Biodegradable Synthetic Organelles Demonstrate ROS Shielding in Human-Complex-I-Deficient Fibroblasts

Biodegradable, semipermeable nanoreactors that are capable of undergoing cellular integration and, subsequently, function as synthetic organelles represent an exciting therapeutic technology. Polymersomal nanoreactors have been investigated as a suitable candidate for the engineering of such a system, with the chemical versatility and structural robustness required for such a demanding application. Although steps have been taken to demonstrate this capacity, there has yet to be a system presented with biochemically robust data showing therapeutic efficacy in primary human cells. The reason for this shortfall is the absence of essential criteria of the polymersomes tested so far; biodegradability, intrinsic semipermeability, and a biomedically relevant setting. Herein, we present enzyme-loaded, biodegradable poly(ethylene glycol)-block-poly(caprolactone-gradient-trimethylene carbonate) (PEG-PCLgTMC) polymersomal nanoreactors, readily fabricated using the biocompatible direct hydration methodology. Physical characterization of PEG-PCLgTMC polymersomes highlights their semipermeable membrane and ability to shield enzymatic cargo. Surface modification with cell-penetrating peptides (CPPs) directs cellular integration of enzyme-loaded PEG-PCLgTMC nanoreactors in a controlled fashion. Using HEK293T cells and human skin fibroblasts we demonstrate that biocompatible catalase nanoreactors successfully perform as a synthetic organelle, imparting activity-dependent antioxidant (reactive-oxygen-species-shielding, ROS-shielding) capacity to cells. Notably, for the first time, patient-derived human-complex-I-deficient primary fibroblasts are effectively protected against the toxicity of exogenous H2O2 by the action of internalized enzyme-loaded nanoreactors, showcasing this system in a therapeutically relevant context

Mechanisms of signal transduction in the exocrine pancreas

Contains fulltext : mmubn000001_059724102.pdf (publisher's version ) (Open Access)Promotor : J. de PontVII, 126 p

Beta-adrenergic relaxation in mesenteric resistance arteries of spontaneously hypertensive and Wistar-Kyoto rats: the role of precontraction and intracellular Ca2+.

Contains fulltext : 23432___.PDF (publisher's version ) (Open Access

Synchronized Ca2+ signaling by intercellular propagation of Ca2+ action potentials in NRK fibroblasts

Contains fulltext : 24954___.PDF (publisher's version ) (Open Access

Photo-induction and automated quantification of reversible mitochondrial permeability transition pore opening in primary mouse myotubes

Contains fulltext : 136017.pdf (publisher's version ) (Open Access)Opening of the mitochondrial permeability transition pore (mPTP) is involved in various cellular processes including apoptosis induction. Two distinct states of mPTP opening have been identified allowing the transfer of molecules with a molecular weight <1500 Da or <300 Da. The latter state is considered to be reversible and suggested to play a role in normal cell physiology. Here we present a strategy combining live-cell imaging and computer-assisted image processing allowing spatial visualization and quantitative analysis of reversible mPTP openings ("DeltaPsi flickering") in primary mouse myotubes. The latter were stained with the photosensitive cation TMRM, which partitions between the cytosol and mitochondrial matrix as a function of mitochondrial membrane potential (DeltaPsi). Controlled illumination of TMRM-stained primary mouse myotubes induced DeltaPsi flickering in particular parts of the cell ("flickering domains"). A novel quantitative automated analysis was developed and validated to detect and quantify the frequency, size, and location of individual DeltaPsi flickering events in myotubes

Mitochondrial Morphofunction in Mammalian Cells

Contains fulltext : 204156.pdf (publisher's version ) (Open Access)Significance: In addition to their classical role in cellular ATP production, mitochondria are of key relevance in various (patho)physiological mechanisms including second messenger signaling, neuro-transduction, immune responses and death induction. Recent Advances: Within cells, mitochondria are motile and display temporal changes in internal and external structure ("mitochondrial dynamics"). During the last decade, substantial empirical and in silico evidence was presented demonstrating that mitochondrial dynamics impacts on mitochondrial function and vice versa. Critical Issues: However, a comprehensive and quantitative understanding of the bidirectional links between mitochondrial external shape, internal structure and function ("morphofunction") is still lacking. The latter particularly hampers our understanding of the functional properties and behavior of individual mitochondrial within single living cells. Future Directions: In this review we discuss the concept of mitochondrial morphofunction in mammalian cells, primarily using experimental evidence obtained within the last decade. The topic is introduced by briefly presenting the central role of mitochondria in cell physiology and the importance of the mitochondrial electron transport chain (ETC) therein. Next, we summarize in detail how mitochondrial (ultra)structure is controlled and discuss empirical evidence regarding the equivalence of mitochondrial (ultra)structure and function. Finally, we provide a brief summary of how mitochondrial morphofunction can be quantified at the level of single cells and mitochondria, how mitochondrial ultrastructure/volume impacts on mitochondrial bioreactions and intramitochondrial protein diffusion, and how mitochondrial morphofunction can be targeted by small molecules

Differences in uptake, storage and release properties between inositol trisphosphate-sensitive and -intensitive Ca2+ stores in permeabilized pancreatic acinar cells

Contains fulltext : 21807___.PDF (publisher's version ) (Open Access

Calcium oscillations in melanotrope cells of Xenopus laevis are differentially regulated by cAMP-dependent and cAMP-independent mechanisms

Contains fulltext : 23458___.PDF (publisher's version ) (Open Access

Cholecystokinin-stimulated enzyme secretion from dispersed rabbit pancreatic acinar cells : phosphorylation-dependent changes in potency and efficacy

Contains fulltext : 21666___.PDF (publisher's version ) (Open Access

Mitochondrial dynamics in human NADH:ubiquinone oxidoreductase deficiency.

Contains fulltext : 79621.pdf (publisher's version ) (Closed access)Mitochondrial NADH:ubiquinone oxidoreductase or complex I (CI) is a frequently affected enzyme in cases of mitochondrial disorders. However, the cytopathological mechanism of the associated pediatric syndromes is poorly understood. Evidence in the literature suggests a connection between mitochondrial metabolism and morphology. Previous quantitative analysis of mitochondrial structure in cultured fibroblasts of 14 patients revealed that mitochondria were fragmented and/or less branched in patients with severe CI deficiency. These patient cells also displayed greatly increased levels of reactive oxygen species (ROS) and marked aberrations in mitochondrial and cellular Ca(2+)/ATP handling upon hormone stimulation. Here, we discuss the interrelationship between these parameters and demonstrate that the hormone-induced increase in mitochondrial Ca(2+) and ATP concentration, as well as the rate of cytosolic Ca(2+) removal, are not related to mitochondrial length and/or degree of branching, but decrease as a function of the number of mitochondria per cell. This suggests that the amount of mitochondria, and not their shape, is important for Ca(2+)-induced stimulation of mitochondrial ATP generation to feed cytosolic ATP-demanding processes