Lecture 59/12/12how viral protease is activated?Once viral envelope proteins are readyOne product of GAGOne product of POLEach will be fragmented into several pieces before virus becomes infectiousGAG & POL poly proteins are delivered to sites of viral assembly with viral RNA that is packaged in virus. The insertion of envelope proteins and these proteins at these sites are what provide conformational change that cause the budding the be initiated.When protease becomes functional?When some of the GAG proteins come into contact with POL, there is a self-catalysis process. The conformation these proteins adopt, causes the viral protease to be generated because 1 GAG – POL could act on another GAG-POL to release viral proteaseOnce proteases have been shed into site, the viral maturation process is completed.6 – 8 * 10^-9 m between space of membranes (not stained) [60-80 A]ELECTRON MICROSCOPYObserving membrane with microscope/ staining:Slice cell as thin as you can.1) tissue incubated with glutaraldehyde(aldehyde cause covalent bonding between neighboring macromolecules – mainly proteins. Cells and tissues die. Become sort of frozen skeletons.2) incubate with OsO4 (osmium tetroxide)The stain is added to tissue to provide differences in densities to be seen by microscope)Osmium will bind to macromolecules differentially.Once tissue is fixed and stained, view with microscope.3)Embed tissue in plastic.Expoxy Resin: blocks of “plastic” to form. Sliced and those slices are placed on microscope. Bombarded with electron beams and beneath has electron screen.Looking at fluorescent screen. Electrons interact with screen, depending on the different densities.Initial interpretation from electromicrograph: 1925Membrane consists of lipid bilayerProteins are said to be laid as sheets – not correctOsmium is a result of interaction with proteins on surface of membraneWhat is real vs what is artificially created?Membrane Lipids:Majority of lipids are phospholipids1. Fatty acids2. Phosphate group3. GlycerolFatty Acids: not found free in plasma membrane. Attached to larger groupsLong chain of Hydrocarbons. Forming single and double bonds.Hydrogen covalently linked to carbonsAcidic, carboxylic end.*If double bonds exist, kinks form unsaturated fatty aciddouble bonds are important for fluidityGlycerol attaches to fatty acid tail. 3 OH groups are sites of attachment. Fatty acids are attached to 2 of the 3 OH groupsPhosphate attaches to glycerol. sometimes molecules will attach to the phosphate including:Choline (CH2)3 – N+ - CH2 - CH2 – OHOverall name: Phosphotidyl CholineAMPHIPATHIC: 2 solubility properties. Polar head (hydrophilic) [can interact with oxygens in water] and nonpolar tail (hydrophobic) [excluded from water]why water form high energy bonds [oxygen and hydrogen]: hydrogen bonding. Provides stability.Has to be able to break hydrogen bonding to interact with water.Charged end of molecule can form bonds with water – therefore, soluble.Hydrophobic- chains section cannot form bonds with waterLipid BilayerLangmuir, 1917Gortel & Grendel, 1925: plasma membranes consist of 2 layers of lipids with orientation of “bilayer”.Pour oil or layer of water, oil will spread. These are monomolecular films spread over surface of water.How they form?Charged, carboxylic end, will interact with surface end of water. Hydrophilic end will be sticking outside of water.Using trough, squeezing area together, layer pushes back and if squeezed enough, will buckle. Examined pressure /force of film or oil that it exerts.Established background“squeezing” molecules together – creating force. Kinks then realized.Using Langmuir Trough,1917G&G: used 1 cubic mm of blood. Known number of RBCs in the volume and were able to measure radii of RBC’s (number and dimensions) [using light microscope]“bags of hemoglobin” surrounded by plasma membrane. No internal organelles. No nuclei. Eukaryotic.Able to calculate SA of individual cell and total SA of all cellscells extract lipid with acetone (finds total amount of lipid from cells)take extract to Langmuir trough, acetone evaporates and lipids of membrane remain.Measure SAFound out the SA was 2x the amount of a single membrane MUST BE LIPID BILAYER1 cc of blood = 5 * 10^9 cells.Centrifuge blood so cells platelet to bottom and separate from plasma. Will occupy 50% of total. Rest will be plasma.Volume of cells condenses to .5 cc.Only assumption can make: cells are spherical.Because of polarity of groups in membrane , average length of fatty acid should be 30 A. each polar head: 15A. 2 Polar heads. Total: 60 A8/31/12eukaryotes, prokaryotes, viruses17th century, Percivall Pott MD- Diseases linked to certain occupations- British- Neurological disorders with workers in paint- Cancer of scrotum of younger men- Scrotum houses testicles- Connection drawn with men working as chimney sweeps- Chimney sweeps usually were children as they were smaller and could fit into the chimneys- Soot- Linked agent (soot) to form of cancer FIRST TIME LINK MADE- lesion manifested itself 10 years after exposure- some sort of memory to cells- soot (hydrocarbon) causes mutation of proliferating cells - Pott’s Disease: circulation disorder in extremities- Pott’s fractureHippocrates- 466 BC in Greece- Keep records, observations established medicine practices- Prognostication, physical changes that alter health of individual, not mystical causeGalen- What humans and animals consist of- 4 humors - Blood, phlegm, yellow bile, black bile - the imbalance of these humors that cause someone to become ill- Continues to influence medicine todayMicroscopes:Light Microscope: larger organelles. TissuesElectron: organellesScanning tunneling: look at structure of atomRobert Hooke:- focusing light on top of object. - Cork: saw formerly living cells. Remnants of cellulose in cell walls- 1665, published book with drawings from microscope observationsVan Leeuwenhoek, 17th century:- textile merchant, observant on density of weaves- 150 microscopes- metal plate with hole- place close to eye and look through lens- first to describe RBCs- ejaculate from animals and described protozoa - member of Royal Society of LondonCell TheoryRobert Brown (1800s)- plant tissue with staining and microscope. Saw “opaque spots”, called them nucleiMatthias Schleiden (1838) & Theodor Schwann (1839): developed cell theoryNuclei associated with structures that are compartment likeSecretory cells:
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