Structural biology of the macroautophagy machinery

Macroautophagy is a conserved degradative process mediated through formation of a unique double- membrane structure, the autophagosome. The discovery of autophagy-related （Atg） genes required for autophagosome formation has led to the characterization of approximately 20 genes mediating this process. Recent structural studies of the Atg proteins have provided the molecular basis for their function. Here we summarize the recent progress in elucidating the structural basis for autophagosome formation.     

Detergent sclerosants at sub-lytic concentrations induce endothelial cell apoptosis through a caspase dependent pathway

To investigate the apoptotic effects of detergent sclerosants sodium tetradecylsulphate (STS) and polidocanol (POL) on endothelial cells at sub-lytic concentrations. Human umbilical vein endothelial cells (HUVECs) were isolated and labelled with antibodies to assess for apoptosis and examined with confocal microscopy and flow cytometry. Isolated HUVECs viability was assessed using propidium iodide staining. Early apoptosis was determined by increased phosphatidylserine exposure by lactadherin binding. Caspase 3, 8, 9 and Bax activation as well as inhibitory assays with Pan Caspase (Z-VAD-FMK) and Bax (BI-6C9) were assessed to identify apoptotic pathways. Porimin activation was used to assess cell membrane permeability. Cell lysis reached almost 100 % with STS at 0.3 % and with POL at 0.6 %. Apoptosis was seen with both STS and POL at concentrations ranging from 0.075 to 0.15 %. PS exposure increased with both STS and POL and exhibited a dose-dependent trend. Active Caspase 3, 8 and 9 but not Bax were increased in HUVECs stimulated with low concentrations of both STS and POL. Inhibitory assays demonstrated Caspase 3, 8, 9 inhibition at low concentrations (0.075 to 0.6 %) with both STS and POL. Both agents increased the activation of porimin at all concentrations. Both sclerosants induced endothelial cell (EC) apoptosis at sub-lytic concentrations through a caspase-dependant pathway. Both agents induced EC oncosis.     

Light induces an increase in the pH of and a decrease in the ammonia concentration in the extrapallial fluid of the giant clam Tridacna squamosa

The objective of this study was to examine whether 12 h of light exposure would lead to an increase in the pH of and a decrease in the concentration of total ammonia in the extrapallial fluid of the giant clam Tridacna squamosa. We also aimed to elucidate indirectly whether movements of ammonia and/or protons (H(+)) occurred between the extrapallial fluid and the outer mantle epithelium. The pH of the extrapallial fluid of T. squamosa exposed to 12 h of light was significantly higher than that of clams exposed to 12 h of darkness. Conversely, the total ammonia concentration in the extrapallial fluid of the former was significantly lower than that of the latter. In addition, the glutamine content in the mantle adjacent to the extrapallial fluid of clams exposed to 12 h of light was significantly greater than that of clams exposed to 12 h of darkness. These results suggest that in the extrapallial fluid of T. squamosa exposed to light, NH(3) combined with H(+) as NH(+)(4) and that NH(+)(4) was transported into the mantle and used as a substrate for glutamine formation. Injection of NH(4)Cl into the extrapallial fluid led to an instantaneous increase in the total ammonia concentration therein, but the total ammonia concentration decreased subsequently and returned to the control value within 1 h. This is in support of the proposition that NH(+)(4) could be transported from the extrapallial fluid to the mantle. Injection of HCl into the extrapallial fluid led to an instantaneous decrease in the pH of the extrapallial fluid. However, there was a significant increase in pH within 1 h in light or darkness, achieving a partial recovery toward the control pH value. The increase in pH within this 1-h period in light or darkness was accompanied by a significant decrease in the total ammonia concentration in the extrapallial fluid, which supports the proposition that H(+) could be transported in combination with NH(3) as NH(+)(4). Therefore, our results prompt a reexamination of the previous proposition that the removal of H(+) by NH(3) can facilitate calcification in molluscs in general and an investigation of the relationship between H(+) removal through NH(+)(4) transport and light-enhanced calcification in T. squamosa.     

The influence of feeding and fasting on plasma metabolites in the dogfish shark (Squalus acanthias)

Dogfish sharks are opportunistic predators, eating large meals at irregular intervals. Here we present a synthesis of data from several previous studies on responses in plasma metabolites after natural feeding and during prolonged fasting (up to 56 days), together with new data on changes in plasma concentrations of amino acids and non-esterified fatty acids. Post-prandial and long-term fasting responses were compared to control sharks fasted for 7 days, a typical inter-meal interval. A feeding frenzy was created in which dogfish were allowed to feed naturally on dead teleosts at two consumed ration levels, 2.6% and 5.5% of body weight. Most responses were more pronounced at the higher ration level. These included increases in urea and TMAO concentrations at 20 h, followed by stability through to 56 days of fasting. Ammonia levels were low and exhibited little short-term response to feeding, but declined to very low values during the extended fast. Glucose and β-hydroxybutyrate both fell after feeding, the latter to a greater and more prolonged extent (up to 60 h), whereas acetoacetate did not change. During prolonged fasting, glucose concentrations were well regulated, but β-hydroxybutyrate increased to 2–3-fold control levels. Total plasma amino acid concentrations increased in a biphasic fashion, with peaks at 6–20 h, and 48–60 h after the meal, followed by homeostasis during the extended fast. Essential and non-essential amino acids generally followed this same pattern, though some exhibited different trends after feeding: taurine, β-alanine, and glycine (decreases or stability), alanine and glutamine (modest prolonged increases), and threonine, serine, asparagine, and valine (much larger short-term increases). Plasma non-esterified fatty acid concentrations declined markedly through 48 h after the 2.6% meal. These data are interpreted in light of companion studies showing elevations in aerobic metabolic rate, urea production, rectal gland function, metabolic base excretion, and activation of ornithine–urea cycle and aerobic enzymes after the meal, and muscle N-depletion but maintenance of osmolality and urea production during long-term fasting.     

Stability and motor adaptation in human arm movements

In control, stability captures the reproducibility of motions and the robustness to environmental and internal perturbations. This paper examines how stability can be evaluated in human movements, and possible mechanisms by which humans ensure stability. First, a measure of stability is introduced, which is simple to apply to human movements and corresponds to Lyapunov exponents. Its application to real data shows that it is able to distinguish effectively between stable and unstable dynamics. A computational model is then used to investigate stability in human arm movements, which takes into account motor output variability and computes the force to perform a task according to an inverse dynamics model. Simulation results suggest that even a large time delay does not affect movement stability as long as the reflex feedback is small relative to muscle elasticity. Simulations are also used to demonstrate that existing learning schemes, using a monotonic antisymmetric update law, cannot compensate for unstable dynamics. An impedance compensation algorithm is introduced to learn unstable dynamics, which produces similar adaptation responses to those found in experiments.     

Identification of p10 as a neurotoxic product generated from the proteolytic cleavage of the neuronal Cdk5 activator

The involvement of cyclin‐dependent kinase‐5 (Cdk5) and p25, the proteolytic fragment of activator p35, has long been implicated in the development of neuron‐fibrillary tangles (NFTs), a hallmark of Alzheimer's disease (AD). Findings in this area over the past decade have been highly controversial and inconclusive. Here we report unprecedented detection of endogenous p10, the smaller proteolytic fragment of the Cdk5 activator p35 in treated primary cortical neurons that underwent significant apoptosis, triggered by proteasome inhibitors MG132 and lactacystin, and protein kinase inhibitor staurosporine (STS). p10 appeared exclusively in the detergent‐resistant fraction made up of nuclear matrix, membrane‐bound organelles, insoluble membrane proteins, and cytoskeletal components. Intriguingly, transient overexpression of p10 in neural cells induced apoptotic morphologies, suggesting that p10 may play an important role in mediating neuronal cell death in neurodegenerative diseases. We demonstrated for the first time that p10‐mediated apoptosis occurred via a caspases‐independent pathway. Furthermore, as p10 may contain the myristoylation signal for p35 which is responsible for binding p35 to several intracellular components and the membrane, all in all these novel results present that the accumulation of p10 to the detergent‐insoluble fraction may be a crucial pathological event to triggering neuronal cell death. J. Cell. Biochem. 111: 1359–1366, 2010. © 2010 Wiley‐Liss, Inc.     

New extensometer to measure in vivo uniaxial mechanical properties of human skin

Biomechanical properties of skin are important for clinical decision making as well as clinical intervention. Measuring these properties in vivo is critical for estimating dimensional behaviour of skin flap or graft after harvest. However, existing methodologies and devices often suffer from lack of standardisation and unwanted peripheral force contribution due to the deformation of surrounding tissues during measurement. This naturally leads to measurement inaccuracies and lack of reproducibility. In order to improve the measurement accuracy, a new portable extensometer, which measures the non-invasive in vivo biomechanical properties of skin, has been designed and constructed. This design incorporates three pads that attach to the skin, including a C-shaped pad to shield the force sensor from peripheral forces. Such design produces data that are significantly closer to in vitro measurements. The results have been verified by finite element analysis, and experiments on rubber sheets and pig skins. This device can be used to obtain biomechanical properties of skin that will aid doctors in measuring skin elasticity and surgical planning, especially in skin flap surgery.     

Non-invasive prediction of skin flap shrinkage: a new concept based on animal experimental evidence

A non-invasive, in vivo method has been developed to predict the skin flap shrinkage (retraction) following a harvest. It involves the use of a novel custom-designed extensometer to measure the force-displacement behaviour of skin and subsequent data analysis to estimate the shrinkage. In validation experiments performed on pigs, this method has been shown to produce results with an average absolute error of 6.0% between the actual and predicted shrinkages. This may be close to what an experienced surgeon would estimate subjectively, thus indicating the potential usefulness of this method to predict flap shrinkage of patient's donor sites.     

A simulation approach to understanding the masticatory process

An analysis of the reduction of food particle sizes during human mastication is presented in terms of the probability of a particle being broken (selection function) and the distribution of fragment sizes produced when it fails (breakage function). Both selection and breakage functions are defined and a general equation produced. Several feasible behaviours for these two variables that have been suggested in the literature are modelled by computer simulation and the results are compared to published breakdown patterns. The conclusions are that selection and breakage functions probably behave very simply with respect to particle size, and that these behaviours could be deduced from an analysis of food particle size distributions and the rate at which particle sizes are reduced per chew.