An antioxidant prevents and reverses calcium-induced uncoupling of rat liver mitochondria

Accumulation of Ca2+ by rat liver mitochondria in the presence of inorganic phosphate results in spontaneous activation of respiration accompanied by a progressive loss of the accumulated cation. The lipid peroxidation inhibitor, ionol, completely prevents and reverses the Ca2+/phosphate-induced loss of accumulated Ca2+ and restores the respiration to state 4 level without having any effect on the rate of Ca2+ accumulation and respiration in the presence of an uncoupler. No correlation between the ionol-dependent loss of Ca2+ and the formation of malonic dialdehyde in mitochondria was found. The measurements of delta psi across the inner mitochondrial membrane during a progressive loss of Ca2+ suggest that the Ca2+/phosphate-induced "uncoupling" is mainly due to the appearance of electrogenic fluxes (but not Ca2+ cycling) which is under control of some products of initial steps of lipid peroxidation.     

The effect of ionizing radiation on the respiration of rat thymocytes

The oligomycin-sensitive component of the rate of oxygen consumption by rat thymocytes increased by 22% after X-irradiation. The postirradiation increase in the thymocyte respiration rate was connected with the impairment of a balance between sodium, potassium and calcium and with the protein synthesis activation, and was independent of lymphokine production by lipoxygenase. Irradiation was ineffective with respect to electron transport in the respiratory chain and electrogenic leakages through the inner membrane of thymocyte mitochondria.     

Polymer-based cell-free expression of ligand-binding family B G-protein coupled receptors without detergents

G-protein coupled receptors (GPCRs) constitute the largest family of intercellular signaling molecules and are estimated to be the target of more than 50% of all modern drugs. As with most integral membrane proteins (IMPs), a major bottleneck in the structural and biochemical analysis of GPCRs is their expression by conventional expression systems. Cell-free (CF) expression provides a relatively new and powerful tool for obtaining preparative amounts of IMPs. However, in the case of GPCRs, insufficient homogeneity of the targeted protein is a problem as the in vitro expression is mainly done with detergents, in which aggregation and solubilization difficulties, as well as problems with proper folding of hydrophilic domains, are common. Here, we report that using CF expression with the help of a fructose-based polymer, NV10 polymer (NVoy), we obtained preparative amounts of homogeneous GPCRs from the three GPCR families. We demonstrate that two GPCR B family members, corticotrophin-releasing factor receptors 1 and 2β are not only solubilized in NVoy but also have functional ligand-binding characteristics with different agonists and antagonists in a detergent-free environment as well. Our findings open new possibilities for functional and structural studies of GPCRs and IMPs in general.     

Positively charged ceramide is a potent inducer of mitochondrial permeabilization

Ceramide-induced cell death is thought to be mediated by change in mitochondrial function, although the precise mechanism is unclear. Proposed models suggest that ceramide induces cell death through interaction with latent binding sites on the outer or inner mitochondrial membranes, followed by an increase in membrane permeability, as an intermediate step in ceramide signal propagation. To investigate these models, we developed a new generation of positively charged ceramides that readily accumulate in isolated and in situ mitochondria. Accumulated, positively charged ceramides increased inner membrane permeability and triggered release of mitochondrial cytochrome c. Furthermore, the positively charged ceramide-induced permeability increase was suppressed by cyclosporin A (60%) and 1,3-dicyclohexylcarbodiimide (90%). These observations suggest that the inner membrane permeability increase is due to activation of specific ion transporters, not the generalized loss of lipid bilayer barrier functions. The difference in sensitivity of ceramide-induced ion fluxes to inhibitors of mitochondrial transporters suggests activation of at least two transport systems: the permeability transition pore and the electrogenic H(+) channel. Our results indicate the presence of specific ceramide targets in the mitochondrial matrix, the occupation of which triggers permeability alterations of the inner and outer mitochondrial membranes. These findings also suggest a novel therapeutic role for positively charged ceramides.     

Genome‐wide SNP analysis unveils genetic structure and phylogeographic history of snow sheep (Ovis nivicola) populations inhabiting the Verkhoyansk Mountains and Momsky Ridge (northeastern Siberia)

Insights into the genetic characteristics of a species provide important information for wildlife conservation programs. Here, we used the OvineSNP50 BeadChip developed for domestic sheep to examine population structure and evaluate genetic diversity of snow sheep (Ovis nivicola) inhabiting Verkhoyansk Range and Momsky Ridge. A total of 1,121 polymorphic SNPs were used to test 80 specimens representing five populations, including four populations of the Verkhoyansk Mountain chain: Kharaulakh Ridge–Tiksi Bay (TIK, n = 22), Orulgan Ridge (ORU, n = 22), the central part of Verkhoyansk Range (VER, n = 15), Suntar‐Khayata Ridge (SKH, n = 13), and Momsky Ridge (MOM, n = 8). We showed that the studied populations were genetically structured according to a geographic pattern. Pairwise F_ST values ranged from 0.044 to 0.205. Admixture analysis identified K = 2 as the most likely number of ancestral populations. A Neighbor‐Net tree showed that TIK was an isolated group related to the main network through ORU. TreeMix analysis revealed that TIK and MOM originated from two different ancestral populations and detected gene flow from MOM to ORU. This was supported by the f3 statistic, which showed that ORU is an admixed population with TIK and MOM/SKH heritage. Genetic diversity in the studied groups was increasing southward. Minimum values of observed (Ho) and expected (He) heterozygosity and allelic richness (Ar) were observed in the most northern population—TIK, and maximum values were observed in the most southern population—SKH. Thus, our results revealed clear genetic structure in the studied populations of snow sheep and showed that TIK has a different origin from MOM, SKH, and VER even though they are conventionally considered a single subspecies known as Yakut snow sheep (Ovis nivicola lydekkeri). Most likely, TIK was an isolated group during the Late Pleistocene glaciations of Verkhoyansk Range.     

Characterization of protein detergent complexes by NMR, light scattering, and analytical ultracentrifugation

Abstract  Bottlenecks in expression, solubilization, purification and crystallization hamper the structural study of integral membrane proteins (IMPs). Successful crystallization is critically dependent on the purity, stability and oligomeric homogeneity of an IMP sample. These characteristics are in turn strongly influenced by the type and concentration of the detergents used in IMP preparation. By utilizing the techniques and analytical tools we earlier developed for the characterization of protein-detergent complexes (PDCs) [21], we demonstrate that for successful protein extraction from E. coli membrane fractions, the solubilizing detergent associates preferentially to IMPs rather than to membrane lipids. Notably, this result is contrary to the generally accepted mechanism of detergent-mediated IMP solubilization. We find that for one particular member of the family of proteins studied (E. coli receptor kinases, which is purified in mixed multimeric states and oligomerizes through its transmembrane region), the protein oligomeric composition is largely unaffected by a 10-fold increase in protein concentration, by alteration of micelle properties through addition of other detergents to the PDC sample, or by a 20-fold variation in the detergent concentration used for solubilization of the IMP from the membrane. We observed that the conditions used for expression of the IMP, which impact protein density in the membrane, has the greatest influence on the IMP oligomeric structure. Finally, we argue that for concentrating PDCs smaller than 30 kDa, stirred concentration cells are less prone to over-concentration of detergent and are therefore more effective than centrifugal ultrafiltration devices.     

Mitochondrially targeted ceramides preferentially promote autophagy, retard cell growth, and induce apoptosis

C(6)-pyridinium (D-erythro-2-N-[6'-(1'-pyridinium)-hexanoyl]sphingosine bromide [LCL29]) is a cationic mitochondrion-targeting ceramide analog that promotes mitochondrial permeabilization and cancer cell death. In this study, we compared the biological effects of that compound with those of D-erythro-C(6)-ceramide, its non-mitochondrion-targeting analog. In MCF7 cells it was found that C(6)-pyridinium ceramide preferentially promoted autophagosome formation and retarded cell growth more extensively than its uncharged analog. This preferential inhibition of cell growth was also observed in breast epithelial cells and other breast cancer cells. In addition, this compound could promote Bax translocation to mitochondria. This redistribution of Bax in MCF7 cells could be blocked by the pan-caspase inhibitor zVAD-fmk but via a Bid-independent signaling pathway. Moreover, C(6)-pyridinium ceramide-induced translocation of Bax to mitochondria led to mitochondrial permeabilization and cell death. Overall, we show that mitochondrial targeting of C(6)-pyridinium ceramide significantly enhances cellular response to this compound.     

The effect of cyclosporin A and oligomycin on the nonspecific permeability of the mitochondria inner membrane

The effect of oligomycin and cyclosporine A on Ca(2+)-induced nonspecific permeability of the inner mitochondrial membrane was under study. Both oligomycin and cyclosporine A were able to prevent the activation of nonspecific permeability; however, but cyclosporine A was the only agent which could restore the initial permeability of the inner mitochondrial membrane. The effect of cyclosporine A was not shown to be mediated through redistribution of Ca2+ ions between different subpopulations of mitochondria.     

The effect of butylated hydroxytoluene, an inhibitor of lipid peroxidation, on the calcium-induced uncoupling of rat liver mitochondria

The accumulation of Ca2+ by rat liver mitochondria in the presence of Pi results in spontaneous activation of respiration, accompanied by progressive loss of the accumulated cation. The lipid peroxidation inhibitor, butylated hydroxytoluene, completely prevents and reverses the loss of accumulated Ca2+ and restores respiration to the state 4 level, but exerts no effect on the rate of Ca2+ accumulation and respiration in the presence of the uncoupler. The strong inhibition by butylated hydroxytoluene of ruthenium red-insensitive Ca2+ efflux has also been observed. No correlation between the BHT-sensitive Ca2+ loss and the formation of malonic dialdehyde in mitochondria has been found. The data obtained suggest that the Ca2+-induced uncoupling of mitochondria is mainly due to the appearance of electrogenic ion fluxes that are controlled by the initial steps of lipid peroxidation.     

Mechanism accounting for the induction of nonspecific permeability of the inner mitochondrial membrane by hydroperoxides

The effect of antioxidants on the nonspecific permeability of the inner mitochondrial membrane induced by cumene hydroperoxide or Ca(2+) has been studied. Butylated hydroxytoluene, butylated hydroxyanisole and 2,2,5,7,8-pentamethyl-6-chromanol, taken at a concentration up to 50 microM, suppress the cumene hydroperoxide-induced accumulation of lipid peroxidation products. In the same range of concentrations, these antioxidants inhibit the activation of nonspecific permeability by cumene hydroperoxide or Ca(2+). Propyl gallate, being less effective under such conditions, fails to affect the induction of nonspecific permeability. Additionally, 2,2,5,7,8-pentamethyl-6-chromanol at a concentration decreasing the accumulation of lipid peroxidation products by 70% has been shown not to increase the lag period of nonspecific permeability induction. Higher antioxidant concentrations, while leading to an increase in the lag period of nonspecific permeability induction, cause but minor suppression of lipid peroxidation. From the results obtained we can assume that free radicals formed in the course of hydroperoxide decomposition or on mitochondrial redox complex interact directly with a system responsible for nonspecific permeability or with regulating components of this system.