Avtor/Urednik		Gregorc, Uroš
Naslov		Vloga membransko vezanih gvanilatnih kinaz v apoptozi
Tip		monografija
Kraj izdaje		Ljubljana
Založnik		Medicinska fakulteta
Leto izdaje		2002
Obseg		str. 58
Jezik		slo
Abstrakt		A group of proteolytic enzymes, caspases, is activated during apoptosis. They in turn cleave different cellular proteins. Activation of caspases and cleavages of their substrates lead to biochemical and morphological changes in apoptotic cells. Membrane associated guanylate kinases (MAGUK), proteins involved in cell-cell connections, are also cleaved by caspases. These cleavages allegedly induce disruption of cell-cell interaction complexes, isolation of apoptotic cells and their death without any consequence for surrounding tissue. For our studies we have choosen four members of MAGUK - two memebers of DLG family (DLG1 and DLG4/PSD-95) and MAGI family (MAGI-2 and -3). Initial studies were performed on in vitro translated, 35S-Met radiolabelled, proteins. We have inserted cDNA for selected MAGUK in mammalian expression vector pcDNA3 (Invitrogen) and translated them with TNT® T7 Coupled Reticulocyte Lysate System (Promega). We have prepared recombinant caspases-3, -6, -7 and -8, and performed cleavage experiments on selected MAGUK. They are all cleaved by caspase-3. While DLG1, MAGI-2 and -3 are cleaved at physiologically significant concentration of caspase-3, DLG4/PSD-95 is cleaved only at high caspase-3 concentrations. No other caspase cleaved any of the investigated MAGUK. We have selected a line of spontaneously transformed human keratinocytes (HaCaT) for in vivo studies. These are epithelial cells and when grown in culture they form monolayers. UV irradiation and staurosporine was used for induction of apoptosis. Progression of apoptosis was monitored by measuring caspase activity in cellular lysates and by PARP (poly (ADP-ribose) polymerase) cleavage. DLG1 cleavage was assayed using Western blots and detection with specific monoclonal antobodies. Caspase activity increased with time as well as amount of cleaved PARP and DLG1. (Abstract truncated at 2000 characters).
Deskriptorji		APOPTOSIS INTERCELLULAR JUNCTIONS PHOSPHOTRANSFERASES CYSTEINE PROTEINASES RETICULOCYTES TRANSLATION, GENETIC RECOMBINANT PROTEINS KERATINOCYTES ULTRAVIOLET RAYS STAUROSPORINE NAD+ ADP-RIBOSYLTRANSFERASE BLOTTING, WESTERN ANTIBODIES, MONOCLONAL