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Biology How Cells are Studied I Optic techniques for cellular and subcellular architecture The Light Microscopy Limit of resolution Scale of cell biology μ m, nm, and A Compound microscopy Types of light microscopy Brightfield microscopy basic form inexpensive and easy for color and fixed specimen and not for living species Phase-contrast microscopy phase plate good for living, unstained specimen Dark field microscopy Fluorescence microscopy fluorescent compounds exciter filter barrier filter Differential -interference -contrast microscopy (DIC) (Nomarski) polarizer analyzer Wollaston prism to produce 3-D image Confocal microscopy to produce 3-D image from a collection of optic sections Sample preparation techniques in light microscopy Fixation Cryoprotection Embedding and sectioning Staining Labeling radioisotope immunolabeling The Electron Microscopy Use a beam of electron to produce an image Two major types of electron microscopy Transmission electron microscopy (TEM) Vacuum system Electron gun Electromagnetic lenses and image formation Photographic system Sample preparation techniques in TEM microscopy Fixation Embedding, Sectioning, and poststaining Electron microscopic autoradiography Negative staining Shadowing Freeze-fracturing Freeze-etching Scanning electron microscopy (SEM): 3 D images Second electrons Sample preparation techniques in SEM microscopy Fixation Postfixation Dehydration Poststaining Mounting Coating with a layer gold or a mixture of gold and palladium. How Cells are Studied II Biochemical Techniques for Cellular and Subcelllular Functions Isolation of cells Source for the best yield fetal or neonatal tissue Disrupting the extracellular matrix and intercellular junctions Proteolytic enzymes Chelating agents Approaches to separate cell types Centrifugation Cell sorter: fluorescence-activated cell sorter What to do with a uniform population of cells For biochemical analysis For cell culture Fractionation of organelles and macromolecules Cell disruption: homogenate Centrifugation Separation by size Separation by size and shape Separation by buoyant density Cell-free system Isolation Reconstitution Chromatography Partition chromatography Column chromatography Ion-exchange chromatography Gel-filtration chromatography Affinity chromatography HPLC Electrophoresis Proteins usually have a net positive or negative charge that reflects the mixture of charged amino acids they contain. If an electric field is applied to a solution containing a protein molecules, the protein will migrate at a rate that depends onits net charge and on its size and shape SDS-PAGE SDS β -mercaptoethanol Coomassie blue Silver stain Western blotting 2-D gel electrophoresis First dimension: isoelectrical focusing Second dimension: SDS-PAGE Analysis of polypeptides Peptide mapping Amino acid sequena Membranes: Their Structure and Function Generalization of membranes They are assembly of lipids and proteins held together by noncovalent interactions. They are dynamic fluid structure. Depending on the source, membranes vary in thickness, in lipid composition and in their ratio of lipid and protein. Functional roles of membranes Define and compartmentalize the cell Serve as the locus of specific functions Control movement of substances into and out of the cell and its compartments Play a role in cell-to-cell communication and detection of external signals Biochemical models of membranes Fluid mosaic model Transmembrane protein structure Three main constituents of membranes Membrane lipids Approximately 50% of mass Lipid bilayers: amphipathic molecules Typical membrane lipids phospholipids glycolipids sphingolipids cholesterol Analysis of membrane lipids Membrane proteins Association with lipids Peripheral membrane proteins and integral membrane proteins Classification of membrane proteins by function Studies of membrane proteins Solubilization, isolation and reconstitution Studies of red blood cell ghosts* Membrane carbohydrates Approximately 2-10 % of mass Confined mainly to the non-cytosolic surface On the extracellular surface of the cells Inward toward the lumen of the compartment Covalent linkage to proteins and lipids Glycoproteins and proteoglycans Glycolipids Analysis of carbohydrate moiety of membranes Lectins Functions of membrane Membrane asymmetry Asymmetric distribution of lipids, proteins and carbohydrates Diffusion in the membranes Transverse diffusion Lateral diffusion Membrane fluidity Lipid bilayer is a two-dimensional fluid Membrane fluidity depends upon its composition Length of hydrocarbon chain and saturation Cholesterol Regulation of membrane fluidity Mobility of membrane proteins Cell fusion experiment TransportAcross Membranes Categories of membrane transport Cellular transport It concerns the exchange of materials between the cells and its environment Intracellular transport It evolves movement of substances across membranes of organelles inside the cell Transcellular transport It involves the movement of a substance in on one side and out on the other side Mechanisms of membrane transport for small molecules Passive Transport: It does not require energy; it occurs because of the tendency for dissolved molecules to move from higher to lower concentrations. 1.) Simple diffusion Factors governing diffusion across lipid bilayers size polarity ionization Kinetics for simple diffusion V=k DX] outside-[X] inside 2.) Facilitated transport Involvement of a membrane transport protein carrier protein channel protein Kinetics for facilitated transport follow Michaelis-Menten kinetics Specificity of transport proteins Examples 3.) Ionophores: They are small hydrophobic molecules that dissolve in lipid bilayers and increase their ion permeability Classes of ionophores mobile ion carriers channel formers Active Transport It requires energy; it takes place against the electrochemical gradient 1.) 3 major functions - uptakes of fuel molecules and nutrients - removal of waste materials, secretory products and sodium ions - maintenance of a constant, optimal internal environment of inorganic ions 2.) Directionality 3.) Kinetics for uncharged molecules for charged molecules 4.) Involvement of membrane potential 5.) Simple versed coupled transport 6.) Energy source 7.) Examples Cellular transports: exocytosis and endocytosis Both involve the sequential formation and fusion of membrane- bounded vesicles Exocytosis: 1.) Steps Packing secretory vesicles Response to extracellular signals Fusion with membrane: recognition sites and Ca ++ Discharge the contents 2.) Membranes asymmetry is maintained through secretion 3.) Two pathways of exocytosis Constitutive exocytosis continuous secretion in all eukaryotic cells Regulated exocytosis extracellular triggers control the secretion in secretory cells: hormones, neurotransmitters or digestive enzymes Endocytosis: 1.) Steps: a complementary process of exocytosis 2.) Two types of endocytosis Pinocytosis: cellular drinking ingestion of fluid and solutes via small vesicles in many cell types Phagocytosis: cellular eating ingestion of macromolecules in specified phagocytic cells 3.) Steps with pinocytosis: Begins at clathrin coated pits Form coated vesicles Shed the coats Fused with endosome Lysosome 4.) Receptor-mediated endocytosis Ligands and cell-surface receptors are involved Example: uptake of cholesterol 5.) Transcytosis Chemistry! Energy Conversion I Mitochondria structure Size Shape Matrix Outer membrane Inner membrane Intermembrane space 5 Stages of respiratory metabolism 1) Glycolysis 2) TCA cycle 3) Electron transport chain 4) Pumping of proton 5) Oxidative phosphorylation The TricarboxylicAcid Cycle: TCAcycle It occurs in mitochondria matrix Substrate: acetyl CoA Products: carbon dioxide and reduced coenzymes, NADH and FADH Reaction involved with TCAcycle Conversion of pyruvate to acetyl coenzyme A decarboxylation and oxidative reaction coenzymeA Entry of acetate into
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