Lecture 1 - Cell & Cell Reaction Flashcards
What is the resolution of LM? TEM? SEM?
LM - 0.2 uM
TEM - 1nM
SEM - 2nM
What is the size of an RBC?
7.0uM (used as a reference)
How thick is a paraffin section on a typical glass slide?
5-12uM
What is the diameter of an average virus?
0.02-0.09uM
What are the steps and reagents in specimen prep for LM?
1) Fixation - 10% neutral buffered formalin (formaldehyde stabilized with methanol)
2) Dehydration - w/ alcohol
3) Rinsing with xylene or chloroform
4) Infiltration with paraffin
Explain H&E.
Hematoxylin - purple/blue, Basophilic, stains proteins
Eosin - red/pink, acidophilic, stain cytoplasm, collagen, elastic fibers
What is formaldehyde used for?
The aldehydereactive group crosslinks proteins.
What is the structure of formaldehyde?
CH2=O
What is the most common fixative for EM?
Glutaraldehyde
What is the function and mechanism of glutaraldehyde?
Crosslinks proteins by forming methylene bridges between polypeptides at reactive side groups. Preserves proteins and nucleoproteins well. Slight rxn w/ lipids.
What is the first step following fixation for TEM specimen prep?
Tissue must be post-fixed in osmium tetroxide
What is the function of osmium tetroxide?
Osmium tetroxide is a harsh oxidative agent that preserves membrane and lipid components.
What is used for dehydration in TEM specimen prep? infiltration?
Dehydration: alchohol and acetone Infiltration: epoxy resin
How are specimens stained for TEM?
With heavy metals (ex uranium, lead salts). These will provide contrast in EM.
What contains the surface detail information for EM?
Secondary electrons: electrons that are low energy emitted from the surface of the specimen (up to a depth of 20A).
What step follows fixation for SEM?
Dehydration with ETOH and critical point drying
What step follows drying for SEM?
Glued onto a specimen stub and given conductive coating (ex gold, gold-palladium)
Explain the typical procedure for immunocytochemistry visualization?
1) Block non-specific protein binding with a protein solution
2) Incubate w/ primary antibody
3) Incubate w/ secondary antibody that contains a visualization label
What is the charge of an acidic dye and what is the charge of the molecule it will stain?
Na+dye- (acidic dye is negative)
What is the charge of a basic dye and what is the charge of the molecule it will stain?
Cl-dye+ (basic dye is positive)
What does Periodic acid Shiff (PAS) stain bind?
Glycogen and various carbohydrate containing molecules
What does Silver stain bind?
Reticular fibers (which are Type III collagen)
How thick is a standard lipid bilayer?
7-10nM
What makes up a cellular membrane?
phospholipids, glycolipids, cholesterol
What are gangliosides? Where are they abundant?
Glycolipids with sialic acid (9-carbon monosaccharide) containing oligosaccharides. Abundant in nerve tissue.
What is the cell surface receptor for cholera toxin and diphtheria toxin?
GM1 ganglioside
Where is GM1 located?
It is asymmetrically distributed in outer membrane leaflet.
Define glycocalyx.
“Sugar coat” commonly associated with the extracytoplasmic aspect of the outer leaflet of the plasma membrane.
Define glycosaminoglycans.
Negatively charge polysaccharides
Define proteoglycans.
Heavily glycosylated glycoproteins.
What are the functions of the glycocalyx?
- Cellular attachment to extracellular matrix components
- Binding of antigens and enzymes to cell surface
- Facilitating cell-cell recognition and interaction
What percentage of the cell membrane is made up by cholesterol?
2%
What is the role of cholesterol in the cell membrane?
Assists in maintaining membrane structural integrity by decreasing mobility of first few CH2 groups on phospholipids. Keeps phospholipid bilayer from becoming too fluid or too rigid (crystallizing).
When a freeze fracture is completed which membrane/ face do most proteins stick to?
P-face (protoplasmic face) - the external surface of the inner leaflet
What is GLUT1 and what form of glucose does it accept?
GLUT1 - a multipass transmembrane protein. It is stereospecific accepts D- but not L-glucose
Where are Na+ and Cl- more concentrated (inside or outside cell) and what is the factor by which they are concentrated?
Outside cell, 10-20X concentrated.
Where is K+ concentrated (inside or outside cell) and what is the factor by which they are concentrated?
Inside cell, 20-40X concentrated.
How does Oubain inhibit the Na+/K+ pump?
It binds to the K+ site and stops the function (cycling) of the enzyme.
How is glucose transported through the intestinal epithelium?
Na+ flows down its gradient and “drags” glucose with it. Receptor on epithelial cells in intestinal lumen binds Na+ and glucose and then changes conformation. At basal side glucose moves down concentration gradient and passively exits through glucose specific carrier.
What is the significance of malignant cell over-expression of 170kD ATPase (P170).
These cells can utilize ATP and pump anticancer drugs out more effectively/quicker.
What causes cystic fibrosis?
Defective CFTR, which is an ATP and cAMP sensitive Cl- channel. In cf patients CFTR is insensitive to cAMP and Cl- flux across the membrane does not occur.
What type of receptors are nicotinic acetylcholine receptors?
Ion channel-linked receptors
What is the common mechanism of catalytic receptors and what are some examples of catalytic receptors?
Single pass transmembrane protein that bind ligand and internal aspect becomes active kinase. Examples include receptors for insulin, EGF, PDGF.
What is the general role of:
- Gs proteins
- Gi proteins
- Gp proteins
Gs: stimulatory (ex activate adenyl cyclase)
Gi: Inhibitory
Gp: Activate phospholipase C
What ion channel is blocked to treat hypertension?
Calcium channels (calcium channel blockers)
What ion channel is blocked to treat epilepsy (seizures)?
Sodium channel (sodium channel blockers)
What ion channel is blocked to treat type II diabetes?
Potassium channel (glipizide)
Toxin that inactivates Na+ channels (occupies Na+ binding sites). Leads to dizziness, tingling about mouth, respiratory failure, death.
Tetrodotoxin
Alters Gs protein so they cannot hydrolyze GTP
Cholera toxin
What is the effect of faulty Gs proteins?
Mental retardation / diminished growth / diminished sexual development
Hereditary condition due to abnormal carrieor proteins that cannot remove cystine from the urine - produces kidney stones.
Cystinurea
Autoimmune disease where IgG antibodies bind to TSH receptors and stimulate thyroid follicular cell constitutively. This leads to enlarged thyroid, eyeball protrusion.
Graves disease
