43 research outputs found
Disruption of Interleukin-1β Autocrine Signaling Rescues Complex I Activity and Improves ROS Levels in Immortalized Epithelial Cells with Impaired Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Function
Patients with cystic fibrosis (CF) have elevated concentration of cytokines in sputum and a general inflammatory condition. In addition, CF cells in culture produce diverse cytokines in excess, including IL-1B. We have previously shown that IL-1B, at low doses (~30 pM), can stimulate the expression of CFTR in T84 colon carcinoma cells, through NF-KB signaling. However, at higher doses (>2.5 ng/ml, ~150 pM), IL-1B inhibit CFTR mRNA expression. On the other hand, by using differential display, we found two genes with reduced expression in CF cells, corresponding to the mitochondrial proteins CISD1 and MTND4. The last is a key subunit for the activity of mitochondrial Complex I (mCx-I); accordingly, we later found a reduced mCx-I activity in CF cells. Here we found that IB3-1 cells (CF cells), cultured in serum-free media, secrete 323±5 pg/ml of IL-1B in 24 h vs 127±3 pg/ml for S9 cells (CFTR-corrected IB3-1 cells). Externally added IL-1B (5 ng/ml) reduces the mCx-I activity and increases the mitochondrial (MitoSOX probe) and cellular (DCFH-DA probe) ROS levels of S9 (CFTR-corrected IB3-1 CF cells) or Caco-2/pRSctrl cells (shRNA control cells) to values comparable to those of IB3-1 or Caco-2/pRS26 cells (shRNA specific for CFTR). Treatments of IB3-1 or Caco-2/pRS26 cells with either IL-1β blocking antibody, IL-1 receptor antagonist, IKK inhibitor III (NF-KB pathway) or SB203580 (p38 MAPK pathway), restored the mCx-I activity. In addition, in IB3-1 or Caco-2/pRS26 cells, IL-1B blocking antibody, IKK inhibitor III or SB203580 reduced the mitochondrial ROS levels by ~50% and the cellular ROS levels near to basal values. The AP-1 inhibitors U0126 (MEK1/2) or SP600125 (JNK1/2/3 inhibitor) had no effects. The results suggest that in these cells IL-1B, through an autocrine effect, acts as a bridge connecting the CFTR with the mCx-I activity and the ROS levels.Fil: Clauzure, Mariangeles. Pontificia Universidad Católica Argentina "Santa María de los Buenos Aires". Instituto de Investigaciones Biomédicas. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas; ArgentinaFil: Valdivieso, Ángel Gabriel. Pontificia Universidad Católica Argentina "Santa María de los Buenos Aires". Instituto de Investigaciones Biomédicas. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas; ArgentinaFil: Massip Copiz, María Macarena. Pontificia Universidad Católica Argentina "Santa María de los Buenos Aires". Instituto de Investigaciones Biomédicas. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas; ArgentinaFil: Schulman, Gustavo. Pontificia Universidad Católica Argentina "Santa María de los Buenos Aires". Instituto de Investigaciones Biomédicas. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas; ArgentinaFil: Teiber, Maria Luz. Pontificia Universidad Católica Argentina "Santa María de los Buenos Aires". Instituto de Investigaciones Biomédicas. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas; ArgentinaFil: Tomás A. Santa-Coloma. Pontificia Universidad Católica Argentina "Santa María de los Buenos Aires". Instituto de Investigaciones Biomédicas. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas; Argentin
Biosilo de residuos de merluza y harina de cebada fermentados con bacterias ácido lácticas seleccionadas
Suitability of Lactococcus lactis Tw34 and Lactobacillus plantarum Lb7 as bio-silage inoculants was studied. Experiments were carried out with a mixture of hake (Merluccius hubbsi) by-products and barley meal fermented during 7 days. A sample acidified with lactic acid was used as control. In biological mixtures, the pH dropped below 5,0 after 2 days of fermentation and remained stable until the end of the experience. Maximum populations (> 109 CFU/g) were reached after 5 days of incubation at 18°C. Water soluble peptides concentration increased during the 7 days of incubation and no significant differences (p < 0,05) were found between the control and both bio-silage. Trichloroacetic acid soluble peptides concentrations of biosilages were higher than the control (p > 0,05). After 7 days, phosphorous concentrations reached 2.26 and 2,42 g/100 g in bio-silages fermented with Lc. lactis Tw34 and Lb. plantarum Lb7, respectively, while control values remains almost stable (1,61 g/100 g). At the end of the experience, inhibitory activity of trypsin was abolished in both bio-silage while in control sample antinutritional factors remained active. The results indicated the feasibility of the use of the selected mixture as substrate for bio-silage production and the effectiveness Lc. lactis Tw34 and Lb. plantarum Lb7 as inoculants.Se estudió la capacidad como inoculantes de biosilo de Lactococcus lactis Tw34 y Lactobacillus plantarum Lb7. Los experimentos se llevaron a cabo con una mezcla de subproductos de merluza (Merluccius hubbsi) y harina de cebada, fermentada durante 7 días. Como control se utilizó una muestra acidificada con ácido láctico. En las mezclas biológicas, el pH disminuyó por debajo de 5,0 después de 2 días de fermentación y permaneció estable hasta el final de la experiencia. La población máxima (>109 UFC/g) se alcanzó después de 5 días de incubación a 18°C. La concentración de péptidos solubles en agua aumentó durante los 7 días de incubación y no hubo diferencias significativas (p < 0,05) entre el control y ambos biosilos. Las concentraciones de péptidos solubles en ácido tricloroacético de los biosilos fueron superiores al control (p > 0,05). Después de 7 días, las concentraciones de fósforo alcanzadas fueron 2,26 y 2,42 g /100 g en los biosilos fermentados con Lc. lactis Tw34 y Lb. plantarum Lb7 respectivamente, mientras que en el control los valores permanecieron casi estables (1,61 g/100 g). Al final de la experiencia, la actividad inhibitoria de tripsina fue suprimida en ambos biosilos mientras que, en el control los factores antinutricionales seguían siendo activos. Los resultados indican la factibilidad del uso de la mezcla seleccionada como sustrato para la producción de biosilo y la eficacia de Lc. lactis Tw34 y Lb. plantarum Lb7 como inoculantes
Unraveling Brazilian Indian population prostate good health: clinical, anthropometric and genetic features
Retrospective canine skin peripheral nerve sheath tumors data with emphasis on histologic, immunohistochemical and prognostic factors
Biosilagem de desperdício de merluza e farinha de cevada fermentados com bactérias ácido lácticas selecionadas
Suitability of Lactococcus lactis Tw34 and Lactobacillus plantarum Lb7 asbio-silage inoculants was studied. Experiments were carried out with a mixtureof hake (Merluccius hubbsi) by-products and barley meal fermentedduring 7 days. A sample acidified with lactic acid was used as control. Inbiological mixtures, the pH dropped below 5,0 after 2 days of fermentationand remained stable until the end of the experience. Maximum populations(> 109 CFU/g) were reached after 5 days of incubation at 18°C. Water solublepeptides concentration increased during the 7 days of incubation andno significant differences (p 0,05). After 7 days, phosphorousconcentrations reached 2.26 and 2,42 g/100 g in bio-silages fermented withLc. lactis Tw34 and Lb. plantarum Lb7, respectively, while control values remainsalmost stable (1,61 g/100 g). At the end of the experience, inhibitoryactivity of trypsin was abolished in both bio-silage while in control sampleantinutritional factors remained active. The results indicated the feasibility ofthe use of the selected mixture as substrate for bio-silage production andthe effectiveness Lc. lactis Tw34 and Lb. plantarum Lb7 as inoculants.Suitability of Lactococcus lactis Tw34 and Lactobacillus plantarum Lb7 as bio-silage inoculants was studied. Experiments were carried out with a mixture of hake (Merluccius hubbsi) by-products and barley meal fermented during 7 days. A sample acidified with lactic acid was used as control. In biological mixtures, the pH dropped below 5,0 after 2 days of fermentation and remained stable until the end of the experience. Maximum populations (> 109 CFU/g) were reached after 5 days of incubation at 18°C. Water soluble peptides concentration increased during the 7 days of incubation and no significant differences (p 0,05). After 7 days, phosphorous concentrations reached 2.26 and 2,42 g/100 g in bio-silages fermented with Lc. lactis Tw34 and Lb. plantarum Lb7, respectively, while control values remains almost stable (1,61 g/100 g). At the end of the experience, inhibitory activity of trypsin was abolished in both bio-silage while in control sample antinutritional factors remained active. The results indicated the feasibility of the use of the selected mixture as substrate for bio-silage production and the effectiveness Lc. lactis Tw34 and Lb. plantarum Lb7 as inoculants.Foi estudada a capacidade de inoculantes em biosilagem de Lactococcus lactis Tw34 y Lactobacillus plantarum Lb7. Os experimentos foram realizados com uma mistura de produtos de merluza (Merluccius hubbsi) e farinha de cevada, fermentada durante 7 dias. Como controle se utilizou uma amostra acidificada com ácido láctico. Nas misturas biológicas, o pH ficou abaixo 5,0 depois de 2 dias de fermentação e permaneceu estável até o final do experimento. A contagem máxima de viáveis (>109 UFC/g) foi alcançada depois de 5 dias de incubação a 18°C. A concentração de peptídeos solúveis em água aumentou durante os 7 dias de incubação e não houve diferença significativa (p 0,05). Depois de 7 dias, as concentrações de fósforo alcançadas foram de 2,26 e 2,42 g/100 g nas silagens fermentadas com Lc. lactis Tw34 e Lb. plantarum Lb7 respectivamente, en quanto que no controle os valores permaneceram quase estáveis (1,61 g/100 g). No final do experimento, a atividade inibitória de tripsina foi suprimida em ambas silagens enquanto que no controle os fatores antinutricionais continuavam ativos. Os resultados indicam a possibilidade do uso de uma mistura selecionada como substrato para a produção de silagem e a capacidade de Lc. lactis Tw34 e Lb. plantarum Lb7 como inoculantes.Fil: Marguet, Emilio Rogelio. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de la Patagonia "San Juan Bosco". Facultad de Ciencias Naturales - Sede Trelew; ArgentinaFil: Vallejo, Marisol. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de la Patagonia "San Juan Bosco". Facultad de Ciencias Naturales - Sede Trelew; ArgentinaFil: Schulman, Gustavo. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de la Patagonia "San Juan Bosco". Facultad de Ciencias Naturales - Sede Trelew; ArgentinaFil: Ibañez, Cecilia Mariel. Universidad Nacional de la Patagonia "San Juan Bosco". Facultad de Ciencias Naturales - Sede Trelew; ArgentinaFil: Ledesma, Pablo Jeronimo. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de la Patagonia "San Juan Bosco". Facultad de Ciencias Naturales - Sede Trelew; ArgentinaFil: Parada, Romina Belén. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de la Patagonia "San Juan Bosco". Facultad de Ciencias Naturales - Sede Trelew; Argentin
Disruption of interleukin-1β autocrine signaling rescues complex I activity and improves ROS levels in immortalized epithelial cells with impaired cystic fibrosis transmembrane conductance regulator (CFTR) function.
Patients with cystic fibrosis (CF) have elevated concentration of cytokines in sputum and a general inflammatory condition. In addition, CF cells in culture produce diverse cytokines in excess, including IL-1β. We have previously shown that IL-1β, at low doses (∼30 pM), can stimulate the expression of CFTR in T84 colon carcinoma cells, through NF-κB signaling. However, at higher doses (>2.5 ng/ml, ∼150 pM), IL-1β inhibit CFTR mRNA expression. On the other hand, by using differential display, we found two genes with reduced expression in CF cells, corresponding to the mitochondrial proteins CISD1 and MTND4. The last is a key subunit for the activity of mitochondrial Complex I (mCx-I); accordingly, we later found a reduced mCx-I activity in CF cells. Here we found that IB3-1 cells (CF cells), cultured in serum-free media, secrete 323±5 pg/ml of IL-1β in 24 h vs 127±3 pg/ml for S9 cells (CFTR-corrected IB3-1 cells). Externally added IL-1β (5 ng/ml) reduces the mCx-I activity and increases the mitochondrial (MitoSOX probe) and cellular (DCFH-DA probe) ROS levels of S9 (CFTR-corrected IB3-1 CF cells) or Caco-2/pRSctrl cells (shRNA control cells) to values comparable to those of IB3-1 or Caco-2/pRS26 cells (shRNA specific for CFTR). Treatments of IB3-1 or Caco-2/pRS26 cells with either IL-1β blocking antibody, IL-1 receptor antagonist, IKK inhibitor III (NF-κB pathway) or SB203580 (p38 MAPK pathway), restored the mCx-I activity. In addition, in IB3-1 or Caco-2/pRS26 cells, IL-1β blocking antibody, IKK inhibitor III or SB203580 reduced the mitochondrial ROS levels by ∼50% and the cellular ROS levels near to basal values. The AP-1 inhibitors U0126 (MEK1/2) or SP600125 (JNK1/2/3 inhibitor) had no effects. The results suggest that in these cells IL-1β, through an autocrine effect, acts as a bridge connecting the CFTR with the mCx-I activity and the ROS levels
The mitochondrial complex I activity is reduced in cells with impaired cystic fibrosis transmembrane conductance regulator (CFTR) function
Abstract: Cystic fibrosis (CF) is a frequent and lethal autosomal recessive disease. It results from different possible mutations in the CFTR gene, which encodes the CFTR chloride channel. We have previously studied the differential expression of genes in CF and CF corrected cell lines, and found a reduced expression of MTND4 in CF cells. MTND4 is a mitochondrial gene encoding the MTND4 subunit of the mitochondrial Complex I (mCx-I). Since this subunit is essential for the assembly and activity of mCx-I, we have now studied whether the activity of this complex was also affected in CF cells. By using Blue Native-PAGE, the in-gel activity (IGA) of the mCx-I was found reduced in CFDE and IB3-1 cells (CF cell lines) compared with CFDE/ 6RepCFTR and S9 cells, respectively (CFDE and IB3-1 cells ectopically expressing wild-type CFTR). Moreover, colon carcinoma
T84 and Caco-2 cells, which express wt-CFTR, either treated with CFTR inhibitors (glibenclamide, CFTR(inh)-172 or GlyH101) or transfected with a CFTR-specific shRNAi, showed a significant reduction on the IGA of mCx-I. The reduction of the mCx-I activity caused by CFTR inhibition under physiological or pathological conditions may have a profound impact on mitochondrial functions of CF and non-CF cells
Graphical summary for IL-1β effects on mCx-I activity and mitochondrial ROS levels in IB3-1 and Caco-2/pRS26.
<p>The figure illustrates the interactions among the different proteins, kinases or small molecules involved in this work. The interactions were drawn by using the software Pathway Studio (v 9, Elsevier). Arrows with the+symbol represent stimulations and those with the -| symbol represent inhibition. Green ellipses: small molecules; red sickle-vertex: kinases; purple rectangle: disease (CF); blue star-vertex: shRNA specific for CFTR. The results obtained with IL-1β blocking Ab or with the receptor inhibitor IL1RN suggest that an autocrine IL-1β signaling is responsible for the reduced mCx-I activity and the increased ROS levels seen in IB3-1 CF cells or in Caco-2/pRS26 cells. Inhibition of NF-κB or p38 MAPK also resulted in increased mCx-I activity and decreased ROS levels. The inhibition of MEK1/2 or JNKs (AP-1 pathway) had no effects. The mechanisms by which CFTR increases IL-1β and IL1-β, p38 MAPKs or NF-κB inhibit mCx-I and increase ROS levels remain to be determined (dotted lines).</p
P38/MAPK1 inhibition.
<p>After 24-1 cells were incubated for 24 h with increasing concentrations of the p38 MAPK inhibitor SB203580 (1, 5, 10 and 20 µM) and mCx-I activity was measured by using BN-PAGE and spectrophotometry. A: mCx-I in-gel activity (IGA) and mCx-III (WB). B: Densitometric quantification and statistical analysis of the results shown in panel A. IGA of mCx-I was calculated as the ratio mCx-I (IGA)/mCx-III (WB). C: Spectrophotometric measurements of the mitochondrial NADH-cytochrome c reductase activity in the same experiments of panel A, expressed as percentage (%) relative to S9 control values. Measurements were performed in triplicate and data were expressed as mean ± SE of three independent experiments (n = 3). *indicates p<0.05 compared with basal IB3-1 cells.</p