OXPHOS Organization and Activity in Mitochondria of Plants with Different Life Strategies.

The study of the supramolecular organization of the mitochondrial oxidative phosphorylation system (OXPHOS) in various eukaryotes has led to the accumulation of a considerable amount of data on the composition, stoichiometry, and architecture of its constituent superstructures. However, the link between the features of system arrangement and the biological characteristics of the studied organisms has been poorly explored. Here, we report a comparative investigation into supramolecular and functional OXPHOS organization in the mitochondria of etiolated shoots of winter wheat (Triticum aestivum L.), maize (Zea mays L.), and pea (Pisum sativum L.). Investigations based on BN-PAGE, in-gel activity assays, and densitometric analysis revealed both similarities and specific OXPHOS features apparently related to the life strategies of each species. Frost-resistant winter wheat was distinguished by highly stable basic I1III2IVa/b respirasomes and V2 dimers, highly active complex I, and labile complex IV, which were probably essential for effective OXPHOS adaptation during hypothermia. Maize, a C4 plant, had the highly stable dimers IV2 and V2, less active complex I, and active alternative NAD(P)H dehydrogenases. The latter fact could contribute to successful chloroplast-mitochondrial cooperation, which is essential for highly efficient photosynthesis in this species. The pea OXPHOS contained detergent-resistant high-molecular respirasomes I1-2III2IVn, highly active complexes IV and V, and stable succinate dehydrogenase, suggesting an active energy metabolism in organelles of this plant. The results and conclusions are in good agreement with the literature data on the respiratory activity of mitochondria from these species and are summarized in a proposed scheme of organization of OXPHOS fragments.

Design of novel water-soluble isoxazole-based antimicrobial agents and evaluation of their cytotoxicity and acute toxicity.

Based on the readily available 3-organyl-5-(chloromethyl)isoxazoles, a number of previously unknown water-soluble conjugates of isoxazoles with thiourea, amino acids, some secondary and tertiary amines, and thioglycolic acid were synthesized. The bacteriostatic activity of aforementioned compounds has been studied against Enterococcus durans B-603, Bacillus subtilis B-407, Rhodococcus qingshengii Ac-2784D, and Escherichia coli B-1238 microorganisms (provided by All-Russian Collection of Microorganisms, VKM). The influence of the nature of the substituents in positions 3 and 5 of the isoxazole ring on the antimicrobial activity of the obtained compounds has been determined. It is found that the highest bacteriostatic effect is observed for compounds containing 4-methoxyphenyl or 5-nitrofuran-2-yl substituents in position 3 of the isoxazole ring as well as methylene group in position 5 bearing residues of l-proline or N-Ac-l-cysteine (5a-d, MIC 0.06-2.5 µg/ml). The leading compounds showed low cytotoxicity on normal human skin fibroblast cells (NAF1nor) and low acute toxicity on mice in comparison with the well-known isoxazole-containing antibiotic oxacillin.

New insights into the organisation of the oxidative phosphorylation system in the example of pea shoot mitochondria.

The physical and functional organisation of the OXPHOS system in mitochondria in vivo remains elusive. At present, different models of OXPHOS arrangement, representing either highly ordered respiratory strings or, vice versa, a set of randomly dispersed supercomplexes and respiratory complexes, have been suggested. In the present study, we examined a supramolecular arrangement of the OXPHOS system in pea shoot mitochondria using digitonin solubilisation of its constituents, which were further analysed by classical BN-related techniques and a multidimensional gel electrophoresis system when required. As a result, in addition to supercomplexes I1III2, I1III2IVn and III2IV1-2, dimer V2, and individual complexes I-V previously detected in plant mitochondria, new OXPHOS structures were also revealed. Of them, (1) a megacomplex (IIxIIIyIVz)n including complex II, (2) respirasomes I2III4IVn with two copies of complex I and dimeric complex III2, (3) a minor new supercomplex IV1Va2 comigrating with I1III2, and (4) a second minor form of ATP synthase, Va, were found. The activity of singular complexes I, IV, and V was higher than the activity of the associated forms. The detection of new supercomplex IV1Va2, along with assemblies I1III2 and I1-2III2-4IVn, prompted us to suggest the occurrence of in vivo oxphosomes comprising complexes I, III2, IV, and V. The putative oxphosome's stoichiometry, historical background, assumed functional significance, and subcompartmental location are discussed herein.

The role of flavin-containing enzymes in mitochondrial membrane hyperpolarization and ROS production in respiring Saccharomyces cerevisiae cells under heat-shock conditions.

Heat shock is known to accelerate mitochondrial ROS production in Saccharomyces cerevisiae cells. But how yeast mitochondria produce ROS under heat-shock condition is not completely clear. Previously, it was shown that ROS production in heat-stressed fermenting yeast cells was accompanied by mitochondrial membrane potential (MMP) increase. In the current investigation the relationship between ROS production and MMP was studied in respiring yeast cells in stationary phase, using diphenyleneiodonium chloride (DPI), an inhibitor of flavin-containing proteins, as well as the mutants deleted for NDE1, NDE2 and NDI1 genes, encoding flavin-containing external and internal NADH dehydrogenases. It was shown that heat shock induced a transient burst in mitochondrial ROS production, which was paralleled by MMP rise. ROS production and MMP was significantly suppressed by DPI addition and deletion of NDE1. The effect of DPI on ROS production and MMP rise was specific for respiring cells. The results obtained suggest that the functioning of mitochondrial flavin-binding enzymes, Nde1p for instance, is required for the hyperpolarization of inner mitochondrial membrane and ROS production in respiring S. cerevisiae cells under heat-shock conditions.

Relation between cell death progression, reactive oxygen species production and mitochondrial membrane potential in fermenting Saccharomyces cerevisiae cells under heat-shock conditions.

Moderate heat shock increased reactive oxygen species (ROS) production that led to cell death in glucose-grown Saccharomyces cerevisiae cells. Conditions that disturb mitochondrial functions such as treatment by uncouplers and petite mutation were shown to inhibit ROS production and protects cell from thermal death. Hence, mitochondria are responsible for ROS production and play an active role in cell death. An increase in ROS production was accompanied by hyperpolarization of inner mitochondrial membrane. All agents suppressing hyperpolarization also suppressed heat-induced ROS production. It was supposed that generation of ROS under moderate heat shock in glucose-grown S. cerevisiae cells is driven by the mitochondrial membrane potential.