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(Answered) physiological systems questions


Hi, I have 7 physiological systems questions that I need desperate help with. I have attached the document containing the 7 questions thank you. If i'm pleases with the answers I also have another 13 potential questions. Please include all working out for the question.;Attached is also the notes for the experiment it is based on.;Attachments Preview;Biosensors practical notes updated version.docx Download Attachment;BioNanotechnology Practical: Ion Channels in Membranes.;Membrane Conduction and Ion Channels;Key Learning Objectives;1. Bilayer lipid membranes (BLM) are the major constituent of cell membranes.;2. BLMs block the passage of ions such as Na+, K+, Cl- and Ca++.;3. Ion channels penetrate cell membranes permitting the passage of ions across the membrane.;4. A convenient tool for studying the ion transport properties is a tethered membrane that is more;stable and easier to study than many other model systems.;5.;Gramicidin is bacterial polypeptide and is an example of an ion channel. In this practical class;you will be asked to fabricate a tethered membrane, insert gramicidin, and measure its;conductance.;6. You will be instructed to form a tethered membrane and to include in it the ion channel;Gramicidin.;7. You will need to measure the conductivity of Gramicidin in membrane and determine he;membrane thickness.;SDx tethered membranes March 2014.;BioNanotechnology Practical: Ion Channels in Membranes.;Background;Cell Membranes;Cell membrane properties control the behaviour of all plants, bacteria and animals. Cell membranes consist;of self-assembled supramolecular structures formed by amphiphiles, or compounds that have polar segments;that strongly attract water and non-polar segments that do not. This results in the non-polar segments being;excluded from the aqueous phase and assembling into bimolecular sheets which eventually form closed;spheres which are the precursors of biological cells. The amphiphiles we are interested in here are known as;lipids and the cell-like structures they form when dispersed in water are known as liposomes. Liposomes can;be 10 nm to hundreds of micrometres in diameter but all have walls that are approximately 4 nm thick, and;are nearly impermeable to ions such as Na+, K+ and Cl-. The 4 nm thick lipid bilayer, that forms the wall of a;liposome is similar to that found in all cell membranes, whether they are from bacteria, plants or animals.;Alterations in membrane ionic permeability are the basis of;Signalling between neurones in the brain, and between neurones in the sympathetic and;autonomic nervous systems.;The senses of sight, sound, taste touch and smell in animals, and related functions in plants;and bacteria.;Mitochondrial metabolism and bioenergetics.;Cell membrane biochemistry is a core discipline within medical research and a core interest of the;Pharmaceutical Industry when searching for drug targets to address a wide range of medical conditions.;Membrane research is a significant component of a current major international research effort focussed on;replacement antibiotics for penicillin which is becoming increasingly ineffective against methicillin resistant;bacterial strains of Staphylococcus Aureus. Compounds that interact with membranes are also important in;understanding the effects of many types of venom, toxins, and some chemical warfare agents..;Tethered membranes;Traditional techniques used to study transmembrane ion transport require the use very small liposomes or;single cells pieced using fragile microelectrodes. Tethered membranes provide a stable planar phospholipid;bilayer over a relatively large surface area (2-3 mm2) that is a convenient alternative tool to study ion;transport in membrane bound ion channels. The tethering of the membrane is achieved using sulphur;chemistry to gold (gold is not totally unreactive and possesses a chemistry with sulphur). Molecular tethers;are thus molecules that possess a sulphur group, polar linkers and a hydrophobic segment that embeds in the;lipid bilayer. The polar linkers allow the existence of an aqueous layer, between the gold electrode and the;membrane. The assembly of a tethered membrane is shown below.;SDx tethered membranes March 2014.;BioNanotechnology Practical: Ion Channels in Membranes.;(a) Ethanol solutions containing 0.4mM;disulphides are exposed to pure fresh gold;for 30 minutes. The molecules collide with;the gold and sulphur-gold bonds form;causing the self assembly of a lipid-spacer;monolayer. In todays practical class 10%;of the molecules are hydrophobic lipidic;anchor groups, and ninety percent are;hydrophilic spacers. This ratio can be;reduced to below 1% tether molecules or;up to 100% tether molecules. The motive;for reducing the fraction of tethers is to;provide more space to incorporate large;channels or to increase the number of;tethers to fabricate a more stable device.;(b) Following the adsorption of the self;assembled monolayer at the gold surface a;further 8ul of 3mM free lipid in ethanol is;allowed to assemble at the surface and then;rinsed with buffer.;(c) Rinsing with buffer causes the mix of;tethered and free lipids to form into a;tethered bilayer, 4nm thick on a 3nm;hydrophilic cushion. The hydrophilic;cushion mimics the inside of a cell and the;lipid bilayer mimics a cell membrane.;Ion Channels;SDx tethered membranes March 2014.;BioNanotechnology Practical: Ion Channels in Membranes.;Ion channels are molecules that create hydrophilic pathways across lipid bilayer membranes permiting ions;to cross otherwise impermeable membranes. Common bacteria such as Pneumonia, Diphtheria, Golden;Staphylococcus and Anthrax are pathogenic because the toxins they produce are ion channels that puncture;the cells of target organisms and collapse their transmembrane potentials.;Gramicidin (gA): Another ion channel, found in the soil bacteria, B. brevis is gramicidin A (See Figure;Below). Being much smaller, with molecular weight of 1882 Da, two molecules end-to-end are required to;span the lipid bilayer. Gramicidin is ion selective and is only conducive to monovalent cations (especially;Na+).;The bacterial ion channel gramicidin (gA). Monomers in the inner and outer leaflets of the bilayer;membrane need to align to form a continuous channel to permit ions to cross the membrane.;(a) Schematic figure of gramicidin A in a tethered membrane. An excitation potential of 20mV a.c. is applied;and the current due to ions being driven back and forth across the membrane is measured.;(b) More detail of gramcidin A showing two gramicidin monmers aligning and forming a conductive dimer.;Beneath the image of the dimer is an end view showing the pore through the centre of gramicidin through;which ions pass.;SDx tethered membranes March 2014.;BioNanotechnology Practical: Ion Channels in Membranes.;Membrane Preparation kit;A six-channel electrode is provided in this practical class that is to be assembled into a flow cell cartridge;(Fig 1A and 2A below). The assembled cartridge plugs into a conductance reader (Fig 2B below) [SDx;tethaPod ], that reads both the membrane conductance and capacitance. A cartridge preparation kit is;supplied by which consists of;individually packaged electrodes pre-coated with tethering chemistry (Fig. 3A below);a flow cell cartridge top containing the gold counter electrode (Fig.2A and 3B below);an alignment jig for use when attaching the electrode to the flow-cell cartridge (Fig. 1A and 3C;below);a silicon rubber pressure pad used when attaching the electrode to the flow cell cartridge (Fig. 3D;below);an aluminium pressure plate used when attaching the electrode to the flow cell cartridge (Fig. 3E;below);a pressure clamp is used when attaching the electrode to the flow cell cartridge (Fig. 1B below);FIGURE 1;FIGURE 2;FIGURE 3;SDx tethered membranes March 2014.;BioNanotechnology Practical: Ion Channels in Membranes.;In addition to the supplied membrane preparation kit you will need;(i) Pair of scissors to open the slide pack;(ii) A 10ul and 100ul pipette and tips to deliver the phospholipid (8l) and rinse with buffer(100l);(iii) Tweezers to remove the slide from the sealed pack.;(iv) Waste bin to collect used
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