3 - Molecular and Cellular Adaptation

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V B
Created by V B almost 4 years ago
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Question Answer
What is a muscle physiologist interested in? The molecular and cellular regulation of skeletal muscle after exercise ie. the black box mechanisms that underpin adaptation to exercise in skeletal muscle
What happens during a molecular adaptation? 1) Signalling/Protein activity 2) Gene expression/transcription (turn the gene on or off) 3) Translation into functional protein
What affects whether a sensor protein is active or inactive? What does this lead to? Phosphorylation The activation of genes transcription factors
Where are messages taken to make proteins? Where does this occur? Ribosomes In the nucleus
How many types of cell are there? ~200
What determines the function of a cell? Which genes are activates/deactivated
What is the first stage of adaptation? Cell signalling/transduction
What is cell signalling/transduction? An environmental cue comes from outside the cell, it responds by altering its behaviour through a series of chemical reactions. This changes gene expression and protein levels to carry out a function.
What creates extracellular molecules? What are they called? Give some examples Exercise Ligands calcium, hormones, insulin, catecholemines, hypoxia, tension, insulin-like growth factor
Ligands include a chain reaction of signalling proteins, what is that chain? Receptor proteins, intracellular signalling transduction proteins and target proteins or transcription factors.
Describe the simplistic cell signalling diagram. 3373c941-273f-48de-9b45-cf7ad14f4913.png (image/png)
What do intracellular signalling proteins act as? Molecular switches
What does a molecular switch do? What is it undertaken by? Causes phosphorylation or dephosphorylation causing proteins to become active or inactive. Protein kinases
How can we assess signalling muscle? How do we stop phosphorylation occuring? Using a muscle biopsy. Homogenise the tissue in a cold lysis buffer or freeze it in liquid nitrogen.
What is protein quantification assay? What does the assay contain? Quantify total protein using commercially available assay kit to standardise protein between samples. Bicinchoninic acid (BCA) & Copper II sulphate
What two reactions does the protein quantification assay rely on? 1) The peptide bonds in protein reduce Cu2+ ions from the copper II sulphate to Cu+. The amount of Cu2+ reduced is proportionate to the amount of protein present in the solution. 2) 2 molecules of bicinchoninic acid chelate with each Cu+ ion forming a purple product that strongly absorbs light at a wavelength of 562mm.
If there is more protein when protein quantification assay is performed, what will happen? more protein absorbs more light so the colour will be darker.
How do you prepare a sample for protein quantification assay? (step 1&2) 1) Add an equal amount of samply to leammi buffer Beta Mercaptoethanol - this removes disulphide bridges between amino acids to linearise and impart the negative charge. 2) SDS also helps linearise and impart the negative charge
How do you prepare a sample for protein quantification assay? (step 3,4 & 5) 3) sample is glycerol heavy 4) add blue dye called bromophenol blue 5) Boil samples to denature the proteins & make them linear
Describe SDS PAGE. SDS is an onionic detergen applied to protein sample linearise proteins & make them evenly -vely charged Acrylamide creates a mesh at different percentages once set allows proteins to be stored based on size Proteins with less mass travel quicker through the gel due to the sieving effect of the gel.
What do you do with the proteins on the gel after SDS PAGE? Transfer them to a nitrocellulose membrane using an electrical charge.
When using SDS PAGE what is used as the control and why? GAPDH It wont change so if it has all been equally loaded the stripe will be equal all the way along.
What is a primary antibody? It is generated when a host species or immune cell culture is exposed to a protein of interest
What do you do with a primary antibody? Inject what you want the antibody for into a mouse to gain more Harvest them and use as a sensitive and specific detection tool which binds directly to the protein
What is a secondary antibody? It is an antibody which is directed at a species specific portion of the primary antibody - an anti mouse secondary will bind to almost any mouse-sourced primary antibody
How do you detect luminol? Horseradish Peroxidase catalyses the oxidation of luminol in the presence of hydrogen peroxide causing the luminol to emit light.
What is a transcription factor? Where does it occur? What does it do? A protein that binds to DNA and therefore enhances.inhibits gene expression In the nucleus Directs RNA polymerase II to the correct gene to be expressed.
What does RNA polymerase do? It is the enzyme that transcribes DNA to mRNA
How do we isolate RNA? Homogenise fresh tissue in tri-reagent Extraction of nucleic acids involves adding chloroform to phenol and thiocyanate and the cells/tissues lysed in an aqueous solution. Centrifuge this and it will seperate the phases
After centrifuging RNA isolation what are the phases presented? ca6e7776-8b9b-4707-a845-0cc9bb2733ad.png (image/png)
What is RNA made up of? What is special about them and why? Nucleic acids which are polar because of their negatively charged phosphate backbone.
Why are nucleic acids soluble in the upper aqueous phase instead of the lower organic phase? Water is polar - more polar than phenol
What is alcohol precipitation? Alcohol precipitates the polar RNA out of the water as it is much less polar. This measures RNA concentration and standardises amounts between samples.
What does PCR detect? A double stranded molecule
What is RT-PCR reverse transcription? Why is this necessary? it forms a double stranded DNA so PCR can detect it. RNA is single stranded so a complementary DNA molecule needs to be created.
What are the steps of a polymerase chain reaction after a double strand has been created? (1-3) 1. cDNA is denatured (95*C) 2. Temp. is decreased (60*C) and primers bind 3. Temp. is increased and the single stranded cDNA that has the primers bound is extended using free nucleotides to create 2 cDNA molecules.
What are the steps of a polymerase chain reaction after a double strand has been created? (4&5) 4. The cycle is repeated 40 times to create almost a billion copies of out target sequence. (Doubles in number everytime it is heated and cooled) 5. SYBR green dye binds to double stranded DNA & once excited by a laser emits light directly proportional to amount of cDNA
Overview: structural, compositional & cellular adaptation Fibre Type: IHC enzymatic staining/immunofluorescent staining for MYH's & RT-PCR for MYH gene expression/SDS PAGE for protein content
Immuno-histochemistry (IHC) fibre type Sections are fixed (PFA) maintains architecture (crosslinks) Permeabilised using triton/detergent, open up pores of cells to allow primary/secondary antibody/flourescent stain
Bioengineered skeletal muscle Mechanical stretch in culture stimulates exercise then we can manipulate it
Summary What are the three pillars of molecular adaptation? 1) Cell signalling (SDS page, western blot) 2) Gene expression (RT-PCR) 3) Total protein content (SDS PAGE, western blot)