate the radiation response of human cancer tissue samples would be of great advantage to the individualization of radiotherapy in cancer patients. However, the effect of radiation on [3H]thymidine incorporation by proliferating cells reflects a composite of cell cycle arrest and induced cell death pathways. Here we consider whether it is feasible to correct for cell cycle effects based on comparison of the effects of radiation and the mitotic inhibitor paclitaxel on [3H]thymidine incorporation. Sixty-two short-term (7-day) cultures of human tumor tissue from 61 patients with melanoma, gynecological cancer, brain cancer, and head and neck cancer, as well as 18 5-day cultures of low passage human tumor cell lines, were irradiated at doses from 2 to 9 Gy, or exposed to paclitaxel (200 nM). [3H]Thymidine incorporation was measured at the end of the incubation. Cell cycle times could be estimated from the paclitaxel data and were 2.7 to 18.6 days for melanomas, 2.5 to >40 days for carcinomas, 3.9 to 39 days for brain tumors, and 1.1 to 3.8 days for cell lines. The effects of radiation on [3H]thymidine incorporation varied widely (0-97% and 0-99% inhibition for 2 and 9 Gy, respectively), and in 23 of the clinical samples, but in none of the cell lines, radiation caused significantly greater inhibition of [3H]thymidine incorporation than paclitaxel (p < 0.05). We argue that that these differences reflect radiation-induced cell loss from G1 phase and/or S phase. Responses of short-term cultures of clinical tumor material to radiation, with appropriate correction for cell cycle effects, might have the potential to provide information on radiation-induced cell death in individual patients.


Assuntos
Interfase/efeitos da radiação , Neoplasias/patologia , Morte Celular/efeitos dos fármacos , Morte Celular/efeitos da radiação , Humanos , Interfase/efeitos dos fármacos , Paclitaxel/farmacologia , Células Tumorais Cultivadas
13.
J Palliat Care ; 20(1): 28-31, 2004.
Artigo em Inglês | MEDLINE | ID: mdl-15132073

RESUMO

Dysphagia from mechanical esophageal obstruction in patients with advanced malignancy is a common and debilitating symptom in patients referred to palliative care services. Relief of such dysphagia is often attempted by insertion of an esophageal stent in the hope that this will improve the quality of life for these patients. We describe a series of 39 patients who had an esophageal stent inserted under radiologic guidance for malignant dysphagia over an 82-month period. While the stents were clearly effective at relieving dysphagia, they also induced significant comorbidity, in particular, moderate to severe chest pain occurring in 46% and reflux esophagitis in 26% of our non-selected patient group. In addition we found an eight percent mortality rate from esophageal bleeding following stent insertion. When discussing the potential role of esophageal stent insertion with patients under the care of palliative care teams, the frequency and severity of these significant secondary symptoms need to be considered.


Assuntos
Transtornos de Deglutição/cirurgia , Neoplasias Esofágicas/complicações , Estenose Esofágica/complicações , Cuidados Paliativos/métodos , Stents , Adulto , Idoso , Idoso de 80 Anos ou mais , Causas de Morte , Dor no Peito/epidemiologia , Dor no Peito/etiologia , Comorbidade , Transtornos de Deglutição/etiologia , Neoplasias Esofágicas/mortalidade , Estenose Esofágica/mortalidade , Esofagite Péptica/epidemiologia , Esofagite Péptica/etiologia , Humanos , Incidência , Auditoria Médica , Pessoa de Meia-Idade , Nova Zelândia/epidemiologia , Cuidados Paliativos/psicologia , Cuidados Paliativos/normas , Hemorragia Pós-Operatória/etiologia , Hemorragia Pós-Operatória/mortalidade , Qualidade de Vida , Radiologia Intervencionista , Estudos Retrospectivos , Índice de Gravidade de Doença , Stents/efeitos adversos , Stents/normas , Análise de Sobrevida , Resultado do Tratamento
14.
DNA Repair (Amst) ; 3(5): 455-64, 2004 May 04.
Artigo em Inglês | MEDLINE | ID: mdl-15162792

RESUMO

Initial recognition of DNA damage is the crucial but poorly understood first step in DNA repair by the human nucleotide excision repair(NER) and mismatch repair (MMR) systems. Failure by NER or MMR to recognize DNA damage threatens the genetic integrity of the organism and may play a role in carcinogenesis. Both NER and MMR recognize and repair a wide variety of structurally dissimilar lesions against the background of normal DNA. Previous studies have suggested that detection of thermodynamic destabilization of DNA caused by covalent damage and base mismatches is a potential mechanism by which repair pathways with broad specificity such as NER and MMR recognize their substrates. However, both NER and MMR respectively, repair a wide variety of stabilizing and destabilizing covalent DNA lesions and base pair mismatches. A common feature of lesions that are both thermodynamically stabilizing and destabilizing is the alteration of the local DNA flexibility (dynamics). In this review we describe the experimental evidence for altered dynamics from NMR and thermodynamic studies on normal and damaged DNA molecules with respect to recognition by NER and MMR. Based on these data, we propose a model for initial detection of lesions by both NER and MMR that occurs through an indirect readout mechanism of alternative DNA conformations induced by covalent damage and base mismatches.


Assuntos
Pareamento Incorreto de Bases , Dano ao DNA , Reparo do DNA/fisiologia , DNA/química , DNA/metabolismo , Conformação de Ácido Nucleico , Animais , Pareamento Incorreto de Bases/genética , DNA/genética , Dano ao DNA/efeitos dos fármacos , Dano ao DNA/genética , Reparo do DNA/efeitos dos fármacos , Reparo do DNA/genética , Humanos , Modelos Genéticos
15.
Anticancer Drugs ; 15(4): 357-62, 2004 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-15057140

RESUMO

Topoisomerase IIalpha (Topo IIalpha) is an essential nuclear enzyme with a role in the maintenance of DNA topology. Topo IIalpha is a target for several anticancer drugs and the levels of activity of this enzyme have been implicated in the development of drug resistance. Our objective was to identify regulatory transcription factors involved in drug-induced down-regulation of Topo IIalpha. A breast cancer cell line was subjected to a pulsed exposure of doxorubicin and resistant clones propagated. Whole-cell extracts were studied by immunoblotting and RT-PCR for drug-induced changes in the amounts Topo IIalpha, Sp1, Sp3, NF-Y and MDR1. Topo IIalpha levels were reduced in six out of eight cell lines. Of these, three showed concomitant changes in the expression of Sp1 and NF-YA. Thus, we provide the first evidence for roles of Sp1 and NF-Y in bringing about the drug-induced down-regulation of Topo IIalpha gene expression.


Assuntos
Fator de Ligação a CCAAT/biossíntese , DNA Topoisomerases Tipo II/biossíntese , Regulação para Baixo , Fator de Transcrição Sp1/biossíntese , Fatores de Transcrição/biossíntese , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/biossíntese , Antibióticos Antineoplásicos/farmacologia , Antígenos de Neoplasias , Fator de Ligação a CCAAT/genética , Divisão Celular/efeitos dos fármacos , Extratos Celulares , Linhagem Celular Tumoral , DNA Topoisomerases Tipo II/genética , Proteínas de Ligação a DNA , Doxorrubicina/farmacologia , Humanos , Immunoblotting , Fator de Transcrição Sp1/genética , Fatores de Transcrição/genética , Transcrição Gênica , Transfecção
16.
J Am Chem Soc ; 126(2): 583-90, 2004 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-14719957

RESUMO

Molecular dynamics (MD) simulations were conducted for a G[bond]T mismatch-containing DNA decamer, d(CCATGCGTGG)(2), and its Watson-Crick parent sequence, d(CCACGCGTGG)(2). Dynamics in unrestrained MD trajectories were in poor agreement with prior (13)C NMR studies. However, the accuracy of the trajectories was improved by the use of time-averaged interatomic distance restraints derived from (1)H NMR. Postprocess smoothing of the trajectories further improved accuracy. Comparison of restrained and smoothed trajectories of the two DNA molecules revealed distinct differences in dynamics. The major groove width of the mismatched oligomer was more variable over the course of the simulation compared to its parent sequence. Greater variability in helical parameters stretch and opening for the mismatches indicated less kinetically stable base pairing. Interbase helical parameters rise, roll, and tilt were also more variable in certain base steps involving mismatched bases. These dynamic differences between normal and G[bond]T mismatched DNA reflect differences in local flexibility that may play a role in mismatch recognition by the MutS. A potential alternate G[bond]T mismatch binding mode for MutS is also proposed.


Assuntos
Pareamento Incorreto de Bases , DNA/química , Guanidina/química , Ressonância Magnética Nuclear Biomolecular/métodos , Timidina/química , Simulação por Computador , DNA/genética , DNA/metabolismo , Guanidina/metabolismo , Ligação de Hidrogênio , Termodinâmica , Timidina/genética , Timidina/metabolismo
17.
Biochem J ; 374(Pt 3): 723-9, 2003 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-12769819

RESUMO

Topo IIalpha (topoisomerase IIalpha) is a major target of several commonly used anticancer drugs and is subject to down-regulation at the transcriptional level in some drug-resistant cell lines and tumours in response to chemotherapy. Clinical resistance to such drugs has been correlated with down-regulation of topo IIalpha at transcription in some drug-resistant cell lines and tumours. Putative binding sites for a variety of transcription factors, including Sp1 (specificity protein 1) and NF-Y (nuclear factor Y) have previously been identified in the topo IIalpha promoter, but their functional significance and interactions have not been described following exposure to anti-cancer drugs. The binding of these factors to specific putative regulatory elements in the topo IIalpha promoter was studied using electrophoretic-mobility-shift assays. Sp1 was found to bind strongly to both distal and proximal GC-rich elements and NF-Y to ICB1 (the first inverted CCAAT box). The functional significance of transcription-factor binding was studied using transient transfection of HeLa cells using a luciferase reporter driven by a 617-bp minimal promoter containing point mutations in putative regulatory elements. Sp1 and NF-Y were both found to be transcriptional modulators with activator or repressor functions depending on protein/DNA context. Moreover, a functional interaction between Sp1 and NF-Y bound at proximal elements was observed.


Assuntos
Fator de Ligação a CCAAT/fisiologia , DNA Topoisomerases Tipo II/genética , DNA Topoisomerases Tipo II/metabolismo , Regiões Promotoras Genéticas , Fator de Transcrição Sp1/fisiologia , Fatores de Transcrição/fisiologia , Antígenos de Neoplasias , Fator de Ligação a CCAAT/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/fisiologia , Ensaio de Desvio de Mobilidade Eletroforética , Regulação Enzimológica da Expressão Gênica , Genes Reporter/genética , Células HeLa , Humanos , Luciferases/genética , Mutagênese Sítio-Dirigida , Proteínas Recombinantes de Fusão/genética , Fator de Transcrição Sp1/genética , Fator de Transcrição Sp2 , Fatores de Transcrição/genética , Transfecção , Células Tumorais Cultivadas
18.
J Am Chem Soc ; 124(28): 8206-19, 2002 Jul 17.
Artigo em Inglês | MEDLINE | ID: mdl-12105898

RESUMO

Farnesylation is a posttranslational lipid modification in which a 15-carbon farnesyl isoprenoid is linked via a thioether bond to specific cysteine residues of proteins in a reaction catalyzed by protein farnesyltransferase (FTase). We synthesized analogues (3-6) of farnesyl pyrophosphate (FPP) to probe the range of modifications possible to the FPP skeleton which allow for efficient transfer by FTase. Photoaffinity analogues of FPP (5, 6) were prepared by substituting perfluorophenyl azide functional groups for the omega-terminal isoprene of FPP. Substituted anilines replace the omega-terminal isoprene in analogues 3 and 4. Compounds 3-5 were prepared by reductive amination of the appropriate anilines with 8-oxo-geranyl acetate, followed by ester hydrolysis, chlorination, and pyrophosphorylation. Additional substitution of three methylenes for the beta-isoprene of FPP gave photoprobe 6 in nine steps. Preparation of the analogues required TiCl(4)-mediated imine formation prior to NaBH(OAc)(3) reduction for anilines with a pK(a) < 1. The azide moiety was not affected by Ph(3)PCl(2) conversion of allylic alcohols 13-16 into corresponding chlorides 17-20. Analogues 3-6 are efficiently transferred to target N-dansyl-GCVLS peptide substrate by mammalian FTase. Comparison of analogue structures and kinetics of transfer to those of FPP reveals that ring fluorination and para substituents have little effect on the affinity of the analogue pyrophosphate for FTase and its transfer efficiency. These results are also supported with models of the analogue binding modes in the active site of FTase. The transferable azide photoprobe 5 photoinactivates FTase. Transferable analogues 5 and 6 allow the formation of appropriately posttranslationally modified photoreactive peptide probes of isoprene function.


Assuntos
Alquil e Aril Transferases/química , Hemiterpenos , Pentanos , Marcadores de Fotoafinidade/química , Fosfatos de Poli-Isoprenil/química , Alquil e Aril Transferases/metabolismo , Aminação , Compostos de Anilina/química , Compostos de Anilina/metabolismo , Azidas/química , Azidas/metabolismo , Sítios de Ligação , Butadienos/química , Butadienos/metabolismo , Compostos de Dansil/química , Compostos de Dansil/metabolismo , Ativação Enzimática , Cinética , Modelos Moleculares , Oligopeptídeos/química , Oligopeptídeos/metabolismo , Marcadores de Fotoafinidade/síntese química , Fotoquímica , Fosfatos de Poli-Isoprenil/síntese química , Fosfatos de Poli-Isoprenil/metabolismo , Sesquiterpenos , Relação Estrutura-Atividade
19.
J Mol Biol ; 319(1): 191-207, 2002 May 24.
Artigo em Inglês | MEDLINE | ID: mdl-12051946

RESUMO

Detailed description of the characteristics of mismatched DNA that are distinct from normal DNA is vital to the understanding of how mismatch repair proteins are able to recognize and repair these DNA lesions. To this end, we have used nuclear Overhauser effect spectroscopy (NOESY)-based distance restraints and (13)C relaxation measurements to solve the solution structures and measure some of the internal dynamics of the G-T mismatched DNA oligomer d(CCATGCGTGG)(2) (GT) and its parent DNA sequence d(CCACGCGTGG)(2) (GC). In GT, the mismatched G7 is structurally perturbed much more than the mismatched T4 relative to their corresponding bases in GC. The degree of G7 displacement differs from previous high-resolution structures of G-T mismatch-containing B-DNA, suggesting a dependence of G-T mismatch-induced structural perturbation on sequence context. The internal dynamics of GC and GT differ on multiple timescales. The mismatched G7 of GT contains spins that decrease significantly in order in GT compared to GC, while spins in C6, T8, and A3 have significantly higher order in GT compared to GC. Linear correlations between helical parameters of GC and GT and the order of C-1' and aromatic methine carbon atoms relate differences in internal dynamics to the structures quantitatively. The dynamic differences between the normal and mismatched DNA signify changes in local flexibility that may be exploited by the mismatch repair system to bind mismatched DNA preferentially while ignoring normal DNA.


Assuntos
Pareamento Incorreto de Bases/genética , DNA/química , DNA/genética , Ressonância Magnética Nuclear Biomolecular , Conformação de Ácido Nucleico , Sequência de Bases , Isótopos de Carbono , Citosina/química , DNA/síntese química , Reparo do DNA , Guanina/química , Cinética , Modelos Moleculares , Relação Estrutura-Atividade , Termodinâmica , Timina/química
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