Vortex-Induced Formation of Insulin Amyloid Superstructures Probed by Time-Lapse Atomic Force Microscopy and Circular Dichroism Spectroscopy

Misfolding and aggregation of proteins are multipathway processes that result in polymorphism of amyloid fibrils. While agitation is one of the most common means of inducing structural variants of fibrils (the so-called ‘amyloid strains’), there is as yet no mechanistic explanation for this effect. In this study, time-lapse atomic force microscopy has been employed to probe insulin fibrillation upon intensive agitation. At 60 °C, the initial stages of aggregation in agitated samples are similar to those in quiescent solutions; however, in vortexed samples, an abrupt and highly cooperative collapse of early filaments occurs, yielding twisted and laterally aligned aggregates with defined chiroptical properties. In the absence of any detectable birefringence and linear dichroism, the observed strong Cotton effect is attributed to twisted chiral amyloid superstructures. Early fibrils formed in agitated samples, but transferred to quiescent conditions before the collapse event, do not form the superstructures. On the other hand, mature insulin fibrils grown in quiescent samples and later subjected to rapid vortexing transform into clumps of finely broken fibers lacking the superstructural chirality and chiroptical properties of the continuously agitated samples. A generalized mechanism of inducing structural variants of amyloid fibrils by hydrodynamic forces favoring secondary nucleation events over elongation of fibrils is put forward. We propose that competition between low-aspect-ratio and high-aspect-ratio amyloidogenic pathways driven by fluid dynamics may play an important role in promoting distinct amyloid strains.     

A metagenomic approach for the identification and cloning of an endoglucanase from rice straw compost

Over the years, culturable cellulase-producing microorganisms have been isolated from a variety of sources and genes of cellulolytic enzymes have been cloned. Then again, the “great plate count anomaly” phenomenon necessitates a culture-independent metagenomic approach for the isolation of cellulolytic genes from microorganisms in their natural environment. We have constructed a metagenomic library derived from rice straw composts. Of 2739 clones screened, a lambda clone carrying a 12 kb genomic fragment was able to yield a clear zone on an agar plate containing carboxymethyl cellulose (CMC). A 4.7 kb subclone, generated by restriction enzyme digestion, was found to harbor a GH12 cellulase gene, RSC-EG1, encoding 464 amino acids along with two other ORFs. The identified cellulolytic gene showed more than 70% similarity on the amino acid level with cellulase from Micromonospora aurantiaca and Thermobispora sp. Interestingly, RSC-EG1 contains a stretch of approximately 86 amino acids not present in either of these close relatives. Our results demonstrated that RSC-EG1, stable over a wide temperature and pH range, is a novel endoglucanase, and provided the first example of metagenomics approach to isolate cellulolytic gene from rice straw composts.     

Tetramethylphenylenediamine-induced hepatocyte cytotoxicity caused by lysosomal labilisation and redox cycling with oxygen activation

It has already been reported that in vivo muscle necrosis induced by various phenylenediamine derivatives correlated with their in vitro autoxidation rate [9]. Now in a more detailed investigation of the cytotoxic mechanism of a ring-methylated phenylenediamine known as tetramethylphenylenediamine or durenediamine (DD) towards isolated rat hepatocytes has been carried out. Cytotoxicity was preceded by ROS formation which was markedly increased by inactivating DT-diaphorase or catalase but were prevented by a subtoxic concentration of the mitochondrial respiratory inhibitor cyanide. This suggests that ROS generation could be attributed to a futile two-electron redox cycle involving oxidation of phenylenediamine to the corresponding diimine by the mitochondrial electron transfer chain and re-reduction by the DT-diaphorase. Endocytosis inhibitors, lysosomotropic agents or lysosomal protease inhibitors also prevented DD-induced cytotoxicity suggesting that DD-induced ROS caused lysosomal damage and protease activation in hepatocytes. Furthermore preincubation with deferoxamine (a ferric iron chelator) or addition of antioxidants, catalase or ROS scavengers (mannitol, tempol or dimethylsulfoxide) prevented DD cytotoxicity. These results suggest that H(2)O(2) reacts with lysosomal Fe(2+) to form "ROS" which causes lysosomal lipid peroxidation, membrane disruption, protease release and cell death.