Effect of cyclosporine A and oligomycin on non-specific permeability of the inner mitochondrial membrane

The effect of oligomycin and cyclosporine A on the induction of non-specific permeability of the inner mitochondrial membrane by Ca²⁺ was under study. Both oligomycin and cyclosporine A were able to prevent the activation of non-specific permeability, but cyclosporine A was the only agent which could restore initial permeability of the inner mitochondrial membrane. The effect of cyclosporine A was shown not to be mediated through redistribution of Ca²⁺ between different mitochondrial subpopulations     

Ion transport in rat liver mitochondria: the effect of the incubation medium osmolarity

A decrease in the incubation medium osmolarity from 320 to 120 mosM reverses the pH dependence of K+ efflux from rat liver mitochondria. The K+ efflux is no longer inhibited by oligomycin and a free radical scavenger butylhydroxytoluene. At 320 mosM, the addition of carbonyl cyanide 3-chlorophenylhydrazone (CCCP) accelerates the K+ efflux, while EGTA inhibits it. At 120 mosM these CCCP and EGTA effects are reversed. In either case the K+ efflux is inhibited by Mg2+. The decrease in osmolarity changes the ruthenium red-insensitive Ca2+ efflux in the same manner. It has thus been shown that the modification of the mitochondrial structure by changing the incubation medium osmolarity results in a qualitative alteration of the systems regulating the K+ and Ca2+ effluxes.     

Solution NMR Structure and Functional Analysis of the Integral Membrane Protein YgaP from Escherichia coli

The solution NMR structure of the α-helical integral membrane protein YgaP from Escherichia coli in mixed 1,2-diheptanoyl-sn-glycerol-3-phosphocholine/1-myristoyl-2-hydroxy-sn-glycero-3-phospho-(1′-rac-glycerol) micelles is presented. In these micelles, YgaP forms a homodimer with the two transmembrane helices being the dimer interface, whereas the N-terminal cytoplasmic domain includes a rhodanese-fold in accordance to its sequence homology to the rhodanese family of sulfurtransferases. The enzymatic sulfur transfer activity of full-length YgaP as well as of the N-terminal rhodanese domain only was investigated performing a series of titrations with sodium thiosulfate and potassium cyanide monitored by NMR and EPR. The data indicate the thiosulfate concentration-dependent addition of several sulfur atoms to the catalytic Cys-63, which process can be reversed by the addition of potassium cyanide. The catalytic reaction induces thereby conformational changes within the rhodanese domain, as well as on the transmembrane α-helices of YgaP. These results provide insights into a potential mechanism of YgaP during the catalytic thiosulfate activity in vivo.     

Effects of the membrane potential upon the Ca(2+)- and cumene hydroperoxide-induced permeabilization of the inner mitochondrial membrane.

A protonophore-induced delta psi decrease in a 180-140 mV range causes an increase in the lag-period of Ca(2+)-induced mitochondrial permeabilization but has little effect on the cumene hydroperoxide-induced permeability transition of mitochondria. Suppression of the non-specific permeability induction seems to be mediated by an increase in [ADP] in the mitochondrial matrix. A further decrease in delta psi leads to additional suppression of the non-specific permeability as a result of a partial ruthenium red-sensitive efflux of the previously accumulated Ca2+. On the other hand, complete dissipation of delta psi causes immediate induction of the non-specific permeability. It is concluded that only complete dissipation of delta psi caused by H+ leakages may act as a trigger for non-specific permeability induction.     

Investigating the Prehistory of Tungusic Peoples of Siberia and the Amur-Ussuri Region?with Complete mtDNA Genome Sequences and Y-chromosomal Markers

Evenks and Evens, Tungusic-speaking reindeer herders and hunter-gatherers, are spread over a wide area of northern Asia, whereas their linguistic relatives the Udegey, sedentary fishermen and hunter-gatherers, are settled to the south of the lower Amur River. The prehistory and relationships of these Tungusic peoples are as yet poorly investigated, especially with respect to their interactions with neighbouring populations. In this study, we analyse over 500 complete mtDNA genome sequences from nine different Evenk and even subgroups as well as their geographic neighbours from Siberia and their linguistic relatives the Udegey from the Amur-Ussuri region in order to investigate the prehistory of the Tungusic populations. These data are supplemented with analyses of Y-chromosomal haplogroups and STR haplotypes in the Evenks, Evens, and neighbouring Siberian populations. We demonstrate that whereas the North Tungusic Evenks and Evens show evidence of shared ancestry both in the maternal and in the paternal line, this signal has been attenuated by genetic drift and differential gene flow with neighbouring populations, with isolation by distance further shaping the maternal genepool of the Evens. The Udegey, in contrast, appear quite divergent from their linguistic relatives in the maternal line, with a mtDNA haplogroup composition characteristic of populations of the Amur-Ussuri region. Nevertheless, they show affinities with the Evenks, indicating that they might be the result of admixture between local Amur-Ussuri populations and Tungusic populations from the north.     

Ceramide and mitochondria in ischemic brain injury

Sphingolipids are essential structural components of cellular membranes, playing prominent roles in signal transduction that governs cell proliferation, differentiation and apoptosis. Ceramides, a family of distinct molecular species characterized by various acyl chains, are synthesized de novo at the cytosolic side of the endoplasmic reticulum serving as precursors for the biosynthesis of sphingolipids in the Golgi. Recently, mitochondria emerged as an important intracellular compartment of sphingolipid metabolism. Thus, several sphingolipid-metabolizing enzymes were found to be associated with mitochondria, including neutral ceramidase, novel neutral sphingomyelinase, and (dihydro) ceramide synthase, an important ceramide-generating enzyme in de novo ceramide synthesis and recycling pathway. Mitochondrial dysfunction appears to be essential in tissue damage after brain ischemia/reperfusion (IR). Mitochondria are known to be involved in both the necrosis and apoptosis detected in animal models of ischemic stroke, and treatments that ameliorate tissue infarction were associated with better recovery of mitochondrial function. Although mitochondrial injury in stroke has been extensively studied and key mitochondrial functions affected by IR are mainly characterized, the nature of the molecule that causes loss of mitochondrial integrity and function remains obscure. Emerging data indicate a deregulation of ceramide metabolism in mitochondria damaged by IR suggesting that ceramides could play critical roles in cerebral IR-induced mitochondrial damage. This review will examine the experimental evidence supporting the key role of ceramides in mitochondrial dysfunction in cerebral IR and highlight potential targets for development of novel therapeutic approaches for stroke treatment.     

Ion permeability induction by the SH cross-linking reagents in rat liver mitochondria is inhibited by the free radical scavenger,butylhydroxytoluene

The hydrophobic, potentially SH cross-linking reagent, phenylarsine oxide (PhAsO), was found to induce K⁺ and Ca²⁺ effluxes from mitochondria and to accelerate the respiration rate in state 4. The hydrophobic monofunctional electrophilic agent,N-ethylmaleimide, does not exhibit this effect but prevents the action of PhAsO. The polar potentially SH cross-linking reagents (arsenite, diamide) induce ion fluxes only in the presence of P_i. Ion fluxes induced by the SH reagents are inhibited by butylhydroxytoluene (an inhibitor of free radical reactions), andN,N-dicyclohexylcarbodiimide, not by oligomycin. It is inferred that the induction of ion fluxes in mitochondria caused by cross-linking of two juxtaposed SH groups is related to the development of free radical reactions.Abbreviations PhAsO phenylarsine oxide - NEM N-ethylmaleimide - HEPES N-2-hydroxyethylpiperazine-N-ethanesulfonic acid - RR ruthenium red - CCCP carbonyl cyanide-m-chlorophenylhydrazone - BHT butylhydroxytoluene - DCCD N,N-dicyclohexylcarbodiimide - DTNB 5,5-dithio-bis-2-nitrobenzoic acid - diamide diazenedicarboxylic acid-bis-dimethyl-amide - mersalyl O-[3-hydroxymercuri)-2-methoxypropyl) carbamoylphenoxyacetic acid - DTE dithioerythritol     

Application of Mistic to improving the expression and membrane integration of histidine kinase receptors from <Emphasis Type="Italic">Escherichia coli</Emphasis>

Integral membrane proteins have become the focus of interest of many laboratories and structural genomics consortia, but their study is hampered by bottlenecks in production, solubilization, purification and crystallization. In our laboratory we have addressed the problem of high-level protein expression in the membrane of Escherichia coli by use of Mistic, a novel Bacillus subtilis protein, as a fusion partner. In this study we examine the effect of Mistic on protein expression and membrane integration levels of members of the E. coli histidine kinase receptor family. We find that Mistic fusion invariably increases the overall yield by targeting the cargo proteins more efficiently to the membrane and may even replace the signal sequence. Mistic fusion methods will likely be instrumental for high-level expression of other integral membrane proteins.