CELL Biology UNIT 1 - Summary The World of the Cell PDF

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Unit 1 study guide with book and lecture notes...

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01/21/2016 CELL BIO- UNIT ONE EXAM STUDY GUIDE: 

40 questions, chapters 9/10/11

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5 points each

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Techniques & Tools

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Basic unit of life  cell

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Emphasis on eukaryotes- animal & plant cells

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Need to purify/ isolate cells 

Cell culture/ tissue culture (growing cells in culture) microscopy  perturbing cellular functions  cell & organelle fractionate  studying macromolecules (DNA, RNA, protein)

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Most cells can be cultured in the lab w/ some sort of medium 

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Rich mediums: o

9 essential amino acids

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Vitamins

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Peptide and protein growth factors (often supplied by the addition of serum)

Primary cell culture- cells prepared directly from tissues of an organism 

Often display the differentiated properties of the organs from which they were isolated

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Ex:

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Fibroblasts- secrete collagen

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Skeletal muscle cells- fuse to form giant muscle cells that spontaneously contract

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Nerve cells- extend axons that are electrically excitable, form synapses

Disadvantage- primary cells have finite number of doublings

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Cell strain- a lineage of cells originating from 1 initial primary cell culture 

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50-100 doublings

Once they touch each other they stop growing

Transformed cells- usually cells derived from a tumor or cells that have undergone

spontaneous genetic change (oncogenic transformation) 

Ex: o

HeLa cells (human tumor cells) 

o 

Cancer cells- grow layers on top of layers

CHO cells (Chinese hamster ovary cells)

IMMORTAL, cell line o

Can grow forever!

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Grow to higher densities

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Solid surface not required

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Disadvantage- may not accurately represent original cells in tissues ( # of chromosomes in transformed cells is altered, termed aneuploidy)

Fluorescence- activated cell sorter (FACS) separates cells: 

Make it easier to separate cells very quickly

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Different cells carry different marker*

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T cells are basic cells in the immune system w/ 2 different markers on its surface o

Antibody can conjugate to different markers

Hybridomas  used to produce monoclonal antibody 

Generate specific antibodies- how you produce antibodies

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Inject an antigen protein into the mice- immune cells can respond

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Polyclonal antibodies recognize different parts of a protein

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Antibodies recognize specific regions of cells

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Cancer cells grow much faster than normal cells

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Get clones by individually placing them

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Hybridomas are produced by injecting a specific antigen into a mouse, collecting an antibody producing cell from the mouse’s spleen and fusing it with a tumor cell called a myeloma cell

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Microscopy

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1. Magnification- enlarging/zooming in 

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Projection lens + objective lens = magnification

2. Resolution- ability to distinguish between 2 objects 

Beam of any type of radiation cannot be used to probe structures much smaller than its wavelength

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Limits of light microscopy set by wavelength of visible light

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Wavelength of visible light= 0.45 micrometers (violet) – 0.7 micrometers (deep red)

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Resolution (D)- minimum distance between 2 distinguishable objects o

D= 0.61λ/Nsinα

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N is the refractive index of the medium α is the wavelength of angular aperture Nsinα = Numerical aperture (NA)

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λ is the wavelength of incident light

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Resolution is improved by using shorter wavelengths or increasing either N or α.

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Resolution of light microscopy is ~0.2µm

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Shortest wavelength- purple

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Refractive index can be used to make adjustments

3. Sample Preparation

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BRIGHT FIELD MICROSCOPY:

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Bright field microscopy is widely used in the regular lab 

Advantages: simple, inexpensive, no staining required, no dyes, live cell imaging

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Limitations: very low contrast of most biological samples o

Low apparent optical resolution due to the blur of out of focus material

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Optical, physical, and biochemical ways to improve these limitations

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You can see mitosis and other basic movements from a live cell

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PHASE CONTRAST & DIFFERENTIAL INTERFERENCE CONTRAST (DIC) MICROSCOPY:

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Two ways to improve the images

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Phase Contrast- edge of cells are more apparent, some internal structures shown

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Thin layers of cells not thick tissues (one layer)

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Location and movement of larger organelles in live cells

DIC - 3D images from polarized light 

Suited for extremely small details and thick objects

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Thin optical sections through the object for the reconstruction of 3D structure

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Physical & (bio)chemical methods for improving images: -

Sample preparation for straining o

Fix  embed/ section (thick specimen only)  stain

1. Fixation: 

Cross-linking agents: glutaraldehyde & formaldehyde (form covalent bonds w/ free amino groups)

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Partially permeabilizes cells (for staining) 

Permeablize cells with detergent to disrupt cell membrane & let dye reach the nucleic acids & proteins

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Embedding & sectioning (thick specimen only) 

Embedding using wax or resins

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Use a knife to cut into thin sections

H&E staining: o

Hematoxylin: nuclear stain (blue- violet) 

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Eosin: acid that binds to base 

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Base that binds to acids (DNA) Cytoplasm proteins (pink)

FLUORESCENCE MICROSCOPY: 

Improves details of image

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Has a filter to illuminate sample

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Fluorophores- fluorescent dyes or proteins

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Absorb light at a shorter wavelength (excitation)

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Emit light at a longer wavelength (emission)

There are different variations of fluorescence microscopy 1.

Immunofluorescence microscopy: i. Used to detect specific proteins with an antibody to which a fluorescent dye has been covalently attached

1. Inject a protein X into a rabbit (antigen) 2. 8-10 weeks you get a serum containing Ab to protein X 3. Specific bindings occur a. Antibodies- proteins produced by B cells that recognize and bind to foreign antigen (protein) b. Generates antibodies specific to your protein c.

Primary antibody recognizes protein X

d. Fluorescent secondary antibody is used to amplify the signal of a different antigen 2.

Localization of Proteins in living cells: i. GFP- green fluorescent protein ii. GFP contains a serine, tyrosine, and glycine sequence whose side chains spontaneously cyclize to form a green fluorescing chromophore when illuminated w/ blue light iii. Uses recombinant DNA so sample is not killed iv. Idea of tagging specific proteins with GFP so you can view its distribution in a living cell over time

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Confocal laser scanning microscopy (CLSM): i. Ordinary light and fluorescence microscopy requires sectioning (thick specimen); therefore, 3D information is lost ii. If thick specimen are used for conventional light microscopy the image is blurred above and blew the plane of focus iii. CLSM makes it possible to focus on a chosen plane of a thick specimen while rejecting the light that comes from out of focus areas Light from the bottom & the top iv. Generally used with fluorescence optics v. Increases contrast of the images

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Advanced Fluorescence Microscopy

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1. FRET Fluorescence resonance energy transfer

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Measuring the distance between entities, such as proteins o

Can determine if two proteins interact in vitro

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Applications: protein- protein interactions, structure & conformation of proteins

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Uses two fluorescent proteins in which the emission wavelength of the first is the same as the excitation wavelength of the second

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Protein is not fixed and can travel

2. FRAP  Fluorescence recovery after photo-bleaching 

Measuring lateral diffusion rates of integral membrane protein

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Allows the dynamics of the population of molecules to be analyzed after they have been tagged with GFP proteins

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Has revealed how dynamic many components in the cell are

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Application: Measuring lipids movement on cell plasma membrane

3. Ion Sensitive Fluorescent Dyes 

Low [Ca2+] = blue

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Med [Ca2+] = green

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High [Ca2+] = yellow/orange/ red

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Very sensitive to ions

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Protein conformation is dependent on calcium

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Calcium concentrations are very important in cell biology

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Calcium concentrations are low in the cytoplasm

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Can use this dye to determine concentrations in cells

4. TIRF- total internal reflection fluorescence 

Allows fluorescent samples adjacent to a coverslip to be seen with great clarity

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Ability to observe a very thin region of a specimen

5. Super- Resolution microscopy 

Allows for detailed fluorescent images at nanometer resolution

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[200 nanometers is the limit of the light microscopecan’t see proteins]

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Photon activated microscopy o

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Highest intensity in the middle- drops quickly 

Surrounding is blurry b/c intensity drops

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Can activate individual small regions

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Computer can get rid of the surrounding noise

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Very sharp image

ELECTRON MICROSCOPY: 

Offers a much higher resolution of ultrastructure than can be obtained by light microscopy

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Living material can NOT be viewed by electron microscopy

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Shortest wavelength (beam of electrons) = 0.005 nm o

Nsina=0.0305

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D=0.1 nm (2000 X better than light microscope)

1. Transmission electron microscopy (TEM) 

Electrons are emitted from a filament and accelerated in an electric field

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Fixation  Embedding  Sectioning

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Fixation- glutaraldehyde, osmium tetraoxide (binds & stabilizes lipid bilayer; STAIN)

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Embedding- dehydration, solid plastic block

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Sectioning- 50-100 nm thick, since electrons have limited penetrating power

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> atomic number > electron scattering > contrast (darker)

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Simple specimen (proteins or viruses) can be negatively stained with heavy metals form examination

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Thin sections in TEM provide 2D information

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Immuno- electron microscopy o

Specific proteins can be localized by employing specific antibodies associated with a heavy metal marker, such as small gold particles

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Secondary antibodies bind to primary antibodies which bind to proteins

2. Scanning electron microscopy (SEM) 

Reveals the surface features of specimen

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Provides a 3D image!

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Whole cells or unsectioned tissue specimen are fixed, dried and coated w/ a heavy metal such as platinum

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Drugs are commonly used in Cell biology

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Brefeldin A: inhibits Golgi function

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•Cytochalasin A: inhibits actin

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•Nocodazole: inhibits microtubules

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RNA interference (RNAi) 

Makes the drugs protein specific

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Cells use to suppress the expression of genes by either blocking translation of specific mRNAs through miRNAs or degradation of specific mRNAs targeted by small interfering SiRNAs

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System developed as a defense mechanism against invading viruses & has provided researchers with a very powerful tool to experimentally suppress the expression of particular genes and explore the resulting consequences

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Whole cell purification~ 

Disruption must be gentle to preserve the structure & function of organelles o

Ultrasonic vibrations (sonication)

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Tissue homogenizer

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High speed blender

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Force through small pores of a filter

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Swelling of the cells in a hypotonic solution weakens the plasma membrane, making it easier to rupture

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Forms a homogenate or extract

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Homogenate contains population of particles of different density, shape and size due to different sedimentation rates! *

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Organelle purification ~

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Differential Velocity Centrifugation

Yields fractions of party purified organelles that differ in mass & density

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Type of coarse separation on different densities***

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Centrifuge- nuclei comes out first, then mitochondria/chloroplasts/lysosomes, then plasma membrane/fragments of the ER, then ribosomal subunits, finally the cytosol

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Can create isolated purified organelles

Magnetic fractionation

Purification of endosomes

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Utilizes endocytosis o

Endosome w/ iron particle enters cell

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The cell is lysed

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The endosomes with iron particles are put through electro magnet fractionation

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Cell debris is removed and the final product is the endosome w/ iron particle

Immunoaffinity purification of organelles

Use antibodies against organelle specific membrane proteins to purify organelles and vesicles of similar sizes & densities

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Bioinformatics/ Proteomics 

Time of flight is proportional to the square root of the mass/ charge ratio

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Slow  fat

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Fast  skinny

CHAPTER 10- BIOMEMBRANE STRUCTURE

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Biomembrane Functions: 

Selective permeability o

Membrane is selective, small things like CO2 & oxygen can cross but not polar substances like amino acids

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Compartmentalize - Localizing biochemical reactions o

Organelles also have membranes (advantage of eukaryotes)

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Allows for specialization

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Scaffold for biochemical activities

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Transporting solutes

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Signal transduction o

Receive signals from the outside environment- channeled to the nucleus

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Energy transduction

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Intercellular interaction

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Membrane has two layers

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Some proteins have

carbohydrates or lipids associated with them 

Some proteins can bind to

the cytoskeleton 

Membrane is mainly made

up of lipids  

Lipids are Amphiphilic    

Hydrophilic- water loving

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Hydrophobic- hates water

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3 categories of lipids:

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1. Phosphoglycerides

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2. Sphingolipids

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3. Sterols

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1. PHOSPHOGLYERIDES: 

Head group- choline  phosphate  glycerol

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Phosphoglycerate is the longest part

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Double bonds cause a crease in the tail

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Unsaturated- has double bonds & is more stable (solid) o

Saturated- has no double bonds

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Unsaturated tails are more fluid like*

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Cis ( double bonds) have a lower melting point

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Amphipathic*

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Most abundant class of phospholipids in most membranes

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Examples: PE, PI, PS o

Plasmalogens contain one fatty acyl chain attached to glycerol by an ester linkage and one attached by an ether linkage

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PS & PI carry negative charges

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**Saturated fatty acids are straight

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Unsaturated fatty acids have a kink 

Cis fatty acids cause a little kink o

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Trans fatty acids cause a curvature o

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BEST fatty acids!! Has the largest constituents on the same side of the double bond Has the largest constituents on opposite sides of the double bond

Unsaturated is better than saturated o

0 grams trans fat is important because it has double bonds

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Your body cant metabolize trans fat as well

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More double bonds is better for your health- think olive oil

2. SPHINGOLIPIDS: 

Very important lipid in the membrane

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Remember the general structure

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Some sphingolipids are phospholipids, but not all o

If they don’t have a phosphate they are not phospholipids!

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Sphinomyelin (phospholipids) + Glucosylcerebroside (glucose head group) composed head

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All derived from sphingosine, an amino alcohol with a long hydrocarbon chain + contains a long chain fatty acid attached in amide linkage to the sphingosine amino group

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Some sphingolipids are amphipathic glycolipids whose polar head groups are sugars that are not linked via a phosphate group

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3. STEROLS: 

Four ring isoprenoid based hydrocarbon

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No charge

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They have a hydrophobic & hydrophilic group

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Cholesterol- major animal sterol o

Has a hydroxyl substituent on one ring

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Almost entirely hydrocarbon

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Amphipathic (b/c it...