Binding protein dependent secondary

Jacobs, M. H., van der Heide, T., Driessen, A. J. and Konings, W. N. (1996). Glutamate transport in Rhodobacter sphaeroides is mediated by a novel binding protein-dependent secondary transport system, Proc. Natl Acad. Sci. USA, 93, 12 786-12 790. 

As with other in vivo selections, it is critical to validate hits at the end of the selection experiment with a secondary screen. The most common secondary screen used with the Y2H assay is a lacZ screen. If either the DBD or AD fusion is under control of an inducible promoter, this screen can be carried out both under inducing and non-inducing conditions to ensure that transcription activation is protein dependent. Y3H systems, where transcription activation depends on a bridging RNA or small molecule, and reverse Y2H systems, where a third protein is disrupting the interaction, provide a built-in control. One can simply check that transcription activation is in fact dependent on the third component. As with any in vivo selection, the evolved plasmid should be isolated and retransformed into a fresh yeast selection strain to ensure that the phenotype is plasmid-dependent. Ultimately, the interaction will be confirmed with co-immunoprecipitation experiments or other in vitro binding assays [39]. 

Injury to the endothelium and exposure of tissue factor on the subendothelial layer to plasma proteins also activate the blood coagulation cascade, which ultimately activates thrombin and Factor XIII Factor XIII cross-links strands of polymerized fibrin monomers to form a stable clot (the secondary hemostatic plug). The blood coagulation cascade consists of a series of enzymes (such as Factor X), which are inactive until proteolytically cleaved by the preceding enzyme in the cascade. Other proteins (Factor V and Factor VIII) serve as binding proteins, which assemble factor complexes at the site of injury. Ca and y-carboxyglutamate residues in the proteins (formed by a vitamin K-dependent process in the liver) attach the factor complexes to phosphoUpids exposed on platelet membranes. Consequently, thrombus formation is rapidly accelerated and localized to the site of injury. 

Retinol is nearly always present in the food in the form of esters which are hydrolysed in the lumen of the intestine. The retinol released is quite readily absorbed into the mucosal cells where it is re-esterified, chiefly with palmitic acid. The retinyl esters are then transported via the lymphatic system into the portal circulation from which they are removed and stored in the liver. Release of the vitamin from the liver depends on the production by the liver of a special retinolbinding protein (RBP). Production of the retinol-binding protein may be disturbed in diseases of the liver or kidneys or in protein/energy malnutrition. In such circumstances retinol cannot be mobilized from the stores and a secondary deficiency may result. Thus it can be seen that the level of retinol in the general circulation is normally highly regulated and is more or less independent of the body s reserves. 

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