MEASUREMENT OF SUPEROXIDE FORMATION BY MITOCHONDRIAL COMPLEX I OF YARROWIA LIPOLYTICA

Complex I (NADH: ubiquinone oxidoreductase) is generally regarded as one of the major sources of mitochondrial reactive oxygen species (ROS). Mitochondrial membranes from the obligate aerobic yeast Yarrowia lipolytica, as well as the purified and reconstituted enzyme, can be used to measure complex I-dependent generation of superoxide (O-2(center dot-)). The use of isolated complex I excludes interference with other respiratory chain complexes and matrix enzymes during superoxide dismutase-sensitive reduction of acetylated cytochrome c. Alternately. hydrogen peroxide formation can be measured by the Amplex Red/horseradish peroxidase assay. Both methods allow the determination of complex I-generated ROS, depending on substrates (NADH, artificial ubiquinones), membrane potential, and active/deactive transition. ROS production by Yorrowia complex I in the

Differential susceptibility of mitochondrial complex II to inhibition by oxaloacetate in brain and heart

Mitochondrial Complex II is a key mitochondrial enzyme connecting the tricarboxylic acid (TCA) cycle and the electron transport chain. Studies of complex II are clinically important since new roles for this enzyme have recently emerged in cell signalling, cancer biology, immune response and neurodegeneration. Oxaloacetate (OAA) is an intermediate of the TCA cycle and at the same time is an inhibitor of complex II with high affinity (Kd ~ 10− 8 M). Whether or not OAA inhibition of complex II is a physiologically relevant process is a significant, but still controversial topic. We found that complex II from mouse heart and brain tissue has similar affinity to OAA and that only a fraction of the enzyme in isolated mitochondrial membranes (30.2 ± 6.0% and 56.4 ± 5.6% in the heart and brain, respectively) is in the free, active form. Since OAA could bind to complex II during isolation, we established a novel approach to deplete OAA in the homogenates at the early stages of isolation. In heart, this treatment significantly increased the fraction of free enzyme, indicating that OAA binds to complex II during isolation. In brain the OAA-depleting system did not significantly change the amount of free enzyme, indicating that a large fraction of complex II is already in the OAA-bound inactive form. Furthermore, short-term ischemia resulted in a dramatic decline of OAA in tissues, but it did not change the amount of free complex II. Our data show that in brain OAA is an endogenous effector of complex II, potentially capable of modulating the activity of the enzyme.

Honey bee (Apis mellifera) venom induces AIM2 inflammasome activation in human keratinocytes

Following allergen exposure, cytokines and other pro-inflammatory signals play an important role in the immunological cascade leading to allergic sensitization. Inflammasomes sense exogenous and endogenous danger signals and trigger IL-1β and IL-18 activation which in turn shape Th2 responses. Honey bee venom (BV) allergies are very common; however, the local inflammatory cascade leading to the initiation of allergic sensitization is poorly understood. In this study, the local inflammatory cascades in skin after exposure to BV were investigated.

The enteropathogenic Escherichia coli effector NleH inhibits apoptosis induced by Clostridium difficile toxin B

Clostridium difficile is a leading cause of nosocomial infections, causing a spectrum of diseases ranging from diarrhoea to pseudomembranous colitis triggered by a range of virulence factors including C. difficile toxins A (TcdA) and B (TcdB). TcdA and TcdB are monoglucosyltransferases that irreversibly glycosylate small Rho GTPases, inhibiting their ability to interact with their effectors, guanine nucleotide exchange factors, and membrane partners, leading to disruption of downstream signalling pathways and cell death. In addition, TcdB targets the mitochondria, inducing the intrinsic apoptotic pathway resulting in TcdB-mediated apoptosis. Modulation of apoptosis is a common strategy used by infectious agents. Recently, we have shown that the enteropathogenic Escherichia coli (EPEC) type III secretion system effector NleH has a broad-range anti-apoptotic activity. In this study we examined the effects of NleH on cells challenged with TcdB. During infection with wild-type EPEC, NleH inhibited TcdB-induced apoptosis at both low and high toxin concentrations. Transfected nleH1 alone was sufficient to block TcdB-induced cell rounding, nuclear condensation, mitochondrial swelling and lysis, and activation of caspase-3. These results show that NleH acts via a global anti-apoptotic pathway.

Legionella pneumophila secretes a mitochondrial carrier protein during infection

The Mitochondrial Carrier Family (MCF) is a signature group of integral membrane proteins that transport metabolites across the mitochondrial inner membrane in eukaryotes. MCF proteins are characterized by six transmembrane segments that assemble to form a highly-selective channel for metabolite transport. We discovered a novel MCF member, termed Legionellanucleotide carrier Protein (LncP), encoded in the genome of Legionella pneumophila, the causative agent of Legionnaire's disease. LncP was secreted via the bacterial Dot/Icm type IV secretion system into macrophages and assembled in the mitochondrial inner membrane. In a yeast cellular system, LncP induced a dominant-negative phenotype that was rescued by deleting an endogenous ATP carrier. Substrate transport studies on purified LncP reconstituted in liposomes revealed that it catalyzes unidirectional transport and exchange of ATP transport across membranes, thereby supporting a role for LncP as an ATP transporter. A hidden Markov model revealed further MCF proteins in the intracellular pathogens, Legionella longbeachae and Neorickettsia sennetsu, thereby challenging the notion that MCF proteins exist exclusively in eukaryotic organisms.

Genetically monomorphic brown trout (Salmo trutta L.) populations as revealed by mitochondrial DNA, multi-locus and single-locus minisatellite (VNTR) analyses.

Normally, populations of brown trout are genetically highly variable. Two adjacent populations from NW Scotland, which had previously been found to be monomorphic for 46 protein-coding loci, were studied by higher resolution techniques. Analyses of mitochondrial DNA, multilocus DNA fingerprints and eight specific minisatellite loci revealed no genetic variation among individuals or genetic differences between the two populations. Continual low effective population sizes or severe repeated bottlenecks, as a result of low or variable recruitment, probably explain the atypical absence of genetic variation in these trout populations. Growth data do not provide any evidence of a reduction in fitness in trout from these populations.

Differences in mitochondrial and nuclear DNA status of high-density and low-density sperm fractions after density centrifugation preparation

Background: In order to isolate the â??bestâ?? sperm for assisted conception a discontinuous two-step density gradient centrifugation is usually employed. This technique is known to isolate a subpopulation with good motility, morphology and nuclear DNA (nDNA) integrity. As yet its ability to isolate sperm with unfragmented mitochondrial DNA (mtDNA) is unknown. Methods: Semen was obtained from men (n=28) attending our Regional Fertility Centre for infertility investigations. We employed a modified long polymerase chain reaction to study mtDNA and a modified alkaline Comet assay to determine nDNA fragmentation. Results: The high- density fraction displayed significantly more wild type mtDNA (75% of samples) than that of the low- density fraction (25% of samples). In the high-density fraction, there was a higher incidence of single, rather than double or multiple deletions and the deletions were predominantly small scale (0.1-4.0kb). There was a strong correlation between nDNA fragmentation, the number of mtDNA deletions (r=0.7, p

A comparison of mitochondrial and nuclear DNA status in testicular sperm from fertile men and those with obstructive azoospermia

Background: Mitochondria are vital to sperm as their motility powerhouses. They are also the only animal organelles with their own unique genome; encoding subunits for the complexes required for the electron transfer chain. Methods: A modified long PCR technique was used to study mitochondrial DNA (mtDNA) in ejaculated and testicular sperm samples from fertile men (n=11) and testicular sperm from men with obstructive azoospermia (n=25). Nuclear DNA fragmentation was measured by an alkaline single cell gel electrophoresis (COMET) assay. Results: Wild-type mtDNA was detected in only 60% of fertile mens�??�?�¢?? testicular sperm, 50% of their ejaculated sperm and 46% of testicular sperm from men with obstructive azoospermia. The incidence of mitochondrial deletions in testicular sperm of fertile and infertile men was not significantly different but the mean size of the deletions was significantly less in testicular sperm from fertile men compared with men with obstructive azoospermia (p<0.02). Nuclear DNA fragmentation in testicular sperm from fertile men and men with obstructive azoospermia was not significantly different. Conclusion: Multiple mtDNA deletions are common in testicular and ejaculated sperm from both fertile and infertile men. However, in males with obstructive azoospermia the mtDNA deletions in testicular sperm are of a larger scale.