Mcbii recitation 1 membranes 2011 2


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Mcbii recitation 1 membranes 2011 2

  1. 1. Molecular & Cell Biology Recitation Spring, 2011 Recitation Instructors: Prof. Eric Brenner Erik Duboue Nathan Poslusny
  2. 2. How can the recitation help you?Recitation Participation: 5% (Attendance, Discussion, Group Work)Recitation Presentation: 5%Class Paper: 10% =20% Final Grade
  3. 3. Recitation 1:Lipids and Cell Membrane Fluidity
  4. 4. Part 1: Using lipids to help fight cancerBackground: What are the major problems withcurrent chemotherapeutic techniques? •Toxicity to non-target cells •Drug stability •Target cell specificity
  5. 5. Hard tumors are particularly difficult to treatwith chemotherapy. Why? • They have poorly dispersed vascular systems, which reduces exposure to blood- borne drugs. • They are densely fibrous, which serves as a physical barrier. • They have high internal pressures, which means soluble drugs are not easily delivered into the tumor
  6. 6. Is there a “magic bullet” that can target drugs right into cancers that are difficult to treat? If so, what is this new technology called?
  7. 7. Nanomedicine(The medical application of nanotechnology) Nano drugs = 10 to 100 nanometers
  8. 8. Nanomedicine uses nanoparticlesNanoparticles preferentiallyaccumulate at tumor sites becausetumors lack an effective lymphaticdrainage system. Hence, poor vascular structure, such as that found in hard tumors, is advantageous for nanomedicine
  9. 9. So, how can membranes be used in nanomedicine? Liposomes are artificially prepared vesicles made from a lipid bilayer. Liposomes can be filled with drugs, and used to deliver these drugs to cancer cells and other diseased cells or tissues To understand how liposomes are made, we must first review membranes and their characteristic components…
  10. 10. Part 1I: What are keyFeatures ofMembranes? • Sheet-like Structures • Asymmetric Bilipid Leaflets • Closed Boundaries • Lipids and Proteins (but also carbohydrates, and other molecules…) • Non-covalent Assemblies - disassociates and easily reconstituted • Fluid (not random) How do the properties of lipids confer the characteristics of cell membranes?
  11. 11. Basic Structure of a Phospholipid:What does amphipathic mean? *Having both hydrophilic & hydrophobic properties How are membrane components oriented? *It is energetically favorable for amphipathic lipids to form membranes, as a lipid bilayer allows for the hydrophobic tails to be buried & the hydrophilic head groups to interact with either the cytosol or the extracellular space.
  12. 12. Side Chains:Can be Saturated or Unsaturated Saturated fatty acids are “Saturated” with H’s all single bonds form a straight chain Unsaturated fatty acids are missing some H’s double bonds form kinks
  13. 13. What consequence do these different forms have on membrane properties?• Saturated side chains are straight and don’t take up much room, meaning that they can pack tightly together, increasing density and making membranes less-fluid What is healthy vs. unhealthy?Ex. butter (saturated fats) are solid at room temp
  14. 14. Sphingolipids are defined by their sphingosine group, are (mostly) uncharged, and commonly found in neuronal cell membranesWhat kind of lipid is this? (what’s the distinguishing feature?) *sphingolipid, defined by the sphingosine (red)What are the features of the side chains? *saturated What is the overall charge on this lipid? *uncharged
  15. 15. Unsaturated chains“kink”, can’t pack in asdensely made make themembrane more fluid Ex. oils are liquid at room temp
  16. 16. What kind of lipid is this? (what’s the distinguishing feature?) *phospholipidWhat are the features of the side chains? *one saturated, one unsaturatedWhat is the overall charge on this lipid? *it has one (-) & one (+), but overall it is uncharged
  17. 17. This lipid is made of ceramide &glucose What kind of lipid is this? (what is it made of?) *glycolipid (it contains glucose) What are the features of the side chains? *one saturated, one unsaturated What is the overall charge on this lipid? Any +, -, neutral? *it is uncharged Unsaturated fats help maintain membrane fluidity. What other famous lipid also helps?
  18. 18. CholesterolWhat features make it amphipathic? *the ring structure is hydrophobic, and the -OH is hydrophilicWhat is the overall charge on this lipid? *unchargedIs it found mostly inside the bilayer or does it stick out? *it is found inside the bilayerWhat can this structure tell us about how this molecule may work in the membrane?*it helps maintain fluidity by preventing interactions between fatty acid side chai
  19. 19. Lipid Content of Membranes…Varies Among MembranesExamples:Myelinated Nerve Cells: Cholesterol and CerebrosidesLiver Cell Plasma membranes: Cholesterol and Phospholipids
  20. 20. How can we measure the various lipids in a membrane? 2. Remove Lipids, but1. Obtain Sample leave behindTissues, Cell proteins, carbohydrates,Extracts, etc…) etc.) How? Ch PE PCOrganic solvent eg.chloroform/methanol, cyclohexane PSHow detect? thin-layer chromatography, HPLC and more advanced methods
  21. 21. Part II: Techniques How do we study membrane lipids? Prepare "Ghosts"What’s osmotic lysis? Place the cell in a hypotonic solution allowing it to burst
  22. 22. Ghost can be used to study membrane composition Intact cell membranes are impermeable to enzymes and SITS;however, ghosts are leaky, and enzymes and SITS can enter them! RBC Leaky Ghost
  23. 23. How can we assess the asymmetry of thelipid composition of leaflets? *Sequentially use enzymes that selectively degrade different components Sphingomyelinase: degrades sphingomyelin Sea snake venom: contains phospholipases that degrade phosphoglycerides SITS: membrane impermeable compound that binds to -NH3+ (primary amine) groups and then fluoresces
  24. 24. Before treatment Red Blood Cells* Ghosts*Sphingomyelin 26% 26%Phosphatidyl Choline 32% 32%Phosphatidyl 31% 31% Ethanolamine 10% 10%Phosphatidyl Serine 1% 1%Other * These values are expressed as percentages of phospholipids alone; cholesterol and glycolipids are not included.
  25. 25. What do these lipids share in common? *these are all phospholipids
  26. 26. Treatment with sphingomyelinase Sphingomyelin RBC Leaky Ghost sphingomyelin: 26%- sphingomyelin: 26%->0% >6%
  27. 27. Treatment with sphingomyelinase Sphingomyelin RBC Leaky Ghost sphingomyelin: 26%- sphingomyelin: 26%->0% >6%
  28. 28. Sphingomyelin quantification and location Red Blood Cells Leaky Ghosts Sphingomyelin 26% 6% 26% 0% Phosphatidyl Choline 32% 32% Phosphatidyl Ethanolamine 31% 31% Phosphatidyl 10% 10% Serine Other, including Difference= degradation 1% 21% 1% 27% sum of products changesWhich leaflet contains sphingomyelin? *it is in both the inner (6%) & outer(20%) leaflet
  29. 29. Sea snake venom contains phospholipases that degrade phosphoglycerides Red Blood Cells Leaky Ghosts Sphingomyelin 26% 26% Phosphatidyl Choline 32% 9% 32% 0% Phosphatidyl 31% 25% 31% 0% Ethanolamine Phosphatidyl 10% 10% 0% Serine Other, including degradation 1% 30% 1% 74% products Which leaflets contain phosphoglycerides? *the phospholipids are in both the inner (44%) and outer (29%) leaflet
  30. 30. SITS: membrane impermeable compound that binds to - NH3+ groups and then fluoresces Red Blood Cells Leaky Ghosts Fluorescent Signal /+ +++ Where are the primary amines principally located? *in the inner leaflet
  31. 31. Conclusion:The lipid content varies between the leaflets within a membrane Interior Layer Exterior Layer Sphingomyelin 6% 20% Phosphatidyl 9% 23% Choline Phosphatidyl 25% 6% Ethanolamine Phosphatidyl 10% 0 Serine Other 1%
  32. 32. Red Blood Cells Leaky GhostsSphingomyelin 26% 26%Phosphatidyl 32% 9% 32% 0%CholinePhosphatidyl 31% 25% 31% 0%EthanolaminePhosphatidyl 10% 10% 0%SerineOther, including 1% 30% 1% 74%degradationproducts
  33. 33. Now, how do we target……toxic drugs with high specificity to solid tumors, while maintaining drug stability?
  34. 34. LiposomesPreparationHow do you “fill” them with the drug? *Fill liposomes with the pharmaceutical agent by adding it to the buffer before sonicationHow could you purify the liposomes? *purify liposomes using gel filtration (you are selecting against free lipids and giant liposome blobs, so it makes sense to select based on size)
  35. 35. You can also prepare liposomes with planar bilayers Pinhole Side view in chamber
  36. 36. Prepare Planar Bilayers (cont.)a. Place a fine-tip paintbrush into membrane-forming solution.b. Stroke it over a hole (1 mm in diameter)c. Bilayer forms
  37. 37. PEG orothercompound
  38. 38. Her-2 isreceptorin• Her-2 receptor overexpressed ~25% of breast cancer patients• “Trastuzumab”, a recombinant HER-2 antibody conjugated to chemo-loaded lipsomes• Hard tumors have “enhanced permeability and retention effect”. That is liposomes will preferentially “extravasate” in the abnormal blood vessels that occur in tumors.
  39. 39. Dox = doxorubicinClinical Cancer Research (2002) 8: 1172-1181
  40. 40. HomeworkHow would you prepare Anti-Her2 immunoliposomes? - Show individual steps in a flow chart.Also, how does the liposome deliver its contents into a cancer cell once it has arrived at the cancer cell?For reference see - Liposome-based drug delivery in breast cancer treatmPark, J. W. Breast Cancer Research (2002) 4: 95-99