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078 - What would be the result if an inhibitor for the secondary antibody was introduced to a sample viewed with indirect immunofluorescence?
The secondary antibody would not be allowed to bind to the primary antibody and thus the desired target would not be fluorescently labeled. This could lead to misinterpretation of the sample.
079 - How can photobleaching be used to
Photobleaching is used in FRAP in order to view the transport of fluorophore molecules into the bleached region which can help determine the diffusional coefficient for that particular structure.
080 - The dichromatic mirrors will ___________ shorter wavelengths while _____________ longer wavelengths.
081 - List and briefly describe three major drawbacks to fluorescent microscopy?
Quenching: loss of energy from the fluorophore to surrounding molecules;
Photobleaching: irreversible decomposition of the fluorescent molecule;
Distortion in the image: fluorescent molecules out of the focal plane are excited causing blurring
082 - The first filter in an epifluorescent microscope is called the _________ filter and will only allow the ________wavelength to be transmitted while the second filter is called the __________ filter and only allow the ______________ wavelength to be transmitted.
Excitation and Excitation; Emission and Emission
083 - The first pinhole aperture __________ the ____________ while the second pinhole aperture _____________ all but the ________________.
- The first pinhole aperture Scatters the excitation laser while the
- second pinhole aperture Deflects all but the emission light rays
084 - What can be created with the use of
Each plane scanned can be compiled into a 3D image.
085 - How does multiconfocal laser differ
from confocal lasers? Be precise.
Multi-photon lasers use two low energy quanta, that pulse at a Femtoseconds, while confocal uses a single continuous laser. Multiphoton is more focused, higher intensity.
086 - The speed at which confocal microscopy is done causes problems when attempting to record what?
Movement, because the process is too slow.
087 - While ________ penetration and _____________ excitation is possible with multiconfocal microscopy, it has two
major drawbacks. What are they?
penetration and single molecule
excitation is possible with multiconfocal microscopy
- Lasers are much more expensive and the setup is more complex than regular confocal.
088 - For extrinsically activated apoptosis by, what is the pathway after the FasL has bound to its death domain and involves Bid?
Pro-caspase 8 associates with FADD to become Caspase 8, Caspase 8 activates Bid, leading to release of cytochrome c from mitochondria which forms apoptosomes. Apoptosomes activate procaspase 9 to caspase 9 which causes apoptosis.
089 - What is the basic structure for pro-apoptotic BcL-2 proteins and how does it affect anti-apoptotic members?
- Have a helical channel on one termini that allows them to insert into the hydrophobic tail of the anti-apoptotic proteins.
- This insertion renders the anti-apoptotic members inactive.
090 - How does an apoptotic event suppress necrosis from occurring?
Activation of caspase-8 allows it to cleave RIP1. Without RIP1 the cell cant undergo necrosis.
091 - What inhibitor can cause a cell favoring apoptosis to undergo necrosis and does it accomplish this?
Oligomycin causes cell to become apoptotic by blocking synthesis of ATP by the mitochondria.
092 - Mitochondrial swelling that leads to apoptosis is due to what?
The MPTP (Mitochondrial permeability transition pore) remaining open allowing for metabolites and other molecules up to 1.5 kDa to enter the mitochondrial matrix.
093 - Name two inhibitors for the extrinsic apoptotic pathway, the proteins that they affect and the outcome.
- Flipase inhibits caspase 8 which might allow for necrosis to occur and IAP (inhibitors of apoptosis) inhibits caspase-3 which would not complete the cascade and thus apoptosis
- would not occur.
094 - How can the MPTP affect the H+ gradient across the mitochondrial membrane and what are the possible consequences?
If the MPTP remains open, it can cause the H+ to become equalized, stopping ATP synthesis, thus causing cell death.
269 - __________ is an enhanced fluorescence microscopy technique developed by Stafan Hell in 1994, which provides super resolution by selectively deactivating fluorophores to enhance the imaging.
STED (Stimulated Emission Depletion)
270 - Fluoresence is where a molecule ________ a photon of light and becomes __________. Then it emits a photon of
light of __________ wavelength upon relaxation.
2Fluoresence is where a molecule Absorbs a photon of light and becomes Exited Then it emits a photon oflight of Higher wavelength upon relaxation.
271 - What are the differences and purposes of the exciting barrier filter and the emission barrier filter?
The excitation barrier lets you control the wavelength of light that will illuminate and excite the specimen, while the emission barrier filter is used to control the wavelength of light that is emitted by the specimen and observed.
272 - In Indirect Immunofluorescence, what is the purpose of using a secondary antibody with bound fluors, why not simply use a fluorescent primary antibody?
The secondary antibody binding to the primary antibody helps amplify the fluorescence signal.
273 - How can the FRAP technique demonstrate the membrane fluidity model of the plasma membrane?
- A cell surface can be labeled with fluorescent dye, and then a select area can be photo-bleached using a strong laser. Due to the movements in the plasma membrane, the fluorescently labeled unbleached molecules will diffuse into the bleached area, and this can be visible as an increase in intensity
- over the bleached area. Thus proving the membrane fluidity.
274 - ___________ is a fluorescence technique used to study the close distance interactions between molecules by using energy transfer in close proximity.
FRET (Fluorescence resonance energy transfer)
275 - A new technique which changes the
way a laser is scanned over specimens in laser scanning confocal microscopy. This new technique can get images at rates of 30fps. What is this technique called?
276 - Give 3 basic requirements for Fluorescence Resonance Energy Transfer to occur.
The separation between the donor and acceptor must be 2-10 nm
There must be overlap between the emission spectrum of the donor, and absorption spectrum of acceptor
The relative orientations of the acceptor and donor must be adequate to promote the energy transfer.
277 - A certain FRET based sensor contains GFP and RFP that is connected by a linker containing DEVD. Name a caspase whose activity can be detected by using this FRET sensor due to the way it will cleave the DEVD linker region and what will be the result on the FRET sensor.
Caspase-3 will cut the DEVD linker region, and cause the GFP and RFP to separate so no FRET will occur between the two.
278 - Would mammalian cells be resistant
to UV-Light induced apoptosis through the intrinsic pathway if they did not have cytochrome c. Explain why.
Yes the cell would be resistant, because cytocrhome-c mediates apoptosis from signals in the intrinsic pathway. Been proven through experiments with mice.
279 - Describe the general mechanism of
caspase activity and mention any inhibitors.
- Capsases are synthesized in the inactive form as long inactive precursors (pro-caspases), which are then activated in complexes such as the
- apoptosome, or by proteolytic cleavage (pro-domain removed). IAP’s can also
- interact with caspases and inhibit their activity.
280 - You have to treat a patient with a
fast growing prostate tumor which is resistant to DNA damaging chemotherapeutic agents, but not resistant to agents that affect the cell cycle & metabolism. Which of the following genes due to being deleted or inactivated could be causing this selective resistance?
(this multiple choice question has been scrambled)
281 - You are considering treatment of a
leukemic patient with TNF. Upon further analysis you determine that the leukemic cells have an inactivating mutation of caspase-8. Will treatment with TNF be an effective therapy for this patient?
No, because Caspase-8 is the initiator caspase downstream of TNF receptors, so cells with inactive caspase-8 will not undergo apoptosis upon treatment with TNF.
282 - Compare the main structural difference between effector and initiator caspases, and give 2 examples of each.
The initiator caspases have an extended pro-domain which includes adaptor domains. (-2, -8, -9
, and -10)
- Effector caspases are smaller and only contain about 20-20 residues in
- the pro-domain (-3, -6, and -7)
283 - Why is apoptotic cell death more advantageous to multicellular organisms compared to cell death via necrosis?
- In apoptosis, cells are more efficiently removed from tissues by phagocytois, whereas in necrosis, cells release their contents into
- extra-cellular space and cause inflammation.
284 - A polypeptide has been isolated from a toxic plant that localizes to the mitochondria after Endocytosis by the cell. Once inside the cell, the polypeptide forms large channels in the outer membrane of the mitochondria, thus releasing proteins from the intermembrane space into the cytoplasm.
What effect will be observed in relation to apoptosis?
Because of the formation of the large channels in the membrane, cytochrome c will be released and cause apoptosis of the cells that are treated with this toxin.
285 - Describe differences between Tumor Necrosis Factor 1 and 2 receptors, such as where they are found, if they have death domains, and how each is activated.
TNFR-1 contains the death domain, TNFR-2 does not.
TNFR1 is expressed in most tissues, and can be fully activated by both the membrane bound and soluble trimeric forms of TNF,
- TNFR2 is found only in cells of the
- immune system and only respond to the membrane-bound form of the TNF homotrimer.
286 - In response to DNA damage, how does
the activation of p53 effect the progression of the cell cycle and the cell’s survival?
After p53 is activated due to DNA damage, its target genes are expressed. The Cdk inhibitor, p21 causes cell cycle arrest. Other target genes are expressed from the Bcl-2 family (PUMA and Noxa) induce apoptosis.
287 - What role do Bcl-2 family proteins have in regulating apoptosis in mammalian cells?
Pro-apoptotic members of the Bcl-2 family induce apoptosis by promoting release of cytochrome-c from the mitochrondria, thus leading the caspase activation.
The activity of these pro-apoptotic proteins is regulated by anti-apoptotic BH3-only members of the Bcl-2 Family.
288 - Which of the following promote apoptosis and which ones inhibit it? (bcl-2, bax, bcl-XL, bcl-Xs) ?
Bcl-2 and Bcl-XL are inhibiting. Bax, bcl-Xs are promoting.
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