RESUMO
Recurrent Pseudomonas aeruginosa infections involving biofilm formation are frequent in cystic fibrosis, aggravating the respiratory distress. Co-administration of clarithromycin and classical tobramycin could improve the health status of patients. Antibiotic toxicity was assessed on epithelial (CFBE41o(-)) and macrophagic (THP-1) cell lines. Non-toxic concentrations of antibiotics alone or in combination were applied twice daily for 12 days on mature (12-day-old) biofilms of three P. aeruginosa strains, developed either in prokaryotic culture broth [tryptic soy broth (TSB)] or in a eukaryotic cell culture medium (RPMI-FCS) more similar to an in vivo environment. The antibiofilm and bactericidal effects of antibiotics were assessed. No toxicity of tobramycin was observed on eukaryotic cell lines at concentrations up to 500µg/mL, whilst 100µg/mL was selected as the clarithromycin upper safe limit. The amount of biofilm was strongly reduced by 100µg/mL and 500µg/mL tobramycin for each strain in both media, whilst clarithromycin was only effective in RPMI-FBS, with synergistic (PAO1 strain) and additive (PYO2 strain) effects detected when combining tobramycin 4µg/mL and clarithromycin 100µg/mL. Finally, tobramycin at ≥100µg/mL exerted strong bactericidal effects on each strain in both media. Clarithromycin also exerted bactericidal effects on each strain in both media; its effect was weaker than tobramycin in TSB but was similar in RPMI-FBS. Synergistic effects were observed on PAO1 and MUCO biofilms, e.g. when combining tobramycin 4µg/mL and clarithromycin 100µg/mL. These in vitro data show that co-administration of clarithromycin and tobramycin acts synergistically against in vitro P. aeruginosa biofilms.
Assuntos
Antibacterianos/farmacologia , Biofilmes/efeitos dos fármacos , Claritromicina/farmacologia , Sinergismo Farmacológico , Pseudomonas aeruginosa/efeitos dos fármacos , Tobramicina/farmacologia , Animais , Antibacterianos/toxicidade , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Claritromicina/toxicidade , Meios de Cultura/química , Humanos , Viabilidade Microbiana/efeitos dos fármacos , Pseudomonas aeruginosa/fisiologia , Tobramicina/toxicidadeRESUMO
The present work aims at identifying new ion channel modulators able to target mitochondrial ATP-sensitive potassium channels (mitoKATP channels). An innovative approach should consist in fixing a cationic and hydrophobic triphenylphosphonium fragment on the structure of known KATP channel openers. Such phosphonium salts are expected to cross the biological membranes and to accumulate into mitochondria. Previous works revealed that the presence of an (R)-1-hydroxy-2-propylamino chain at the 3-position of 4H-1,2,4-benzothiadiazine 1,1-dioxides KATP channel openers increased, in most cases, the selectivity towards the pancreatic-type (SUR1/Kir6.2) KATP channel. In order to target cardiac mitoKATP channels, we decided to introduce a triphenylphosphonium group through an ester link on the SUR1-selective (R)-7-chloro-3-(1-hydroxy-2-propyl)amino-4H-1,2,4-benzothiadiazine 1,1-dioxide. The new compounds were found to preserve an inhibitory activity on insulin secretion (SUR1-type KATP channel openers) while no clear demonstration of an impact on mitochondria from cardiomyocytes (measurement of oxygen consumption, respiratory parameters and ATP production on H9C2 cells) was observed. However, the most active (inhibition of insulin release) compound 17 was found to penetrate the cardiac cells and to reach mitochondria.
Assuntos
Benzotiadiazinas/química , Benzotiadiazinas/farmacologia , Diazóxido/química , Diazóxido/farmacologia , Canais de Potássio/metabolismo , Animais , Linhagem Celular , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Compostos Organofosforados/química , Compostos Organofosforados/farmacologia , RatosRESUMO
Tau proteins and amyloid-ß (Aß) peptides are the current recognized cerebrospinal fluid (CSF) biomarkers used as an aid in the diagnosis of Alzheimer's disease (AD). However, there is no consensus on their clinical use due to non-qualified cut-off values, probably related to the observed high pre-analytical and analytical variability. Standardized pre-analytical protocols have therefore been proposed. Importantly, these recommend the use of polypropylene collection/sampling tubes while, to date, no broad comparison of these types of tubes has been conducted. In this study, we first compared, as part of a real clinical workflow, the impact of four different collection tubes on the CSF concentration of Aß peptides (Aß42, Aß40) and total (hTau) and phosphorylated (P-Tau181P) tau proteins measured using routine ELISA kits. We then extended this study to 11 polypropylene tubes used by different clinical laboratories, and investigated their plastic polymer composition using differential scanning calorimetry and Fourier Transformed Infrared spectroscopy. Significant concentration variations linked solely to the use of different types of tubes were observed. This was particularly marked for Aß peptides, with >50% disparity occurring in less than five minutes. Polymer composition analysis revealed that most polypropylene tubes were in fact copolymers with at least polyethylene. There was no clear correlation between tube composition and pre-analytical behavior. Our results show that the use of polypropylene tubes does not guarantee satisfactory pre-analytical behavior. They also point to collection/sampling tubes being a major pre-analytical source of variability that could impact the significance of AD biological diagnosis.
Assuntos
Doença de Alzheimer/líquido cefalorraquidiano , Doença de Alzheimer/diagnóstico , Peptídeos beta-Amiloides/líquido cefalorraquidiano , Erros de Diagnóstico , Proteínas tau/líquido cefalorraquidiano , Ensaio de Imunoadsorção Enzimática , Análise de Fourier , Humanos , Fosforilação , Risco , Espectrofotometria Infravermelho , Estatísticas não Paramétricas , Fatores de TempoAssuntos
Doença de Alzheimer/líquido cefalorraquidiano , Peptídeos beta-Amiloides/líquido cefalorraquidiano , Manejo de Espécimes/instrumentação , Proteínas tau/líquido cefalorraquidiano , Doença de Alzheimer/diagnóstico , Biomarcadores/líquido cefalorraquidiano , Varredura Diferencial de Calorimetria , Humanos , Polietileno/química , Polipropilenos/química , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
INTRODUCTION: Activator protein-2 (AP-2) alpha and AP-2gamma transcription factors contribute to ERBB2 gene overexpression in breast cancer. In order to understand the mechanism by which the ERBB2 gene is overexpressed we searched for novel AP-2 interacting factors that contribute to its activity. METHODS: Ku proteins were identified as AP-2alpha interacting proteins by glutathione serine transferase (GST)-pull down followed by mass spectrometry. Transfection of the cells with siRNA, expression vectors and reporter vectors as well as chromatin immunoprecipitation (ChIP) assay were used to ascertain the implication of Ku proteins on ERBB2 expression. RESULTS: Nuclear proteins from BT-474 cells overexpressing AP-2alpha and AP-2gamma were incubated with GST-AP2 or GST coated beads. Among the proteins retained specifically on GST-AP2 coated beads Ku70 and Ku80 proteins were identified by mass spectrometry. The contribution of Ku proteins to ERBB2 gene expression in BT-474 and SKBR3 cell lines was investigated by downregulating Ku proteins through the use of specific siRNAs. Depletion of Ku proteins led to downregulation of ERBB2 mRNA and protein levels. Furthermore, reduction of Ku80 in HCT116 cell line decreased the AP-2alpha activity on a reporter vector containing an AP-2 binding site linked to the ERBB2 core promoter, and transfection of Ku80 increased the activity of AP-2alpha on this promoter. Ku siRNAs also inhibited the activity of this reporter vector in BT-474 and SKBR3 cell lines and the activity of the ERBB2 promoter was further reduced by combining Ku siRNAs with AP-2alpha and AP-2gamma siRNAs. ChIP experiments with chromatin extracted from wild type or AP-2alpha and AP-2gamma or Ku70 siRNA transfected BT-474 cells demonstrated Ku70 recruitment to the ERBB2 proximal promoter in association with AP-2alpha and AP-2gamma. Moreover, Ku70 siRNA like AP-2 siRNAs, greatly reduced PolII recruitment to the ERBB2 proximal promoter. CONCLUSIONS: Ku proteins in interaction with AP-2 (alpha and gamma) contribute to increased ERBB2 mRNA and protein levels in breast cancer cells.
Assuntos
Antígenos Nucleares/metabolismo , Neoplasias da Mama/metabolismo , Proteínas de Ligação a DNA/metabolismo , Receptor ErbB-2/biossíntese , Fator de Transcrição AP-2/metabolismo , Antígenos Nucleares/genética , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/genética , Regulação para Baixo , Feminino , Regulação Neoplásica da Expressão Gênica , Células HCT116 , Humanos , Autoantígeno Ku , Regiões Promotoras Genéticas , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/genética , Receptor ErbB-2/genética , Fator de Transcrição AP-2/genética , Transcrição Gênica , Transfecção , Regulação para CimaRESUMO
Transmissible spongiform encephalopathies are a group of neurodegenerative disorders caused by a posttranslational, conformational change in the cellular isoform of the prion protein (PrP(C)) into an infectious, disease-associated form (PrP(Sc)). Increasing evidence supports a role for PrP(C) in the cellular response to oxidative stress. We investigated the effect of oxidative stress mediated by paraquat exposure on SH-SY5Y neuroblastoma cells. A loss of mitochondrial membrane potential and subsequent reduction in ATP production were demonstrated in untransfected SH-SY5Y cells, an effect that was ameliorated by the expression of PrP(C). Cells expressing either PrP-DeltaOct, which lacks the octapeptide repeats, or PrP-DA, in which the N-terminus is tethered to the membrane, showed increased sensitivity to paraquat compared with cells expressing wild-type PrP(C) as shown by reduced viability, loss of their membrane integrity, and reduced mitochondrial bioenergetic measurements. Exposure of prion-infected mouse SMB15S cells to paraquat resulted in a reduction in viability to levels similar to those seen in the untransfected SH-SY5Y cells. However, "curing" the cells with pentosan sulfate restored the viability to the level observed in the SH-SY5Y cells expressing PrP(C). These data would indicate that the molecular mechanism promoting cellular resistance to oxidative stress had been compromised in the infected SMB15S cells, which could be reinstated upon curing. Our study supports the hypothesis that PrP(C) expression protects cells against paraquat-induced oxidative injury, demonstrates the significance of the N-terminal region of the protein in mediating this protective effect, and also shows that the biochemical consequences of prion infection may be reversed with therapeutic intervention.
Assuntos
Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Oxidantes/intoxicação , Estresse Oxidativo , Paraquat/intoxicação , Príons/farmacologia , Trifosfato de Adenosina/antagonistas & inibidores , Animais , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Resistência a Medicamentos , Metabolismo Energético/efeitos dos fármacos , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Neurônios/metabolismo , Poliéster Sulfúrico de Pentosana/farmacologia , Príons/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/farmacologia , Scrapie/metabolismo , Scrapie/patologia , Scrapie/fisiopatologia , TransfecçãoRESUMO
A synthetic peptide corresponding to the 106-126 amyloidogenic region of the cellular human prion protein (PrP(c)) is useful for in vitro study of prion-induced neuronal cell death. The aim of the present work was to examine the implication of the cellular prion protein in the toxicity mechanism induced by PrP 106-126. The effect of PrP 106-126 was investigated both on human neuroblastoma SH-SY5Y cells and on SH-SY5Y overexpressing murine cellular prions (wtPrP). We show by metabolic assay tests and ATP assays that PrP(c) expression does not modulate the toxicity of the prion peptide. Moreover, we investigated the effect of this peptide on an established non neuronal model, rabbit kidney epithelial A74 cells that express a doxycycline-inducible murine PrP(c) gene. We show for the first time that the prion peptide 106-126 does not exert any toxic effect on this cell line in the presence or absence of doxycycline. Our results show that the PrP 106-126-induced cell alteration is independent of PrP(c) expression. Rather, it seems to act via an interaction with lipidic components of the plasma membrane as strengthened by our results showing the differential susceptibility of neuronal and non neuronal cell lines that significantly differ by their membrane fatty acid composition.
Assuntos
Sistema Nervoso Central/metabolismo , Células Epiteliais/metabolismo , Lipídeos de Membrana/metabolismo , Neurônios/metabolismo , Fragmentos de Peptídeos/toxicidade , Doenças Priônicas/metabolismo , Príons/toxicidade , Animais , Morte Celular/fisiologia , Linhagem Celular Tumoral , Membrana Celular/química , Membrana Celular/metabolismo , Sistema Nervoso Central/patologia , Sistema Nervoso Central/fisiopatologia , Resistência a Medicamentos/fisiologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/patologia , Humanos , Lipídeos de Membrana/química , Camundongos , Degeneração Neural/metabolismo , Degeneração Neural/fisiopatologia , Neurônios/efeitos dos fármacos , Neurônios/patologia , Fragmentos de Peptídeos/metabolismo , Proteínas PrPC/genética , Proteínas PrPC/metabolismo , Doenças Priônicas/fisiopatologia , Príons/metabolismo , Coelhos , Transfecção , Transgenes/genéticaRESUMO
Prion diseases are fatal neurodegenerative disorders characterized by the accumulation in the brain of an abnormally misfolded, protease-resistant, and beta-sheet rich pathogenic isoform (PrP(SC)) of the cellular prion protein (PrP(C)). In the present work, we were interested to study the mode of prion protein interaction with the membrane using the 106-126 peptide and small unilamellar lipid vesicles as model. As previously demonstrated, we showed by MTS assay that PrP 106-126 induces alterations in the human neuroblastoma SH-SY5Y cell line. We demonstrated for the first time by lipid-mixing assay and by the liposome vesicle leakage test that PrP 106-126, a non-tilted peptide, induces liposome fusion thus a potential cell membrane destabilization, as supported by membrane integrity assay (LDH). By circular dichroism (CD) analysis we showed that the fusogenic property of PrP 106-126 in the presence of liposome is associated with a predominantly beta-sheet structure. These data suggest that the fusogenic property associated with a predominant beta-sheet structure exhibited by the prion peptides contributes to the neurotoxicity of these peptides by destabilizing cellular membranes. The latter might be attached at the membrane surface in a parallel orientation as shown by molecular modeling.