RESUMEN
A precision measurement of the differential cross sections dσ/dΩ and the linearly polarized photon asymmetry Σ≡(dσâ¥-dσâ¥)/(dσâ¥+dσâ¥) for the γpâπ0p reaction in the near-threshold region has been performed with a tagged photon beam and almost 4π detector at the Mainz Microtron. The Glasgow-Mainz photon tagging facility along with the Crystal Ball/TAPS multiphoton detector system and a cryogenic liquid hydrogen target were used. These data allowed for a precise determination of the energy dependence of the real parts of the S- and all three P-wave amplitudes for the first time and provide the most stringent test to date of the predictions of chiral perturbation theory and its energy region of agreement with experiment.
RESUMEN
OBJECTIVE: To develop and establish the psychometric properties of an instrument to measure face validity, feasibility and utility of patient questionnaires used during health care. DESIGN: Our instrument, QQ-10, is a 10-item self-completed questionnaire, which was developed during the evaluation of another questionnaire (ePAQ-PF), to assess patients' views on questionnaire use during health care. SETTING: Urogynaecology Department, Royal Hallamshire Hospital, Sheffield, UK. PARTICIPANTS: The Sheffield maternity patient user group identified 10 key themes relating to patients' views on using questionnaires; these themes translated into 10 statements, each using the same 5-point Likert response scale. INTERVENTION: Not applicable. Outcome Measures Principal component analysis established the factor structure of our instrument. Internal reliability was assessed using Cronbach's alpha. Construct validity was assessed using Spearman's rho. RESULTS: Factor analysis yielded two meaningful factors: Value and Burden, both achieving Cronbach's alpha scores >0.7. Significant correlations were found between scores for Value and communication experience and between scores for Burden and barriers to health care. CONCLUSIONS: Our instrument offers a valid, reliable measure of patients' views relating to value and burden of using health-related quality of life questionnaires. Its two domains show good internal reliability and with its free text items, it may offer a valuable, standardized assessment of face validity and utility of other questionnaires used in health care.
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Atención a la Salud/métodos , Calidad de Vida , Encuestas y Cuestionarios , Adulto , Anciano , Anciano de 80 o más Años , Análisis Factorial , Femenino , Humanos , Masculino , Persona de Mediana Edad , Satisfacción del Paciente , Psicometría , Reproducibilidad de los Resultados , Adulto JovenAsunto(s)
Cesárea/efectos adversos , Fascitis Necrotizante/cirugía , Infección Puerperal/cirugía , Enfermedades Uterinas/cirugía , Adulto , Fascitis Necrotizante/tratamiento farmacológico , Fascitis Necrotizante/etiología , Femenino , Humanos , Embarazo , Infección Puerperal/tratamiento farmacológico , Infección Puerperal/etiología , Enfermedades Uterinas/tratamiento farmacológico , Enfermedades Uterinas/etiología , Adulto JovenAsunto(s)
Aborto Terapéutico/métodos , Embarazo Abdominal/terapia , Abortivos no Esteroideos/uso terapéutico , Terapia Combinada , Femenino , Humanos , Metotrexato/uso terapéutico , Embarazo , Embarazo Abdominal/diagnóstico por imagen , Ultrasonografía Intervencional/métodos , Ultrasonografía Prenatal/métodos , Adulto JovenRESUMEN
Sema7A is a recently described member of the semaphorin family that is associated with the cell surface via a glycophosphatidylinositol linkage. This study examined the mRNA expression and biological properties of this protein. Although the expression of Sema7A was demonstrated in lymphoid and myeloid cells, no stimulation of cytokine production or proliferation was evident in B or T cells. In contrast, Sema7A is an extremely potent monocyte activator, stimulating chemotaxis at 0.1 pm and inflammatory cytokine production (interleukin-1 (IL-1beta), tumour necrosis factor-alpha (TNF-alpha), IL-6 and IL-8) and superoxide release at 1-10 pm. Sema7A is less effective at stimulating neutrophils. Sema7A also significantly increases granulocyte-macrophage colony-stimulating factor (GM-CSF) production from monocytes but has no consistent effect on IL-10, IL-12 or IL-18. Sema7A can also induce monocytes toward a dendritic cell morphology. Sema7A is expressed in monocytes and probably released through proteolysis and acts as a very potent autocrine activator of these cells.
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Antígenos CD/farmacología , Glicoproteínas/farmacología , Lipoproteínas/farmacología , Monocitos/inmunología , Semaforinas , Animales , Antígenos CD/análisis , Antígenos CD/genética , Células CHO , Células Cultivadas , Quimiotaxis de Leucocito , Clonación Molecular , Cricetinae , Citocinas/biosíntesis , Relación Dosis-Respuesta a Droga , Citometría de Flujo , Proteínas Ligadas a GPI , Glicoproteínas/genética , Humanos , Lipoproteínas/genética , Monocitos/efectos de los fármacos , Neutrófilos/efectos de los fármacos , Neutrófilos/inmunología , ARN Mensajero/biosíntesis , Superóxidos/metabolismo , Polimerasa Taq/metabolismoRESUMEN
This study is an exploratory investigation of the regional timing of cortical activity associated with verbal working memory function. ERP activity was obtained from a single subject using a 124-channel sensor array during a task requiring the monitoring of imageable words for occasional targets. Distributed cortical activity was estimated every 2.5 ms with high spatial resolution using real head, boundary element modelling of non-target activity. High-resolution structural MRI was used for segmentation of tissue boundaries and co-registration to the scalp electrode array. The inverse solution was constrained to the cortical surface. Cortical activity was observed in regions commonly associated with verbal working memory function. This included: the occipital pole (early visual processing); the superior temporal and inferior parietal gyrus bilaterally and the left angular gyrus (visual and phonological word processing); the dorsal lateral occipital gyrus (spatial processing); and aspects of the bilateral superior parietal lobe (imagery and episodic verbal memory). Activity was also observed in lateral and superior prefrontal regions associated with working memory control of sensorimotor processes. The pattern of cortical activity was relatively stable over time, with variations in the extent and amplitude of contributing local source activations. By contrast, the pattern of concomitant scalp topography varied considerably over time, reflecting the linear summation effects of volume conduction that often confound dipolar source modelling.
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Corteza Cerebral/fisiología , Memoria/fisiología , Red Nerviosa/fisiología , Adulto , Mapeo Encefálico/métodos , Potenciales Evocados/fisiología , Humanos , Imagen por Resonancia Magnética/métodos , Masculino , Estimulación Luminosa/métodosRESUMEN
From two related series of 2-(alkylthio)-pyrimidones, a novel series of 1-((amidolinked)-alkyl)-pyrimidones has been designed as nanomolar inhibitors of human lipoprotein-associated phospholipase A2. These compounds show greatly enhanced activity in isolated plasma. Selected derivatives such as compounds 51 and 52 are orally active with a good duration of action.
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Inhibidores Enzimáticos/farmacología , Lipoproteínas/metabolismo , Fosfolipasas A/antagonistas & inhibidores , Pirimidinonas/farmacología , Administración Oral , Animales , Inhibidores Enzimáticos/administración & dosificación , Inhibidores Enzimáticos/química , Estructura Molecular , Fosfolipasas A/metabolismo , Fosfolipasas A2 , Pirimidinonas/administración & dosificación , Pirimidinonas/química , ConejosRESUMEN
A weak HTS hit at the CCR2B receptor has been converted into a potent antagonist by array SAR studies. Selectivity over the closely related CCR5 receptor is also achieved.
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Amidas/química , Amidas/farmacología , Quimiocina CCL2/metabolismo , Indoles/farmacología , Piperidinas/farmacología , Receptores de Quimiocina/antagonistas & inhibidores , Amidas/síntesis química , Amidas/metabolismo , Quimiocina CCL2/química , Quimiotaxis de Leucocito/efectos de los fármacos , Humanos , Indoles/síntesis química , Indoles/química , Indoles/metabolismo , Cinética , Ligandos , Estructura Molecular , Monocitos/efectos de los fármacos , Piperidinas/síntesis química , Piperidinas/química , Piperidinas/metabolismo , Receptores CCR2 , Receptores CCR5/metabolismo , Receptores de Quimiocina/química , Receptores de Quimiocina/metabolismo , Relación Estructura-ActividadRESUMEN
Fractalkine is a recently identified chemokine that exhibits cell adhesion and chemoattractive properties. It represents a unique member of the chemokine superfamily because it is located predominantly in the brain in which it is expressed constitutively on specific subsets of neurons. To elucidate the possible role of neuronally expressed fractalkine in the inflammatory response to neuronal injury, we have analyzed the regulation of fractalkine mRNA expression and protein cleavage under conditions of neurotoxicity. We observed that mRNA encoding fractalkine is unaffected by experimental ischemic stroke (permanent middle cerebral artery occlusion) in the rat. Similarly, in vitro, levels of fractalkine mRNA were unaffected by ensuing excitotoxicity. However, when analyzed at the protein level, we found that fractalkine is rapidly cleaved from cultured neurons in response to an excitotoxic stimulus. More specifically, fractalkine cleavage preceded actual neuronal death by 2-3 hr, and, when evaluated functionally, fractalkine represented the principal chemokine released from the neurons into the culture medium upon an excitotoxic stimulus to promote chemotaxis of primary microglial and monocytic cells. We further demonstrate that cleavage of neuron-derived, chemoattractive fractalkine can be prevented by inhibition of matrix metalloproteases. These data strongly suggest that dynamic proteolytic cleavage of fractalkine from neuronal membranes in response to a neurotoxic insult, and subsequent chemoattraction of reactive immune cells, may represent an early event in the inflammatory response to neuronal injury.
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Membrana Celular/metabolismo , Quimiocinas CX3C/metabolismo , Encefalitis/metabolismo , Infarto de la Arteria Cerebral Media/metabolismo , Proteínas de la Membrana/metabolismo , Neuronas/metabolismo , Fenilalanina/análogos & derivados , Animales , Animales Recién Nacidos , Encéfalo/irrigación sanguínea , Encéfalo/efectos de los fármacos , Encéfalo/fisiopatología , Membrana Celular/efectos de los fármacos , Células Cultivadas , Quimiocina CX3CL1 , Quimiocinas CX3C/genética , Quimiocinas CX3C/farmacología , Quimiotaxis/efectos de los fármacos , Quimiotaxis/fisiología , Medios de Cultivo Condicionados/análisis , Medios de Cultivo Condicionados/metabolismo , Modelos Animales de Enfermedad , Encefalitis/etiología , Endotelio Vascular/citología , Endotelio Vascular/metabolismo , Ácido Glutámico/toxicidad , Infarto de la Arteria Cerebral Media/complicaciones , Interleucina-1/genética , Interleucina-1/metabolismo , Inhibidores de la Metaloproteinasa de la Matriz , Metaloproteinasas de la Matriz/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/farmacología , Microglía/citología , Microglía/efectos de los fármacos , Monocitos/citología , Monocitos/efectos de los fármacos , Neuronas/citología , Neuronas/efectos de los fármacos , Fenilalanina/farmacología , Inhibidores de Proteasas/farmacología , ARN Mensajero/metabolismo , Ratas , Tiofenos/farmacología , Transfección , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Recombinant fractalkine possesses both chemoattractive and adhesive properties in vitro. Previous studies have demonstrated an upregulation of this molecule on the membranes of activated human endothelial cells and hypothesised that fractalkine plays a role in the recruitment and adherence of monocytes to the activated endothelium. Here we present data analysing both the adhesive and chemoattractive properties of this chemokine expressed by activated human umbilical vein endothelial cells. We demonstrate that both recombinant fractalkine and endogenously produced fractalkine function as adhesion molecules, tethering monocytes to the endothelium. However, our data demonstrate that although recombinant fractalkine has the potential to function as a potent monocyte chemoattractant, the endogenous fractalkine cleaved from activated human umbilical vein endothelial cells is not responsible for the observed chemotaxis in this model. Instead, we show that monocyte chemoattractant protein-1 (MCP-1), secreted from the activated human umbilical vein endothelial cells, is responsible for the chemotaxis of these monocytes.
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Quimiocinas CX3C , Quimiocinas CXC/fisiología , Endotelio Vascular/fisiología , Proteínas de la Membrana/fisiología , Monocitos/citología , Western Blotting , Adhesión Celular/efectos de los fármacos , Línea Celular , Quimiocina CX3CL1 , Quimiocinas CXC/genética , Quimiocinas CXC/farmacología , Factores Quimiotácticos/fisiología , Quimiotaxis/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Humanos , Proteínas de la Membrana/genética , Proteínas de la Membrana/farmacología , Monocitos/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Recombinantes/farmacología , Factores de Tiempo , Factor de Necrosis Tumoral alfa/farmacología , Venas Umbilicales/citología , Venas Umbilicales/metabolismoRESUMEN
Human CC chemokine receptor 1 (CCR1) has been proposed as a receptor for CKbeta8. To obtain conclusive evidence, binding-displacement studies of 125I-CKbeta8 (25-99) were performed on membranes of Chinese hamster ovary cells expressing human CCR1. The Ic50 for displacement of 125I-CKbeta8 (25-99) with CKbeta8 (25-99) was 0.22 nM. The longer forms of CKbeta8 (24-99 and 1-99) also displaced 125I-CKbeta8, with Ic50 values of 6.5 and 16 nM, respectively. Displacement profiles of 125I-CKbeta8 (25-99) on freshly prepared human monocytes indicated that CCR1 was the major receptor for CKbeta8. We conclude that CCR1 is a receptor for different-length CKbeta8 and that CKbeta8 (25-99) has a similar affinity for CCR1 as macrophage inflammatory protein-1alpha (MIP-1alpha). The longer variants of CKbeta8 are significantly less potent than CKbeta8 (25-99) and MIP-1a on CCR1 and monocytes (P < 0.05).
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Quimiocinas CC/metabolismo , Receptores de Quimiocina/metabolismo , Animales , Unión Competitiva , Células CHO , Cricetinae , Humanos , Radioisótopos de Yodo , Monocitos/metabolismo , Péptidos/metabolismo , Receptores CCR1 , TransfecciónRESUMEN
A novel and potent azetidinone inhibitor of the lipoprotein-associated phospholipase A2 (Lp-PLA2), i.e. platelet-activating factor acetylhydrolase, is described for the first time. This inhibitor, SB-222657 (Ki=40+/-3 nM, kobs/[I]=6. 6x10(5) M-1.s-1), is inactive against paraoxonase, is a poor inhibitor of lecithin:cholesterol acyltransferase and has been used to investigate the role of Lp-PLA2 in the oxidative modification of lipoproteins. Although pretreatment with SB-222657 did not affect the kinetics of low-density lipoprotein (LDL) oxidation by Cu2+ or an azo free-radical generator as determined by assay of lipid hydroperoxides (LOOHs), conjugated dienes and thiobarbituric acid-reacting substances, in both cases it inhibited the elevation in lysophosphatidylcholine content. Moreover, the significantly increased monocyte chemoattractant activity found in a non-esterified fatty acid fraction from LDL oxidized by Cu2+ was also prevented by pretreatment with SB-222657, with an IC50 value of 5.0+/-0.4 nM. The less potent diastereoisomer of SB-222657, SB-223777 (Ki=6.3+/-0.5 microM, kobs/[I]=1.6x10(4) M-1.s-1), was found to be significantly less active in both assays. Thus, in addition to generating lysophosphatidylcholine, a known biologically active lipid, these results demonstrate that Lp-PLA2 is capable of generating oxidized non-esterified fatty acid moieties that are also bioactive. These findings are consistent with our proposal that Lp-PLA2 has a predominantly pro-inflammatory role in atherogenesis. Finally, similar studies have demonstrated that a different situation exists during the oxidation of high-density lipoprotein, with enzyme(s) other than Lp-PLA2 apparently being responsible for generating lysophosphatidylcholine.
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Azetidinas/farmacología , Inhibidores Enzimáticos/farmacología , Lipoproteínas LDL/metabolismo , Fosfolipasas A/metabolismo , Sulfóxidos/farmacología , 1-Alquil-2-acetilglicerofosfocolina Esterasa , Arildialquilfosfatasa , Quimiotaxis de Leucocito/efectos de los fármacos , Esterasas/antagonistas & inhibidores , Humanos , Oxidación-Reducción , Fosfatidilcolina-Esterol O-Aciltransferasa/antagonistas & inhibidores , Fosfolipasas A/antagonistas & inhibidores , Fosfolipasas A2 , Unión ProteicaRESUMEN
A new CC chemokine, designated CKbeta-8 or myeloid progenitor inhibitor factor-1, was recently identified in a large scale sequencing effort and was cloned from a human aortic endothelial library. CKbeta-8 cDNA encodes a signal sequence of 21 amino acids, followed by a 99-amino acid predicted mature form. CKbeta-8 was expressed and purified from a baculovirus insect cell expression system, which resulted in the identification of different N-terminal variants of the secreted chemokine. The three major forms (containing amino acids 1-99, 24-99, and 25-99 of the secreted chemokine) showed a large variation in potency. CKbeta-8 activated both monocytes and eosinophils to mobilize intracellular calcium; however, the shortest form of CKbeta-8 (25-99) was >2 orders of magnitude more potent than the longest form. Cross-desensitization experiments in both monocytes and eosinophils suggested that the CCR1 receptor was probably the predominant receptor that mediates this chemokine's physiologic response. However, incomplete desensitization was encountered in both cell systems, suggesting involvement of an additional receptor(s). Interestingly, the short form of CKbeta-8 was the most potent chemotactic chemokine that we have ever evaluated in the monocyte system (EC50 = 54 pM). However, in contrast to its action on monocytes, CKbeta-8 was a very poor chemotactic factor for eosinophils.
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Quimiocinas CC/química , Quimiocinas CC/aislamiento & purificación , Secuencia de Aminoácidos , Calcio/metabolismo , Quimiocinas CC/genética , Quimiocinas CC/metabolismo , Factores Quimiotácticos Eosinófilos/fisiología , Quimiotaxis de Leucocito/efectos de los fármacos , Eosinófilos/inmunología , Eosinófilos/metabolismo , Humanos , Datos de Secuencia Molecular , Proteínas Quimioatrayentes de Monocitos/fisiología , Monocitos/inmunología , Monocitos/metabolismo , Isoformas de Proteínas/química , Isoformas de Proteínas/inmunología , Isoformas de Proteínas/aislamiento & purificación , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacologíaRESUMEN
Using expressed sequence tag (EST) homology screening, a new human serine dependent phospholipase A2 (HSD-PLA2) was identified that has 40% amino acid identity with human low density lipoprotein-associated phospholipase A2 (LDL-PLA2). HSD-PLA2 has very recently been purified and cloned from brain tissue but named PAF-AH II. However, because the homology with LDL-PLA2 suggested a broader substrate specificity than simply platelet activating factor (PAF), we have further characterized this enzyme using baculovirus-expressed protein. The recombinant enzyme, which was purified 21-fold to homogeneity, had a molecular mass of 44kDa and possessed a specific activity of 35 micromol min-1 mg-1 when assayed against PAF. Activity could also be measured using 1-decanoyl-2-(4-nitrophenylglutaryl) phosphate (DNGP) as substrate. Like LDL-PLA2, HSD-PLA2 was able to hydrolyse oxidatively modified phosphatidylcholines when supplemented to human LDL prior to copper-stimulated oxidation. A GXSXG motif evident from sequence information and inhibition of its activity by 3,4, dichloroisocoumarin, diisopropyl fluorophosphate (DFP) and diethyl p-nitrophenyl phosphate (DENP) confirm that the enzyme is serine dependent. Moreover, sequence comparison indicates the HSD-PLA2 probable active site triad positions are shared with LDL-PLA2 and a C. elegans homologue, suggesting that these sequences comprise members of a new enzyme family. Although clearly structurally related with similar substrate specificities further work reported here shows HSD-PLA2 and LDL-PLA2 to be different with respect to chromosomal localization and tissue distribution.
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Fosfolipasas A/metabolismo , Fosfolípidos/metabolismo , Factor de Activación Plaquetaria/metabolismo , Secuencia de Aminoácidos , Animales , Encéfalo/enzimología , Caenorhabditis elegans , Línea Celular , Cromatografía de Afinidad , Cromatografía en Gel , Cromatografía por Intercambio Iónico , Clonación Molecular , Secuencia de Consenso , Humanos , Cinética , Datos de Secuencia Molecular , Especificidad de Órganos , Oxidación-Reducción , Fosfolipasas A/química , Fosfolipasas A/aislamiento & purificación , Fosfolipasas A2 , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Spodoptera , Especificidad por Sustrato , TransfecciónRESUMEN
Eotaxin has been found to bind exclusively to a single chemokine receptor, CCR3. Using expression sequence tag screening of an activated monocyte library, a second chemokine has been identified; it was expressed and purified from a Drosophila cell culture system and appears to only activate CCR3. Eotaxin-2, MPIF-2, or CKbeta-6, is a human CC chemokine with low amino acid sequence identity to other chemokines. Eotaxin-2 promotes chemotaxis and Ca2+ mobilization in human eosinophils but not in neutrophils or monocytes. Cross-desensitization calcium mobilization experiments using purified eosinophils indicate that eotaxin and MCP-4, but not RANTES, MIP-1alpha, or MCP-3, can completely cross-desensitize the calcium response to eotaxin-2 on these cells, indicating that eotaxin-2 shares the same receptor used by eotaxin and MCP-4. Eotaxin-2 was the most potent eosinophil chemoattractant of all the chemokines tested. Eotaxin-2 also displaced 125I-eotaxin bound to the cloned CCR3 stably expressed in CHO cells (CHO-CCR3) and to freshly isolated human eosinophils with affinities similar to eotaxin and MCP-4. 125I-Eotaxin-2 binds with high affinity to eosinophils and both eotaxin and cold eotaxin-2 displace the ligand with equal affinity. Eotaxin and eotaxin-2 promote a Ca2+ transient in RBL-2H3 cells stably transfected with CCR3 (RBL-2H3-CCR3) and both ligands cross-desensitized the response of the other but not the response to LTD4. The data indicate that eotaxin-2 is a potent eosinophil chemotactic chemokine exerting its activity solely through the CCR3 receptor.
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Quimiocinas CC , Quimiocinas/fisiología , Eosinófilos/fisiología , Receptores de Quimiocina/metabolismo , Secuencia de Aminoácidos , Animales , Unión Competitiva , Células CHO/metabolismo , Calcio/metabolismo , Movimiento Celular/fisiología , Quimiocina CCL11 , Quimiocina CCL24 , Quimiocina CCL8 , Quimiocinas/genética , Quimiocinas/aislamiento & purificación , Clonación Molecular , Cricetinae , Citocinas/genética , ADN Complementario/genética , Eosinófilos/efectos de los fármacos , Eosinófilos/metabolismo , Humanos , Datos de Secuencia Molecular , Proteínas Quimioatrayentes de Monocitos/genética , Ratas , Receptores CCR3 , Receptores de Quimiocina/fisiologíaRESUMEN
Here we describe the characterization of a novel human CC chemokine, tentatively named monocyte chemotactic protein (MCP-4). This chemokine was detected by random sequencing of expressed sequence tags in cDNA libraries. The full-length cDNA revealed an open reading frame for a 98-amino acid residue protein, and a sequence alignment with known CC chemokines showed high levels of similarity (59-62%) with MCP-1, MCP-3, and eotaxin. MCP-4 cDNA was cloned into Drosophila S2 cells, and the mature protein (residues 24-98) was purified from the conditioned medium. Recombinant MCP-4 induced a potent chemotactic response (EC50 = 2.88 +/- 0.15 nM) and a transient rise in cytosolic calcium concentration in fresh human peripheral blood monocytes but not in neutrophils. Binding studies in monocytes showed that MCP-4 and MCP-3 were very potent in displacing high affinity binding of 125I-MCP-1 (IC50 for MCP-4, MCP-3, and unlabeled MCP-1 of 2.1 +/- 1.4, 0.85-1.6, and 0.7 +/- 0.2 nM respectively), suggesting that all three chemokines interact with the CC chemokine receptor-2 (MCP-1 receptor). This was confirmed in binding studies with Chinese hamster ovary cells, stably transfected with the CC chemokine 2B receptor. Northern blot analysis in extracts of normal human tissues showed expression of mRNA for MCP-4 in small intestine, thymus, and colon, but the level of protein expression was too low to be detected in Western blot analysis. However, expression of MCP-4 protein was demonstrated by immunohistochemistry in human atherosclerotic lesion and found to be associated with endothelial cells and macrophages.
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Proteínas Quimioatrayentes de Monocitos/metabolismo , Receptores de Quimiocina , Receptores de Citocinas/metabolismo , Secuencia de Aminoácidos , Animales , Arteriosclerosis/metabolismo , Unión Competitiva , Western Blotting , Células CHO , Calcio/metabolismo , Clonación Molecular , Cricetinae , Humanos , Datos de Secuencia Molecular , Proteínas Quimioatrayentes de Monocitos/análisis , Proteínas Quimioatrayentes de Monocitos/farmacología , ARN Mensajero/análisis , Receptores CCR2 , Proteínas Recombinantes/biosíntesisRESUMEN
A novel LDL-associated phospholipase A2 (LDL-PLA2) has been purified to homogeneity from human LDL obtained from plasma apheresis. This enzyme has activity toward both oxidized phosphatidylcholine and platelet activating factor (PAF). A simple purification procedure involving detergent solubilization and affinity and ion exchange chromatography has been devised. Vmax and Km for the purified enzyme are 170 micromol.min-1.mg-1 and 12 micromol/L, respectively. Extensive peptide sequence from LDL-PLA2 facilitated identification of an expressed sequence tag partial cDNA. This has led to cloning and expression of active protein in baculovirus. A lipase motif is also evident from sequence information, indicating that the enzyme is serine dependent. Inhibition by diethyl p-nitrophenyl phosphate and 3,4-dichloroisocoumarin and insensitivity to EDTA, Ca2+, and sulfhydryl reagents confirm that the enzyme is indeed a serine-dependent hydrolase. The protein is extensively glycosylated, and the glycosylation site has been identified. Antibodies to this LDL-PLA2 have been raised and used to show that this enzyme is responsible for >95% of the phospholipase activity associated with LDL. Inhibition of LDL-PLA2 before oxidation of LDL reduces both lysophosphatidylcholine content and monocyte chemoattractant ability of the resulting oxidized LDL. Lysophosphatidylcholine production and monocyte chemoattractant ability can be restored by addition of physiological quantities of pure LDL-PLA2.
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Clonación Molecular , Lipoproteínas LDL/metabolismo , Lipoproteínas/metabolismo , Fosfolipasas A/genética , Fosfolipasas A/aislamiento & purificación , Serina/metabolismo , Secuencia de Aminoácidos , Estenosis de la Válvula Aórtica/genética , Baculoviridae/metabolismo , Secuencia de Bases , Humanos , Datos de Secuencia Molecular , Oxidación-Reducción , Fosfolipasas A/metabolismo , Fosfolipasas A2RESUMEN
In this study, the effects of a series of phorbol esters with different spectra of biological activities and different patterns of activation of the isoenzymes of protein kinase C (PKC) have been studied in human neutrophils. The aim was to gain more information on which isoenzymes of PKC are involved in neutrophil activation, specifically inhibition of fMet-Leu-Phe (fMLP)-stimulated bivalent cation influx and stimulation of O2-. release (either alone or potentiation of the response to fMLP). Prior addition of both phorbol 12-myristate 13-acetate (PMA) and sapintoxin A (SAPA) inhibited fMLP-stimulated Mn2+ influx. Higher concentrations of resiniferatoxin (RX) were also inhibitory, inhibition being more apparent at longer preincubation times. However, 12-deoxyphorbol 13-O-phenylacetate (DOPPA) showed only a slight inhibitory effect and required a prolonged preincubation. PMA, SAPA and RX, but not DOPPA, stimulated O2-. release by themselves. Lower concentrations of PMA, SAPA and RX, which were ineffective alone, considerably potentiated O2-. release stimulated by fMLP, whereas DOPPA had little or no effect. These results rule out a major role for PKC-delta (not activated by SAPA) and PKC-beta 1 (activated by DOPPA), but suggest the involvement of RX kinase in addition to PKC in the inhibition of fMLP-stimulated Mn2+ influx and potentiation of fMLP-stimulated O2-. release. However, when the cytosolic free Ca2+ concentration ([Ca2+]i) was elevated with the Ca2+ ionophore ionomycin, DOPPA was able to stimulate O2-. release, which probably reflects the known Ca2+ requirement for activation of PKC-beta 1 by DOPPA in vitro. The effects of the other phorbols were also enhanced when [Ca2+]i was elevated; all of the phorbols synergize, to variable extents, with Ca2+ to activate PKC in vitro. Enhancement of RX-stimulated O2- release by elevation of [Ca2+]i was unexpected, since RX kinase has been reported to be inhibited by high concentrations of Ca2+ in vitro. Finally, use of fura-2 and SK&F 96365 to manipulate the fMLP-stimulated rise in [Ca2+]i showed that when fMLP was able to evoke its normal rise in [Ca2+]i (to a peak of 700-900 nM), O2-. release was potentiated by PMA, SAPA and RX. However, when fMLP was only able to evoke a small increase in [Ca2+]i (to a peak of 400 nM), potentiation by PMA was unaffected but potentiation by SAPA and RX was considerably reduced. This observation agrees with published data demonstrating that activation of PKC in vitro by SAPA is more Ca(2+)-dependent than activation by PMA.(ABSTRACT TRUNCATED AT 400 WORDS)