Ca(2+)-triggered exocytosis is a hallmark of neurosecretory granules, but the cellular pathway leading to the assembly of these regulated exocytotic carriers is poorly understood. Here we used the pituitary AtT-20 cell line to study the biogenesis of regulated exocytotic carriers involved in peptide hormone secretion. We show that immature secretory granules (ISGs) freshly budded from the trans-Golgi network (TGN) exhibit characteristics of unregulated exocytotic carriers. During a subsequent maturation period they undergo an important switch to become regulated exocytotic carriers. We have identified a novel sorting pathway responsible for this transition. The SNARE proteins, VAMP4 and synaptotagmin IV (Syt IV), enter ISGs initially but are sorted away during maturation. Sorting is achieved by vesicle budding from the ISGs, because it can be inhibited by brefeldin A (BFA). Inhibition of this sorting pathway with BFA arrested the maturing granules in a state that responded poorly to stimuli, suggesting that the transition to regulated exocytotic carriers requires the removal of a putative inhibitor. In support of this, we found that overexpression of Syt IV reduced the stimulus-responsiveness of maturing granules. We conclude that secretory granules undergo a switch from unregulated to regulated secretory carriers during biogenesis. The existence of such a switch may provide a mechanism for cells to modulate their secretory activities under different physiological conditions.

] i increase is essential for many cellular functions and is required for the phagocyte Fc␥R-induced oxidative burst (6, 7).Many lines of evidence in both human and murine systems indicate that tyrosine phosphorylation events are critical for phagocyte Fc␥R functions, including phagocytosis (8 -10). In addition, in many systems examined, tyrosine kinase activity is required for the receptor-induced rise in [Ca 2ϩ ] i (presumably through tyrosine phosphorylation of phospholipase C␥1 and generation of inositol 1,4,5-trisphosphate). However, the role of [Ca 2ϩ ] i in Fc␥ receptor-mediated phagocytosis has been controversial. For example, work in murine macrophage cell lines suggests that transients in [Ca 2ϩ ] i are not essential for phagocytosis of antibody-opsonized erythrocytes (EA) (11-13). In contrast, phagocytosis of EA by human neutrophils is significantly impaired by chelation of intracellular calcium and abrogation of [Ca 2ϩ ] i transients (14,15 ] i -dependent or independent fashion (19). These observations, coupled with the recent data of Stendahl and co-workers (20) that [Ca 2ϩ ] i storage organelles accumulate at contact sites during phagocytosis in human neu-

To what extent will KSR limit combination inventions in biotech?

These experiments evaluate overexpression of basic fibroblast growth factor (FGF-2) as a treatment to decrease photoreceptor cell death in the constant light damage model of retinal degeneration. Increased FGF-2 expression was accomplished by gene transfer to the retina via injection into the subretinal space of a recombinant adeno-associated virus (rAAV), containing the FGF-2 gene. Rescue effects were assessed by morphometry and electroretinographic analysis.