Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 112
Filtrar
Más filtros

Banco de datos
Tipo del documento
Intervalo de año de publicación
1.
Appetite ; 182: 106417, 2023 Mar 01.
Artículo en Inglés | MEDLINE | ID: mdl-36521648

RESUMEN

PURPOSE: Dietary behaviors differ between socio-economic groups and are one key determinant of health inequalities. Psychological factors such as attitudes are assumed to underlie the relation between inequality and dietary behaviors, but this assumption has rarely been tested empirically. We focus on a specific food group shown as detrimental to health: processed meat. METHODS: In two representative international surveys (Survey 1: N = 10,226 participants from nine European countries - Austria, France, Germany, Italy, Netherlands, Poland, Russia, Spain, UK; Survey 2: N = 9149 participants from the same countries, except not including Austria and the Netherlands), participants reported inequality indicators (education, income), processed meat consumption as well as their attitudes toward nutrition and food. PRINCIPAL RESULTS: There were diverging relationships between indicators of inequality and processed meat consumption: the higher the educational attainment, the lower the consumption of processed meat (rSurvey1 = -0.062, p < .001; rSurvey2 = -0.071, p < .001). At the same time, higher income was related to higher processed meat consumption (rSurvey1 = 0.088, p < .001; rSurvey2 = 0.152, p < .001). A path model showed that four of seven attitude factors mediated the relation between education and processed meat consumption (i.e., indifference toward nutrition and food, preference for regional and fresh food, processed food consumption, health efforts); none of the attitude factors mediated the relation between income and overall processed meat consumption. CONCLUSIONS: Processed meats are consumed very frequently across European countries. The relation between inequality and processed meat consumption is heterogeneous and partially mediated by attitudes. More research is needed to better understand how psychological factors explain social inequality in nutrition behaviors and health in general.


Asunto(s)
Dieta , Carne , Humanos , Renta , Europa (Continente) , Escolaridad
2.
Anal Chem ; 91(17): 11030-11037, 2019 09 03.
Artículo en Inglés | MEDLINE | ID: mdl-31365232

RESUMEN

Macacine herpesvirus or B Virus (BV) is a zoonotic agent that leads to high mortality rates in humans if transmitted and untreated. Here, BV is used as a test case to establish a two-step procedure for developing high throughput serological assays based on synthetic peptides. In step 1, peptide microarray analysis of 42 monkey sera (30 of them tested BV positive by ELISA) revealed 1148 responses against 369 different peptides. The latter could be grouped into 142 different antibody target regions (ATRs) in six different glycoproteins (gB, gC, gD, gG, gH, and gL) of BV. The high number of newly detected ATRs was made possible inter alia by a new preanalytical protocol that reduced unspecific binding of serum components to the cellulose-based matrix of the microarray. In step 2, soluble peptides corresponding to eight ATRs of particularly high antigenicity were synthesized and coupled to fluorescently labeled beads, which were subsequently employed in immunochemical bead flow assays. Their outcome mirrored the ELISA results used as reference. Hence, convenient, fast, and economical screening of arbitrarily large macaque colonies for BV infection is now possible. The study demonstrates that a technology platform switch from two-dimensional high-resolution peptide arrays used for epitope discovery to a readily available bead array platform for serology applications is feasible.


Asunto(s)
Anticuerpos Antivirales/sangre , Epítopos/sangre , Infecciones por Herpesviridae/veterinaria , Herpesvirus Cercopitecino 1/inmunología , Enfermedades de los Primates/diagnóstico , Proteínas Virales/sangre , Animales , Sitios de Unión , Epítopos/química , Infecciones por Herpesviridae/diagnóstico , Infecciones por Herpesviridae/inmunología , Infecciones por Herpesviridae/virología , Herpesvirus Cercopitecino 1/genética , Humanos , Sueros Inmunes/química , Inmunoconjugados/química , Macaca mulatta/inmunología , Macaca mulatta/virología , Modelos Moleculares , Enfermedades de los Primates/inmunología , Enfermedades de los Primates/virología , Análisis por Matrices de Proteínas/instrumentación , Análisis por Matrices de Proteínas/métodos , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Proteínas Virales/química
3.
J Bacteriol ; 198(1): 138-46, 2016 01 01.
Artículo en Inglés | MEDLINE | ID: mdl-26324453

RESUMEN

UNLABELLED: High levels of the universal bacterial second messenger cyclic di-GMP (c-di-GMP) promote the establishment of surface-attached growth in many bacteria. Not only can c-di-GMP bind to nucleic acids and directly control gene expression, but it also binds to a diverse array of proteins of specialized functions and orchestrates their activity. Since its development in the early 1990s, the synthetic peptide array technique has become a powerful tool for high-throughput approaches and was successfully applied to investigate the binding specificity of protein-ligand interactions. In this study, we used peptide arrays to uncover the c-di-GMP binding site of a Pseudomonas aeruginosa protein (PA3740) that was isolated in a chemical proteomics approach. PA3740 was shown to bind c-di-GMP with a high affinity, and peptide arrays uncovered LKKALKKQTNLR to be a putative c-di-GMP binding motif. Most interestingly, different from the previously identified c-di-GMP binding motif of the PilZ domain (RXXXR) or the I site of diguanylate cyclases (RXXD), two leucine residues and a glutamine residue and not the charged amino acids provided the key residues of the binding sequence. Those three amino acids are highly conserved across PA3740 homologs, and their singular exchange to alanine reduced c-di-GMP binding within the full-length protein. IMPORTANCE: In many bacterial pathogens the universal bacterial second messenger c-di-GMP governs the switch from the planktonic, motile mode of growth to the sessile, biofilm mode of growth. Bacteria adapt their intracellular c-di-GMP levels to a variety of environmental challenges. Several classes of c-di-GMP binding proteins have been structurally characterized, and diverse c-di-GMP binding domains have been identified. Nevertheless, for several c-di-GMP receptors, the binding motif remains to be determined. Here we show that the use of a synthetic peptide array allowed the identification of a c-di-GMP binding motif of a putative c-di-GMP receptor protein in the opportunistic pathogen P. aeruginosa. The application of synthetic peptide arrays will facilitate the search for additional c-di-GMP receptor proteins and aid in the characterization of c-di-GMP binding motifs.


Asunto(s)
GMP Cíclico/análogos & derivados , Análisis por Matrices de Proteínas/métodos , Pseudomonas aeruginosa/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Secuencia de Consenso , GMP Cíclico/química , GMP Cíclico/genética , GMP Cíclico/metabolismo , Datos de Secuencia Molecular , Estructura Molecular , Movimiento , Unión Proteica , Estructura Terciaria de Proteína
4.
J Proteome Res ; 13(12): 5956-64, 2014 Dec 05.
Artículo en Inglés | MEDLINE | ID: mdl-25318615

RESUMEN

Urokinase plasminogen activator receptor (uPAR) and the epithelial integrin αvß6 are thought to individually play critical roles in cancer metastasis. These observations have been highlighted by the recent discovery (by proteomics) of an interaction between these two molecules, which are also both implicated in the epithelial-mesenchymal transition (EMT) that facilitates escape of cells from tissue barriers and is a common signature of cancer metastases. In this study, orthogonal in cellulo and in vitro functional proteomic approaches were used to better characterize the uPAR·αvß6 interaction. Proximity ligation assays (PLA) confirmed the uPAR·αvß6 interaction on OVCA429 (ovarian cancer line) and four different colon cancer cell lines including positive controls in cells with de novo ß6 subunit expression. PLA studies were then validated using peptide arrays, which also identified potential physical sites of uPAR interaction with αvß6, as well as verifying interactions with other known uPAR ligands (e.g., uPA, vitronectin) and individual integrin subunits (i.e., αv, ß1, ß3, and ß6 alone). Our data suggest that interaction with uPAR requires expression of the complete αß heterodimer (e.g., αvß6), not individual subunits (i.e., αv, ß1, ß3, or ß6). Finally, using in silico structural analyses in concert with these functional proteomics studies, we propose and demonstrate that the most likely unique sites of interaction between αvß6 and uPAR are located in uPAR domains II and III.


Asunto(s)
Antígenos de Neoplasias/metabolismo , Integrinas/metabolismo , Receptores del Activador de Plasminógeno Tipo Uroquinasa/metabolismo , Secuencia de Aminoácidos , Antígenos de Neoplasias/química , Línea Celular Tumoral , Transición Epitelial-Mesenquimal , Humanos , Integrinas/química , Datos de Secuencia Molecular , Dominios y Motivos de Interacción de Proteínas , Mapeo de Interacción de Proteínas , Proteómica , Receptores del Activador de Plasminógeno Tipo Uroquinasa/química
5.
Mol Microbiol ; 90(6): 1216-32, 2013 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-24127899

RESUMEN

The ubiquitous second messenger c-di-GMP regulates the switching of bacterial lifestyles from motility to sessility and acute to chronic virulence to adjust bacterial fitness to altered environmental conditions. Conventionally, EAL proteins being c-di-GMP phosphodiesterases promote motility and acute virulence phenotypes such as invasion into epithelial cells and inhibit biofilm formation. We report here that in contradiction, the EAL-like protein STM1697 of Salmonella typhimurium suppresses motility, invasion into HT-29 epithelial cell line and secretion of the type three secretion system 1 effector protein SipA, whereas it promotes rdar biofilm formation and CsgD expression. STM1697 can, however, functionally replace the EAL-like protein STM1344 and vice versa, whereby both proteins neither degrade nor bind c-di-GMP. Like STM1344, STM1697 suppresses the transcription of class 2 and class 3 flagella regulon genes by binding to FlhD, a component of the master regulator of the flagella regulon FlhD4 C2 and act additively under numerous conditions. Interestingly, the interaction interface of STM1697 with FlhD2 is distinct from its paralogue STM1344. We predict that the stand alone EAL domain proteins STM1697 and STM1344 belong to a subclass of EAL domain proteins in S. typhimurium, which are all involved in motility, biofilm and virulence regulation through interaction with proteins that regulate flagella function.


Asunto(s)
Proteínas Bacterianas/metabolismo , Biopelículas/crecimiento & desarrollo , Flagelos/fisiología , Salmonella typhimurium/fisiología , Salmonella typhimurium/patogenicidad , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Flagelos/genética , Regulación Bacteriana de la Expresión Génica , Genes Bacterianos , Células HT29 , Humanos , Proteínas de Microfilamentos/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Movimiento , Fenotipo , Hidrolasas Diéster Fosfóricas/metabolismo , Conformación Proteica , Infecciones por Salmonella , Salmonella typhimurium/genética , Virulencia
6.
BMC Biotechnol ; 14: 68, 2014 Jul 17.
Artículo en Inglés | MEDLINE | ID: mdl-25033887

RESUMEN

BACKGROUND: Tuberculosis is the leading cause of death due to bacterial infections worldwide, mainly caused by Mycobacterium tuberculosis. The antigen 85 complex comprises a set of major secreted proteins of M. tuberculosis, which are potential biomarkers for diagnostic. RESULTS: In this work, the first human single chain fragment variable (scFv) antibodies specific for the tuberculosis biomarker 85 B were selected by phage display from naïve antibody gene libraries (HAL7/8). Produced as scFv-Fc in mammalian cells, these antibodies were further characterized and analysed for specificity and applicability in different tuberculosis antigen detection assays. Sandwich detection of recombinant 85 B was successful in enzyme linked immunosorbent assay (ELISA), lateral flow immunoassay and immunoblot. Whereas detection of M. tuberculosis cell extracts and culture filtrates was only possible in direct ELISA and immunoblot assays. It was found that the conformation of 85 B, depending on sample treatment, influenced antigen detection. CONCLUSIONS: Recombinant antibodies, selected by phage display, may be applicable for 85 B detection in various assays. These antibodies are candidates for the development of future point of care tuberculosis diagnostic kits. Using 85 B as a biomarker, the antigen conformation influenced by sample treatment is important.


Asunto(s)
Aciltransferasas/inmunología , Antígenos Bacterianos/inmunología , Proteínas Bacterianas/inmunología , Mycobacterium tuberculosis/metabolismo , Anticuerpos de Cadena Única/metabolismo , Aciltransferasas/análisis , Aciltransferasas/química , Secuencia de Aminoácidos , Antígenos Bacterianos/análisis , Antígenos Bacterianos/química , Proteínas Bacterianas/análisis , Proteínas Bacterianas/química , Ensayo de Inmunoadsorción Enzimática , Mapeo Epitopo , Epítopos/química , Epítopos/inmunología , Epítopos/metabolismo , Biblioteca de Genes , Humanos , Biblioteca de Péptidos , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Anticuerpos de Cadena Única/genética , Anticuerpos de Cadena Única/inmunología
7.
Microb Cell Fact ; 13: 17, 2014 Jan 29.
Artículo en Inglés | MEDLINE | ID: mdl-24475978

RESUMEN

BACKGROUND: The nuclear export of unspliced and partially spliced HIV-1 mRNA is mediated by the recognition of a leucine-rich nuclear export signal (NES) in the HIV Rev protein by the host protein CRM1/Exportin1. This makes the CRM1-Rev complex an attractive target for the development of new antiviral drugs. Here we tested the anti-HIV efficacy of ratjadone A, a CRM1 inhibitor derived from myxobacteria. RESULTS: Ratjadone A inhibits HIV infection in vitro in a dose-dependent manner with EC50 values at the nanomolar range. The inhibitory effect of ratjadone A occurs around 12 hours post-infection and is specific for the Rev/CRM1-mediated nuclear export pathway. By using a drug affinity responsive target stability (DARTS) assay we could demonstrate that ratjadone A interferes with the formation of the CRM1-Rev-NES complex by binding to CRM1 but not to Rev. CONCLUSION: Ratjadone A exhibits strong anti-HIV activity but low selectivity due to toxic effects. Although this limits its potential use as a therapeutic drug, further studies with derivatives of ratjadones might help to overcome these difficulties in the future.


Asunto(s)
Infecciones por VIH/prevención & control , VIH-1/metabolismo , Carioferinas/metabolismo , Myxococcales/metabolismo , Pironas/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Productos del Gen rev del Virus de la Inmunodeficiencia Humana/metabolismo , Transporte Activo de Núcleo Celular/efectos de los fármacos , Antivirales/farmacología , Línea Celular , Proteína p24 del Núcleo del VIH/metabolismo , Humanos , Carioferinas/antagonistas & inhibidores , Unión Proteica , Pironas/química , Pironas/farmacología , ARN Mensajero/metabolismo , Receptores Citoplasmáticos y Nucleares/antagonistas & inhibidores , Productos del Gen rev del Virus de la Inmunodeficiencia Humana/antagonistas & inhibidores , Proteína Exportina 1
8.
Health Expect ; 17(3): 376-87, 2014 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-22390229

RESUMEN

BACKGROUND: Cardiovascular diseases are the number one cause of death and a source of chronic disability. OBJECTIVES: To assess recognition of and reaction to symptoms of heart attack and stroke, and how recognition is related to the frequency of consulting physicians and other information sources. DESIGN: Face-to-face computer-assisted personal interviews. PARTICIPANTS: Representative sample of 10,228 persons in Austria, France, Germany, Italy, the Netherlands, Poland, Russia, Spain and UK, aged 14-98. MAIN OUTCOME VARIABLES: Recognition of heart attack and stroke symptoms and proper reaction to symptoms. RESULTS: Chest pain was the only heart attack symptom recognized by more than 50% of participants. Eight percent knew no symptoms. Of 14 stroke symptoms, none was recognized by more than 50% of participants; 19% could not identify any symptom. For both heart attack and stroke, Germans and Austrians recognized the largest number of symptoms. Persons in Italy, Poland, Russia and Spain knew only about half as many symptoms as in Germany or Austria. Only 51% of Europeans would call an ambulance when someone suffers a stroke, the fewest (33 and 34%) in Germany and Austria. In most countries, people who consulted their physician more frequently had no better recognition of heart attack or stroke symptoms. CONCLUSIONS: The majority of persons in nine European countries recognize few heart attack and stroke symptoms; many do not know how to react. This low level of knowledge constitutes a major health risk and likely leads to delay in treatment, contributing to the high mortality and morbidity from these diseases.


Asunto(s)
Conocimientos, Actitudes y Práctica en Salud , Paro Cardíaco/diagnóstico , Accidente Cerebrovascular/diagnóstico , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Intervalos de Confianza , Europa (Continente) , Femenino , Humanos , Entrevistas como Asunto , Masculino , Persona de Mediana Edad , Investigación Cualitativa , Derivación y Consulta/estadística & datos numéricos , Encuestas y Cuestionarios , Adulto Joven
9.
J Biol Chem ; 287(46): 39158-70, 2012 Nov 09.
Artículo en Inglés | MEDLINE | ID: mdl-22948154

RESUMEN

Carcinoembryonic antigen-related cell adhesion molecule 3 (CEACAM3) is a phagocytic receptor on human granulocytes, which mediates the opsonin-independent recognition and internalization of a restricted set of Gram-negative bacteria such as Neisseria gonorrhoeae. In an unbiased screen using a SH2 domain microarray we identified the SH2 domain of growth factor receptor-bound protein 14 (Grb14) as a novel binding partner of CEACAM3. Biochemical assays and microscopic studies demonstrated that the Grb14 SH2 domain promoted the rapid recruitment of this adaptor protein to the immunoreceptor-based activation motif (ITAM)-like sequence within the cytoplasmic domain of CEACAM3. Furthermore, FRET-FLIM analyses confirmed the direct association of Grb14 and CEACAM3 in intact cells at the sites of bacteria-host cell contact. Knockdown of endogenous Grb14 by RNA interference as well as Grb14 overexpression indicate an inhibitory role for this adapter protein in CEACAM3-mediated phagocytosis. Therefore, Grb14 is the first negative regulator of CEACAM3-initiated bacterial phagocytosis and might help to focus granulocyte responses to the subcellular sites of pathogen-host cell contact.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/fisiología , Antígeno Carcinoembrionario/metabolismo , Neisseria gonorrhoeae/metabolismo , Fagocitosis , Citoplasma/metabolismo , Transferencia Resonante de Energía de Fluorescencia/métodos , Granulocitos/citología , Células HEK293 , Células HeLa , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos , Fosforilación , Fosfotirosina/química , Estructura Terciaria de Proteína , Tirosina/química
10.
Mol Med ; 18: 1499-508, 2013 Feb 08.
Artículo en Inglés | MEDLINE | ID: mdl-23269976

RESUMEN

Our previously reported phase I clinical trial with the allogeneic gene-modified tumor cell line RCC-26/CD80/IL-2 showed that vaccination was well tolerated and feasible in metastatic renal cell carcinoma (RCC) patients. Substantial disease stabilization was observed in most patients despite a high tumor burden at study entry. To investigate alterations in immune responses that might contribute to this effect, we performed an extended immune monitoring that included analysis of reactivity against multiple antigens, cytokine/chemokine changes in serum and determination of the frequencies of immune suppressor cell populations, including natural regulatory T cells (nTregs) and myeloid-derived suppressor cell subsets (MDSCs). An overall immune response capacity to virus-derived control peptides was present in 100% of patients before vaccination. Vaccine-induced immune responses to tumor-associated antigens occurred in 75% of patients, demonstrating the potent immune stimulatory capacity of this generic vaccine. Furthermore, some patients reacted to peptide epitopes of antigens not expressed by the vaccine, showing that epitope-spreading occurred in vivo. Frequencies of nTregs and MDSCs were comparable to healthy donors at the beginning of study. A significant decrease of nTregs was detected after vaccination (p = 0.012). High immune response rates, decreased frequencies of nTregs and a mixed T helper 1/T helper 2 (T(H)1/T(H)2)-like cytokine pattern support the applicability of this RCC generic vaccine for use in combination therapies.


Asunto(s)
Vacunas contra el Cáncer/inmunología , Carcinoma de Células Renales/inmunología , Carcinoma de Células Renales/patología , Inmunidad/inmunología , Neoplasias Renales/inmunología , Linfocitos T Reguladores/inmunología , Vacunación , Carcinoma de Células Renales/sangre , Carcinoma de Células Renales/prevención & control , Citocinas/biosíntesis , Citocinas/sangre , Humanos , Neoplasias Renales/sangre , Neoplasias Renales/patología , Neoplasias Renales/prevención & control , Recuento de Linfocitos , Linfocitos Infiltrantes de Tumor/inmunología , Células Mieloides/inmunología , Células Mieloides/patología , Metástasis de la Neoplasia , Estadificación de Neoplasias , Péptidos/inmunología , Análisis de Supervivencia , Células TH1/inmunología , Células Th2/inmunología , Factores de Tiempo , Resultado del Tratamiento
11.
Nat Chem Biol ; 8(1): 93-101, 2011 Nov 20.
Artículo en Inglés | MEDLINE | ID: mdl-22101602

RESUMEN

Several lines of evidence indicate that prefibrillar assemblies of amyloid-ß (Aß) polypeptides, such as soluble oligomers or protofibrils, rather than mature, end-stage amyloid fibrils cause neuronal dysfunction and memory impairment in Alzheimer's disease. These findings suggest that reducing the prevalence of transient intermediates by small molecule-mediated stimulation of amyloid polymerization might decrease toxicity. Here we demonstrate the acceleration of Aß fibrillogenesis through the action of the orcein-related small molecule O4, which directly binds to hydrophobic amino acid residues in Aß peptides and stabilizes the self-assembly of seeding-competent, ß-sheet-rich protofibrils and fibrils. Notably, the O4-mediated acceleration of amyloid fibril formation efficiently decreases the concentration of small, toxic Aß oligomers in complex, heterogeneous aggregation reactions. In addition, O4 treatment suppresses inhibition of long-term potentiation by Aß oligomers in hippocampal brain slices. These results support the hypothesis that small, diffusible prefibrillar amyloid species rather than mature fibrillar aggregates are toxic for mammalian cells.


Asunto(s)
Amiloide/química , Oxazinas/química , Fragmentos de Péptidos/química , Secuencia de Aminoácidos , Amiloide/toxicidad , Amiloide/ultraestructura , Línea Celular Tumoral , Hipocampo/química , Hipocampo/efectos de los fármacos , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Microscopía Electrónica de Transmisión , Modelos Moleculares , Fragmentos de Péptidos/toxicidad , Fragmentos de Péptidos/ultraestructura , Estructura Secundaria de Proteína , Transmisión Sináptica
12.
Microb Cell Fact ; 12: 85, 2013 Sep 24.
Artículo en Inglés | MEDLINE | ID: mdl-24063434

RESUMEN

BACKGROUND: Drug-resistance and therapy failure due to drug-drug interactions are the main challenges in current treatment against Human Immunodeficiency Virus (HIV) infection. As such, there is a continuous need for the development of new and more potent anti-HIV drugs. Here we established a high-throughput screen based on the highly permissive TZM-bl cell line to identify novel HIV inhibitors. The assay allows discriminating compounds acting on early and/or late steps of the HIV replication cycle. RESULTS: The platform was used to screen a unique library of secondary metabolites derived from myxobacteria. Several hits with good anti-HIV profiles were identified. Five of the initial hits were tested for their antiviral potency. Four myxobacterial compounds, sulfangolid C, soraphen F, epothilon D and spirangien B, showed EC50 values in the nM range with SI > 15. Interestingly, we found a high amount of overlapping hits compared with a previous screen for Hepatitis C Virus (HCV) using the same library. CONCLUSION: The unique structures and mode-of-actions of these natural compounds make myxobacteria an attractive source of chemicals for the development of broad-spectrum antivirals. Further biological and structural studies of our initial hits might help recognize smaller drug-like derivatives that in turn could be synthesized and further optimized.


Asunto(s)
Fármacos Anti-VIH/aislamiento & purificación , Fármacos Anti-VIH/farmacología , Myxococcales/química , Resistencia a Medicamentos , Ensayos Analíticos de Alto Rendimiento/métodos , Humanos
13.
RNA Biol ; 10(11): 1661-9, 2013 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-24418890

RESUMEN

Processing bodies (P-bodies) are cytoplasmatic mRNP granules containing non-translating mRNAs and proteins from the mRNA decay and silencing machineries. The mechanism of P-body assembly has been typically addressed by depleting P-body components. Here we apply a complementary approach and establish an automated cell-based assay platform to screen for molecules affecting P-body assembly. From a unique library of compounds derived from myxobacteria, 30 specifically inhibited P-body assembly. Gephyronic acid A (GA), a eukaryotic protein synthesis inhibitor, showed the strongest effect. GA also inhibited, under stress conditions, phosphorylation of eIF2α and stress granule formation. Other hits uncovered interesting novel links between P-body assembly, lipid metabolism, and internal organelle physiology. The obtained results provide a chemical toolbox to manipulate P-body assembly and function.


Asunto(s)
Gránulos Citoplasmáticos/metabolismo , Descubrimiento de Drogas , Myxococcales/química , Ribonucleoproteínas Citoplasmáticas Pequeñas/metabolismo , Bibliotecas de Moléculas Pequeñas , Línea Celular Tumoral , Cicloheximida/farmacología , Gránulos Citoplasmáticos/efectos de los fármacos , Factor 2 Eucariótico de Iniciación/metabolismo , Ácidos Grasos Monoinsaturados/farmacología , Células HeLa , Humanos , Metabolismo de los Lípidos , Myxococcales/metabolismo , Fosforilación , Puromicina/farmacología , Estabilidad del ARN
14.
J Biol Chem ; 286(11): 9555-66, 2011 Mar 18.
Artículo en Inglés | MEDLINE | ID: mdl-21216968

RESUMEN

Carcinoembryonic antigen-related cell adhesion molecule 3 (CEACAM3) is an immunoglobulin-related receptor expressed on human granulocytes. CEACAM3 functions as a single chain phagocytotic receptor recognizing gram-negative bacteria such as Neisseria gonorrhoeae, which possess CEACAM-binding adhesins on their surface. The cytoplasmic domain of CEACAM3 contains an immunoreceptor tyrosine-based activation motif (ITAM)-like sequence that is phosphorylated upon receptor engagement. Here we show that the SH2 domains of the regulatory subunit of phosphatidylinositol 3'-kinase (PI3K) bind to tyrosine residue 230 of CEACAM3 in a phosphorylation-dependent manner. PI3K is rapidly recruited and directly associates with CEACAM3 upon bacterial binding as shown by FRET analysis. Although PI3K activity is not required for efficient uptake of the bacteria by CEACAM3-transfected cells or primary human granulocytes, it is critical for the stimulated production of reactive oxygen species by infected phagocytes and the intracellular degradation of CEACAM-binding bacteria. Together, our results highlight the ability of CEACAM3 to coordinate signaling events that not only mediate bacterial uptake, but also trigger the killing of internalized pathogens.


Asunto(s)
Antígeno Carcinoembrionario/metabolismo , Gonorrea/metabolismo , Granulocitos/metabolismo , Neisseria gonorrhoeae/metabolismo , Fagocitosis/fisiología , Fosfatidilinositol 3-Quinasas/metabolismo , Estallido Respiratorio/fisiología , Adhesinas Bacterianas/metabolismo , Antígeno Carcinoembrionario/genética , Gonorrea/genética , Células HEK293 , Humanos , Fosfatidilinositol 3-Quinasas/genética , Fosforilación/fisiología , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/fisiología , Dominios Homologos src
15.
J Biol Chem ; 286(10): 8414-8424, 2011 Mar 11.
Artículo en Inglés | MEDLINE | ID: mdl-21183679

RESUMEN

To develop a novel attenuation strategy applicable to all influenza A viruses, we targeted the highly conserved protein-protein interaction of the viral polymerase subunits PA and PB1. We postulated that impaired binding between PA and PB1 would negatively affect trimeric polymerase complex formation, leading to reduced viral replication efficiency in vivo. As proof of concept, we introduced single or multiple amino acid substitutions into the protein-protein-binding domains of either PB1 or PA, or both, to decrease binding affinity and polymerase activity substantially. As expected, upon generation of recombinant influenza A viruses (SC35M strain) containing these mutations, many pseudo-revertants appeared that partially restored PA-PB1 binding and polymerase activity. These polymerase assembly mutants displayed drastic attenuation in cell culture and mice. The attenuation of the polymerase assembly mutants was maintained in IFNα/ß receptor knock-out mice. As exemplified using a H5N1 polymerase assembly mutant, this attenuation strategy can be also applied to other highly pathogenic influenza A virus strains. Thus, we provide proof of principle that targeted mutation of the highly conserved interaction domains of PA and PB1 represents a novel strategy to attenuate influenza A viruses.


Asunto(s)
Subtipo H5N1 del Virus de la Influenza A/enzimología , Subtipo H7N7 del Virus de la Influenza A/enzimología , Gripe Humana/enzimología , Mutación , ARN Polimerasa Dependiente del ARN/metabolismo , Proteínas Virales/metabolismo , Animales , Perros , Humanos , Subtipo H5N1 del Virus de la Influenza A/genética , Subtipo H5N1 del Virus de la Influenza A/inmunología , Subtipo H7N7 del Virus de la Influenza A/genética , Subtipo H7N7 del Virus de la Influenza A/inmunología , Vacunas contra la Influenza/biosíntesis , Vacunas contra la Influenza/genética , Vacunas contra la Influenza/inmunología , Gripe Humana/genética , Gripe Humana/inmunología , Interferón-alfa/genética , Interferón-alfa/inmunología , Interferón-alfa/metabolismo , Interferón beta/genética , Interferón beta/inmunología , Interferón beta/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , ARN Polimerasa Dependiente del ARN/genética , ARN Polimerasa Dependiente del ARN/inmunología , Vacunas Atenuadas/biosíntesis , Vacunas Atenuadas/genética , Vacunas Atenuadas/inmunología , Proteínas Virales/genética , Proteínas Virales/inmunología , Replicación Viral/genética , Replicación Viral/inmunología
16.
Chembiochem ; 13(3): 409-15, 2012 Feb 13.
Artículo en Inglés | MEDLINE | ID: mdl-22271561

RESUMEN

Elevated expression of interleukin-8 (IL-8) has been implicated in inflammatory diseases, in tumor growth, and in angiogenesis. The aim of this study was to identify natural or synthetic compounds that suppress IL-8 production in response to interleukin-1 (IL-1), the natural inflammatory stimulus of the IL-8 gene. We therefore developed an IL-1-inducible cell-based screening assay by stable integration of an IL-8 reporter gene into HeLa S3 cells. The screening of heterogeneous compound libraries revealed several compounds that displayed an inhibitory effect on the reporter gene expression. Following hit validation, we focused on the most efficient compound, spirangien A, and its chemical derivate spirangien M522. Detailed analysis shows that both compounds are potent inhibitors of the endogenous IL-8 gene transcription. Furthermore, both compounds decelerate the phosphorylation and degradation of IκBα, the key regulator of the IL-1-stimulated NF-κB signaling pathway. Our study has identified the two spirangiens A and M522 as potent inhibitors of IL-1/NF-κB-mediated IL-8 gene expression.


Asunto(s)
Acetales/farmacología , Ácidos Grasos Insaturados/farmacología , Interleucina-8/antagonistas & inhibidores , Myxococcales/química , Piranos/farmacología , Compuestos de Espiro/farmacología , Acetales/química , Relación Dosis-Respuesta a Droga , Ácidos Grasos Insaturados/química , Perfilación de la Expresión Génica , Células HeLa , Humanos , Proteínas I-kappa B/antagonistas & inhibidores , Proteínas I-kappa B/metabolismo , Interleucina-8/genética , Fosforilación/efectos de los fármacos , Piranos/química , ARN Mensajero/antagonistas & inhibidores , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Compuestos de Espiro/química , Transcripción Genética/efectos de los fármacos , Transcripción Genética/genética
17.
FASEB J ; 25(3): 894-906, 2011 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-21106938

RESUMEN

Macrophage migration inhibitory factor (MIF) is a cytokine that mediates inflammatory diseases. MIF promotes atherogenic leukocyte recruitment through a promiscuous, yet highly affine, interaction with CXCR2 and CXCR4. Binding to CXCR2 is dependent on a pseudo-(E)LR motif in MIF, but a second interaction site has been elusive. Here we identified an N-like loop in MIF, suggesting that MIF binding to CXCR2 follows the 2-site binding mode of bona fide chemokines. For MIF, the model predicts interactions between the N-like loop and the CXCR2 N domain (site 1) and pseudo-(E)LR and extracellular loops (ELs) of CXCR2 (site 2). Applying biophysical and peptide array analysis, we demonstrated an interaction between MIF and the CXCR2 N domain, which was pseudo-(E)LR independent. Peptide array analysis also indicated that the pseudo-(E)LR motif is responsible for MIF binding to EL2 and 3. Notably, peptides MIF-(40-49) and MIF-(47-56), representing N-like-loop-derived peptides, but not a scrambled control peptide, significantly blocked MIF/CXCR2 binding, MIF-mediated monocyte arrest under flow on aortic endothelial cells in vitro (IC(50): 1.24×10(-6) M), and MIF-dependent monocyte adhesion to atherosclerotic mouse carotid arteries in vivo. Thus, the N-like loop in MIF is critical for MIF's noncognate interaction with CXCR2 and proatherogenic functions. The 2-site binding model that explains chemokine receptor activation also applies to MIF.


Asunto(s)
Aterosclerosis/metabolismo , Células Endoteliales/fisiología , Oxidorreductasas Intramoleculares/metabolismo , Factores Inhibidores de la Migración de Macrófagos/metabolismo , Receptores de Interleucina-8B/química , Receptores de Interleucina-8B/metabolismo , Animales , Aorta/citología , Apolipoproteínas E/genética , Aterosclerosis/patología , Aterosclerosis/fisiopatología , Sitios de Unión/fisiología , Unión Competitiva/fisiología , Dicroismo Circular , Células Endoteliales/citología , Células HEK293 , Humanos , Interleucina-8/metabolismo , Oxidorreductasas Intramoleculares/genética , Factores Inhibidores de la Migración de Macrófagos/genética , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Monocitos/citología , Unión Proteica/fisiología , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Receptores Inmunológicos/química , Receptores Inmunológicos/genética , Receptores Inmunológicos/metabolismo , Receptores de Interleucina-8B/genética , Relación Estructura-Actividad
18.
Microb Cell Fact ; 11: 52, 2012 Apr 30.
Artículo en Inglés | MEDLINE | ID: mdl-22545867

RESUMEN

Myxobacteria are amongst the top producers of natural products. The diversity and unique structural properties of their secondary metabolites is what make these social microbes highly attractive for drug discovery. Screening of products derived from these bacteria has revealed a puzzling amount of hits against infectious and non-infectious human diseases. Preying mainly on other bacteria and fungi, why would these ancient hunters manufacture compounds beneficial for us? The answer may be the targeting of shared processes and structural features conserved throughout evolution.


Asunto(s)
Productos Biológicos/metabolismo , Myxococcales/metabolismo , Descubrimiento de Drogas , Myxococcales/genética , Myxococcales/crecimiento & desarrollo , Esporas Bacterianas/crecimiento & desarrollo , Esporas Bacterianas/metabolismo
19.
Ann Fam Med ; 15(3): 280, 2017 May.
Artículo en Inglés | MEDLINE | ID: mdl-28483900
20.
Mol Cell Proteomics ; 9(1): 1-10, 2010 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-19674966

RESUMEN

Protein affinity reagents (PARs), most commonly antibodies, are essential reagents for protein characterization in basic research, biotechnology, and diagnostics as well as the fastest growing class of therapeutics. Large numbers of PARs are available commercially; however, their quality is often uncertain. In addition, currently available PARs cover only a fraction of the human proteome, and their cost is prohibitive for proteome scale applications. This situation has triggered several initiatives involving large scale generation and validation of antibodies, for example the Swedish Human Protein Atlas and the German Antibody Factory. Antibodies targeting specific subproteomes are being pursued by members of Human Proteome Organisation (plasma and liver proteome projects) and the United States National Cancer Institute (cancer-associated antigens). ProteomeBinders, a European consortium, aims to set up a resource of consistently quality-controlled protein-binding reagents for the whole human proteome. An ultimate PAR database resource would allow consumers to visit one on-line warehouse and find all available affinity reagents from different providers together with documentation that facilitates easy comparison of their cost and quality. However, in contrast to, for example, nucleotide databases among which data are synchronized between the major data providers, current PAR producers, quality control centers, and commercial companies all use incompatible formats, hindering data exchange. Here we propose Proteomics Standards Initiative (PSI)-PAR as a global community standard format for the representation and exchange of protein affinity reagent data. The PSI-PAR format is maintained by the Human Proteome Organisation PSI and was developed within the context of ProteomeBinders by building on a mature proteomics standard format, PSI-molecular interaction, which is a widely accepted and established community standard for molecular interaction data. Further information and documentation are available on the PSI-PAR web site.


Asunto(s)
Bases de Datos de Proteínas/normas , Proteoma/análisis , Sistemas de Administración de Bases de Datos/normas , Humanos , Cooperación Internacional , Proteómica/métodos , Terminología como Asunto
SELECCIÓN DE REFERENCIAS
DETALLE DE LA BÚSQUEDA