Knockdown of DAPK1 attenuates IL-1B-induced extracellular matrix degradation and inflammatory response in osteoarthritis chondrocytes via regulating the p38 MAPK-signaling pathway

Objective To reveal the possible effects of death-associated protein kinase 1 (DAPK1) on the progression of osteoarthritis (OA) and the potential underlying mechanism. Methods : The expression of DAPK1 in OA and normal samples and interleukin (IL)-1β-stimulated chondrocytes was analyzed by quantitative real-time polymerase chain reaction and Immunoblot assay. Cell viability, proliferation, and apoptosis in DAPK1-knockdown cells stimulated with IL-1β were detected by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution, 5-ethynyl-2β-deoxyuridine staining and flow cytometry. The chondrocyte degradation and inflammatory response in IL-1β-induced chondrocytes were investigated by Immunoblot analysis and enzyme-linked-immunosorbent serologic assay. In addition, the effect of DAPK1 on p38 mitogen-activated protein kinase (MAPK) activation was analyzed by immunoblot assay. Results : This study revealed that DAPK1 was highly expressed in OA patients and IL-1β-induced chondrocytes. Down-regulation of DAPK1 enhanced IL-1β-induced chondrocyte proliferation. DAPK1 knockdown inhibited IL-1β-induced chondrocyte degradation. In addition, DAPK1 depletion inhibited IL-1β-induced chondrocyte inflammation. Mechanically, it was revealed that down--regulation of DAPK1 could inhibit the p38 MAPK pathway, and therefore affected progression of OA. Conclusion : DAPK1 knockdown attenuates IL-1β-induced extracellular matrix degradation and inflammatory response in OA chondrocytes by regulating the p38 MAPK pathway (AU)

IGF-1 modulates gene expression of proteins involved in inflammation, cytoskeleton, and liver architecture

Even though the liver synthesizes most of circulating IGF-1, it lacks its receptor under physiological conditions. However, according to previous studies, a damaged liver expresses the receptor. For this reason, herein, we examine hepatic histology and expression of genes encoding proteins of the cytoskeleton, extracellular matrix, and cell-cell molecules and inflammation-related proteins. A partial IGF-1 deficiency murine model was used to investigate IGF-1's effects on liver by comparing wild-type controls, heterozygous igf1+/-, and heterozygous mice treated with IGF-1 for 10 days. Histology, microarray for mRNA gene expression, RT-qPCR, and lipid peroxidation were assessed. Microarray analyses revealed significant underexpression of igf1 in heterozygous mice compared to control mice, restoring normal liver expression after treatment, which then normalized its circulating levels. IGF-1 receptor mRNA was overexpressed in Hz mice liver, while treated mice displayed a similar expression to that of the controls. Heterozygous mice showed overexpression of several genes encoding proteins related to inflammatory and acute-phase proteins and underexpression or overexpression of genes which coded for extracellular matrix, cytoskeleton, and cell junction components. Histology revealed an altered hepatic architecture. In addition, liver oxidative damage was found increased in the heterozygous group. The mere IGF-1 partial deficiency is associated with relevant alterations of the hepatic architecture and expression of genes involved in cytoskeleton, hepatocyte polarity, cell junctions, and extracellular matrix proteins. Moreover, it induces hepatic expression of the IGF-1 receptor and elevated acute-phase and inflammation mediators, which all resulted in liver oxidative damage (AU)

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Estudio del riesgo cardiovascular en pacientes con bloqueo del sistema renina-angiotensina a través del proteoma de las vesículas extracelulares circulantes / Cardiovascular risk study in patients with renin-angiotensin system blockade by means of the proteone of circulating extracellular vesicles

Introducción: Las vesículas extracelulares (EV) son liberadas al torrente circulatorio por determinados tipos celulares a consecuencia de procesos de transporte, activación o muerte celular. El recuento de EV circulantes de origen plaquetario y endotelial ha demostrado tener un importante papel como biomarcador de riesgo cardiovascular. Por lo tanto, el proteoma de estas EV podría reflejar los procesos celulares subyacentes en pacientes con hipertensión arterial y albuminuria. Material y métodos: El contenido proteico de las EV circulantes se ha analizado mediante cromatografía líquida acoplada a espectrometría de masas. Las EV han sido aisladas mediante un protocolo de ultracentrifugación optimizado para evitar la contaminación de proteínas del plasma. La pureza de la fracción aislada ha sido verificada mediante microscopia electrónica y confocal, y por citometría de flujo. Resultados: En este estudio mostramos un método de gran rendimiento y pureza para obtener extractos proteicos de EV circulantes de pacientes hipertensos con/sin albuminuria para análisis proteómico. Además, aportamos el proteoma de referencia de EV de estos pacientes, compuesto por 2.463 proteínas, y demostramos que las proteínas transportadas por estas vesículas están relacionadas con procesos cruciales involucrados en el riesgo cardiovascular asociado. Conclusión: El proteoma de las EV circulantes constituye una interesante fuente de indicadores para la evaluación de procesos relacionados con el riesgo cardiovascular en pacientes hipertensos con bloqueo del sistema renina-angiotensina

Introduction: Extracellular vesicles (EVs) are released to the bloodstream by certain cell types due to transport, activation and cell death processes. Blood count of EVs from platelet and endothelial origin has been proved to be a cardiovascular risk biomarker. Thus, EVs proteome might reflect the underlying cellular processes in hypertensive patients with albuminuria. Material and methods: Protein content of circulating EVs was analyzed by liquid chromatography coupled to mass spectrometry. EVs were isolated by an ultracentrifugation protocol optimized in order to avoid contamination by blood plasma proteins. Purity of the isolated fraction was verified by electronic and confocal microscopy, and by flow cytometry. Results: We hereby show a method to isolate circulating EVs from hypertensive patients with/without albuminuria with high yield and purity. Besides, we provide a reference proteome of the EVs of these patients, composed of 2,463 proteins, and prove that the proteins carried by these vesicles are associated with crucial processes involved in the inherent cardiovascular risk. Conclusion: The proteome of circulating EVs is an interesting source of indicators in the evaluation of cardiovascular risk in hypertensive patients with renin-angiotensin system blockage

Síndrome de Fraser: nueva mutación en el gen FREM2 / Fraser syndrome caused by a new mutation in the FREM2 gene

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Molecular markers of cell adhesion in ameloblastomas. An update

Ameloblastoma is the most common odontogenic tumor of epithelial origin, and though it is of a benign nature, it frequently infiltrates the bone, has a high rate of recurrence and could potentially become malignant. Cellular adhesion potentially plays an important role in the manifestation of these characteristics and in the tumor biology of ameloblastomas. Losses of cell-cell and extracellular matrix adhesion and cohesion are among the first events that occur in the invasion and growth of tumors of epithelial origin. The present review includes a description of the molecules that are involved in cell adhesion as reported for various types of ameloblastomas and discusses the possible roles of these molecules in the biological behaviors of this odontogenic tumor. Knowledge of the complex mechanisms in which these molecules play a role is critical for the research and discovery of future therapeutic targets

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Extracellular DNase activity of Cryptococcus neoformans and Cryptococcus gattii

Se ha estudiado la actividad extracelular de la enzima ADNasa en 73 cepas de Cryptococcus neoformans y 12 cepas de C. gattii. Para medir dicha actividad se utilizó la técnica de aparición de halos claros en agar DNasa con y sin verde metilo. Todas las cepas analizadas produjeron DNasa extracelular sin observar diferencias significativas entre C. neoformans y C. gattii. La producción de DNasa fue mayor en los aislamientos clínicos (valor medio del halo: 6,2mm) que en los recogidos de medio ambiente (valor medio del halo: 2,9mm). Mediante ensayo en gel PAGE con ADN como sustrato, se pudo estimar el peso molecular del enzima en 31kD. Estos resultados permiten apoyar la hipótesis de que la ADNasa extracelular podría ser un factor de patogenicidad implicado en la virulencia en el complejo de especies C. neoformans-C. gattii(AU)

Extracellular DNase activity was studied in 73 strains of Cryptococcus neoformans and 12 strains of Cryptococcus gattii. DNase activity was measured by DNase agar clearance with and without Methyl Green. All strains tested showed extracellular DNase activity and no significant difference was found betweenC. neoformans and C. gattii strains. DNase production was higher in strains from clinical origin (average radius of 6.2mm) than among environmental strains (average radius of 2.9mm). The extracellular enzyme may be detected by DNA substrate PAGE assays and its molecular weight was estimated at 31kD. These results suggest that extracellular DNase could be considered as a virulence factor involved in C. neoformans–C. gattii species complex pathogenicity(AU)

Raquitismo hipofosfatémico familiar / Familial hypophosphatemic rachitism

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Transcriptome and secretome analyses of the adaptive response of Pseudomonas aeruginosa to suboptimal growth temperature

Pseudomonas aeruginosa is an opportunistic pathogen involved in several diseases, including cystic fibrosis and nosocomial infections. Although the behavior of this bacterium at 37 degrees C has been intensively studied, little is known about its capacity to adapt and survive at suboptimal temperatures, such as those encountered in hospitals. In this work, transcriptomic and proteomic analyses were used to identify factors that allow P. aeruginosa to become established at room temperature (close to 25 degrees C) and thus facilitate host infections. Since the virulence of this pathogen is multifactorial and dependent on the extracellular release of toxins and degradative enzymes targeted to the host by several secretory systems, the study focused on genes activated at 25 degrees C, namely, those encoding either components of the secretory machinery or secreted proteins. These observations were enhanced by 2D-PAGE analyses, which showed that the production of effectors from type I and type II secretion systems (respectively, proteases AprA and PrpL) and of a hemolysin co-regulated protein (Hcp) related to the type VI secretion system was specifically stimulated when the growth temperature was lowered from 37 to 25 degrees C. The results provide a fundamental basis for investigating the processes that allow P. aeruginosa to adapt to suboptimal growth temperatures and which thereby promote nosocomial infection (AU)

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Nuevas perspectivas terapéuticas de la fibrosis hepática / No disponible

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Distrofia corneal granular autosómica dominante causada por mutación del gen TGFBI en una familia mexicana / Autosomal dominant granular corneal dystrophy caused by a TGFBI gene mutation in a mexican family

Objetivo: Las distrofias corneales son un grupo dealteraciones hereditarias en las que una acumulaciónprogresiva de material amiloide, hialino o mixtoen las distintas capas corneales produce disminuciónde la transparencia corneal. Se describen lascaracterísticas clínicas y los estudios molecularesdel gen TGFBI en una paciente Mexicana con unadistrofia corneal estromal de tipo granular.Métodos: Examen oftalmológico completo, caracterizaciónfenotípica de la distrofia corneal, y análisisdel gen TGFBI por reacción en cadena de lapolimerasa (PCR) y por secuenciación nucleotídica,en DNA de la propósita y de un hermano afectado.Resultados: Las lesiones corneales observadas en lapaciente fueron compatibles con el diagnóstico dedistrofia corneal estromal de tipo granular (clásica).No se observaron lesiones en las otras capas corneales.El análisis del gen TGFBI en DNA de la pacientey de un hermano afectado reveló una mutaciónpuntual, de adenina a guanina, en el exón 14 de TGFBIque origina un cambio de histidina a arginina enel aminoácido 626 (H626R) de la proteína TGFBI.Conclusiones: Éste es el primer caso en el que sedemuestra que una distrofia corneal granular es distrocausadapor la mutación H626R en TGFBI. Estamutación ha sido reportada consistentemente en ladistrofia estromal de tipo empalizada, clínicamentediferente a la granular. Nuestros datos indican queexisten excepciones en la aparente correlacióngenotipo-fenoitipo establecida en el grupo de distrofiascorneales asociadas a mutación en el genTGFBI

Objective: To describe the clinical data and the ;;results of molecular analyses of the TGFBI gene in ;;a patient with classic granular stromal corneal dystrophy ;;(type I). ;;Methods: A female patient aged 60-years complaining ;;of a long-standing decrease of visual acuity ;;bilaterally associated with photophobia and foreign ;;body sensation, underwent a complete ophthalmologic ;;examination. Molecular analyses of DNA ;;from the patient and from an affected brother included ;;PCR amplification of exons 4, 11, 12, and 14 of ;;the TGFBI gene and direct automated sequencing ;;of the PCR products. ;;Results: The affected patient showed a pattern of ;;corneal stromal lesions that was compatible with a ;;diagnosis of classic granular dystrophy. No involvement ;;of other corneal layers was evident. Molecular ;;analysis disclosed a point mutation in exon 14 of ;;the TGFBI gene which consisted of an adenine to ;;guanine change at nucleotide position 1924, predicting ;;a substitution of arginine instead of histidine at ;;residue 626 of the TGFBI protein (H626R). An ;;identical mutation was detected in DNA from her ;;affected brother. Conclusions: This is the first time that a case of ;;stromal granular dystrophy has been demonstrated ;;to be caused by the H626R mutation, a molecular ;;defect classically detected in the phenotypically ;;distinct lattice corneal dystrophy. Our data indicate ;;that the same molecular defects in the TGFBI gene ;;lead to different phenotypes in stromal dystrophies, ;;thus expanding the genotypic-phenotypic spectrum ;;in this group of corneal diseases

Expresión de proteínas relacionadas con resistencia a múltiples fármacos (MDR-proteínas) en tumores sólidos / Expresssion of multidrug resistance (MDR)-associated proteins in solid tumors

Las causas de que una célula tumoral sea resistente a la quimioterapia son muchas y de variada naturaleza. El motivo del presente trabajo es realizar una revisión y una puesta al día de una de estas posibles causas, en concreto, la expresión de proteínas relacionadas con la resistencia a múltiples fármacos (AU)

The causes of drug resistance in tumor cells vary widely. The present study aims to provide an update of multidrug resistance in tumor cells and, in particular, of multidrug resistance-associated proteins (AU)

Bases fisiológicas de la regeneración ósea I. Histología y fisiología del tejido óseo / Physiological bases of bone regeneration I. Histology and physiology of bone tissue

El hueso es el único tejido del organismo capaz de regenerarse, permitiendo la restitutio ad integrum tras el trauma. Cuando se produce una fractura, se coloca un implante osteointegrado o se realiza un injerto para aumentar el sustrato óseo antes de la inserción de implantes, lo que se pretende es la regeneración ósea, es decir, la formación de hueso nuevo que, tras un proceso de remodelado, sea idéntico al preexistente.El hueso es un tejido dinámico en constante formación y reabsorción. Este fenómeno equilibrado, denominado proceso de remodelado, permite la renovación de un 5-15 % del hueso total al año en condiciones normales (1). El remodelado óseo consisteen la reabsorción de una cantidad determinada de hueso llevada a cabo por los osteoclastos, así como la formación de la matriz osteoide por los osteoblastos y su posterior mineralización. Este fenómeno tiene lugar en pequeñas áreas de la cortical o de la superficie trabecular, llamadas “unidades básicas de remodelado óseo”.La actuación terapéutica en los campos de la Traumatología y Ortopedia, Cirugía Oral y Maxilofacial e Implantología, se asienta sobre los principios biológicos de la regeneración ósea, en los que están implicados células, matriz extracelular y señales osteoinductivas. El objetivo de este trabajo es realizar una puesta al día de los conocimientos actuales sobre los mecanismos bioquímicos y fisiológicos de la regeneración ósea, resaltando de manera especial el papel que en ella juegan las células y las proteínas de la matriz ósea

Bone is the only body tissue capable of regeneration, allowing the restitutio ad integrum following trauma. In the event of a fracture or bone graft, new bone is formed, which following the remodeling process is identical to the pre-existing.Bone is a dynamic tissue in constant formation and resorption. This balanced phenomena, known as the remodeling process, allows the renovation of 5-15% of the total bone mass per year under normal conditions (1). Bone remodeling consists of the resorption of a certain amount of bone by osteoclasts, likewise the formation of osteoid matrix by osteoblasts, and its subsequentmineralization. This phenomenon occurs in small areas of the cortical bone or the trabecular surface, called “Basic Multicellular Units” (BMU). Treatment in Traumatology, Orthopedics, Implantology, and Maxillofacial and Oral Surgery, is based on the biologic principals of bone regeneration, in which cells, extracellular matrix, and osteoinductive signals are involved.The aim of this paper is to provide an up date on current knowledge on the biochemical and physiological mechanisms of bone regeneration, paying particular attention to the role played by the cells and proteins of the bone matrix

Síndrome de Kindler: aportación de un caso / Kindler syndrome: presentation of a case

El síndrome de Kindler, es una enfermedad muy poco frecuente debida a mutaciones que originan defectos en la unión actina-matriz extracelular. Suele cursar con ampollas acrales desde el nacimiento en zonas más expuestas a los traumatismos, fotosensibilidad marcada que mejora con la edad y desarrollo de poiquilodermia y atrofia cutánea. Con relativa frecuencia se describe afectación de mucosas y degeneración maligna

Kindler syndrome is a very rare disease caused by mutations resulting in defects in the extracellular matrix-actin link. It usually presents with acral blistering from birth in trauma-prone areas, pronounced photosensitivity that improves with age and the development of poikiloderma and cutaneous atrophy. Mucosal involvement and degeneration have been described with relative frequency

Desarrollo de un protocolo de análisis de la expresión génica mediante «differential display» que reduce el número de falsos positivos / The development of a protocol for the analysis of genetic expression through «differential display», as a means to reducing the number of false positives

Entre los métodos empleados para los análisis de la expresión de genes, el método de "differential display" ha sido ampliamente utilizado y, a pesar del uso extendido de los "microarrays", es aún un método válido para el análisis con muestras cuyo transcriptoma es desconocido. Con el objeto de reducir el elevado número de falsos positivos que genera esta técnica, hemos optimizado el protocolo para reducir la posibilidad de generar falsos positivos. En primer lugar, hemos marcado radiactivamente el cebador oligo-dT con lo que los fragmentos de DNA identificados son extremos 3'-UTR de RNAm. Por muestra hemos realizado dos transcripciones inversas y dos reacciones de PCR en cada una de ellas. Para seleccionar un fragmento de DNA, debía estar diferencialmente expresado en las 4 reacciones de PCR. Por último, todos los fragmentos fueron clonados y secuenciados por triplicado. Estas modificaciones al protocolo nos ha permitido identificar 5 genes expresados diferencialmente entre células epiteliales de intestino en estado proliferativo y diferenciado

The analysis of genetic expression, the differential display (DD) method has been widely used, but inspite of the extensive use of the «microarrays» method, it is still to be considered as a valid method for the analysis of samples whose transcriptone is not known. In this work, an attempt has been made to reduce the high number of false positives generated by this technique by optimising method protocol. As a preliminary step, we radioactively marked the oligo dT primer with which the fragments of identified DNA were extreme 3'-UTR of mRNA. For each sample two inverse transcriptions and two PCR reactions were performed. Only fragments of DNA that are expressed differentially in all 4 PCR reactions should be selected. Finally, all of the fragments were cloned and sequenced in triplicate. These protocol modifications have allowed us to identify 5 differentially expressed genes, in intestinal epithelial cells in both proliferative and differentiated states

Colicin S8 export: extracellular and cytoplasmic colicin are different

The properties of colicin S8 are different for the cytoplasmic, periplasmic and extracellular protein. Interactions with its specific receptors reflect this. Active cell extracts separate into a non-anionic along with an anionic fraction by DEAE-Sephacell chromatography. Previously, we have purified cell-associated colicin S8 as an aggregation of highly related polypeptides; cytoplasmic colicin S8 seems to be post-translationally processed into an aggregation of polypeptides of molecular mass ranging from 45,000 Da to 60,000 Da. We suggest that a conformational change to colicin S8 may occur related to the export process (AU)

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Aumento de la expresión del proteoglicano condroitín-sulfato, fibronectina y fibroblastos en la estenosis hipertrófica de píloro / Increase of the chondroitin-sulfate proteoglycan, fibronectin and fibroblasts in infantile hypertrophic pyloric stenosis

Introducción. La estenosis hipertrófica de píloro (EHP) se caracteriza por la hipertrofia de la capa muscular del píloro. Su etiología permanece todavía desconocida. En los últimos años ha sido publicado algún trabajo que estudia la matriz extracelular (MEC) en la EHP. Nuestro objetivo fue investigar la expresión de dos moléculas de MEC: el proteoglicano condroitín-sulfato (PGCS) y la fibronectina (FN), así como la de los fibroplalstos. Material y métodos. Las biopsias delpíloro fueron obtenidas de 33 pacientes con EHP y 12 controles. Se utilizó inmunohistoquímica indirecta usando anticuerpos monoclonales dirigidos contra el PGCS, la FN y los fibroplastos. Los resultados fueron expresados mediante una escala semicuantitativa, como sigue: fuerte (++), moderada (+), débil (+/-) y ausente (-). Resultados. Se desmostró que la inmunorreactividad para el PGCS se localizaba en el tejido conjuntivo de los septos, y la de la FN en el espacio pericelular. Ambas moléculas estaban muy aumentadas en la capa muscular del píloro con EHP con relación a la capa muscular de los píloros control. También demostramos un marcado aumento en la expresión del número de fibroplastos en la capa muscular del píloro con EHP. Aunque la mayor expresión se localizó en los septos, también observamos gran número entre las células de músculo liso. Conclusiones: Sugerimos que la EHP, no sólo se caracteriza por la hipertrofia de la capa muscular, sino también por el aumento de varias moléculas de la MEC, como el PGCS y la FN. También sugerimos que el aumento de fibroplastos podría explicar la mayor expresión de estas moléculas de MEC en la capa muscular del píloro con EHP (AU)