RESUMEN
Marine environments are expected to be one of the most affected ecosystems by climate change, notably with increasing ocean temperature and ocean acidification. In marine environments, microbial communities provide important ecosystem services ensuring biogeochemical cycles. They are threatened by the modification of environmental parameters induced by climate change that, in turn, affect their activities. Microbial mats, ensuring important ecosystem services in coastal areas, are well-organized communities of diverse microorganisms representing accurate microbial models. It is hypothesized that their microbial diversity and metabolic versatility will reveal various adaptation strategies in response to climate change. Thus, understanding how climate change affects microbial mats will provide valuable information on microbial behaviour and functioning in changed environment. Experimental ecology, based on mesocosm approaches, provides the opportunity to control physical-chemical parameters, as close as possible to those observed in the environment. The exposure of microbial mats to physical-chemical conditions mimicking the climate change predictions will help to decipher the modification of the microbial community structure and function in response to it. Here, we present how to expose microbial mats, following a mesocosm approach, to study the impact of climate change on microbial community.
RESUMEN
This study aimed to determine the effect of the climatic change on the phototrophic communities of hypersaline microbial mats. Ocean acidification and warming were simulated alone and together on microbial mats placed into mesocosms. As expected, the temperature in the warming treatments increased by 4 °C from the initial temperature. Surprisingly, no significance difference was observed between the water pH of the different treatments despite of a decrease of 0.4 unit pH in the water reserves of acidification treatments. The salinity increased on the warming treatments and the dissolved oxygen concentration increased and was higher on the acidification treatments. A total of 37 pigments were identified belonging to chlorophylls, carotenes and xanthophylls families. The higher abundance of unknown chlorophyll molecules called chlorophyll derivatives was observed in the acidification alone treatment with a decrease in chlorophyll a abundance. This change in pigmentary composition was accompanied by a higher production of bound extracellular carbohydrates but didn't affect the photosynthetic efficiency of the microbial mats. A careful analysis of the absorption properties of these molecules indicated that these chlorophyll derivatives were likely bacteriochlorophyll c contained in the chlorosomes of green anoxygenic phototroph bacteria. Two hypotheses can be drawn from these results: 1/ the phototrophic communities of the microbial mats were modified under acidification treatment leading to a higher relative abundance of green anoxygenic bacteria, or 2/ the highest availability of CO2 in the environment has led to a shift in the metabolism of green anoxygenic bacteria being more competitive than other phototrophs.
Asunto(s)
Bacterioclorofilas , Cambio Climático , Clorofila , Clorofila A , Humanos , Concentración de Iones de Hidrógeno , Agua de MarRESUMEN
Acquired and inherited thrombophilia have both been reported to be associated with an increased risk of obstetric complications in early or later stages of pregnancy. Annexin A2 (ANXA2) is strongly expressed in vascular and placental tissues and plays a crucial role in fibrinolysis. The aim of the present study was to evaluate the prevalence of antibodies directed against ANXA2 in patients with recurrent miscarriage or obstetric complications. Anti-ANXA2 antibodies (aANXA2) were detected by ELISA in the sera from 46 women with obstetric morbidity, mainly recurrent miscarriage. The cut-off value for positivity was defined as 3 standard deviations above the mean optical density (OD) obtained in the sera from 42 female blood donors. The prevalence of aANXA2 in patients and healthy individuals was 15.2% and 2.3%, respectively. A statistically significant difference was observed between the 2 groups in terms of aANXA2 IgG titers (p=0.01). The highest aANXA2 levels were observed in sera from 2 patients with recurrent miscarriage and one patient with preeclampsia. aANXA2 could play a role in thrombotic mechanisms leading to recurrent pregnancy loss and placental vascular disease. Further studies are needed to determine whether ANXA2 is critical for maintenance of placental integrity.
Asunto(s)
Aborto Habitual/epidemiología , Anexina A2/inmunología , Mortinato/epidemiología , Trombofilia/epidemiología , Adolescente , Adulto , Anexina A5/inmunología , Anticuerpos Antifosfolípidos/sangre , Estudios de Casos y Controles , Femenino , Francia/epidemiología , Humanos , Inmunidad Humoral , Morbilidad , Embarazo , Prevalencia , Estudios Retrospectivos , Adulto JovenRESUMEN
AIMS: To evaluate vascular expression of annexin A2 (ANXA2) and its subunit S100A10 in lupus nephritis (LN). METHODS: The present histological study included 14 patients with LN and 11 controls (patients with non-lupus kidney diseases). Kidney biopsies from patients with lupus were scored for lupus glomerulonephritis (according to the International Society of Nephrology/Renal Pathology Society 2003 classification) and vascular lesions (such as microthrombi and antiphospholipid syndrome nephropathy (APSN)). ANXA2 and S100A10 expression in glomerular and peritubular capillaries was evaluated by immunohistochemistry on tissue sections. The staining intensity score ranged from 0 (no expression) to 4 (intense expression). RESULTS: In patients with LN, the median age (range) at first kidney biopsy was 36 (18-49). Vascular lesions were observed in six patients (including two with APSN). We observed intense expression of ANXA2 in glomerular and peritubular capillaries while expression of S100A10 was weaker. However, one of the patients with APSN showed strong S100A10 expression. Patients with LN and controls differed significantly in terms of S100A10 expression in peritubular capillaries. We also observed a statistical difference between patients who had LN with renal vascular lesions and those without renal vascular lesions in terms of ANXA2 expression in peritubular capillaries. CONCLUSIONS: The presence of vascular lesions in LN appears to be associated with significant differences in the vascular expression of ANXA2. Vascular expression of ANXA2 was somewhat higher in LN. Vascular expression of S100A10 was somewhat lower in LN (except one of the two patients with APSN). Further studies of ANXA2's putative value as a biomarker of active LN or of vascular lesions in LN are required.
Asunto(s)
Anexina A2/metabolismo , Síndrome Antifosfolípido/metabolismo , Glomérulos Renales/metabolismo , Nefritis Lúpica/metabolismo , Proteínas S100/metabolismo , Adolescente , Adulto , Anciano , Biomarcadores/metabolismo , Capilares/metabolismo , Femenino , Francia , Humanos , Inmunohistoquímica , Riñón/metabolismo , Riñón/fisiopatología , Masculino , Persona de Mediana Edad , Adulto JovenRESUMEN
Annexin A2 (ANXA2, an endothelial cell receptor for plasminogen and tissue plasminogen activator) has been identified as a new autoantigen in antiphospholipid syndrome (APS). The aim of the present study was to evaluate the presence of antibodies against the N-terminal domain of annexin A2 (ANXA2) in primary APS (PAPS). By using a synthetic peptide corresponding to the 31N-terminal amino acids of ANXA2 (ANXA2(N31)) as an antigen, we performed an enzyme-linked immunosorbent assay (ELISA) to measure anti-ANXA2(N31) IgG and IgM antibodies in the serum of PAPS patients (n=19), systemic lupus erythematosus (SLE) patients (n=50) and healthy blood donors (n=106). We did not find any statistically differences between the three groups in terms of IgG and IgM anti-ANXA2(N31) titres. Elevated IgG anti-ANXA2(N31) titres were not observed in the serum of PAPS or SLE patients who had previously tested positive for anti-ANXA2 antibodies. Thus, the ANXA2 N-terminal domain does not appear to be the target antigen for anti-ANXA2 antibodies in APS.
Asunto(s)
Anexina A2/inmunología , Síndrome Antifosfolípido/inmunología , Autoanticuerpos/sangre , Adolescente , Adulto , Anciano , Síndrome Antifosfolípido/sangre , Autoantígenos/inmunología , Estudios de Casos y Controles , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Inmunoglobulina G/sangre , Inmunoglobulina M/sangre , Lupus Eritematoso Sistémico/sangre , Lupus Eritematoso Sistémico/inmunología , Masculino , Persona de Mediana Edad , Estudios RetrospectivosRESUMEN
BACKGROUND: The incidence of morbid obesity is increasing in France; adjustable gastric banding has become the most common surgical treatment. PATIENTS: We report seven cases of patients who presented with gastric erosion as a complication of gastric banding; this occurred at a mean interval of 4 years following the initial bariatric procedure. RESULTS: In six cases, repair was performed laparoscopically; one case required conversion to an open laparotomy approach. There was no mortality but morbidity occurred in 57% of cases: pleural effusion (two) and wound abscess (two). CONCLUSION: Gastric erosion and migration of adjustable gastric rings can occur at a long interval after laparoscopic gastric banding. Long-term follow-up is necessary in all such patients.
Asunto(s)
Migración de Cuerpo Extraño/cirugía , Gastroplastia/instrumentación , Estómago , Adulto , Anciano , Femenino , Humanos , Masculino , Persona de Mediana EdadRESUMEN
OBJECTIVES: The objective of this study were (1) to evaluate the prevalence of anti-annexin II antibodies in patients with various autoimmune diseases and antiphospholipid syndrome and (2) to correlate anti-annexin II antibodies with anti-phospholipid antibodies. MATERIALS AND METHODS: Anti-annexin II antibodies and anti-phospholipid were detected, using an enzyme-linked immunosorbent assay, in the serum of patients with primary antiphospholipid syndrome (n = 16), systemic lupus erythematosus (n = 53), primary Sjögren syndrome (n = 71), systemic sclerosis (n = 17), systemic vasculitis (n = 18), and rheumatoid arthritis (n = 119). Healthy blood donors (n = 99) were used as controls. RESULTS: Anti-annexin II antibodies were significantly more prevalent in patients with connective tissue diseases (8.5%), especially antiphospholipid syndrome (14.8%) and rheumatoid arthritis (10%), than in controls (2%). An inverse correlation was observed between anti-annexin II antibodies and antiphospholipid antibodies. CONCLUSION: Annexin II can be recognized by antibodies in serum from patients with systemic autoimmune disorders. Further studies are required to determine the clinical significance of anti-annexin II antibodies in rheumatoid arthritis and to determine their diagnostic value in discriminating clinical subgroups of patients with antiphospholipid syndrome.
Asunto(s)
Anexina A2/inmunología , Síndrome Antifosfolípido/inmunología , Autoanticuerpos/sangre , Autoantígenos/inmunología , Adulto , Anciano , Anticuerpos Antifosfolípidos/sangre , Anticuerpos Antifosfolípidos/inmunología , Síndrome Antifosfolípido/sangre , Enfermedades Autoinmunes/sangre , Enfermedades Autoinmunes/inmunología , Femenino , Humanos , Masculino , Persona de Mediana EdadRESUMEN
Vascular and/or valvular calcifications in patients with chronic kidney disease (CKD) appear to indicate a poor prognosis in terms of overall survival and cardiovascular morbidity and mortality. Inflammation and oxidative stress represent new features of the arterial and/or valvular calcification process. However, only limited observational and epidemiological data are available in these areas. Therefore, the link between inflammation, oxidation and vascular and/or valvular calcifications deserves careful consideration in CKD patients, since they may become targets for the development of new therapeutic strategies.
Asunto(s)
Calcinosis/etiología , Enfermedades Renales/complicaciones , Estrés Oxidativo , Enfermedades Vasculares/etiología , Calcinosis/metabolismo , Niño , Enfermedad Crónica , Humanos , Inflamación/complicaciones , Enfermedades Renales/metabolismo , Enfermedades Vasculares/metabolismoRESUMEN
Cardiovascular diseases are the main cause of mortality in the western world. It is widely accepted that atherosclerosis, the first etiology, is influenced by free radicals and the oxidizing stress that they cause. In the oxidative theory of atherosclerosis, the atheromatous lesion is initiated by oxidation of two density lipoproteins (LDL), a process still known as lipid peroxidation. Oxidized LDL have many effects on the cells of the vessel wall which, provide an explanation to most of the cellular and tissular changes observed in the plaque. The vascular complications of hypercholesterolaemia, diabetes, hyperhomocysteinemia, hypertension and smoking may, in part, be secondary to oxidizing stress that impairs endothelial function and modify the lipids in the intima of the vessels. The aim of this paper is to review the modes of free radical production, to determine the role of oxidizing stress in the development of atherosclerosis and to show how the different risk factors may initiate atheroma through oxidizing stress.
Asunto(s)
Arteriosclerosis/complicaciones , Arteriosclerosis/fisiopatología , Enfermedades Cardiovasculares/fisiopatología , Radicales Libres/efectos adversos , Estrés Oxidativo , Humanos , Factores de RiesgoRESUMEN
Atherosclerosis includes a series of cellular and molecular responses characteristic of an inflammatory disease. We provide evidence that cupric-ion-oxidized LDL (CuLDL) or endothelial cell-oxidized LDL (ELDL) induced the activation by Tyr-phosphorylation of JAK2, one of the Janus kinase involved upstream of STATs in the JAK/STAT pathway of cytokine transduction. Oxidized LDL (OxLDL) also initiated STAT1 and STAT3 Tyr-phosphorylation and translocation to the nucleus, with a more marked effect for the extensively modified CuLDL. Genistein, a nonspecific Tyr-kinase inhibitor, and AG490, a specific inhibitor of JAKs, markedly prevented the CuLDL-induced enhancement of STAT1 and STAT3 Tyr-phosphorylation and DNA-binding activity, suggesting that JAKs are the main kinases involved in STATs' activation by oxidized LDL. In addition, the lipid extract of CuLDL increased the intracellular levels of lipid peroxidation products and the Tyr-phosphorylation of JAK2, STAT1, and STAT3, whereas the antioxidant vitamin E prevented all these effects. These results demonstrate that OxLDL induces the activation by Tyr-phosphorylation of JAK2, STAT1, and STAT3 by generation of an intracellular oxidative stress by means of its lipid peroxidation products, and thus include JAK2 within the range of oxidative stress-activated kinases.
Asunto(s)
Lipoproteínas LDL/farmacología , Estrés Oxidativo , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas , Línea Celular , Núcleo Celular/metabolismo , Cobre/química , ADN/metabolismo , Proteínas de Unión al ADN/metabolismo , Endotelio Vascular/metabolismo , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Fibroblastos , Genisteína/farmacología , Humanos , Janus Quinasa 2 , Fosforilación , Fosfotirosina/metabolismo , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Factor de Transcripción STAT1 , Factor de Transcripción STAT3 , Transducción de Señal , Transactivadores/metabolismo , Tirfostinos/farmacologíaRESUMEN
UV-A irradiation caused a dose-dependent decrease in cellular oxygen consumption (56%) and ATP content (65%) in human NCTC 2544 keratinocytes, one hour after treatment. This effect was partially reversed by maintaining the irradiated cells in normal culture conditions for 24 h. Using malate/glutamate or succinate as substrates for mitochondrial electron transport, the oxygen uptake of digitonin-permeabilised cells was greatly inhibited following UV-A exposure. These results strongly suggest that UV-A irradiation affects the state 3 respiration of the mitochondria. However, under identical conditions, UV-A exposure did not reduce the mitochondrial transmembrane potential. The antioxidant, vitamin E inhibited UV-A-induced lipid peroxidation, but did not significantly prevent the UV-A-mediated changes in cellular respiration nor the decrease in ATP content, suggesting that these effects were not the result of UV-A dependent lipid peroxidation. UV-A irradiation also led to an increase in MnSOD gene expression 24 hours after treatment, indicating that the mitochondrial protection system was enhanced in response to UV-A treatment. These findings provide evidence that impairment of mitochondrial respiratory activity is one of the early results of UV-A irradiation for light doses much lower than the minimal erythemal dose.
Asunto(s)
Respiración de la Célula/efectos de la radiación , Mitocondrias/efectos de la radiación , Adenosina Trifosfato/metabolismo , Antioxidantes , Línea Celular , Respiración de la Célula/fisiología , Humanos , Líquido Intracelular/metabolismo , Membranas Intracelulares/fisiología , Membranas Intracelulares/efectos de la radiación , Queratinocitos/citología , Queratinocitos/efectos de la radiación , Potenciales de la Membrana/efectos de la radiación , Mitocondrias/fisiología , Superóxido Dismutasa/metabolismo , Rayos UltravioletaRESUMEN
Endogenous ceramide (CER) was generated by treatment of cultured fibroblasts with sphingomyelinase (SMase) from Bacillus cereus. A 30 min treatment with 0.1-0.3 U/ml SMase induced a dose-dependent increase in the intracellular level of CER. The activation of the transcription factors signal transducer and activator of transcription (STAT) 1 and STAT3 by SMase was investigated by determination of the phosphorylation state by immunoblot, and of DNA binding activity by electrophoretic mobility shift assay. SMase treatment induced a dose-dependent Tyr-phosphorylation of STAT1/3. SMase also enhanced STAT1/3 DNA binding activity in a dose-dependent manner. Concomitantly, SMase enhanced the Tyr-phosphorylation of Janus kinase (JAK) 2, a Tyr-kinase localized upstream of STATs in the JAK/STAT pathway. The Tyr-kinase inhibitor genistein and the JAK inhibitor AG490 both prevented JAK2 Tyr-phosphorylation, together with STAT1 and STAT3 Tyr-phosphorylation and binding activity. The SMase-induced increase in STAT1/3 binding activity was prevented by methyl-beta-cyclodextrin, a cholesterol binding agent that causes a loss of compartmentalization of the molecules located in caveolae. This increase was also prevented by the MEK inhibitor PD98059, thus demonstrating the role of the MEK/ERK pathway in this system. Besides ERK, SMase activated other signaling kinases such as JNK and p38. Exogenous natural CER also activated STAT1/3 binding activity, which indicates that most probably, endogenous CER is the second messenger involved in the effect of SMase. These results describe a crosstalk between the SMase/CER and the JAK/STAT signaling pathways and include JAK2 within the range of CER-activated intracellular kinases.
Asunto(s)
Ceramidas/biosíntesis , Proteínas de Unión al ADN/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas , Transducción de Señal , Transactivadores/metabolismo , beta-Ciclodextrinas , Línea Celular , Membrana Celular/metabolismo , Ciclodextrinas/farmacología , Proteínas de Unión al ADN/genética , Relación Dosis-Respuesta a Droga , Activación Enzimática , Fibroblastos/metabolismo , Humanos , Janus Quinasa 2 , Fosforilación , Factor de Transcripción STAT1 , Factor de Transcripción STAT3 , Esfingomielina Fosfodiesterasa/metabolismo , Esfingomielina Fosfodiesterasa/farmacología , Transactivadores/genética , Tirosina/metabolismoRESUMEN
Exposure of human keratinocytes to UVA radiation induced an increase in ceramide (CER) intracellular content, with a dose-dependent effect within the range of 4-9 J/cm(2). The production of CER reached a maximum 2 h after UVA irradiation. The increase of CER was proportional to the intracellular content of reactive oxygen species, was prevented by the antioxidant vitamin E, and enhanced by the prooxidant buthionine-sulfoximine, suggesting the involvement of an oxidative stress. UVA decreased both neutral and acid sphingomyelinase activities measured in vitro. A direct cleavage of sphingomyelin to CER by UVA, recently described, was not observed under our experimental conditions. We also show that, downstream of CER, UVA activated the Ser/Thr kinases ERK, JNK, and p38. Since ceramide has been shown to play a role in stress kinase activation, our results provide a possible mechanism for UVA-induced activation of stress kinases via ceramide formation. However, the actual mechanisms whereby CER is produced in cultured cells under UVA exposure remain to be specified.
Asunto(s)
Ceramidas/efectos de la radiación , Proteínas Quinasas JNK Activadas por Mitógenos , Rayos Ultravioleta , Línea Celular , Ceramidas/metabolismo , Relación Dosis-Respuesta en la Radiación , Activación Enzimática/efectos de la radiación , Humanos , MAP Quinasa Quinasa 4 , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Estrés Oxidativo , Especies Reactivas de Oxígeno/metabolismo , Esfingomielina Fosfodiesterasa/metabolismo , Esfingomielinas/metabolismo , Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
It is now well established that oxidized LDL (OxLDL) is involved in the progression of the atheromatous plaque via several mechanisms, including its cytotoxicity toward the arterial wall. Our study demonstrates that a 4-h incubation of cultured human fibroblasts with 25-75 microg/ml OxLDL induced a dose-dependent increase in the intracellular levels of reactive oxygen species (ROS) and lipid peroxidation end products (TBARS). This effect was markedly prevented by the antioxidant vitamin E. The lipid extract of OxLDL partially reproduced the action of the LDL particle itself. Concomitantly, OxLDL enhanced the DNA binding activity of p53 measured by electrophoretic mobility shift assay, and the intracellular protein level of p53 determined by immunoblot analysis. Cycloheximide prevented the OxLDL-induced augmentation in both p53 binding activity and intracellular level. Again, the lipid extract of OxLDL reproduced the effect of OxLDL on p53 binding activity, whereas vitamin E prevented it. These results indicate that OxLDL initiates an intracellular oxidative stress by means of its lipid peroxidation products, leading to the activation of the tumour suppressor p53 by enhancement of p53 protein synthesis. This effect might be related to the cytotoxic effect of OxLDL since the activation of p53 is known to lead to cell cycle arrest, necrosis or apoptosis.
Asunto(s)
Fibroblastos/efectos de los fármacos , Lipoproteínas LDL/farmacología , Estrés Oxidativo/fisiología , Proteína p53 Supresora de Tumor/metabolismo , Sitios de Unión/efectos de los fármacos , Células Cultivadas , Cicloheximida/farmacología , ADN/biosíntesis , ADN/efectos de los fármacos , ADN/metabolismo , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Fibroblastos/fisiología , Genes Supresores de Tumor/fisiología , Humanos , Peroxidación de Lípido , Especies Reactivas de Oxígeno/metabolismo , Sustancias Reactivas al Ácido Tiobarbitúrico/metabolismo , Proteína p53 Supresora de Tumor/fisiología , Vitamina E/farmacologíaRESUMEN
The endocytotic pathway is profoundly altered by the UVA-induced photosensitization of HS 68 fibroblasts by the fluoroquinolone (FQ) antibiotics lomefloxacin, BAYy 3118, norfloxacin and ciprofloxacin, which preferentially localize in lysosomes. The endocytosis of low-density lipoproteins (LDL) loaded with two carbocyanine dyes compatible for effective Forster-type resonance energy transfer (FRET), namely 3,3'-dioctadecyloxacarbocyanine perchlorate (DiO) as the donor and 1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) as the acceptor, has been used as a model system. Binding of LDL to their cell surface receptors is impaired by irradiation with 10 J cm(-2) of UVA and/or treatment with 250 microM BAYy 3118 during 2 h. Perturbation of the plasma membrane by the FQ is revealed by the change in the rate of exchange of DiO from the LDL to the cell membrane as compared to untreated cells. The lysosomal degradation of LDL, demonstrated by the disappearance of FRET between DiO and DiI, is partly inhibited by the FQ. The actin filament network, involved in the fusion of mature endosomes with lysosomes, is readily destroyed upon photosensitization with the four FQ. However, actin depolymerization can be avoided by incubation of the cells with trans-epoxysuccinyl-1-leucylamido-(4-guanidino)butane, an inhibitor of lysosomal cathepsins prior to FQ photosensitization. All these data suggest that several components of the endocytotic pathway are impaired by photosensitization with these FQ.
Asunto(s)
Antiinfecciosos/efectos adversos , Endocitosis/efectos de los fármacos , Fluoroquinolonas , Trastornos por Fotosensibilidad/inducido químicamente , Células Cultivadas , Endocitosis/efectos de la radiación , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Fibroblastos/efectos de la radiación , Humanos , Lipoproteínas LDL/metabolismo , Trastornos por Fotosensibilidad/metabolismo , Espectrometría de FluorescenciaRESUMEN
The effect of ultraviolet A (UVA) radiation on the DNA-binding activity of the transcription factor STAT1 was studied by electromobility shift assay in the human keratinocyte cell line NCTC 2544. The STAT1-binding activity exhibited a biphasic pattern as a function of UVA doses. For UVA doses lower than 0.6 J/cm(2), a dose-dependent increase in STAT1 activity was observed. In a second phase, with higher UVA doses (1.5 to 9 J/cm(2)), the activity decreased and reached control value at 6 J/cm2. The enhancement of STAT1 activity was transient, peaked at 1 h after UV irradiation, and regularly decreased to control value 24 h after UV. Genistein, a tyrosine kinase inhibitor, H7, a serine/threonine kinase inhibitor, and PD 98059, a MEK inhibitor, prevented the UVA-induced enhancement of STAT1-binding activity, suggesting the involvement of Tyr, Ser/Thr kinases, and MEK in the observed effect. Immunoblot analysis directly demonstrated that the amount of Tyr-phosphorylated STAT1 was parallel to its DNA-binding activity. Immunoblot analysis also demonstrated the nuclear transport of STAT1 after UVA irradiation at low doses. At high doses, a decrease in the STAT1 level was observed both in the cytoplasmic and the nuclear compartments, suggesting that the inactivation was due to a degradation process. UVA irradiation initiated a dose-dependent increase in lipid peroxidation products and reactive oxygen species. Furthermore, the involvement of the oxidative stress in the UVA-induced effect on STAT1 activity is suggested by the protective action of the antioxidants alpha-tocopherol and N-acetylcysteine on both the activation phase (UVA doses lower than 1.5 J/cm(2)) and the inhibitory phase. By contrast, the pro-oxidant drug buthionine sulfoximine enhanced the effect of UVA on STAT1-binding activity. Since STATs are known as transducers of cytokine action, the enhancement of STAT1 activity by low doses of UVA might be related to the proinflammatory effect of solar radiations at the skin level.
Asunto(s)
Proteínas de Unión al ADN/efectos de la radiación , Queratinocitos/efectos de la radiación , Fosfotirosina/biosíntesis , Procesamiento Proteico-Postraduccional/efectos de la radiación , Proteínas Tirosina Quinasas/metabolismo , Transactivadores/efectos de la radiación , Transcripción Genética/efectos de la radiación , Rayos Ultravioleta , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/farmacología , Transporte Biológico/efectos de la radiación , ADN/metabolismo , Proteínas de Unión al ADN/metabolismo , Relación Dosis-Respuesta en la Radiación , Inhibidores Enzimáticos/farmacología , Flavonoides/farmacología , Genisteína/farmacología , Humanos , Queratinocitos/metabolismo , Peroxidación de Lípido , MAP Quinasa Quinasa 1 , Quinasas de Proteína Quinasa Activadas por Mitógenos/antagonistas & inhibidores , Fosforilación/efectos de la radiación , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Especies Reactivas de Oxígeno , Factor de Transcripción STAT1 , Sustancias Reactivas al Ácido Tiobarbitúrico/análisis , Transactivadores/metabolismoRESUMEN
A 48-h incubation of cultured human fibroblasts with 5 x 10(-5) M oleic acid or polyunsaturated fatty acids (PUFA) from the (n-6) (linoleic, gamma-linolenic and arachidonic acids) or (n-3) (alpha-linolenic and eicosapentaenoic acids) series resulted in an enrichment of the cells with the introduced fatty acid. Cell enrichment with PUFA initiated a rise in the intracellular level of reactive oxygen species (ROS) and lipid peroxidation products (thiobarbituric reactive substances TBARS). Simultaneously, cell enrichment with all the studied PUFA induced an increase in AP1 and NFkappaB binding activity measured by electrophoretic mobility shift assay, whereas no significant effect was observed with the monounsaturated oleic acid. Furthermore, the antioxidants vitamin E (alpha-tocopherol) and N-acetyl cysteine prevented both the arachidonic acid-induced increase in intracellular ROS and TBARS, and the activation of AP1 and NFkappaB. These results indicate that the accumulation of PUFA from (n-6) and (n-3) series elicited an intracellular oxidative stress, resulting in the activation of oxidative stress-responsive transcription factors such as AP1 and NFkappaB.
Asunto(s)
Ácidos Grasos Insaturados/farmacología , FN-kappa B/metabolismo , Estrés Oxidativo/fisiología , Factor de Transcripción AP-1/metabolismo , Vitamina E/farmacología , Ácido Araquidónico/metabolismo , Línea Celular , Medios de Cultivo , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Humanos , Cinética , Ácido Oléico/farmacología , Estrés Oxidativo/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismoRESUMEN
The effect of lipopolysaccharide (LPS, endotoxin) on low density lipoprotein (LDL) oxidative modification by copper ions, endothelial and smooth muscle cells was studied by determination of the level of lipid peroxidation products (thiobarbituric acid reactive substances or TBARS), the diene level and the electrophoretic mobility of the LDL particle. LPS 25-75 microg/ml induced a dose-dependent increase in LDL oxidation by copper ions, endothelial and smooth muscle cells. At 75 microg LPS/ml, the TBARS content was 1.9, 1.6, and 1.8-fold increased, respectively. The LDL degradation by J774 macrophage-like cells was concomitantly stimulated. Preincubation of the LDL particle with LPS induced a marked increase in the subsequent LDL oxidative modification either by copper ions or by endothelial and smooth muscle cells. In addition, pretreatment of endothelial and smooth muscle cells with LPS also induced an enhancement of LDL oxidative modification performed in the absence of LPS. This effect was accompanied by a parallel increase in superoxide anion release by the cells. These results point at one of the mechanisms involved in the described association between bacterial infection and acute myocardial infarction as well as coronary heart disease.
Asunto(s)
Cobre/farmacología , Endotelio Vascular/metabolismo , Endotoxinas/farmacología , Escherichia coli , Lipopolisacáridos/farmacología , Lipoproteínas LDL/metabolismo , Músculo Liso Vascular/metabolismo , Células Cultivadas , Relación Dosis-Respuesta a Droga , Macrófagos/metabolismo , Oxidación-Reducción , Sustancias Reactivas al Ácido Tiobarbitúrico/metabolismoRESUMEN
The effect of cupric ion-oxidized low density lipoprotein (Cu-LDL) or endothelial cell-oxidized LDL (E-LDL) on STAT1 and STAT3 (signal transducers and activators of transcription) DNA binding activity was investigated by electrophoretic mobility shift assay in human endothelial cells. Both oxidized LDL enhanced STAT1 and STAT3 binding to their respective consensus binding sites. Furthermore, the activation of STATs was proportional to the oxidation degree of LDL in that the highly oxidized Cu-LDL exhibited a more marked effect than E-LDL. Oxidized LDL induced an intracellular oxidative stress, as shown by the increase in the intracellular level of lipid peroxidation products (thiobarbituric acid-reactive substances) and in the level of reactive oxygen species, measured by the fluorescence of dichlorofluorescein diacetate. The binding activity of STAT1 and STAT3 paralleled these two parameters, which suggests that it is dependent upon the redox state of the cell. The activation of STATs by oxidized LDL was almost completely inhibited by the lipophilic antioxidant vitamin E, and partially antagonized by the hydrophilic thiol-containing compound N-acetylcysteine, suggesting that the oxidative stress induced by oxidized LDL is involved in the observed phenomenon. Furthermore, the lipid extract of Cu-LDL also activated STAT1 and STAT3. Since the STAT pathway plays a key role in cytokine and growth factor signal transduction, the activation of STATs by oxidized LDL might be related to their proinflammatory and fibroproliferative effect in the atherosclerotic plaque.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Lipoproteínas LDL/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Transactivadores/metabolismo , Proteínas de Unión al ADN/genética , Humanos , Factor de Transcripción STAT1 , Factor de Transcripción STAT3 , Transactivadores/genéticaRESUMEN
Oxidative modification of low-density lipoprotein (LDL) is an important feature in the initiation and progression of atherosclerosis. LDL modification by endothelial cells was studied after supplementation of the cells with oleic acid and polyunsaturated fatty acids (PUFA) of the n-6 and n-3 series. In terms of the lipid peroxidation product [thiobarbituric acid reactive substances (TBARS)] content and diene level of the LDL particle, oleic acid had no significant effect, and linoleic acid was poorly effective. Gamma linolenic acid (C18:3,n-6) and arachidonic acid (C20:4,n-6) increased by about 1.6-1.9-fold the cell-mediated LDL modification. PUFA from the n-3 series, alpha linolenic acid (C18:3,n-3), eicosapentaenoic acid (C20:5,n-3) and docosahexaenoic acid (C22:6,n-3), induced a less marked effect (1. 3-1.6-fold increase). The relative electrophoretic mobility of the LDL particle and its degradation by macrophages were enhanced in parallel. Concomitantly, PUFA stimulated superoxide anion secretion by endothelial cells. The intracellular TBARS content was also increased by PUFA. Comparison of PUFA from the two series indicates a good correlation between LDL oxidative modification, superoxide anion secretion and intracellular lipid peroxidation. The lipophilic antioxidant vitamin E decreased the basal as well as the PUFA-stimulated LDL peroxidation. These results indicate that PUFAs with a high degree of unsaturation of the n-6 and n-3 series could accelerate cell-mediated LDL peroxidation and thus aggravate the atherosclerotic process.