Electrophysiological study of the somatic muscle cells of ascaris suum

The electrophysiological properties of muscle cells in the nematode Ascaris suum have been studied extensively (review DeBell 1965). However, details of the ionic mechanisms regulating the spontaneous activity of the somatic muscle cell membrane are still poorly understood. The study described in this thesis, used the patch-clamp technique to examine ion channels in the soma membrane of the muscle cells. In addition, two-electrode voltage-clamp was used to observe the membrane currents of the muscle cell somata.The patch-clamp experiments demonstrated the presence of a high-conductance chloride channel (200pS) spontaneously active at the resting potential. This channel was voltage sensitive. When the patch was depolarized the mean open time of the channel and the probability of opening were both reduced. An additional feature of this voltage sensitivity was the appearance of sub-conductance levels when the patch was depolarized.In patches that contained more than one channel the proportion of time spent with 1,2,3...N channels open was analysed in terms of the binominal distribution. The results indicated that the binomial distribution was not a good approximation to the data. From this analysis it was concluded that either the channels in the same patch did not have the same probability of opening, or that channel openings were not independent of each other.Experiments showed that the probability of chloride channel opening was dependent on the concentration of intracellular calcium. Increasing the concentration of calcium led to an increase in the probability of channel opening. The significance of the calcium sensitivity remains unknown. It was proposed that these channels were responsible for the high resting permeability to chloride.The voltage-clamp experiments demonstrated the presence of two currents activated by membrane depolarization. When the muscle cells were bathed in Ringers containing calcium, depolarization activated an inward current, followed by a large outward current. The inward current increased in amplitude when the calciun concentration of the bathing solution was increased, and was blocked by lanthanizn. The outward current was activated by steps to +55 mV from a holding potential of -35 mV. This current had a steep rise and slow decay and was found to be carried by potassium ions. Depolarizing steps of increased amplitude, increased the outward current amplitude and decreased the time to peak of the current. Experiments were carried out to determine the kinetics of the outward current.Two blockers of potassiun currents were tried in Ascaris, these were TEA and 4-AP. TEA [69 mM] was used in the study of the inward current and blocked most of the outward current. Bath application of 4-AP [5 mM] blocked a fast-transient component of the outward current. The current remaining after 4-AP application had a slow rise time, and a slow decay approximated by a single exponential, with a time constant of 1.1 s. The 4-AP resistant current shewed less steady-state inactivation than the gross outward current. Computer analysis was used to subtract the 4-AP resistant outward current from the gross outward current. The subtracted current represented the current blocked by 4-AP. The decay of the 4-AP blocked current was approximated by a single exponential, with a time constant of 10.4 ms. The function of both of these potassium conductances was thought to be to repolarize the cell after a spike

Molecular control of compound exocytosis: A key role for VAMP8

Exocytosis is the process of fusion of a membrane-bound vesicle with the cell membrane and subsequent release of the vesicle content to the outside. It is now widely accepted that SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) proteins are key components in the molecular machinery of exocytosis. SNARE proteins on the vesicle membrane selectively form complexes with specific SNAREs on the cell membrane. In a variant of exocytosis, called compound exocytosis, secretory vesicles still fuse with the cell membrane but vesicle-to-vesicle fusion enhances secretory output. Two types of compound exocytosis occur, either vesicles fuse with each other and then fuse with the cell membrane, or a vesicle fuses with the cell membrane and then becomes a target for further vesicles to fuse with it. It is expected that SNAREs are important for vesicle-to-vesicle fusion but the mechanism(s) that control these processes is unknown. In our recent paper (Behrendorff et al. 2011) we provide evidence that VAMP8 (a Q-SNARE) is essential in regulating compound exocytosis. Here we discuss the implications of our findings with reference to a new model for the control of vesicle-to-vesicle fusion

Ami-The Chemist's Amanuensis

Poster presented at the VSMF Symposium held at the Unilever Centre on 2011-01-17The Ami project was a six month Rapid Innovation project sponsored by JISC to explore the Virtual Research Environment space. The project brainstormed with chemists and decided to investigate ways to facilitate monitoring and collection of experimental data. A frequently encountered use-case was identified of how the chemist reaches the end of an experiment, but finds an unexpected result. The ability to replay events can significantly help make sense of how things progressed. The project therefore concentrated on collecting a variety of dimensions of ancillary data – data that would not normally be collected due to practicality constraints. There were three main areas of investigation: 1) Development of a monitoring tool using infrared and ultrasonic sensors; 2) Time-lapse motion video capture (for example, videoing 5 seconds in every 60); and 3) Activity-driven video monitoring of the fume cupboard environs. The Ami client application was developed to control these separate logging functions. The application builds up a timeline of the events in the experiment and around the fume cupboard. The videos and data logs can then be reviewed after the experiment in order to help the chemist determine the exact timings and conditions used. The project experimented with ways in which a Microsoft Kinect could be used in a laboratory setting. Investigations suggest that it would not be an ideal device for controlling a mouse, but it shows promise for usages such as manipulating virtual molecules.Ami was a six month project under the “JISC Rapid Innovation Grants 10/09” programme, Grants for the Virtual Research Environment - Rapid Innovation funding call. [http://www.jisc.ac.uk/fundingopportunities/funding_calls/2009/10/vreri.aspx

HCO3- transport through anoctamin/transmembrane protein ANO1/TMEM16A, in pancreatic acinar cells, regulates luminal pH

The identification of ANO1/TMEM16A as the likely calcium-dependent chloride channel of exocrine glands has led to a more detailed understanding of its biophysical properties. This includes a calcium-dependent change in channel selectivity and evidence that HCO3-permeability can be significant. Here we use freshly isolated pancreatic acini that preserve the luminal structure to measure intraluminal pH and test the idea that ANO1/TMEM16A contributes to luminal pH balance. Our data show that, under physiologically relevant stimulation with 10 pm cholesystokinin, the luminal acid load that results from the exocytic fusion of zymogen granules is significantly blunted by HCO3-buffer in comparison with HEPES, and that this is blocked by the specific TMEM16A inhibitor T16inh-A01. Furthermore, in a model of acute pancreatitis, we observed substantive luminal acidification and provide evidence that ANO1/TMEM16A acts to attenuate this pH shift. We conclude that ANO1/TMEM16A is a significant pathway in pancreatic acinar cells for HCO3-secretion into the lumen

Spatial aspects of Ca2+ signalling in pancreatic acinar cells

Secretory cells do not only respond to an agonist with a simple rise in [Ca2+]i. It is now clear that complex patterns of [Ca2+]i elevation in terms of space and time are observed in many cell types and that these patterns may be a cellular mechanism for the regulation of different responses. Ca2+ signalling in exocrine cells of the pancreas promotes the secretion of digestive enzymes and fluid. It has been shown that at high concentrations of agonist (acetylcholine or cholecystokinin) the [Ca2+]i response is initiated in the secretory pole of the cell before spreading across the whole cell. This site of initiation of the [Ca2+]i elevation is in the region where exocytotic release of enzymes occurs and is also the site of a Ca(2+)-dependent chloride channel thought to be crucially important for fluid secretion. Lower concentrations of agonist elicit [Ca2+]i oscillations with complex repetitive patterns characteristic of each agonist. At physiological agonist concentrations, we have recently described repetitive short-lasting Ca2+ spikes that are spatially restricted to the secretory pole of the cell. In addition to these spikes, cholecystokinin also promotes slow transient Ca2+ rises that result in a global rise in Ca2+. The inositol trisphosphate (InsP3) receptor plays a crucial role in all of these various agonist responses, most of which can be reproduced by the infusion of InsP3 into the cell. The high InsP3-sensitivity of the secretory pole is postulated to be due to a localization of high-affinity InsP3 receptors. We speculate that in response to cholecystokinin the short-lasting spikes elicit exocytosis from a small 'available pool' of vesicles and that the broader oscillations induce both exocytosis and cell changes that involve movement of vesicles into this 'available pool'

Multiple Paths Forward: Diversifying Mathematics as a Strategy for College Success (Executive Summary)

This executive summary outlines key findings from a report on how colleges are creating math pathways that are aligned with students' programs of study

Cost-effectiveness of a patient-centred approach to managing multimorbidity in primary care:a pragmatic cluster randomised controlled trial

Objective Patients with multiple chronic health conditions are often managed in a disjointed fashion in primary care, with annual review clinic appointments offered separately for each condition. This study aimed to determine the cost-effectiveness of the 3D intervention, which was developed to improve the system of care. Design Economic evaluation conducted alongside a pragmatic cluster-randomised trial. Setting General practices in three centres in England and Scotland. Participants 797 adults with three or more chronic conditions were randomised to the 3D intervention, while 749 participants were randomised to receive usual care. Intervention The 3D approach: comprehensive 6-monthly general practitioner consultations, supported by medication reviews and nurse appointments. Primary and secondary outcome measures The primary economic evaluation assessed the cost per quality-adjusted life year (QALY) gained from the perspective of the National Health Service (NHS) and personal social services (PSS). Costs were related to changes in a range of secondary outcomes (QALYs accrued by both participants and carers, and deaths) in a cost-consequences analysis from the perspectives of the NHS/PSS, patients/carers and productivity losses. Results Very small increases were found in both QALYs (adjusted mean difference 0.007 (-0.009 to 0.023)) and costs (adjusted mean difference 126 pound (-739 pound to 991)) pound in the intervention arm compared with usual care after 15 months. The incremental cost-effectiveness ratio was 18 pound 499, with a 50.8% chance of being cost-effective at a willingness-to-pay threshold of 20 pound 000 per QALY (55.8% at 30 pound 000 per QALY). Conclusions The small differences in costs and outcomes were consistent with chance, and the uncertainty was substantial; therefore, the evidence for the cost-effectiveness of the 3D approach from the NHS/PSS perspective should be considered equivocal