Fusion pore regulation in peptidergic vesicles

Regulated exocytosis, which involves fusion of secretory vesicles with the plasma membrane, is an important mode of communication between cells. In this process, signalling molecules that are stored in secretory vesicles are released into the extracellular space. During the initial stage of fusion, the interior of the vesicle is connected to the exterior of the cell with a narrow, channel-like structure: the fusion pore. It was long believed that the fusion pore is a short-lived intermediate state leading irreversibly to fusion pore dilation. However, recent results show that the diameter of the fusion pore can fluctuate, suggesting that the fusion pore is a subject of stabilization. A possible mechanism is addressed in this article, involving the local anisotropicity of membrane constituents that can stabilize the fusion pore. The molecular nature of such a stable fusion pore to predict how interacting molecules (proteins and/or lipids) mediate changes that affect the stability of the fusion pore and exocytosis is also considered. The fusion pore likely attains stability via multiple mechanisms, which include the shape of the lipid and protein membrane constituents and the interactions between them.     

Microbial 16S rRNA Ion Tag and community metagenome sequencing using the Ion Torrent (PGM) Platform

Here we demonstrate a cost effective and scalable microbial ecology sequencing platform using the Ion Torrent Personal Genome Machine (PGM). We assessed both PCR amplified 16S rRNA and shotgun metagenomic approaches and generated 100,000+ to 1,000,000+ reads using 'post-light' based sequencing technology within different sized semi-conductor chips. Further development of Golay barcoded Ion Tags allowed multiplex analyses of microbial communities with substantially reduced costs compared with platforms such as 454/GS-FLX. Using these protocols we assessed the bacterial and archaeal dynamics within covered anaerobic digesters used to treat piggery wastes. Analysis of these sequence data showed that these novel methanogenic waste treatment systems are dominated by bacterial taxa, in particular Clostridium, Synergistia and Bacteroides that were maintained as a stable community over extended time periods. Archaeal community dynamics were more stochastic with the key methanogenic taxa more difficult to resolve, principally due to the poor congruence seen between community structures generated either by nested PCR or metagenomic approaches for archaeal analyses. Our results show that for microbial community structure and function analyses, the PGM platform provides a low cost, scalable and high throughput solution for both Tag sequencing and metagenomic analyses.     

Teaching an old dogma new tricks: twenty years of Shc adaptor signalling

Shc (Src homology and collagen homology) proteins are considered prototypical signalling adaptors in mammalian cells. Consisting of four unique members, ShcA, B, C and D, and multiple splice isoforms, the family is represented in nearly every cell type in the body, where it engages in an array of fundamental processes to transduce environmental stimuli. Two decades of investigation have begun to illuminate the mechanisms of the flagship ShcA protein, whereas much remains to be learned about the newest discovery, ShcD. It is clear, however, that the distinctive modular architecture of Shc proteins, their promiscuous phosphotyrosine-based interactions with a multitude of membrane receptors, involvement in central cascades including MAPK (mitogen-activated protein kinase) and Akt, and unconventional contributions to oxidative stress and apoptosis all require intricate regulation, and underlie diverse physiological function. From early cardiovascular development and neuronal differentiation to lifespan determination and tumorigenesis, Shc adaptors have proven to be more ubiquitous, versatile and dynamic than their structures alone suggest.     

The smell of cooperation: rats increase helpful behaviour when receiving odour cues of a conspecific performing a cooperative task

Reciprocity can explain cooperative behaviour among non-kin, where individuals help others depending on their experience in previous interactions. Norway rats (Rattus norvegicus) cooperate reciprocally according to direct and generalized reciprocity. In a sequence of four consecutive experiments, we show that odour cues from a cooperating conspecific are sufficient to induce the altruistic help of rats in a food-exchange task. When rats were enabled to help a non-cooperative partner while receiving olfactory information from a rat helping a conspecific in a different room, they helped their non-cooperative partner as if it was a cooperative one. We further show that the cues inducing altruistic behaviour are released during the act of cooperation and do not depend on the identity of the cue provider. Remarkably, olfactory cues seem to be more important for cooperation decisions than experiencing a cooperative act per se. This suggests that rats may signal their cooperation propensity to social partners, which increases their chances to receive help in return.     

Copper-1,10-Phenanthroline-Induced Apoptosis in Liver Carcinoma Bel-7402 Cells Associates with Copper Overload,Reactive Oxygen Species Production,Glutathione Depletion and Oxidative DNA Damage

The mechanism of cytotoxicity on liver carcinoma Bel-7402 cells induced by copper-1,10-phenanthroline, Cu(OP)₂, has been studied. Cell viability and apoptotic rate were examined in cells treated with Cu(OP)₂ or Cu²⁺ alone. It was found that the apoptosis induced by Cu(OP)₂ could not be induced by Cu²⁺ or OP alone in our experimental conditions. Total copper content in cells was measured by atomic absorption spectrophotometry, and the abnormal elevation of intracellular copper transported by lipophilic OP ligand may play the role of initial factor in the apoptosis, which caused subsequent redox state changes in cells. Intracellular levels of reactive oxygen species (ROS) were detected by fluorescent probe 2′,7′-dichlorofluorescein diacetate (DCFH-DA). Reduced (GSH) and total glutathione (GSSG + GSH) were determined by High-performance liquid chromatography (HPLC) after derivatization, and the ratios of GSH/GSSG were subsequently calculated. The overproduction of ROS and the decreased GSH/GSSG ratio were observed in cells which represented the occurrence of oxidative stress in the apoptosis. Oxidative DNA damage was also found in cells treated with Cu(OP)₂ in the early stage of the apoptosis, and it suggests that the activation of DNA repair system may be involved in the pathway of the apoptosis induced by Cu(OP)₂.     

The first structure of dipeptidyl-peptidase III provides insight into the catalytic mechanism and mode of substrate binding

Dipeptidyl-peptidases III (DPP III) are zinc-dependent enzymes that specifically cleave the first two amino acids from the N terminus of different length peptides. In mammals, DPP III is associated with important physiological functions and is a potential biomarker for certain types of cancer. Here, we present the 1.95-A crystal structure of yeast DPP III representing the prototype for the M49 family of metallopeptidases. It shows a novel fold with two domains forming a wide cleft containing the catalytic metal ion. DPP III exhibits no overall similarity to other metallopeptidases, such as thermolysin and neprilysin, but zinc coordination and catalytically important residues are structurally conserved. Substrate recognition is accomplished by a binding site for the N terminus of the peptide at an appropriate distance from the metal center and by a series of conserved arginine residues anchoring the C termini of different length substrates.     

Basic energetic parameters of Acanthamoeba castellanii mitochondria and their resistance to oxidative stress

The purpose of this study was establishing the basic energetic parameters of amoeba Acanthamoeba castellanii mitochondria respiring with malate and their response to oxidative stress caused by hydrogen peroxide in the presence of Fe(2+) ions. It appeared that, contrary to a previous report (Trocha LK, Stobienia O (2007) Acta Biochim Polon 54: 797), H(2)O(2)-treated mitochondria of A. castellanii did not display any substantial impairment. No marked changes in cytochrome pathway activity were found, as in the presence of an inhibitor of alternative oxidase no effects were observed on the rates of uncoupled and phosphorylating respiration and on coupling parameters. Only in the absence of the alternative oxidase inhibitor, non-phosphorylating respiration progressively decreased with increasing concentration of H(2)O(2), while the coupling parameters (respiratory control ratio and ADP/O ratio) slightly improved, which may indicate some inactivation of the alternative oxidase. Moreover, our results show no change in membrane potential, Ca(2+) uptake and accumulation ability, mitochondrial outer membrane integrity and cytochrome c release for 0.5-25 mM H(2)O(2)-treated versus control (H(2)O(2)-untreated) mitochondria. These results indicate that short (5 min) incubation of A. castellanii mitochondria with H(2)O(2) in the presence of Fe(2+) does not damage their basic energetics.     

Selective delivery of 2-hydroxy APA to Trypanosoma brucei using the melamine motif

Trypanosoma brucei, the parasite that causes human African trypanosomiasis, is auxotrophic for purines and has specialist nucleoside transporters to import these metabolites. In particular, the P2 aminopurine transporter can also selectively accumulate melamine derivatives. In this Letter, we report the coupling of the melamine moiety to 2-hydroxy APA, a potent ornithine decarboxylase inhibitor, with the aim of selectively delivering this compound to the parasite. The best compound described here shows an increased in vitro trypanocidal activity compared with the parent.