Tour-de-Force

Tour-de-Force: Interplay between Mitochondria and Cell Cycle Progression Fall 2007transport chain. Although they both inhibit mitochondrial activity, these inhibitors have different effects onmitochondria and the cell.Ion Trap Tandem Mass Spectrometry analysisTandem mass spectrometry is a more advanced version of the single mass spectrometry. In ion traptandem mass spectrometry, ions are trapped at the same place and several separation steps occur overtime. Several spectrometry measurements can be performed on the mixture, whereas in original versiononly one type of measurement would be possible.JC-1 Dual-Emission Potential-Sensitive ProbeThis is a potential sensitive probe with a dual-emission that can be used to measure mitochondrialmembrane potential. The probe is green when the mitochondrial membrane potential is low and it turnsred at higher membrane potentials. The ratio of the two colors is a measure of mitochondrial membranepotential. This probe specifically measures membrane potential and is not affected by other mitochondrialfactors.Kinase AssayProtein kinases phosphorylate other proteins by catalyzing the addition of a phosphate group to a certainlocation of the proteins. As regulation by phosphorylation is very significant in different functions of the cell,measuring activity of these kinases can prove to be important. In this technique, a certain kinase protein isintroduced to the medium together with ATP and the substrate that the kinase is known to act upon.Phosphate groups are tagged with radioactive or fluorescent tags and become active once incorporatedinto the substrate. Measurements of the activity of these tags indicate protein kinase activity.Knockout/Inhibition of Cyclins/CDKsA knockout of a protein is used to inhibit its activity. The cell is transfected by a plasmid or a DNAconstruct. This transfected construct recombines with the gene of interest. A sequence from the gene istransferred into the construct and thus a part of the original gene is deleted. The deletion of the sequenceprevents proteins from being translated. Even if the proteins are translated, they are non-functional. Thistechnique is used widely to study proteins whose functions are unidentified.Mass SpectrometryMass spectrometry can be used to identify compounds in a given mixture to determine the structure of thecompound as well as to measure the amount of compound in that mixture. The basis of this technique ismeasuring the mass to charge ratio of ions.MitoSOX Red ProbeROS is inevitable as a side product of cellular respiration in aerobic organisms. O 2-superoxide ispredominant in mitochondria and its presence initiates a cascades producing more ROS, includinghydroxyl peroxide and hydroxyl radical.To monitor O 2-in living cells, hydroethidine (HE), which is a reduced form of the nucleic acidintercalator ethidium, is used as a indicator of ROS. Once HE is oxidized, it exhibits weak cytosolic bluefluorescence. However, the probe also binds to nucleic acids, which results in staining the nuclei andparticularly the nucleoli by red fluorescence. The MitoSOX Red mitochondrial superoxide indicatorconsists of HE covalently bound to a triphosphonium cation via a hexyl carbon chain. The phoshoniumgroup is positively charged and targets HE analog to mitochondria. Here it accumulates as a function ofmitochondrial membrane potential after which it exhibits red fluorescence due to oxidation and binding tonucleic acids in the mitochondria.So far, ethidium has been postulated as the oxidation product of HE. Recent findings, however,indicate that 2-hydroxyethidium is the oxidation product of HE. The conventional wavelength that is usedto indicate ethidium was 510 nm. By using this wavelength is was hard to distinguish between ethidiumand 2-hydroxyethidium and therefore it is more significant to use a wavelength of 396 nm to excite theMitoSOX red probe. The MitoSOX Red probe with 396 nm excitation can be used to selectivelymonitor O 2 - in the mitochondria.SCI 332 Advanced Molecular Cell Biology Research Proposal 98