RESUMEN
Intravenous lipid emulsions (ILE) have demonstrated advantages including prevention of essential fatty acid (EFA) deficiency; however, too much EFA can down regulate fatty acid elongation leading to an imbalance of nutritional compounds in plasma and cell membranes. An olive oil-based ILE containing long-chain triacylglycerols (LCT) with a low content (20 %) of PUFA was administered for home parenteral nutrition (HPN) and compared with a conventional soyabean oil-based ILE (PUFA content, 60 %). Thirteen patients (26-92 years) with stable intestinal failure were randomised after a 1-month run-in period with a medium-chain triacylglycerols-LCT-based ILE, to receive 3 months of HPN with either olive oil- (n 6) or soyabean oil-based (n 7) ILE. The nutritional impact and safety of HPN, oral intakes and absorption rates, phospholipid fatty acids in plasma and lymphocyte cell membrane were assessed. The only clinical event reported was one case of pneumonia (soya group). In both groups, 20 : 3n-9:20 : 4n-6 ratios remained within normal ranges (0.03-0.07). There was a significant increase of gamma-linolenic acid (gamma-LA) in plasma and lymphocyte cell membrane (P=0.02) and of oleic acid in plasma (P<0.01) in the olive compared with the soya group. A significant correlation was found between gamma-LA (day 90 - day 0) in plasma and PUFA parenteral intakes (P=0.02), but neither with fat intakes nor with fat absorption rates. In conclusion, plasma and lymphocyte EFA pattern remained in normal ranges without EFA deficiency with both lipid emulsions, despite a lower content of n-3 and n-6 series with the olive oil-based ILE.
Asunto(s)
Enfermedades Intestinales/terapia , Nutrición Parenteral en el Domicilio/métodos , Aceites de Plantas/administración & dosificación , Aceite de Soja/administración & dosificación , Adulto , Anciano , Anciano de 80 o más Años , Índice de Masa Corporal , Membrana Celular/química , Grasas Insaturadas en la Dieta/metabolismo , Método Doble Ciego , Emulsiones/administración & dosificación , Ácidos Grasos Insaturados/administración & dosificación , Femenino , Humanos , Infusiones Intravenosas , Hepatopatías/metabolismo , Linfocitos/química , Masculino , Persona de Mediana Edad , Ácido Oléico/sangre , Aceite de Oliva , Ácido gammalinolénico/análisis , Ácido gammalinolénico/sangreRESUMEN
It has been claimed that lipid emulsions with a restricted linoleic acid content can improve the safety of total parenteral nutrition (TPN). The tolerability of TPN and its effects on the metabolism of fatty acids were assessed in this prospective, double-blind, randomised study comparing an olive/soyabean oil long-chain triacylglycerol (LCT) with a medium-chain triacylglycerol (MCT)/LCT; 50:50 (w) based lipid emulsion in two groups (O and M, respectively; eleven per group) of severely burned patients. After resuscitation (48-72 h), patients received TPN providing 147 kJ/kg per d (35 kcal/kg per d) with fat (1.3 g/kg per d) for 6 d Plasma fatty acids, laboratory parameters including liver function tests, and plasma cytokines were assessed before and after TPN. Adverse events encountered during TPN and the clinical outcomes of patients within the subsequent 6 months were recorded. With both lipid emulsions, the conversion of linoleic acid in its higher derivatives (di-homo-gamma-linolenic acid) improved and essential fatty acid deficiency did not appear. Abnormalities of liver function tests occurred more frequently in the M (nine) than in the O (three) group (P = 0.04, Suissa-Shuster test). Seven patients (four from group O and three from group M) died as a consequence of severe sepsis 3-37 d after completion of the 6 d TPN period. When compared with the surviving patients, those who died were older (P = 0.01) and hyperglycaemic at baseline (P < 0.001), and their plasma IL-6 levels continued to increase (P < 0.04). Although fatty acid metabolism and TPN tolerability were similar with both lipid emulsions, the preservation of liver function noted with the use of the olive oil-based lipid emulsions deserves confirmation.
Asunto(s)
Quemaduras/terapia , Emulsiones Grasas Intravenosas/administración & dosificación , Ácido Linoleico/administración & dosificación , Nutrición Parenteral Total/métodos , Aceites de Plantas/química , Triglicéridos/administración & dosificación , Adulto , Glucemia/análisis , Quemaduras/sangre , Colesterol/sangre , Método Doble Ciego , Ácidos Grasos/sangre , Femenino , Humanos , Ácido Linoleico/sangre , Pruebas de Función Hepática , Masculino , Persona de Mediana Edad , Aceite de Oliva , Nutrición Parenteral Total/efectos adversos , Fosfolípidos/sangre , Estudios Prospectivos , Triglicéridos/sangreRESUMEN
Type II secreted phospholipase A(2) (sPLA(2)) releases precursors of important inflammatory lipid mediators from phospholipids. Some observations have indicated that the sPLA(2), which has been implicated in chronic inflammatory conditions such as arthritis, contributes to atherosclerosis in the arterial wall. sPLA(2) was not detected in control vascular smooth muscle cells (VSMC). Treatment of VSMC with agents that increase intracellular cAMP (eg, forskolin, dibutyryl [db]-cAMP) resulted in a time- and concentration-dependent increase in sPLA(2) gene expression. Semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR) showed a marked dose-dependent inhibition of forskolin-induced mRNA by protein kinase A inhibitor. Electrophoretic mobility shift analysis of nuclear proteins from forskolin-treated and db-cAMP-treated VSMC with C/EBP consensus oligonucleotides and C/EBP oligonucleotides from the rat promoter revealed greater binding than in control VSMC. Incubation of VSMC with H89, a specific protein kinase inhibitor, also blocked the binding of nuclear C/EBP to the C/EBP site of the rat promoter induced by db-cAMP and forskolin. Binding was unchanged with the use of CRE consensus oligonucleotides. Antibodies revealed the specific formation of C/EBP/DNA complexes, the majority of which were supershifted by C/EBP-ss and -delta antibodies. Functional activation of C/EBP was confirmed by a luciferase reporter gene assay. A construct comprising 4 tandem repeat copies of the C/EBP element from the rat sPLA(2) promoter linked to luciferase was transcriptionally activated in VSMC by cotransfection with expression vector for the protein kinase A catalytic subunit. It was also significantly activated in transfected VSMC treated by forskolin or db-cAMP. H89 inhibited this activations. We therefore conclude that the increases in sPLA(2) mRNA and enzyme activity produced by cAMP-elevating agents is controlled by a mechanism involving nuclear C/EBP-ss and -delta acting through a protein kinase A signaling pathway.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/farmacología , Músculo Liso Vascular/efectos de los fármacos , Fosfolipasas A/genética , Sulfonamidas , Factores de Transcripción , Animales , Bucladesina/farmacología , Proteína beta Potenciadora de Unión a CCAAT/genética , Proteína beta Potenciadora de Unión a CCAAT/farmacología , Proteína delta de Unión al Potenciador CCAAT , Proteínas Potenciadoras de Unión a CCAAT/genética , Proteínas Potenciadoras de Unión a CCAAT/farmacología , Células Cultivadas , Colforsina/farmacología , Proteína Quinasa Tipo II Dependiente de AMP Cíclico , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Inducción Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Isoquinolinas/farmacología , Luciferasas/genética , Masculino , Músculo Liso Vascular/enzimología , Oligonucleótidos/farmacología , Fosfolipasas A/biosíntesis , ARN Mensajero/análisis , Ratas , Ratas Wistar , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Transcripción Genética/efectos de los fármacos , TransfecciónRESUMEN
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.
Asunto(s)
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
Modulation of cytosolic phospholipase A(2) (cPLA(2)) activity by sphingomyelin (SPH), ceramide (Cer), and cholesterol (Chol) was investigated in CHO-2B cells activated by the calcium ionophore A23187 and epinephrine. Chol depletion of CHO-2B cells by treatment with methyl-beta-cyclodextrin (5 mm) resulted in the inhibition of the release of arachidonic acid whereas the restoration of the level by Chol-loaded cyclodextrin relieved inhibition. Conversion of CHO-2B cellular SPH to Cer by Staphylococcus aureus sphingomyelinase enhanced endogenous cPLA(2) activation as well as uptake by cells of C2- and C6-ceramide analogs. These results were confirmed in vitro with purified human recombinant cPLA(2) acting on a model phospholipid substrate. The enzyme activity was inhibited by SPH but reactivated by Cer as well as by Chol added to glycerophospholipid liposomal substrates containing SPH. The results of this study, which combine in situ and in vivo experimental approaches, indicate that membrane microdomains enriched in SPH and Chol play a role in the modulation of the activity of cPLA2 and in arachidonic acid-derived mediator production.
Asunto(s)
Colesterol/farmacología , Lípidos de la Membrana/metabolismo , Fosfolipasas A/metabolismo , Esfingolípidos/farmacología , Animales , Células CHO , Calcimicina , Ceramidas/farmacología , Cricetinae , Citosol/enzimología , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Epinefrina/farmacología , Ácidos Grasos/análisis , Ácidos Grasos/metabolismo , Humanos , Ionóforos/farmacología , Membrana Dobles de Lípidos/metabolismo , Liposomas/metabolismo , Fosfolipasas A/antagonistas & inhibidores , Fosfolipasas A/efectos de los fármacos , Fosfolípidos/metabolismo , Ratas , Receptores Adrenérgicos alfa 2 , Proteínas Recombinantes , Esfingosina/análogos & derivados , Esfingosina/farmacologíaRESUMEN
BACKGROUND & AIMS: Hereditary hemochromatosis is associated with C282Y homozygosity. Some heterozygotes may also present with abnormal iron parameters. However, the precise role of H63D and C282Y mutations in iron overload is poorly understood. We investigated the level of expression of the mutated and unmutated HFE alleles in these heterozygous patients. METHODS: We studied the expression of HFE messenger RNAs in peripheral blood mononuclear cells from 34 heterozygotes using reverse-transcription polymerase chain reaction (PCR) followed by enzymatic digestion or sequence analysis of the PCR products, which allows relative quantification of mutated and unmutated transcripts. HFE proteins were quantified by Western blotting in Epstein-Barr virus-immortalized lymphocyte extracts from 2 C282Y and H63D homozygotes and a compound heterozygote. RESULTS: (187C > G; H63D) mutated transcripts predominated in H63D and compound heterozygotes and the normal transcripts in C282Y heterozygotes. The amount of HFE protein was increased in the H63D homozygotes and the compound heterozygote compared with the C282Y homozygotes. In addition, we found a new mutation at codon 282 (C282S) associated with severe iron overload. CONCLUSIONS: We demonstrate the existence of differential allelic expression of the HFE alleles, suggesting that the (187C > G; H63D) mutation plays a role in the disease expression in H63D heterozygotes, in particular when associated with environmental or host factors.
Asunto(s)
Antígenos HLA/genética , Hemocromatosis/genética , Antígenos de Histocompatibilidad Clase I/genética , Proteínas de la Membrana , Transcripción Genética , Adulto , Sustitución de Aminoácidos , Codón , Duodeno/inmunología , Duodeno/patología , Femenino , Ferritinas/sangre , Amplificación de Genes , Genes MHC Clase I , Genotipo , Hemocromatosis/inmunología , Hemocromatosis/patología , Proteína de la Hemocromatosis , Heterocigoto , Humanos , Hígado/inmunología , Hígado/patología , Linfocitos/inmunología , Masculino , Persona de Mediana Edad , Mutación Puntual , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Angiotensin II (Ang II) binds to specific G protein-coupled receptors and is mitogenic in Chinese hamster ovary (CHO) cells stably expressing a rat vascular angiotensin II type 1A receptor (CHO-AT(1A)). Cyclin D1 protein expression is regulated by mitogens, and its assembly with the cyclin-dependent kinases induces phosphorylation of the retinoblastoma protein pRb, a critical step in G(1) to S phase cell cycle progression contributing to the proliferative responses. In the present study, we found that in CHO-AT(1A) cells, Ang II induced a rapid and reversible tyrosine phosphorylation of various intracellular proteins including the protein-tyrosine phosphatase SHP-2. Ang II also induced cyclin D1 protein expression in a phosphatidylinositol 3-kinase and mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK)-dependent manner. Using a pharmacological and a co-transfection approach, we found that p21(ras), Raf-1, phosphatidylinositol 3-kinase and also the catalytic activity of SHP-2 and its Src homology 2 domains are required for cyclin D1 promoter/reporter gene activation by Ang II through the regulation of MAPK/ERK activity. Our findings suggest for the first time that SHP-2 could play an important role in the regulation of a gene involved in the control of cell cycle progression resulting from stimulation of a G protein-coupled receptor independently of epidermal growth factor receptor transactivation.
Asunto(s)
Angiotensina II/metabolismo , Ciclina D1/genética , Regulación de la Expresión Génica , Regiones Promotoras Genéticas , Proteínas Tirosina Fosfatasas/fisiología , Receptores de Angiotensina/biosíntesis , Dominios Homologos src , Animales , Células CHO , Catálisis , Cricetinae , Fase G1 , Humanos , Péptidos y Proteínas de Señalización Intracelular , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteína Tirosina Fosfatasa no Receptora Tipo 1 , Proteína Tirosina Fosfatasa no Receptora Tipo 11 , Proteína Tirosina Fosfatasa no Receptora Tipo 6 , Proteínas Proto-Oncogénicas c-raf/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Ratas , Receptor de Angiotensina Tipo 1 , Receptores de Angiotensina/genética , Fase S , Proteínas Tirosina Fosfatasas con Dominio SH2 , Activación TranscripcionalRESUMEN
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
BACKGROUND: Automated electrophoresis combined with enzymatic cholesterol staining might improve routine assessment of LDL- and HDL-cholesterol (LDLC and HDLC), as an alternative to the Friedewald equation and precipitation. A new method (Hydrasys; SEBIA) that adapts the cholesterol esterase/cholesterol oxidase reaction within urea-free gels was evaluated. METHODS: Fresh sera from 725 subjects (512 dyslipidemics) were analyzed by electrophoresis, in parallel with sequential ultracentrifugation, beta-quantification, calculation, and precipitation. RESULTS: Electrophoresis was linear up to 4 g/L cholesterol, with a detection limit of 0.042 g/L cholesterol/band. Within-run, between-run, between-batch, and between-operator imprecision (CVs) were 1.6%, 2.0%, 1.5%, and 2.7% for LDLC, and 3.9%, 4.3%, 5.5%, and 4.9% for HDLC, and remained unchanged up to 6.3 g/L plasma triglycerides (TGs). Precision decreased with very low HDLC (<0.25 g/L). Serum storage for 3-7 days at +4 or -80 degrees C did not interfere significantly with the assay. Agreement with beta-quantification was stable for LDLC up to 5.07 g/L (r = 0.94), even at TG concentrations >4 g/L (r = 0.91). Bias (2.88% +/- 12%) and total error (7.84%) were unchanged at TG concentrations up to 18.5 g/L. Electrophoresis predicted National Cholesterol Education Program cut-points with <0.04 g/L error, exactly and appropriately classified 79% and 96% of the subjects, and divided by 2.4 (all subjects) and 5.8 (TGs >1.5 g/L) the percentage of subjects underestimated by calculation. One-half of the patients with TGs >4 g/L had LDLC >1.30 g/L. For HDLC, correlation was better with precipitation (r = 0.87) than ultracentrifugation (r = 0.76). Error (-0.10% +/- 26%) increased when HDLC decreased (<0.35 g/L). Direct assessment of the LDLC/HDLC ratio detected 45% more high-risk subjects than the calculation/precipitation combination. CONCLUSIONS: Electrophoresis provides reliable quantification of LDLC, improving precision, accuracy, and concordance over calculation, particularly with increasing plasma TGs. Implementation of methods to detect low cholesterol concentrations could extend the applications for HDLC assessment.
Asunto(s)
HDL-Colesterol/sangre , LDL-Colesterol/sangre , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Apolipoproteínas E/sangre , Bilirrubina/análisis , Precipitación Química , Niño , Colesterol Oxidasa , Colorimetría , Electroforesis en Gel de Agar , Femenino , Hemoglobinas/análisis , Heparina de Bajo-Peso-Molecular/sangre , Humanos , Lipoproteína(a)/sangre , Masculino , Persona de Mediana Edad , Estudios Prospectivos , Sensibilidad y Especificidad , Esterol Esterasa , UltracentrifugaciónRESUMEN
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.
Asunto(s)
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/metabolismoRESUMEN
BACKGROUND: A new intravenous lipid emulsion (ILE) prepared from a mixture of soybean and olive oils contains only long-chain triacylglycerols, with a low proportion (20%) of polyunsaturated fatty acids and 60% monounsaturated fatty acids. OBJECTIVE: The goal of this randomized, double-blind clinical trial was to assess in children the efficacy and safety of this new ILE compared with a control group receiving a soybean-oil emulsion. DESIGN: Eighteen children received for 2 mo 24% of nonprotein energy (1.80 g kg (-)(1) d(-)(1)) either as the new ILE or a soybean oil-based emulsion. Assessments were performed on days -30, 0, 30, and 60 and the changes (day 60 - day 0) assessed by analysis of variance. RESULTS: There were no significant differences in triacylglycerol, apolipoproteins A-I and B, or HDL cholesterol between the 2 groups, whereas total and LDL cholesterol were higher in the soybean oil group on day 60. The pattern of 20:4n-6 in erythrocyte membranes did not change significantly, nor did the ratio of 20:3n-9 to 20:4n-6. On day 60, 18:1n-9 was significantly higher in the olive oil group, the ratio of Sigma(n)-6 > C(18) + 18:3n-6 to 18:2n-6 was 2.20 +/- 0.09 in the olive oil group and 1.33 +/- 0.16 in the soybean-oil group, and Sigma(n)-3 > C(18) was 3.83 +/- 0.30 in the olive oil group and 4. 03 +/- 0.33 in the soybean-oil group. The peroxidation index was lower after the olive oil treatment. CONCLUSIONS: The olive oil-based emulsion was well tolerated, maintained a normal EFA status, and may be more suitable for prevention of lipid peroxidation than the soybean-oil-based emulsion.
Asunto(s)
Grasas Insaturadas en la Dieta/farmacología , Emulsiones Grasas Intravenosas/farmacología , Nutrición Parenteral , Aceites de Plantas/farmacología , Niño , Preescolar , Grasas Insaturadas en la Dieta/efectos adversos , Método Doble Ciego , Emulsiones Grasas Intravenosas/efectos adversos , Femenino , Humanos , Lactante , Masculino , Aceite de Oliva , Aceites de Plantas/efectos adversosRESUMEN
Type II-secreted phospholipase A(2) (type II-sPLA(2)) is expressed in smooth muscle cells during atherosclerosis or in response to interleukin-1beta. The present study shows that the induction of type II-sPLA(2) gene by interleukin-1beta requires activation of the NFkappaB pathway and cytosolic PLA(2)/PPARgamma pathway, which are both necessary to achieve the transcriptional process. Interleukin-1beta induced type II-sPLA(2) gene dose- and time-dependently and increased the binding of NFkappaB to a specific site of type II-sPLA(2) promoter. This effect was abolished by proteinase inhibitors that block the proteasome machinery and NFkappaB nuclear translocation. Type II-sPLA(2) induction was also obtained by free arachidonic acid and was blocked by either AACOCF(3), a specific cytosolic-PLA(2) inhibitor, PD98059, a mitogen-activated protein kinase kinase inhibitor which prevents cytosolic PLA(2) activation, or nordihydroguaiaretic acid, a lipoxygenase inhibitor, but not by the cyclooxygenase inhibitor indomethacin, suggesting a role for a lipoxygenase product. Type II-sPLA(2) induction was obtained after treatment of the cells by 15-deoxy-Delta(12,14)-dehydroprostaglandin J(2), carbaprostacyclin, and 9-hydroxyoctadecadienoic acid, which are ligands of peroxisome proliferator-activated receptor (PPAR) gamma, whereas PPARalpha ligands were ineffective. Interleukin-1beta as well as PPARgamma-ligands stimulated the activity of a reporter gene containing PPARgamma-binding sites in its promoter. Binding of both NFkappaB and PPARgamma to their promoter is required to stimulate the transcriptional process since inhibitors of each class block interleukin-1beta-induced type II-sPLA(2) gene activation. We therefore suggest that NFkappaB and PPARgamma cooperate at the enhanceosome-coactivator level to turn on transcription of the proinflammatory type II-sPLA(2) gene.
Asunto(s)
Interleucina-1/farmacología , Músculo Liso Vascular/efectos de los fármacos , FN-kappa B/metabolismo , Fosfolipasas A/genética , Receptores Citoplasmáticos y Nucleares/metabolismo , Factores de Transcripción/metabolismo , Animales , Secuencia de Bases , Transporte Biológico , Células Cultivadas , Ceramidas/metabolismo , Cicloheximida/farmacología , Cartilla de ADN , Dactinomicina/farmacología , Relación Dosis-Respuesta a Droga , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Masculino , Músculo Liso Vascular/citología , Músculo Liso Vascular/enzimología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Esfingomielina Fosfodiesterasa/metabolismo , Activación TranscripcionalAsunto(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
CHO transfectants expressing the three subtypes of rat alpha2 adrenergic receptors (alpha2AR): alpha2D, alpha2B, alpha2C were studied to compare the transduction pathways leading to the receptor-mediated stimulation of phospholipase A2 (PLA2) in the corresponding cell lines CHO-2D, CHO-2B, CHO-2C. The alpha2B subtype stimulated the arachidonic acid (AA) release after incubation of the cells with 1 microM epinephrine, whereas alpha2D and alpha2C gave no stimulation. Calcium ionophore A23187 (1 microM) increased the release by a factor of 2-4 in the three strains. When cells were incubated with both epinephrine and Ca2+ ionophore, the AA release differed greatly between cell lines with strong potentiation in CHO-2B (2-3 times greater than Ca2+ ionophore alone), moderate potentiation in CHO-2D, and no potentiation in CHO-2C. The three cell lines each inhibited adenylylcyclase with similar efficiencies when 1 microM epinephrine was used as the agonist. The potentiation depended on both alpha2AR and Gi proteins since yohimbine and pertussis toxin inhibited the process. Pretreatment of CHO-2B cells with MAFP which inhibits both cytosolic and Ca2+-independent PLA2, reduced the release of AA induced by epinephrine+Ca2+ ionophore to basal value, whereas bromoenol lactone, a specific Ca2+-independent PLA2 inhibitor, had no effect. Preincubation of the cells with the intracellular calcium chelator BAPTA gave a dose-dependent inhibition of the arachidonic acid (AA) release. In CHO cells expressing the angiotensin II type 1 receptor, coupled to a Gq protein, the agonist (10-7 M) produced maximal AA release: there was no extra increase when angiotensin and Ca2+ ionophore were added together. There was no increase in the amount of inositol 1,4, 5-triphosphate following stimulation of CHO-2B, -2C, -2D cells with 1 microM epinephrine. Epinephrine led to greater phosphorylation of cPLA2, resulting in an electrophoretic mobility shift for all three cell lines, so inadequate p42/44 MAPKs stimulation was not responsible for the weaker stimulation of cPLA2 in CHO-2C cells. Therefore, the stimulation of cPLA2 by Gi proteins presumably involves another unknown mechanism. The differential stimulation of cPLA2 in these transfectants will be of value to study the actual involvement of the transduction pathways leading to maximal cPLA2 stimulation.
Asunto(s)
Ácido Araquidónico/metabolismo , Receptores Adrenérgicos alfa 2/efectos de los fármacos , Animales , Ácido Araquidónico/biosíntesis , Células CHO , Calcimicina/farmacología , Cricetinae , AMP Cíclico/biosíntesis , Epinefrina/farmacología , Toxina del Pertussis , Fosfolipasas A/antagonistas & inhibidores , Fosfolipasas A/metabolismo , Fosfolipasas A2 , Ratas , Receptores Adrenérgicos alfa 2/metabolismo , Transfección , Factores de Virulencia de Bordetella/farmacología , Yohimbina/farmacologíaRESUMEN
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
Secretory type II phospholipase A2 (sPLA2) is inhibited by sphingomyelin (SPH); cholesterol either mixed with the model glycerophospholipid substrate or added to the assay medium as separated liposomes counteracts this inhibition efficiently. The inhibition of fatty acid release assayed by quantitative gas chromatography-MS is observed when SPH is added to erythrocyte membranes as the substrate instead of a readily hydrolysable phosphatidylethanolamine/phosphatidylserine model mixture. Hydrolysis of SPH by Staphylococcus aureus sphingomyelinase suppresses its inhibitory potency. The addition of cholesterol to SPH liposomes with a 1:1 stoichiometry relieves completely the inhibition of sPLA2 exerted by SPH. The mechanism of inhibition suggested by the binding assay is that sPLA2 binds with affinity to the SPH interface, after either phase segregation at the assay temperature or on the pure SPH liposomes added to the incubation medium. Cholesterol is shown to suppress the binding affinity of the enzyme for the SPH interface. A model for inhibition is suggested in which binding of the sphingosine moiety is competitive for sPLA2 (inhibition) or for cholesterol (release of the enzyme).
Asunto(s)
Colesterol/farmacología , Fosfolipasas A/antagonistas & inhibidores , Esfingomielinas/farmacología , Animales , Interacciones Farmacológicas , Membrana Eritrocítica/efectos de los fármacos , Membrana Eritrocítica/enzimología , Fibroblastos/efectos de los fármacos , Fibroblastos/enzimología , Fosfolipasas A2 Grupo II , Hidrólisis , Liposomas , Ratones , Fosfolipasas A2 , Staphylococcus aureus , TransfecciónRESUMEN
Lyso-phospholipids exert a major injurious effect on lung cell membranes during Acute Respiratory Distress Syndrome (ARDS), but the mechanisms leading to their in vivo generation are still unknown. Intratracheal administration of LPS to guinea pigs induced the secretion of type II secretory phospholipase A2 (sPLA2-II) accompanied by a marked increase in fatty acid and lyso-phosphatidylcholine (lyso-PC) levels in the bronchoalveolar lavage fluid (BALF). Administration of LY311727, a specific sPLA2-II inhibitor, reduced by 60% the mass of free fatty acid and lyso-PC content in BALF. Gas chromatography/mass spectrometry analysis revealed that palmitic acid and palmitoyl-2-lyso-PC were the predominant lipid derivatives released in BALF. A similar pattern was observed after the intratracheal administration of recombinant guinea pig (r-GP) sPLA2-II and was accompanied by a 50-60% loss of surfactant phospholipid content, suggesting that surfactant is a major lung target of sPLA2-II. In confirmation, r-GP sPLA2-II was able to hydrolyze surfactant phospholipids in vitro. This hydrolysis was inhibited by surfactant protein A (SP-A) through a direct and selective protein-protein interaction between SP-A and sPLA2-II. Hence, our study reports an in vivo direct causal relationship between sPLA2-II and early surfactant degradation and a new process of regulation for sPLA2-II activity. Anti-sPLA2-II strategy may represent a novel therapeutic approach in lung injury, such as ARDS.
Asunto(s)
Enfermedades Pulmonares/fisiopatología , Lisofosfatidilcolinas/metabolismo , Fosfolipasas A/metabolismo , Proteolípidos/metabolismo , Surfactantes Pulmonares/metabolismo , Enfermedad Aguda , Animales , Técnicas Biosensibles , Líquido del Lavado Bronquioalveolar/química , Ácidos Grasos/metabolismo , Fosfolipasas A2 Grupo II , Cobayas , Hidrólisis , Indoles/farmacología , Lipopolisacáridos/farmacología , Masculino , Ácido Palmítico/metabolismo , Fosfolipasas A/antagonistas & inhibidores , Fosfolipasas A2 , Unión Proteica , Proteína A Asociada a Surfactante Pulmonar , Proteínas Asociadas a Surfactante PulmonarRESUMEN
In this study, the effects of caffeine on lipoprotein lipase (LPL) gene expression were investigated in the 3T3-F442A preadipocyte cell line during the adipocyte differentiation process by determining LPL enzymatic activity and its messenger RNA (mRNA) level. The results demonstrate that caffeine acts on the gene expression of LPL, an early marker of adipocyte differentiation. It has a biphasic action: it increases gene expression in terms of mRNA when it is added to preadipocytes during the early stage of differentiation, but this is accompanied by a reduction of enzymatic activity. On the other hand, when caffeine is added for long periods during differentiation and/or when it is added to mature adipocytes, it induces marked inhibition of mRNA levels, correlated with a marked reduction of secreted enzymatic activity. The inhibitory effect of caffeine on LPL mRNA level can be reproduced by theophylline, a phosphodiesterase inhibitor, and by dibutyryl cyclic AMP, a non-metabolizable analog of cyclic AMP. However, the effect of caffeine and theophylline lasts longer than that of cyclic AMP, suggesting that a mechanism other than inhibition of cyclic AMP hydrolysis may be involved in the action of caffeine.
Asunto(s)
Adipocitos/enzimología , Cafeína/farmacología , Diferenciación Celular/fisiología , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Lipoproteína Lipasa/biosíntesis , Triglicéridos/metabolismo , Células 3T3 , Adipocitos/citología , Adipocitos/efectos de los fármacos , Animales , Bucladesina/farmacología , Diferenciación Celular/efectos de los fármacos , Línea Celular , Cinética , Ratones , ARN Mensajero/biosíntesis , ARN Mensajero/metabolismo , Teofilina/farmacología , Factores de TiempoRESUMEN
Oxidative damage, which plays a major role in the early stages of atherosclerosis, is associated with arachidonic acid (AA) release in vascular smooth muscle cells (VSMC) as in other cell types. In this study, H2O2 was used to investigate mechanisms of AA release from VSMC on oxidative stress. Cell treatment with H2O2 inhibited AA incorporation in an inverse relationship to prolonged H2O2-induced AA release. Identical kinetics of inhibition of AA incorporation and AA release were observed after cell treatment with AlF4-, a process not involving phospholipase A2 (PLA2) activation as recently described (A. Cane, M. Breton, G. Béréziat, and O. Colard. Biochem. Pharmacol. 53: 327-337, 1997). AA release was not specific, since oleic acid also increased in the extracellular medium of cells treated with H2O2 or AlF4- as measured by gas chromatography-mass spectrometry. In contrast, AA and oleic acid cell content decreased after cell treatment. Oleoyl and arachidonoyl acyl-CoA synthases and acyltransferases, assayed using a cell-free system, were not significantly modified. In contrast, a good correlation was observed between decreases in AA acylation and cell ATP content. The decrease in ATP content is only partially accounted for by mitochondrial damage as assayed by rhodamine 123 assay. We conclude that oxidant-induced arachidonate release results from impairment of fatty acid esterification and that ATP availability is probably responsible for free AA accumulation on oxidative stress by preventing its reesterification and/or transmembrane transport.