RESUMEN
Great hope is set in the use of mesenchymal stem cells for gene therapy and regenerative medicine. Since the frequency of this subpopulation of stem cells in bone marrow is low, mesenchymal stem cells are expanded ex vivo and manipulated prior to experimental or clinical use. Different methods for isolation and expansion are available, but the particular effect on the stem cell character is unclear. While the isolation of mesenchymal stem cells by density centrifugation followed by selection of the plastic adherent fraction is frequently used, the composition of expansion media differs. Thus, in the present study we cultured mesenchymal stem cells isolated from five healthy young volunteers in three widely used expansion media and performed a detailed analysis of the effect on morphology, proliferation, clonogenicity, passaging, differentiation and senescence. By this way we clearly show that the type of expansion medium used determines the stem cell character and time of senescence which is critical for future gene therapeutic and regenerative approaches using mesenchymal stem cells.
Asunto(s)
Proliferación Celular/efectos de los fármacos , Medios de Cultivo/química , Células Madre Mesenquimatosas/citología , Adulto , Diferenciación Celular , Células Cultivadas , Femenino , Terapia Genética , Humanos , Masculino , Trasplante de Células Madre Mesenquimatosas , Adulto JovenRESUMEN
The neuronal adaptor protein Fe65 is involved in brain development, Alzheimer disease amyloid precursor protein (APP) signaling, and proteolytic processing of APP. It contains three protein-protein interaction domains, one WW domain, and a unique tandem array of phosphotyrosine-binding (PTB) domains. The N-terminal PTB domain (Fe65-PTB1) was shown to interact with a variety of proteins, including the low density lipoprotein receptor-related protein (LRP-1), the ApoEr2 receptor, and the histone acetyltransferase Tip60. We have determined the crystal structures of human Fe65-PTB1 in its apo- and in a phosphate-bound form at 2.2 and 2.7A resolution, respectively. The overall fold shows a PTB-typical pleckstrin homology domain superfold. Although Fe65-PTB1 has been classified on an evolutionary basis as a Dab-like PTB domain, it contains attributes of other PTB domain subfamilies. The phosphotyrosine-binding pocket resembles IRS-like PTB domains, and the bound phosphate occupies the binding site of the phosphotyrosine (Tyr(P)) within the canonical NPXpY recognition motif. In addition Fe65-PTB1 contains a loop insertion between helix alpha2 and strand beta2(alpha2/beta2 loop) similar to members of the Shc-like PTB domain subfamily. The structural comparison with the Dab1-PTB domain reveals a putative phospholipid-binding site opposite the peptide binding pocket. We suggest Fe65-PTB1 to interact with its target proteins involved in translocation and signaling of APP in a phosphorylation-dependent manner.
Asunto(s)
Proteínas del Tejido Nervioso/química , Neuronas/metabolismo , Proteínas Nucleares/química , Fosfotirosina/química , Secuencia de Aminoácidos , Cristalografía por Rayos X , Evolución Molecular , Humanos , Conformación Molecular , Datos de Secuencia Molecular , Fosforilación , Pliegue de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Homología de Secuencia de Aminoácido , Transducción de SeñalRESUMEN
BACKGROUND AND OBJECTIVE: Experimental data have demonstrated controversial results regarding loop diuretics and their influence on the pulmonary vasculature. The aim of this pilot study was to compare the effect of torasemide versus furosemide on systemic and pulmonary haemodynamics in patients with secondary pulmonary hypertension. METHODS: Twenty-one patients were enrolled in this double-blind, randomized trial: the furosemide group (n = 11) received 40 mg intravenously (IV) and 80 mg orally whereas the torasemide group (n = 10) received 20 mg IV and 20 mg orally. Haemodynamic variables were documented and endothelin-1 and arterial angiotensin-II plasma levels were simultaneously analysed at baseline (T0), 5 minutes after IV administration (T1), at baseline prior to oral administration (T2), and 60 minutes after oral administration (T3). RESULTS: Cardiac output (relative treatment effect over time between groups; p = 0.03) increased significantly in the torasemide group compared with the furosemide group. In the furosemide group, a significant increase in arterial angiotensin-II (AT-II) plasma levels was observed compared with the torasemide group (relative treatment effect over time between groups; p = 0.031). CONCLUSION: Torasemide increased cardiac output (relative treatment effect over the time), whereas treatment with furosemide significantly increased arterial AT-II plasma levels. A possible explanation for these findings might be activation of the renin-angiotensin system by furosemide. However, the underlying pathomechanism remains to be established and evidence from an adequately powered trial is needed to determine if furosemide aggravates cardiac function by increasing AT-II plasma levels.