Marco Galleguillos

Contenido de glicosaminoglicanos del líquido sinovial de la articulación metacarpofalángica de caballos castrados y yeguas de diferentes edades

Resumen en: Total concentration of synovial fluid glycosaminoglycan (GAGs) and a fraction of it (GAGsT) which correspond to different GAGs from hyaluronic acid or su...

ATP-DIPHOSPHOPHYDROLASE ACTIVITY IN RAT HEART TISSUE

Extracellular nucleotides interact with specific receptors on the cell surface and are locally metabolized by ecto‐nucleotidases. Biochemical characterization of the ATPase and ADPase activities detected in rat heart sarcolemma, under conditions where mitochondrial ATPase and adenylate kinase were blocked, supports our proposal that both activities correspond to a single enzyme, known as ATP‐diphosphohydrolase or apyrase. The physiological function of this enzyme could be dephosphorylation of the nucleotides present in the interstitial heart compartment acting together with 5′‐nucleotidase. Both hydrolytic activities have similarities in: sarcolemma localization, bivalent metal ion dependence, optimum pH, effect of several amino acid residue modifiers, competitive inhibition of nucleotide analogs, and broad nucleoside di‐and triphosphate specificity. The ATPase activity could not be separated from the ADPase either through isoelectrofocusing or electrophoresis under acid conditions.

Contenido de glicosaminoglicanos, aldehídos y proteínas en el líquido sinovial de la articulación metacarpofalángica equina normal y alterada

El objetivo del trabajo fue relacionar la severidad del cuadro osteoartrictico con la concentracion de moleculas de lamatriz extracelular del cartilago en el liquido sinovial. Par ello se midieron las concentraciones de proteina total y de grupos aldehidos como productos de degradacion del colageno. Ademas, se midio la concentracion de glicosaminoglicanos (GAGs) del liquido sinovial en su valor total (GAGs)

Nitric oxide synthase activity in brain tissues from llama fetuses submitted to hypoxemia.

The fetal llama (Lama glama; a species adapted to live in chronic hypoxia in the highlands of the Andes) did not increase cerebral blood flow and reduce the brain oxygen uptake during hypoxemia. Although nitric oxide (NO) is a normal mediator in the regulation of vascular tone and synaptic transmission, NO overproduction by hypoxemia could produce neuronal damage. We hypothesized that nitric oxide synthase (NOS) activity is either maintained or reduced in the central nervous system of the llama fetuses submitted to chronic hypoxemia. Approximately 85% of the Ca(2+)-dependent NOS activity was soluble, at least 12% was associated with the mitochondrial fraction, and less than 5% remains associated with microsomes. To understand the role of NO in chronic hypoxemia, we determined the effect of 24-h hypoxemia on NOS activity in the central nervous system. No changes in activity or the subcellular distribution of NOS activity in brain tissues after hypoxemia were found. We proposed that the lack of changes in NOS activity in the llama under hypoxemia could be a cytoprotective mechanism inherent to the llama, against possible toxic effects of NO.

Characterization of ATP-diphosphohydrolase from rat mammary gland

ATP-diphosphohydrolase (or apyrase) hydrolyses nucleoside di- and triphosphates in the presence of millimolar concentration of divalent cations. It is insensitive towards sulfhydryl and aliphatic hydroxyl-selective reagents and to specific inhibitors of ATPases. We present further evidence that ATPase and ADPase activities present in rat mammary gland correspond to apyrase. Two kinetic approaches have been employed, competition plot and chemical modification with group-selective reagents. The M(r) of these activities was determined by 60Co radiation-inactivation. The kinetic approaches employed, competition plot (which discriminate whether competitive reactions occur at the same site) and chemical modification, point to the presence of a single protein which hydrolyses ATP and ADP. The similar M(r) values of ATPase and ADPase activities also support this proposal. ATPase and ADPase activities of mammary gland show a similar sensitivity or insensitivity towards several chemical modifiers. These results suggest that this enzyme is ATP-diphosphohydrolase, also known as apyrase. The results obtained are compared with the ones obtained by us and other authors with the enzyme isolated from other sources.

Detección de glicosaminoglicanos de la matriz del cartílago articular en el líquido sinovial de carpo equino con fractura en esquirla

La fractura en esquirla del carpo es una patologia frecuente e inhabilitante en los equinos de competencia, constituyendo la mayor causa de invalidez y retiro de ejemplares desde las pistas de carrera. Entre los componentes articulares afectados se encuentra el cartilago articular, formado por una abundante matriz extracelular (MEC) de colageno tipo 11 y de proteoglicanos (Todhunter 1996). Los proteoglicanos son responsables de gran parte de las propiedades fisicoquimicas del tejido (Bayliss y col 1999), en especial el agrecan que es el mas abundante (aproximadamente 90 %) y el de mayor tamano (3 x 106 Da) (Alberts y col 1994, Hedlund y col 1999). El agrecan en el cartilago adulto esta formado por una proteina central a la que se unen covalentemente los glicosarninoglicanos (GAGs): condroitin -6 -sulfato y queratan sulfato que son moleculas polianionicas, cuya repulsion y capacidad para retener agua proporciona al cartilago su propiedad de amortiguar los impactos (McIlwraith 1996). En la MEC del cartilago articular, el agrecan forma estructuras macromoleculares a traves de la union no covalente de hasta 100 unidades de agrecan a una cadena de acido hialuronico (Alberts y col 1994, Tulamo y col 1996), que. Aseguran la retencion del agrecan y por tanto, la de agua dentro de la MEC del cartilago (Maroudas y col 1998). El incremento de la actividad de metaloproteinasas de matriz (MMPs) y de agrecanasas provoca la perdida de moleculas de agrecan y de colageno tipo 11, caracteristico del proceso artritico, afectando inicialmente la superficie articular para luego progresar hacia zonas mas profundas del cartilago (Nagase y Kashiwagi 2003). Por esto, se han desarrollado trabajos en equino cuyo objetivo ha sido detectar biomarcadores que relacionen la concentracion de GAGs totales, queratan sulfato, hidroxiprolina o la actividad de MMPs del liquido sinovial con el dano del cartilago en el proceso osteoartritico (Todhunter y col 1997, McIlwraith 2005, Van den Boom y col 2005). Se ha establecido una relacion entre la severidad del dano del cartilago articular con la actividad de las MMPs y con el contenido de hidroxiprolina en el liquido sinovial, aunque dicha relacion ha sido contradictoria para el caso de los GAGs (Fuller y col 2001, Van den Boom y col 2005). El objetivo de este trabajo fue evaluar la progresion del dano de la MEC del cartilago articular en un proceso de naturaleza inflamatoria, a traves de la concentracion de GAGs distintos del acido hialuronico (GAGsS) en el liquido sinovial de carpo, considerando dos periodos diferentes desde el diagnostico de fractura en esquirla hasta la artroscopia. La presencia del trozo osteocondral en la articulacion generaria un dano mecanico que provocaria la persistencia del proceso inflamatorio y de la actividad de metaloproteinasas, efecto que se podria amplificar si el ejemplar es sometido a trauma fisico. Como hipotesis de trabajo se planteo la relacion directa entre el incremento de GAGsS del liquido sinovial y la persistencia de la fractura en esquirla, lo que demostraria la necesidad de dar reposo a los ejemplares afectados y de realizar a la brevedad la artroscopia con el fin de eliminar el trozo osteocondral.

Glycosaminoglycans (GAGs) determination in healthy and damaged equine articular cartilage

espanolRESUMEN: El proposito de este estudio fue establecer la existencia de alguna diferencia en el contenido de GAG entre las superficies articulares que soportan y no soportan peso, comparando articulaciones metacarpofalangicas equinas macroscopicamente sanas y danadas. Las muestras de cartilago se obtuvieron desde la superficie de apoyo de articulaciones macroscopicamente sanas (N1; n=10) y de aquellas macroscopicamente danadas (P1; n=10). Adicionalmente, en una localizacion dorsoproximal se obtuvieron muestras de la superficie del cartilago sin apoyo, desde articulaciones sanas (N2; n=10) y danadas (P2; n=10). Los GAG fueron extraidos desde 100 mg de cartilago de cada muestra y cuantificados mediante el metodo safranina-O que mide la carga anionica total y por el metodo del carbazol que mide el contenido de acido uronico. Ambos metodos fueron capaces de medir el contenido de GAG no encontrandose diferencias entre zonas intraarticulacion (1 y 2). Al comparar el contenido de GAG entre articulaciones sanas y danadas, ambos metodos evidenciaron una disminucion significativa de GAG en las articulaciones danadas (1 y 2). Estos resultados muestran que en un proceso patologico articular cronico se afecta el cartilago en su conjunto y no solo aquellas zonas que muestran lesiones macroscopicas al soportar mayor impacto mecanico. EnglishABSTRACT: The purpose of this study was to establish if there was any difference in the GAGs content between loaded and unloaded surfaces of the joint. Furthermore, the results were compared between macroscopically healthy and damaged joints. Cartilage samples were obtained from two different zones of the equine metacarpophalangeal joint (metacarpal condyles). Samples were collected from the loaded surface of macroscopically healthy joints (N1; n=10) and from macroscopically damaged cartilage (P1; n=10). Additionally, cartilage samples were collected from unloaded areas at the most dorso-proximal zone of the joint in macroscopically healthy joints (N2; n=10) and from the macroscopically pathological joints but without damaged cartilage on the site of sampling (P2; n=10). The GAGs were extracted from 100 mg of cartilage of each sample and quantified through the safranine - O method that measured the total anionic charges, and through the carbazole method that measured the uronic acid content. Both methods measured the GAGs content, showing no differences between intra-joint zones (1 and 2), but when the GAGs content was compared between healthy and pathological joints, both methods showed a significantly decreased GAGs content in the damaged joints (1 and 2). These results show that the whole articular cartilage could be affected in a chronic pathological process and is not only a local process occurring in the macroscopically damaged cartilage associated with the loaded area.

Actividad glutatión S-transferasa en la membrana sinovial de la articulación metacarpofalángica equina normal y alterada

Resumen es: La glutation S-transferasa (GST) se ha descrito en las fracciones citosolica y microsomal de celulas de diferentes tejidos, en los que cataliza la co...

Efecto de la edad sobre el tamaño de los proteoglicanos del cartílago articular equino

SUMMARY The aim of this study was to evaluate the effect of age on proteoglycans size in equine articular cartilage. Cartilage samples were obtained from metacarpophalangeal joints of crossbred equines inmediatelly after slaughter, and alloted in three groups according to the equine’s age: 1.5-2 years (n = 7); 4-5 years (n = 6) and over 10 years (n = 4). The cartilage extracts (100 mg /pair joints) were pooled for each age group and fractionated by exclution and ion exchange gel chromatography. Proteoglycan content of each fraction was determined by colorimetric reaction with safranine-O. The elution profile of Sepharose CL-2B, showed that proteoglycans appear as an polydisperse population of different size and similar molecular mass per age group. The size of proteoglycans decreased with age. A similar result was obtained measuring molecular mass and hydrodynamic radio values of proteoglycans when maximal absorbance values of each age group were used. These results show an inverse relationship between age and proteoglycans size of equine articular cartilage, which could affect the cartilage ability to undergo reversible compression.

ATP-Diphosphohydrolase Activity from Mammary Gland

ATP-diphosphohydrolase (EC 3.6.1.5), also known as apyrase, has been found in rat mammary gland and several other animal tissuesanimal tissues1,2,3,4. The main kinetic characteristics of this enzyme are the broad specificity towards nucleoside di- and triphosphates and the dependence of either Ca2+ or Mg2+. These characteristics differentiate apyrase from the Ca2+transport ATPase and the Golgi UDPase described in mammary gland1,4. The Ca2+ transport enzyme isolated from plasma membrane of mammary carcinoma cells requires Mg2+ in addition to micromolar concentrations of Ca2+, it is calmodulin dependent and has a Mr of 150 kDa by SDS/PAGE5. The UDPase activity6, considered a Golgi apparatus marker, is specific for UDP, GDP and IDP and the optimum pH (below 7.0) is lower than the optimum for apyrase from animal tissues1. We have proposed that the function of this plasma membrane enzyme in mammary gland might be related to the extracellular nucleotide metabolism7. The action of apyrase, which removes Pi from ATP and ADP, and 5′-nucleotidase which finally produces adenosine, could play a role in the scavenging of purine ring ensuring its reincorporation into the cell. Furthermore this enzyme might have an important function in the modulation of extracellular ATP-induced changes of intracellular calcium activity, mediating a P2-type purinergic receptor, recently described in mammary gland epithelial cells8. If these proposed functions are correct, apyrase location in the plasma membrane should be as an ecto-enzyme. It has been shown by Carraway et al.9 in mammary gland and its tumours the presence of an ecto-ATPase activity stimulated by either Mg2+ or Ca2+.