ABSTRACT
Con el objetivo de aislar y caracterizar parcialmente las enzimas ribonucleasas (RNasas) contenidas en el látex de Calotropis procera y Pedilanthus tithymaloides, se colectaron muestras de plantas adultas. Las proteínas solubles fueron extraídas con acetato de sodio y centrifugación a 16.000 x g durante 15 min y fraccionadas por cromatografía de intercambio iónico. Se estimó la masa molecular a través de ecuaciones de regresión lineal. Se realizaron pruebas de glicosilación. En ambas especies, las proteínas con actividad RNasa presentaron una masa molecular entre 28 y 30 kDa. No existe evidencia de proteínas glicosiladas en el látex de C. procera. En P. tithymaloides la RNasa es una proteína glicosilada.
In order to isolate and characterize partially ribonucleases (RNases) enzymes contained in the latex from Calotropis procera and Pedilanthus tithymaloides, samples were collected from mature plants. Soluble proteins were extracted with sodium acetate and centrifugation at 16,000 xg for 15 min and fractionated by ion exchange chromatography. Molecular mass was estimated by linear regression equations. Glycosylation tests were conducted. In both species, proteins with RNase activity showed a molecular mass between 28 and 30 kDa. No evidence of glycosylated proteins in latex from C. procera. In P. tithymaloides, RNase may be a glycosylated protein.
Subject(s)
Calotropis/enzymology , Euphorbiaceae/enzymology , Latex/chemistry , Ribonucleases/isolation & purification , Ribonucleases/metabolism , Calotropis/chemistry , Euphorbiaceae/chemistry , GlycosylationABSTRACT
PURPOSE: To obtain a decellularized tracheal scaffold associating traditional approaches with the novel light-emitting diode (LED) proposal. METHODS: This study was performed with New Zealand adult rabbits weighing 3.0 - 4.0 kg. Different protocols (22) were used combining physical (agitation and LED irradiation), chemical (SDS and Triton X-100 detergents), and enzymatic methods (DNase and RNase). RESULTS: Generally, the cells surrounding soft tissues were successfully removed, but none protocol removed cells from the tracheal cartilage. However, longer protocols were more effective. The cost-benefits relation of the enzymatic processes was not favorable. It was possible to find out that the cartilaginous tissue submitted to the irradiation with LED 630nm and 475 nm showed an increased number of gaps without cells, but several cells were observed to be still present. CONCLUSION: The light-emitting diode is a promising tool for decellularization of soft tissues. .
Subject(s)
Animals , Rabbits , Light , Tissue Scaffolds , Tissue Engineering/methods , Trachea/ultrastructure , Deoxyribonucleases/metabolism , Detergents/pharmacology , Extracellular Matrix/ultrastructure , Ribonucleases/metabolism , Trachea/drug effects , Trachea/enzymologyABSTRACT
The traditional light microscopy has limitations for precise growth assays of malaria parasites in culture or for assessment of new compounds for antimalarial activity; the speed and high reproducibility of flow cytometry can overcome these limitations. A flow cytometric method using PicoGreen, a DNA-binding fluorochrome, was developed with optimal precision suitable for performing growth assays of low-parasitemia field isolates. In addition, intra- and inter-person reproducibility of the flow cytometric and the microscopic method were compared in order to quantitatively demonstrate the improved precision. RNase treatment contributed to the precision of the flow cytometric measurements by enhancing the signal-to-noise ratios. Coefficients of variation of the method were smaller than 10% for 0.1% or higher parasitemia samples. The intra- and inter-person coefficients of variation of the flow cytometric method were three to six times smaller than those of the microscopic method. The flow cytometric method developed in this study yielded substantially more precise results than the microscopic method, allowing determination of parasitemia levels of 0.1% or higher, with coefficients of variation smaller than 10%. Thus, the PicoGreen method could be a reliable high sensitivity assay for analysis of low parasitemia samples and might be applied to a high throughput system testing antimalarial drug activity.
Subject(s)
Humans , Flow Cytometry , Fluorescent Dyes/chemistry , Microscopy , Organic Chemicals/chemistry , Parasitemia/diagnosis , Plasmodium falciparum/isolation & purification , Reproducibility of Results , Ribonucleases/metabolism , Signal-To-Noise RatioABSTRACT
In the mid-eighties of the last century, extracellular-proteolipid complexes have been identified in tumor patients and circulating RNA was suggested to represent a specific secretory product of cancer cells. The presence of specific types of RNA in a variety of cancer types proved to be useful in cancer diagnosis. It has been suggested that extracellular RNA and DNA are not inert molecules, but contain biological activities. Recent data have demonstrated that extracellular RNA is likely to present the up to now undefined “natural foreign surface”, serving as an initiating factor in blood coagulation in vivo. Yet, extracellular RNA seems to have even more functions. Investigations on blood-brain-barrier have shown that extracellular RNA mediates endothelial permeability. Ample success has been achieved in administrating RNase in different animal models of vascular diseases, thereby significantly delaying thrombus formation and reducing cerebral edema formation with neuroprotection in acute stroke models. Furthermore, extracellular mammalian RNA was found to decrease tumor yield in a murine model system, suggesting that extracellular RNA might trigger immune response. Finally, extracellular nucleic acids were identified as danger signals involved in innate immunity related to neutrophil-mediated bacterial killing and haemocyte activation and coagulation in the insects. Thus, a new area of research on extracellular RNA functions with promising future perspectives just started in the field of inflammation and immunity.
Subject(s)
Animals , Blood Coagulation , Extracellular Space/enzymology , Extracellular Space/metabolism , Humans , Immunity, Innate , Inflammation/blood , Inflammation/enzymology , Inflammation/immunology , Inflammation/pathology , RNA/metabolism , Ribonucleases/metabolismABSTRACT
The brain is particularly vulnerable to oxygen free radicals, and these radicals have been implicated in the pathology of several neurological disorders. In this study, the modulation of TNF-related apoptosis-inducing ligand (TRAIL) expression by oxidative stress was shown in LN215 cells, an astroglioma cell line. Hydrogen peroxide (H2O2) treatment increased TRAIL expression in LN215 cells and H2O2-induced TRAIL augmented apoptosis in Peer cells, a cell line sensitive to TRAIL- mediated cell death. Our findings suggest that the upregulation of TRAIL in astroglial cells may abrogate immune cell effector functions.
Subject(s)
Humans , Up-Regulation , TNF-Related Apoptosis-Inducing Ligand/biosynthesis , T-Lymphocytes/metabolism , Ribonucleases/metabolism , Oxidative Stress , Immunosuppressive Agents/pharmacology , Hydrogen Peroxide/pharmacology , Gene Expression Regulation, Neoplastic , Cyclosporine/pharmacology , Cell Line, Tumor , Astrocytes/metabolism , Apoptosis , Hypoxia , Allergy and ImmunologyABSTRACT
En países subdesarrollados la mayoría de la población está expuesta a la deficiencia proteínica en la alimentación y consecuentemente al síndrome de prekwashiorkor, acompañado de bajas defensas del organismo. En estas condiciones aumenta la sensibilidad del humano a los agentes ambientales, cuyo número crece día a día y plantea un serio problema de salud, fundamentando la búsqueda de la fórmula alimentaria ópitma como medida protectora. El propósito de este trabajo es corroborar si la proteína de la alimentación puede fungir como antídoto de la acción tóxica de la tioacetamida que recae sobre el hígado y, al igual que la inanición proteínica, afecta la estructura y las funciones de los hepatocitos. El estudio se realizó a nivel subcelular (lisosomas) en aspectos enzimático (enzimas marcadoras-fosfatasa ácida, ribonucleasa ácida y catepsina) y morfológico. Se encontró: la tiocetamida en condiciones de aporte normal proteínico (18,5%) en la dieta provoca cambios moderados característicos (necrosis centrolobular, alteración del patrón enzimático y de la permeabilidad de membranas lisosomales). En deficiencia proteínica (4%) la afección es mucho más severa histológica y enzimáticamente (necrosis extensa, infiltración periportal grasa, alteración del patrón enzimático, permeabilidad de membranas lisosomal y celular). En condiciones de alimentación rica en proteína (44.5%) los cambios histológicos son discretos, sin afección enzimática ni estructural. Se concluye: el aporte elevado de proteína en la dieta surte en ratas efecto de antídoto de la acción hepatotóxica de la tioacetamida, hecho que plantea la posible utilidad de la proteína dietética en la protección del organismo contra la contaminación ambiental