RESUMEN
Warming climate increases the risk for harmful leaf temperatures in terrestrial plants, causing heat stress and loss of productivity. The heat sensitivity may be particularly high in equatorial tropical tree species adapted to a thermally stable climate. Thermal thresholds of the photosynthetic system of sun-exposed leaves were investigated in three tropical montane tree species native to Rwanda with different growth and water use strategies (Harungana montana, Syzygium guineense and Entandrophragma exselsum). Measurements of chlorophyll fluorescence, leaf gas exchange, morphology, chemistry and temperature were made at three common gardens along an elevation/temperature gradient. Heat tolerance acclimated to maximum leaf temperature (Tleaf ) across the species. At the warmest sites, the thermal threshold for normal function of photosystem II was exceeded in the species with the highest Tleaf despite their higher heat tolerance. This was not the case in the species with the highest transpiration rates and lowest Tleaf . The results point to two differently effective strategies for managing thermal stress: tolerance through physiological adjustment of leaf osmolality and thylakoid membrane lipid composition, or avoidance through morphological adaptation and transpiratory cooling. More severe photosynthetic heat stress in low-transpiring montane climax species may result in a competitive disadvantage compared to high-transpiring pioneer species with more efficient leaf cooling.
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Termotolerancia , Árboles , Aclimatación , Fotosíntesis , Hojas de la Planta , Temperatura , Clima TropicalRESUMEN
It is unclear whether the additional conduit to supplement bilateral internal thoracic arteries (BITA) influences the patient outcome in coronary surgery. This retrospective study compared long-term survival of patients undergoing left-sided BITA grafting in which the third conduit to the right coronary system (RCA) was either vein graft (SVG) or gastroepiploic artery (GEA). From 1989 to 2014, 1432 consecutive patients underwent left-sided revascularization with BITA associated with SVG (nâ¯=â¯599) or GEA (nâ¯=â¯833) to RCA. Propensity score was calculated by logistic regression model and patients were matched 1 to 1 leading to 2 groups of 320 matched patients. The primary end point was the overall mortality from any cause. GEA was used in significantly lower risk patients. The 30-day mortality was 1.6% without influence of the graft configuration. Postoperative follow-up was 13.6 ± 6.6 years and was 94% complete. The significant difference in patients' survival observed at 20 years in favor of GEA in unmatched groups (48 ± 4% vs 33 ± 6%, P < 0.001) was not confirmed in matched groups (41 ± 7% vs 36 ± 7%, Pâ¯=â¯0.112). In multivariable Cox model analysis, the conduit used to RCA did not influence the long-term survival in matched groups, like no other graft configuration or operative parameter. Only complete revascularization remained predictor of survival (Pâ¯=â¯0.016), with age (P < 0.0001), diabetes status (Pâ¯=â¯0.007), and left ventricle ejection fraction (P < 0.0001). Long-term survival in patients undergoing BITA grafting is not affected by using GEA as third arterial conduit in alternative to SVG. Further studies are necessary to assess its impact on long-term cardiac events.
Asunto(s)
Enfermedad de la Arteria Coronaria , Arteria Gastroepiploica , Arterias Mamarias , Puente de Arteria Coronaria/efectos adversos , Enfermedad de la Arteria Coronaria/diagnóstico por imagen , Enfermedad de la Arteria Coronaria/cirugía , Arteria Gastroepiploica/cirugía , Arteria Gastroepiploica/trasplante , Humanos , Arterias Mamarias/cirugía , Arterias Mamarias/trasplante , Estudios Retrospectivos , Resultado del TratamientoRESUMEN
BACKGROUND: The benefit of arterial revascularization in coronary surgery remains controversial. The incremental value of additional grafts to the left internal thoracic artery (ITA) has been mainly assessed according to the number of arterial grafts, possibly limiting the detection of its actual impact. We analyzed the influence of the number of distal arterial anastomoses (DAA) performed on late mortality in patients having received from one to three arterial grafts. METHODS: Retrospective review of 3685 primary isolated coronary artery bypass grafting (CABG) performed from 1989 to 2014 was conducted with a 13-year mean follow-up. One arterial graft (SITA) was used in 969 patients, two arterial grafts, ITA or gastroepiploic artery (GEA), in 1883 patients (BITA: 1644; SITA+GEA: 239), and three arterial grafts in 833 patients (BITA+GEA). Totally, 795 patients (22%) received one DAA, 1142 patients (31%) two, 1337 patients (36%) three, and 411 patients (11%) four or more. A sub-group analysis was done in the 2104 patients with 3-vessel disease who received at least 2 arterial grafts. RESULTS: In this series the early mortality was 1.6% and it was not influenced by the surgical technique. Late mortality was significantly influenced by age, gender, heart failure, LV ejection fraction, diabetes status, complete revascularization, number of arterial grafts, number of DAA, both ITA, sequential ITA graft, GEA graft. In multivariable analysis with Cox regression model, the number of DAA was the only technical significant independent prognosis factor of late survival (p < 0.0001), predominant over both ITA, complete revascularization and number of arterial grafts. The impact of the number of DAA on survival was found discriminant from 1 to 3; after 3 there was no more additional effect. In 3-vessel disease patients who received at least 2 arterial grafts, the number of DAA remained a significant independent prognosis factor of late survival (p < 0.0001). CONCLUSIONS: The number of distal arterial anastomoses is an independent predictor of long-term survival, predominant over the number of arterial grafts and the completeness of the revascularization; higher the number, better the late survival. It is a strong support of the extensive use of arterial grafting in CABG.
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Puente de Arteria Coronaria/mortalidad , Vasos Coronarios/cirugía , Anciano , Anastomosis Quirúrgica , Femenino , Estudios de Seguimiento , Arteria Gastroepiploica/trasplante , Humanos , Masculino , Arterias Mamarias/trasplante , Persona de Mediana Edad , Complicaciones Posoperatorias/mortalidad , Modelos de Riesgos Proporcionales , Estudios Retrospectivos , Tasa de Supervivencia , Resultado del TratamientoRESUMEN
Identification of blood-based biomarkers of Alzheimer's disease (AD) remains a challenge. Neuropathological studies have identified enlarged endosomes in post-mortem brains as the earliest cellular change associated to AD. Here the presence of enlarged endosomes was investigated in peripheral blood mononuclear cells from 48 biologically defined AD patients (25 with mild cognitive impairment and 23 with dementia (AD-D)), and 23 age-matched healthy controls using immunocytochemistry and confocal microscopy. The volume and number of endosomes were not significantly different between AD and controls. However, the percentage of cells containing enlarged endosomes was significantly higher in the AD-D group as compared with controls. Furthermore, endosomal volumes significantly correlated to [C(11)]PiB cortical index measured by positron emission tomography in the AD group, independently of the APOE genotype, but not to the levels of amyloid-beta, tau and phosphorylated tau measured in the cerebrospinal fluid. Importantly, we confirmed the presence of enlarged endosomes in fibroblasts from six unrelated AD-D patients as compared with five cognitively normal controls. This study is the first, to our knowledge, to report morphological alterations of the endosomal compartment in peripheral cells from AD patients correlated to amyloid load that will now be evaluated as a possible biomarker.
Asunto(s)
Enfermedad de Alzheimer/patología , Endosomas/patología , Fibroblastos/patología , Leucocitos Mononucleares/fisiología , Anciano , Anciano de 80 o más Años , Enfermedad de Alzheimer/sangre , Enfermedad de Alzheimer/diagnóstico , Péptidos beta-Amiloides/líquido cefalorraquídeo , Apolipoproteínas E/genética , Biomarcadores/sangre , Biomarcadores/líquido cefalorraquídeo , Biomarcadores/metabolismo , Estudios de Casos y Controles , Disfunción Cognitiva/sangre , Disfunción Cognitiva/diagnóstico , Disfunción Cognitiva/fisiopatología , Femenino , Humanos , Masculino , Microscopía Confocal , Persona de Mediana Edad , Neuroimagen , Tomografía de Emisión de Positrones , Proteínas tau/líquido cefalorraquídeoRESUMEN
INTRODUCTION: The increased prevalence of the sporadic form of Alzheimer's disease (AD) has become a significant health issue in the elderly population. The need for early diagnosis is imperative because this, along with the development of novel therapeutic treatments, would permit the rapid and perhaps more efficient treatment of these debilitating disorders early on. BACKGROUND: Over the last decade, the potential use of certain biomarkers in the cerebrospinal fluid (CSF), and more recently, in the plasma has been investigated. Among the candidates studied includes the neurotoxic amyloid beta peptide and the Tau protein. However, although these two proteins have been clearly shown to be directly related to the pathophysiology of this disorder, it has proven difficult to establish a clear relationship between plasma or CSF levels of Abeta and Tau and the incidence and severity of AD in patients. This is due in part to differences in methodologies related to the detection sensitivity, as well as the variations in the biological data and consequent interpretation of the biochemical and biological data. Peripheral cells, in particular platelets and skin fibroblasts, could be an alternative solution as peripheral biological markers for the early diagnosis of AD. These cells are easily accessible from patients. Furthermore, they would provide a means not only to validate potential therapeutic strategies, but also to study the mechanisms involved in the development of AD, including APP processing. PERSPECTIVES: A combined strategy using both a fundamental mechanistic and an analytical approach of patient peripheral cells will allow the identification of new biological markers for AD, and hence permit immediate therapeutic strategies to be implemented.
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Enfermedad de Alzheimer/diagnóstico , Biomarcadores , Enfermedad de Alzheimer/líquido cefalorraquídeo , Enfermedad de Alzheimer/patología , Enfermedad de Alzheimer/fisiopatología , Enfermedad de Alzheimer/psicología , Péptidos beta-Amiloides/líquido cefalorraquídeo , Péptidos beta-Amiloides/metabolismo , Humanos , Nervios Periféricos/patologíaRESUMEN
A patient, admitted to intensive care unit and sedated with propofol (Diprivan), had repeatedly presented severe decreased of measured bicarbonates without metabolic acidosis and with physiologic values of calculated bicarbonates. Consecutively to the administration of high doses of propofol, an important lactescence of samples had been observed and the tests realised showed that lactescence play a key role in the interference with the measurement of bicarbonates (as described in the literature). So, in the course of propofol administration, it would be advised to interpret with caution the results of measured bicarbonates to not conclude too quickly on a case of "propofol syndrome".
Asunto(s)
Bicarbonatos/sangre , Hipnóticos y Sedantes/administración & dosificación , Propofol/administración & dosificación , Anciano , Errores Diagnósticos , Humanos , Masculino , Reproducibilidad de los ResultadosRESUMEN
We report the case of a newborn presenting a cyanosis after the birth with a good general state. Congenital methemoglobinemia is a rare disease which is characterized by a brutal appearance, in early infancy, of a bluish skin color not regressing with oxygen inspiration, and by a good general state. It is due to the recessive autosomal NADH-cytochrome b5 reductase (EC. 1.6.2.2) deficiency. This enzyme normally allows the reduction of the physiologically formed methemoglobinemia. Two forms of congenital methemoglobinemia have to be distinguished: the benign form (type I) and the severe form (type II).
Asunto(s)
Cianosis/etiología , Metahemoglobinemia/congénito , Metahemoglobinemia/complicaciones , Humanos , Recién Nacido , MasculinoRESUMEN
BACKGROUND: A woman with a past history of allergy to artichoke presented with two episodes of immediate allergic reactions, one of which was a severe anaphylactic shock after eating two types of health foods containing inulin. RESULTS: Dot blot assay techniques identified specific IgEs to artichoke, to yoghurt F, and to a heated BSA + inulin product. Dot blot inhibition techniques confirmed the anti-inulin specificity of specific IgE. CONCLUSIONS: The absence of a positive reaction to an unheated milk-inulin mixture indicates the probability of protein-inulin binding. There is no cross-reactivity with the carbohydrates of the glycosylated allergens.
Asunto(s)
Anafilaxia/inmunología , Inmunoglobulina E/inmunología , Inulina/efectos adversos , Cynara scolymus/efectos adversos , Cynara scolymus/inmunología , Femenino , Alimentos Orgánicos/efectos adversos , Humanos , Immunoblotting , Inulina/inmunología , Persona de Mediana Edad , Yogur/efectos adversosRESUMEN
Secreted phospholipases A(2) is a family of small molecular weight and calcium-dependent enzymes of which the members list is presently growing. Among these enzymes, the synovial type IIA and the type V phospholipases A(2) are involved in inflammation. Although their actual mechanism is still a subject of debate, new therapeutic strategies can result from the knowledge of the regulations of their gene expression. The human genes of the type IIA and type V phospholipases A(2) are located on the chromosome 1 at close positions and transcribed in reverse orientations. These genes can therefore be regulated by common elements but only the regulation of the type IIA phospholipase A(2) gene expression has been extensively studied. Pro-inflammatory cytokines upregulate while the growth factors downregulate the type IIA phospholipase A(2) gene expression. Interleukin-6 and interleukin-1beta exert their effects at least partially at the transcriptional level. The transcriptional regulation of the type IIA phospholipase A(2) gene is cell- and species-specific. The activity of the human promoter is controlled by the CAAT-enhancer binding protein (C/EBP) factors while that of the rat promoter is regulated by nuclear factor kappaB (NF-kappaB) and C/EBPs. Furthermore, the human promoter is constitutively repressed in hepatocytes by single strand DNA binding proteins whose effects are relieved by C/EBP factors while the glucocorticoid receptor interacts with C/EBPs in chondrocytes to achieve full basal and interleukin-1beta-stimulated transcription activity. Other factors like CTF/NF1 and Sp1 might be involved in the regulation of both the rat and human promoter. Peroxisome proliferator-activated receptors could contribute to the stimulation of the rat promoter by NF-kappaB in vascular smooth muscle cells. The study of the coactivators and coinhibitors associated to these transcription factors will give a better understanding of the diversity and complexity of the transcriptional regulations of the type IIA phospholipase A(2) gene.
Asunto(s)
Regulación Enzimológica de la Expresión Génica , Inflamación/enzimología , Fosfolipasas A/genética , Animales , Secuencia de Bases , Sitios de Unión , Citocinas/metabolismo , Sustancias de Crecimiento/metabolismo , Humanos , Datos de Secuencia Molecular , Fosfolipasas A/metabolismo , Regiones Promotoras Genéticas , Alineación de Secuencia , Transducción de Señal , TATA Box , Factores de Transcripción/metabolismo , Transcripción GenéticaRESUMEN
The activity of the [-831; +103] promoter of the human cyclooxygenase-2 gene in cultured rabbit chondrocytes is stimulated 2.9 +/- 0.3-fold by interleukin-1beta and this stimulation depends on [-132; -124] C/EBP binding-and [-223; -214] NF-kappaB binding-sites. The C/EBPbeta and C/EBPdelta factors bind to the [-132; -124] sequence. The [-61; -53] sequence is also recognized by C/EBPbeta and C/EBPdelta as well as USF. Mutation of the whole [-61; -53] sequence abolished the stimulation of transcription but single mutations of the C/EBP or USF site did not alter the activity of the promoter, suggesting that the factors bound to the proximal [-61; -53] sequence interact with different members of the general transcription machinery. The [-223; -214] site binds only the p50/p50 homodimer and a non-rel-related protein, but not the transcriptionally active heterodimer p50/p65. The p50/p50 homodimer could interact with the C/EBP family members bound to the [-132; -124] sequence for full stimulation of the COX-2 transcription by interleukin-1beta in chondrocytes. By contrast, the [-448; -449] sequence binds with a low affinity both the p50/p50 homodimeric and p50/p65 heterodimeric forms of NF-kappaB but has no role in the regulation of the human COX-2 promoter in chondrocytes.
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Proteína beta Potenciadora de Unión a CCAAT/fisiología , Proteínas Potenciadoras de Unión a CCAAT/fisiología , Cartílago Articular/metabolismo , Condrocitos/metabolismo , Interleucina-1/metabolismo , Isoenzimas/genética , Prostaglandina-Endoperóxido Sintasas/genética , Factores de Transcripción , Transcripción Genética , Animales , Secuencia de Bases , Sitios de Unión , Proteína delta de Unión al Potenciador CCAAT , Núcleo Celular/metabolismo , Células Cultivadas , Cloranfenicol O-Acetiltransferasa/metabolismo , Ciclooxigenasa 2 , Femenino , Eliminación de Gen , Regulación de la Expresión Génica , Humanos , Cinética , Proteínas de la Membrana , Modelos Biológicos , Datos de Secuencia Molecular , Mutagénesis , Plásmidos/metabolismo , Regiones Promotoras Genéticas , Unión Proteica , Proteínas Proto-Oncogénicas c-rel/metabolismo , Conejos , Homología de Secuencia de Ácido NucleicoRESUMEN
Secreted type IIA phospholipase A(2), which is involved in arachidonic acid release, is abundantly produced by chondrocytes and secreted in the synovial fluids of patients affected by rheumatoid arthritis. Transfection experiments showed that interleukin-1beta stimulates the phospholipase A(2) [-1614; +20] promoter activity by 6-7-fold and that the [-210; -176] fragment is critical for this stimulation. CAAT enhancer-binding protein (C/EBP) beta and C/EBPdelta transcription factors bind to this element as shown by bandshift experiments. Interleukin-1beta increased the levels of C/EBPdelta mRNA as soon as 2 h and up to 24 h without affecting those of C/EBPbeta. Higher amounts of C/EBPdelta proteins correlate with the stimulation of C/EBPdelta mRNA. Mutations or 5' deletions in the upstream [-247; -210] region reduced by 2-fold the basal and interleukin-1beta-stimulated transcription activities. Two types of factors bind to overlapping sequences on this fragment: NF1-like proteins and the glucocorticoid receptor. The glucocorticoid receptor is responsible for a moderate stimulation of the promoter activity by dexamethasone and may interact with C/EBP factors to achieve a full transcription activity in basal conditions and in the presence of interleukin-1beta. A [-114; -85] proximal regulatory element forms three complexes in bandshift experiments, the slowest mobility one involving the Sp1 zinc finger factor. Mutation of this sequence reduced to 2-fold the stimulation of the promoter activity by interleukin-1beta or the C/EBP factors. Induction of the transcription of secreted type IIA phospholipase A(2) gene by interleukin-1beta in chondrocytes absolutely requires C/EBPbeta and C/EBPdelta factors but does not involve NF-kappaB.
Asunto(s)
Regulación Enzimológica de la Expresión Génica/fisiología , Interleucina-1/fisiología , Fosfolipasas A/genética , Transcripción Genética/fisiología , Animales , Secuencia de Bases , Proteínas Potenciadoras de Unión a CCAAT , Células Cultivadas , Condrocitos/citología , Condrocitos/enzimología , Condrocitos/metabolismo , Cartilla de ADN , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/fisiología , Femenino , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Nucleares/fisiología , Fosfolipasas A2 , Regiones Promotoras Genéticas , Unión Proteica , Conejos , Receptores de Glucocorticoides/metabolismo , Secuencias Reguladoras de Ácidos Nucleicos , Factor de Transcripción Sp1/metabolismoRESUMEN
The 85 kDa cytosolic phospholipase A2 (cPLA2) plays a key role in liberating arachidonic acid from the sn-2 position of membrane phospholipids. When activated by extracellular stimuli, cPLA2 undergoes calcium-dependent translocation from cytosol to membrane sites which are still a matter of debate. In order to evaluate the effect of plasma membrane association on cPLA2 activation, we constructed chimeras of cPLA2 constitutively targeted to the plasma membrane by the N-terminal targeting sequence of the protein tyrosine kinase Lck (Lck-cPLA2) or the C-terminal targeting signal of K-Ras4B (cPLA2-Ras). Constitutive expression of these chimeras in Chinese hamster ovary cells overproducing the alpha2B adrenergic receptor (CHO-2B cells) did not affect the basal release of [3H]arachidonic acid, indicating that constitutive association of cPLA2 with cellular membranes did not ensure the hydrolysis of membrane phospholipids. However, Lck-cPLA2 increased [3H]arachidonic acid release in response to receptor stimulation and to increased intracellular calcium, whereas cPLA2-Ras inhibited it, compared with parental CHO-2B cells and CHO-2B cells producing comparable amounts of recombinant wild-type cPLA2. The lack of stimulation of cPLA2-Ras was not due to a decreased enzymatic activity as measured using an exogenous substrate, or to a decreased phosphorylation of the protein. These results show that the plasma membrane is a suitable site for cPLA2 activation when orientated correctly.
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Fosfolipasas A/química , Fosfolipasas A/metabolismo , Secuencia de Aminoácidos , Animales , Ácido Araquidónico/metabolismo , Sitios de Unión/genética , Células CHO , Membrana Celular/enzimología , Secuencia Conservada , Cricetinae , Citosol/enzimología , Activación Enzimática , Humanos , Técnicas In Vitro , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/química , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/genética , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/metabolismo , Ratones , Datos de Secuencia Molecular , Peso Molecular , Fosfolipasas A/genética , Fosfolipasas A2 , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas ras/química , Proteínas ras/genética , Proteínas ras/metabolismoAsunto(s)
Membrana Celular/metabolismo , Proteínas de Unión al GTP/metabolismo , Fosfolipasas A/metabolismo , Receptores de Superficie Celular/metabolismo , Animales , Células CHO , Calcimicina/farmacología , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Cricetinae , Citosol/enzimología , Activación Enzimática , Epinefrina/farmacología , Fosfolipasas A2 , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , TransfecciónRESUMEN
We have previously shown that the promoter of the type IIa secreted phospholipase A2 gene contains a strong positive regulatory proximal element [-125 to -85] element B. Mutation of this element abolishes the activation of the phospholipase A2 promoter by C/EBPbeta in HepG2 cells. Liver nuclear proteins form three major and two minor complexes with this element. The [-107 to -99] 5'-GACCACGCC-3' sequence is critical for the formation of these complexes and the activity of the promoter. Although the sequence of element B is highly similar to those of Sp1 binding sites, it does not bind Sp1 or other zinc-finger proteins. Each major complex contains a single protein, the molecular masses of these proteins being 100, 90 and 75 kDa. These proteins have the same nucleotide requirements for binding, with the cytosines at positions -102, -100, -99 and the adenosine at -103 being the most important nucleotides. The activity of the phospholipase A2 promoter in HeLa cells was lower than in HepG2 cells, and was correlated with the absence of complex 3 in HeLa cell nuclear extracts. Our results suggest different roles for the proteins bound to the 5'-GACCACGCC-3' sequence. In particular, the 75-kDa protein which forms the third complex is critical for the activity of the promoter of the secretory phospholipase A2 gene.
Asunto(s)
Fosfolipasas A/genética , Regiones Promotoras Genéticas , Factor de Transcripción Sp1/metabolismo , Animales , Secuencia de Bases , Línea Celular , Núcleo Celular/metabolismo , Regulación Enzimológica de la Expresión Génica , Humanos , Fosfolipasas A2 , Unión Proteica , Ratas , Factor de Transcripción Sp1/genética , Dedos de ZincRESUMEN
The A3243G mutation of mitochondrial DNA is associated to the MELAS syndrome and to transmitted forms of diabetes mellitus. This mutation exists in a heteroplasmic state and can be present at a minor and hardly detectable level. The aim was to design a method which could be applied to large series of samples and could provide rapid, sensitive and quantitative detection of this mutation in the wild-type mitochondrial DNA background. The ability of ligation detection reaction (LDR) to satisfy these objectives was evaluated. Ligation detection reaction was performed on a model template composed of mixtures of various proportions of plasmids bearing the wild-type or mutant mitochondrial DNA sequence. Radiolabelled or fluorescent primers and the wild-type and mutant LDR products were separated by electrophoresis on conventional denaturating gel or on an Applied Biosystem 373. The ratios of mutant/wild-type products were consistent with the initial ratios of the plasmids in the template. The sensitivity and accuracy of the fluorescence and isotopic detection methods were similar. The detection limit of mutant DNA was 10% of total mitochondrial DNA. The percentage of mutant DNA in DNA samples extracted from leukocytes of 19 patients having the mutation at different levels, was evaluated by fluorescent or isotopic LDR.
Asunto(s)
ADN Mitocondrial/genética , Síndrome MELAS/genética , Mutación Puntual , Secuencia de Bases , ADN/sangre , Cartilla de ADN , Humanos , Leucocitos , Datos de Secuencia Molecular , Plásmidos , Reacción en Cadena de la Polimerasa/métodos , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Polimerasa Taq , Moldes GenéticosRESUMEN
Tracheal epithelial cells and skin fibroblasts from different cystic fibrosis (CF) patients bearing the deltaF508 mutation of cystic fibrosis transmembrane conductance regulator (CFTR) released more arachidonic acid in response to bradykinin than do other CF and normal cells. Immortalized tracheal epithelial cell lines were used as models to study the mechanisms of this dysregulation. An 85 kD cytosolic phospholipase A2 (cPLA2) was found in these cells and bradykinin increased its binding to membranes of deltaF508 cells (CFT-2) but not to those of a double heterozygous CF cells (CFT-1), or of control cells (NT-1). The expression of G alpha(q)/11 protein was also increased in deltaF508 cells, with increased stimulation of phosphatidylinositol diphosphate-specific phospholipase C (PLC) by bradykinin, and an early, transient activation of mitogen-activated protein (MAP) kinase. As the binding of cPLA2 to membranes is Ca2+-dependent, the increased coupling to PLC could cause the hypersensitivity to bradykinin. Comparison of the effects of bradykinin to those observed with thapsigargin, an inhibitor of calcium reuptake, suggests that the increase of intracellular calcium is not the only mechanism involved in arachidonic acid release by bradykinin in deltaF508 cells. The lack of effect of calcium ionophore A23187 or TPA on arachidonic acid release from any of the cell lines suggested that activation needs a PKC-independent cPLA2 phosphorylation step, perhaps via MAP kinase activation. The binding of cPLA2 to membranes after bradykinin stimulation still occurred in CFT2 cells (deltaF508) homogenized in EDTA, suggesting that a membrane component plus increased intracellular calcium influenced cPLA2 anchoring to membranes. The defective processing of deltaF508 CFTR seems to increase cPLA2 stimulation by bradykinin, since the bradykinin-stimulated release of arachidonic acid is reversed by growing cells at 28 degrees C for 48 h. The deltaF508 mutation of CFTR appears to increase the stimulation of cPLA2 by Gq-mediated receptors in a PKC-independent and MAP kinase-dependent manner. Hence normal CFTR, or normally processed deltaF508 CFTR, inhibit cPLA2 stimulation. The greater reactivity of deltaF508 CFTR cells to inflammatory mediators might be part of the increased sensitivity of CF patients to lung inflammation.
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Bradiquinina/farmacología , Fibrosis Quística/enzimología , Fosfolipasas A/metabolismo , Piel/enzimología , Tráquea/enzimología , Línea Celular , Citosol , Epitelio/enzimología , Fibroblastos/enzimología , Humanos , Fosfolipasas A2 , Transducción de Señal/efectos de los fármacos , Regulación hacia ArribaRESUMEN
We previously reported that the type II secreted phospholipase A2 (sPLA2) promoter from positions (-326 to +20) ([-326;+20] promoter) is negatively regulated by two adjacent regulatory elements, C (-210 to -176) and D (-247 to -210). This study examines in greater detail the way in which this negative regulation operates. Successive 5' deletions of the [-326;+20] type II sPLA2 promoter indicated that the region upstream of position -195 inhibits the transcription activity sixfold in HepG2 cells but not in HeLa cells. Although the whole [-326;-176] region decreased the activity of a heterologous thymidine kinase promoter, this effect was orientation and position sensitive. C/EBP beta, C/EBP alpha, and C/EBP delta, which bind to element C, prevented the inhibition of promoter activity. Electrophoretic mobility shift experiments identified the binding of NF1-like proteins to the [-225;-218] site, which overlaps an insulin response-like sequence, 5'-TGTTTTG-3'. This sequence bound a factor which also recognized the promoters of the apolipoproteins C-III and A-II. Substitutions preventing the binding of this factor or the NF1-like proteins did not increase the transcription activity, but substitution in the [-217;-204] sequence blocked the transcription inhibition. This sequence did not bind any double-strand binding factor, but its antisense strand is critical for the binding of single-strand binding proteins to the [-232;-191] region. We therefore suggest that these single-strand binding proteins are involved in the inhibitory mechanism.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Regulación Enzimológica de la Expresión Génica/genética , Proteínas Nucleares/metabolismo , Fosfolipasas A/genética , Regiones Promotoras Genéticas/genética , Animales , Apolipoproteínas/genética , Secuencia de Bases , Proteínas Potenciadoras de Unión a CCAAT , Carcinoma Hepatocelular , Sondas de ADN , ADN Recombinante/metabolismo , Humanos , Hígado/metabolismo , Neoplasias Hepáticas , Datos de Secuencia Molecular , Neurofibromina 1 , Fosfolipasas A2 , Proteínas/metabolismo , Ratas , Eliminación de Secuencia/genética , Factores de Transcripción/metabolismo , Transcripción Genética/fisiología , Transfección , Células Tumorales CultivadasRESUMEN
Large amounts of type II-secreted phospholipase A2 (type II sPLA2) are secreted into inflammatory synovial fluid and they are believed to induce the synthesis of lipid mediators by articular chondrocytes. Preliminary experiments showed that insulin-like growth factor-I, which counteracts cartilage degradation in arthritis, inhibits interleukin-1beta-induced type II sPLA2 gene expression in rabbit articular chondrocytes (Berenbaum, F., G. Thomas, S. Poiraudeau, G. Bereziat, M.T. Corvol, and J. Masliah. 1994. FEBS Lett. 340: 51-55). The present study showed that IL-1beta induced the sustained synthesis of prostaglandin E2 and a parallel increase in type II sPLA2 gene expression (assessed by enzymatic activity and Northern blot analysis), but no increase in cytosolic PLA2 gene expression (assessed by Northern and Western blot analysis) or cytosolic PLA2 activity in rabbit articular chondrocytes. IGF-I inhibited both IL-1beta-stimulated PGE2 synthesis and type II sPLA2 gene expression, but had no effect on cytosolic PLA2 gene expression. Nuclear run-on experiments revealed that IL-1beta stimulated the transcription rate of type II sPLA2 gene, giving rise to long-lived mRNA in cells treated with actinomycin D. IGF-I did not affect transcription rate, suggesting that it acts as a post-transcriptional step. Sucrose density gradient analysis of the translation step showed no effect of IGF-I on the entry of type II sPLA2 mRNA into the polysomal pool or on its distribution into the various polysomal complexes, suggesting that IGF-I does not act on the translation of the mRNA. Lastly, IGF-I strongly decreased the half-life of IL-1beta-induced type II sPLA2 mRNA (from 92 to 12 h), suggesting that IGF-I destabilizes mRNA. These data demonstrate that IL-1beta stimulates the transcription rate of the type II sPLA2 gene and gives rise to a very stable mRNA. In contrast, IGF-I decreases the half-life of the type II sPLA2 message.
Asunto(s)
Cartílago Articular/efectos de los fármacos , Cartílago Articular/metabolismo , Factor I del Crecimiento Similar a la Insulina/farmacología , Interleucina-1/farmacología , Fosfolipasas A/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Cartílago Articular/citología , Células Cultivadas , ADN Complementario/genética , Dinoprostona/biosíntesis , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Humanos , Cinética , Datos de Secuencia Molecular , Fosfolipasas A/clasificación , Fosfolipasas A/metabolismo , Fosfolipasas A2 , ARN Mensajero/genética , ARN Mensajero/metabolismo , Conejos , Proteínas Recombinantes/farmacología , Ribosomas/efectos de los fármacos , Ribosomas/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
Toxoplasmosis is a serious opportunistic infection in patients with acquired immunodeficiency syndrome (AIDS). The lung is a major site of infection after the central nervous system. The aim of the study was to assess the polymerase chain reaction (PCR) and cell culture for the detection of Toxoplasma gondii. One hundred and thirty two human immunodeficiency virus (HIV)-infected patients with respiratory manifestations, who underwent fibreoptic bronchoalveolar lavage, were investigated. Detection of Toxoplasma gondii was compared using three techniques: Giemsa staining; polymerase chain reaction with specific primers derived from the P30 gene; and culture on the MRC5 cell line. Toxoplasma gondii was detected in the same four samples by all three techniques. We conclude that PCR adds little to conventional (and cheaper) tools already used to diagnose pulmonary toxoplasmosis.
Asunto(s)
Infecciones Oportunistas Relacionadas con el SIDA/parasitología , Enfermedades Pulmonares Parasitarias/diagnóstico , Toxoplasmosis/diagnóstico , Infecciones Oportunistas Relacionadas con el SIDA/diagnóstico , Animales , Colorantes Azulados , Líquido del Lavado Bronquioalveolar/parasitología , Línea Celular , Células Cultivadas , Estudios de Evaluación como Asunto , Humanos , Reacción en Cadena de la Polimerasa/economía , Coloración y Etiquetado , Toxoplasma/aislamiento & purificaciónRESUMEN
Diagnosis of Pneumocystis carinii pneumonia is based on the identification of the various stages of the parasite in lung samples by standard staining techniques. We therefore assessed the value of the PCR for detection of P. carinii in bronchoalveolar lavage, induced sputum, and blood samples relative to that of standard staining techniques.