Accumulation of dodecyltriphenylphosphonium in mitochondria induces their swelling and ROS-dependent growth inhibition in yeast

Hydrophobic cations with delocalized charge are used to deliver drugs to mitochondria. However, micromolar concentrations of such compounds could be toxic due to their excessive accumulation in mitochondria. We studied possible pathophysiological effects of one such cation, i.e. dodecyltriphenylphosphonium (C₁₂-TPP), in the yeast Saccharomyces cerevisiae. First, we found that C₁₂-TPP induces high-amplitude mitochondrial swelling. The swelling can be prevented by addition of protonophorous uncoupler FCCP or antioxidant alpha-tocopherol, but not other tested antioxidants (N-acetylcysteine and Trolox). Second, FCCP prevents ROS-sensitive fluorescent dye (dichlorofluorescein diacetate) staining of yeast treated with C₁₂-TPP. We also showed that all tested antioxidants partially restore the growth inhibited by C₁₂-TPP. The latter points that ROS rather than the mitochondria swelling limit the growth rate.     

Peculiarities of the Mechanism of Interactions of Catalytic Antibodies with Organophosphorus Substrates

Catalytic antibodies are a promising model for creating highly specific biocatalysts with predetermined activity. However, in order to realize the directed change or improve their properties, it is necessary to understand the basics of catalysis and the specificity of interactions with substrates. In the present work, a structural and functional study of the Fab fragment of antibody A5 and a comparative analysis of its properties with antibody A17 have been carried out. These antibodies were previously selected for their ability to interact with organophosphorus compounds via covalent catalysis. It has been established that antibody A5 has exceptional specificity for phosphonate X with bimolecular reaction rate constants of 510 ± 20 and 390 ± 20 min^–1M^–1 for kappa and lambda variants, respectively. 3D-Modeling of antibody A5 structure made it possible to establish that the reaction residue L-Y33 is located on the surface of the active site, in contrast to the A17 antibody, in which the reaction residue L-Y37 is located at the bottom of a deep hydrophobic pocket. To investigate a detailed mechanism of the reaction, A5 antibody mutants with replacements L-R51W and H-F100W were created, which made it possible to perform stopped-flow kinetics. Tryptophan mutants were obtained as Fab fragments in the expression system of the methylotrophic yeast species Pichia pastoris. It has been established that the effectiveness of their interaction with phosphonate X is comparable to the wild-type antibody. Using the data of the stopped-flow kinetics method, significant conformational changes were established in the phosphonate modification process. The reaction was found to proceed using the induced-fit mechanism; the kinetic parameters of the elementary stages of the process have been calculated. The results present the prospects for the further improvement of antibody-based biocatalysts.     

Investigation of activation of phosphate groups in mono- and oligonucleotides with mesitoyl chloride.

It has been demonstrated with the use of 31P NMR pulsed spectroscopy that the reaction of mesitoyl chloride (MsCOCl) both with terminal and internucleotide phosphate groups pA, d(MeOTr)TpT and dpTpT (Ac) proceeds in a quantitative fashion within less than 2 min at 0 degrees C with the respective mixed anhydrides being thereby formed. The anhydrides of phosphomonoesters are resistant, unlike those of phosphodiesters which may be readily split by water, alcohol or amine without the internucleotide bonds being broken. Treatment of poly(U) with an excess of MsCOCl leads to rapid cyclization followed by formation of phosphotriesters. A comparatively easy hydrolysis leads to partial cleavage and isomerization of internucleotide bonds. A similar treatment of UpC showed that about 20% of the internucleotide bonds are cleaved, the remaining UpC being a mixture of approximately equal amounts of 3'-5'- and 2'-5'-isomers.     

Modification of DNA with dihydrazide of succinic acid for preparation of hybridization probes

A two-step chemical method of introduction of nonradioactive labels in DNA was proposed. At first step DNA is modified by succinic dihydrazide at pH 5.0 and 95 degrees C, or at pH 4.5 and 37 degrees C in presence of sodium bisulfite. Then FITC or biotin are joined to the hydrazide groups. DNA modified in this way were shown to be effective hybridisation probes.     

Aspekte der Situationserkennung an Fermentationsprozessen

The establishment of an on- or off-line-optimal-controlled fermentation process requires a system of situation identification — process rearrangement. The choice of the structure and the mathematical model of the situation is difficult. To characterize the situation it is necessary to determine the process-specific vector which regards the major features of the processes. The determination of the structur and the vector which regards the major features of the fermentation process has been demonstrated by several examples.