Rab3D is critical for secretory granule maturation in PC12 cells.

Neuropeptide- and hormone-containing secretory granules (SGs) are synthesized at the trans-Golgi network (TGN) as immature secretory granules (ISGs) and complete their maturation in the F-actin-rich cell cortex. This maturation process is characterized by acidification-dependent processing of cargo proteins, condensation of the SG matrix and removal of membrane and proteins not destined to mature secretory granules (MSGs). Here we addressed a potential role of Rab3 isoforms in these maturation steps by expressing their nucleotide-binding deficient mutants in PC12 cells. Our data show that the presence of Rab3D(N135I) decreases the restriction of maturing SGs to the F-actin-rich cell cortex, blocks the removal of the endoprotease furin from SGs and impedes the processing of the luminal SG protein secretogranin II. This strongly suggests that Rab3D is implicated in the subcellular localization and maturation of ISGs

Roles of Myosin Va and Rab3D in Membrane Remodeling of Immature Secretory Granules

Neuroendocrine secretory granules (SGs) are formed at the trans-Golgi network (TGN) as immature intermediates. In PC12 cells, these immature SGs (ISGs) are transported within seconds to the cell cortex, where they move along actin filaments and complete maturation. This maturation process comprises acidification-dependent processing of cargo proteins, condensation of the SG matrix, and removal of membrane and proteins not destined to mature SGs (MSGs) into ISG-derived vesicles (IDVs). We investigated the roles of myosin Va and Rab3 isoforms in the maturation of ISGs in neuroendocrine PC12 cells. The expression of dominant-negative mutants of myosin Va or Rab3D blocked the removal of the endoprotease furin from ISGs. Furthermore, expression of mutant Rab3D, but not of mutant myosin Va, impaired cargo processing of SGs. In conclusion, our data suggest an implication of myosin Va and Rab3D in the maturation of SGs where they participate in overlapping but not identical tasks

Discovery and progress in our understanding of the regulated secretory pathway in neuroendocrine cells

In this review we start with a historical perspective beginning with the early morphological work done almost 50 years ago. The importance of these pioneering studies is underscored by our brief summary of the key questions addressed by subsequent research into the mechanism of secretion. We then highlight important advances in our understanding of the formation and maturation of neuroendocrine secretory granules, first using in vitro reconstitution systems, then most recently biochemical approaches, and finally genetic manipulations in vitro and in vivo

Novel electrohydrodynamic preparation of porous chitosan particles for drug delivery.

Uniform spherical chitosan particles of size <10 microm in diameter are important in drug delivery applications due to their excellent biocompability and biodegradability. A high concentration of chitosan in the particles can help to control the release of drugs and methods for processing high viscosity chitosan solutions are therefore required. In principle, any type of polymer, whether hydrophobic or hydrophilic, can be electrosprayed to obtain monodisperse particles of diameter <10 microm. In practice, however, electrospraying of biopolymers having viscosities of >100 mPa s results in particles >10 microm diameter. In this study, by reducing surface tension of a high viscosity chitosan suspension, it was found that smaller diameter particles could be prepared. Chitosan solutions were electrosprayed in the stable cone-jet mode to systematically study the relationship between particle diameter, viscosity and surface tension. Increasing viscosity resulted in larger diameter particles with a broad size distribution, but decreasing surface tension had the opposite effect. Results show that a chitosan solution having a viscosity of approximately 80 mPa s can be used to prepare chitosan particles of diameter approximately 2.5 microm which on drying reduced to particles of 500 nm

Novel electrohydrodynamic preparation of porous chitosan particles for drug delivery.

Uniform spherical chitosan particles of size 100 mPa s results in particles >10 microm diameter. In this study, by reducing surface tension of a high viscosity chitosan suspension, it was found that smaller diameter particles could be prepared. Chitosan solutions were electrosprayed in the stable cone-jet mode to systematically study the relationship between particle diameter, viscosity and surface tension. Increasing viscosity resulted in larger diameter particles with a broad size distribution, but decreasing surface tension had the opposite effect. Results show that a chitosan solution having a viscosity of approximately 80 mPa s can be used to prepare chitosan particles of diameter approximately 2.5 microm which on drying reduced to particles of 500 nm

Proteomic profiling of the brain of mice with experimental cerebral malaria

Cerebral malaria (CM) is a severe neurological complication of malaria infection in both adults and children. In pursuit of effective treatment of CM, clinical studies, postmortem analysis and animal models have been employed to understand the pathology and identify effective interventions. In this study, a shotgun proteomics analysis was conducted to profile the proteomic signature of the brain tissue of mice with experimental cerebral malaria (ECM) in order to further understand the underlying pathology. To identify CM-associated response, proteomic signatures of the brains of C57/Bl6N mice infected with P. berghei ANKA that developed neurological syndrome were compared to those of mice infected with P. berghei NK65 that developed equally high parasite burdens without neurological signs, and to those of non-infected mice. The results show that the CM-associated response in mice that developed neurological signs comprise mainly acute-phase reaction and coagulation cascade activation, and indicate the leakage of plasma proteins into the brain parenchyma.Cerebral malaria (CM) remains a major cause of death in children. The majority of these deaths occur in sub-Saharan Africa. Even with adequate access to treatment, mortality remains high and neurological sequelae can be found in up to 20% of survivors. No adjuvant treatment to date has been shown to reduce mortality and the pathophysiology of CM is largely unknown. Experimental cerebral malaria (ECM) is a well-established model that may contribute to identify and test druggable targets. In this study we have identified the disruption of the blood-brain barrier following inflammatory and vascular injury as a mechanism of disease. In this study we report a number of proteins that could be validated as potential biomarkers of ECM. Further studies, will be required to validate the clinical relevance of these biomarkers in human CM