Biophysical and Bioanalytical Analysis of the Iron-ome in Mitochondria Isolated from Saccharomyces cerevisiae

An integrative biophysical and bioanalytical approach to studying the Fe distribution in isolated mitochondria was developed. This procedure involved large-scale growths, the inclusion of a chelator in isolation buffers and an anaerobic isolation protocol. Electron microscopy confirmed that mitochondrial membranes were intact and that samples were largely devoid of contaminants. The Fe-ome-the sum of all Fe species in mitochondria--was studied using a combination of EPR, Mossbauer Spectroscopy, Electron Absorption, ICP-MS and Protein analysis. Isolated mitochondria were packed prior to analysis to improve the S/N ratio. The residual buffer content of sample pellets was determined by use of a radio-labeled buffer. There was essentially no difference in the packing efficiency of mitochondria isolated from respiring and fermenting cells. The determined packing factor, 0.80, was used to calculate concentrations of individual species in neat mitochondria. The Fe-omes of mitochondria isolated from cells grown on respiring, respirofermenting and fermenting media were determined. Neat mitochondria contained ~ 750 mM Fe, regardless of whether the cells had been grown on respiring or fermenting media. The Fe distribution of respirofermenting samples (which can undergo respiration and fermentation simultaneously) was nearly identical to that of respiring mitochondria. Fermenting samples had a very different Fe-distribution. Nearly 40 % of the iron in respiring mitochondria was present in respiratory complexes including cytochrome c, cytochrome bc1, succinate dehydrogenase, and cytochrome c oxidase. Fermenting mitochondria contain an Fe-ome dominated by non-protein centers. Approximately 80 % of the Fe was present as a combination of nonheme HS Fe2+, nonheme Fe3+ and Fe3+ nanoparticles. These centers were present in roughly equal amounts. The remaining 20 % of the Fe was present as respiratory complexes which have concentrations ~ 1/2 to 1/3 that of respiring mitochondria. A model is presented in which the nonheme HS Fe2+ species serves as a feedstock for Fe/S and heme biosynthesis. When the cell is growing on respiring media, this metabolic reservoir diminishes as respiratory complexes are constantly synthesized. Under fermentative growth, the metabolic pool increases due to the reduced demand for respiration-related prosthetic groups

A Composite of Multiple Signals Distinguishes Causal Variants in Regions of Positive Selection

The human genome contains hundreds of regions whose patterns of genetic variation indicate recent positive natural selection, yet for most the underlying gene and the advantageous mutation remain unknown. We developed a method, composite of multiple signals (CMS), that combines tests for multiple signals of selection and increases resolution by up to 100-fold. By applying CMS to candidate regions from the International Haplotype Map, we localized population-specific selective signals to 55 kilobases (median), identifying known and novel causal variants. CMS can not just identify individual loci but implicates precise variants selected by evolution.Organismic and Evolutionary BiologyOther Research Uni

Yeast mitochondrial RNase P, RNase Z and the RNA degradosome are part of a stable supercomplex

Initial steps in the synthesis of functional tRNAs require 5′- and 3′-processing of precursor tRNAs (pre-tRNAs), which in yeast mitochondria are achieved by two endonucleases, RNase P and RNase Z. In this study, using a combination of detergent-free Blue Native Gel Electrophoresis, proteomics and in vitro testing of pre-tRNA maturation, we reveal the physical association of these plus other mitochondrial activities in a large, stable complex of 136 proteins. It contains a total of seven proteins involved in RNA processing including RNase P and RNase Z, five out of six subunits of the mitochondrial RNA degradosome, components of the fatty acid synthesis pathway, translation, metabolism and protein folding. At the RNA level, there are the small and large rRNA subunits and RNase P RNA. Surprisingly, this complex is absent in an oar1Δ deletion mutant of the type II fatty acid synthesis pathway, supporting a recently published functional link between pre-tRNA processing and the FAS II pathway—apparently by integration into a large complex as we demonstrate here. Finally, the question of mt-RNase P localization within mitochondria was investigated, by GFP-tracing of a known protein subunit (Rpm2p). We find that about equal fractions of RNase P are soluble versus membrane-attached

Induction of Biogenic Magnetization and Redox Control by a Component of the Target of Rapamycin Complex 1 Signaling Pathway

Most organisms are simply diamagnetic, while magnetotactic bacteria and migratory animals are among organisms that exploit magnetism. Biogenic magnetization not only is of fundamental interest, but also has industrial potential. However, the key factor(s) that enable biogenic magnetization in coordination with other cellular functions and metabolism remain unknown. To address the requirements for induction and the application of synthetic bio-magnetism, we explored the creation of magnetism in a simple model organism. Cell magnetization was first observed by attraction towards a magnet when normally diamagnetic yeast Saccharomyces cerevisiae were grown with ferric citrate. The magnetization was further enhanced by genetic modification of iron homeostasis and introduction of ferritin. The acquired magnetizable properties enabled the cells to be attracted to a magnet, and be trapped by a magnetic column. Superconducting quantum interference device (SQUID) magnetometry confirmed and quantitatively characterized the acquired paramagnetism. Electron microscopy and energy-dispersive X-ray spectroscopy showed electron-dense iron-containing aggregates within the magnetized cells. Magnetization-based screening of gene knockouts identified Tco89p, a component of TORC1 (Target of rapamycin complex 1), as important for magnetization; loss of TCO89 and treatment with rapamycin reduced magnetization in a TCO89-dependent manner. The TCO89 expression level positively correlated with magnetization, enabling inducible magnetization. Several carbon metabolism genes were also shown to affect magnetization. Redox mediators indicated that TCO89 alters the intracellular redox to an oxidized state in a dose-dependent manner. Taken together, we demonstrated that synthetic induction of magnetization is possible and that the key factors are local redox control through carbon metabolism and iron supply

Proton inhibition of unitary currents of vanilloid receptors

Protons, which are released during inflammation and injury, regulate many receptors and ion channels involved in pain transduction, including capsaicin channels (transient receptor potential vanilloid receptors 1). Whereas extracellular acidification both sensitizes and directly activates the channel, it also causes concomitant reduction of the unitary current amplitudes. Here, we investigate the mechanisms and molecular basis of this inhibitory effect of protons on channel conductance. Single-channel recordings showed that the unitary current amplitudes decreased with extracellular pH in a dose-dependent manner, consistent with a model in which protons bind to a site within the channel with an apparent pKa of ∼6. The inhibition was voltage dependent, ∼65% at −60 mV and 37% at +60 mV when pH was reduced from 7.4 to 5.5. The unitary current amplitudes reached saturation at [K+] ≥ 1 M, and notably the maximum amplitudes did not converge with different pHs, inconsistent with a blockade model based on surface charge screening or competitive inhibition of permeating ions. Mutagenesis experiments uncovered two acidic residues critical for proton inhibition, one located at the pore entrance and the other on the pore helix. Based on homology to the KcsA structure, the two acidic residues, along with another basic residue also on the pore helix, could form a triad interacting with each other through extensive hydrogen bonds and electrostatic contacts, suggesting that protons may mediate the interactions between the selectivity filter and pore helix, thereby altering the local structure in the filter region and consequently the conductance of the channel

Elevated Levels of the Vesicular Monoamine Transporter and a Novel Repetitive Behavior in the Drosophila Model of Fragile X Syndrome

Fragile X Syndrome (FXS) is characterized by mental impairment and autism in humans, and it often features hyperactivity and repetitive behaviors. The mechanisms for the disease, however, remain poorly understood. Here we report that the dfmr1 mutant in the Drosophila model of FXS grooms excessively, which may be regulated differentially by two signaling pathways. Blocking metabotropic glutamate receptor signaling enhances grooming in dfmr1 mutant flies, whereas blocking the vesicular monoamine transporter (VMAT) suppresses excessive grooming. dfmr1 mutant flies also exhibit elevated levels of VMAT mRNA and protein. These results suggest that enhanced monoamine signaling correlates with repetitive behaviors and hyperactivity associated with FXS

Physical Activity Patterns of the Spanish Population Are Mostly Determined by Sex and Age: Findings in the ANIBES Study

Background Representative data for the Spanish population regarding physical activity (PA) behaviors are scarce and seldom comparable due to methodological inconsistencies. Aim Our objectives were to describe the PA behavior by means of the standardized self-reported International Physical Activity Questionnaire (IPAQ) and to know the proportion of the Spanish population meeting and not meeting international PA recommendations. Material and Methods PA was assessed using the IPAQ in a representative sample of 2285 individuals (males, 50.4%) aged 9–75 years and living in municipalities of at least 2,000 inhabitants. Data were analyzed according to: age groups 9–12, 13–17, 18–64, and 65–75 years; sex; geographical distribution; locality size and educational levels. Results Mean total PA was 868.8±660.9 min/wk, mean vigorous PA 146.4±254.1 min/wk, and mean moderate PA 398.1±408.0 min/wk, showing significant differences between sexes (p<0.05). Children performed higher moderate-vigorous PA than adolescents and seniors (p<0.05), and adults than adolescents and seniors (p<0.05). Compared to recommendations, 36.2%of adults performed <150 min/week of moderate PA, 65.4% <75 min/week of vigorous PA and 27.0%did not perform any PA at all, presenting significant differences between sexes (p<0.05). A total of 55.4%of children and adolescents performed less than 420 min/week of MVPA, being higher in the later (62.6%) than in the former (48.4%). Highest non-compliance was observed in adolescent females (86.5%). Conclusion Sex and age are the main influencing factors on PA in the Spanish population. Males engage in more vigorous and light PA overall, whereas females perform more moderate PA. PA behavior differs between age groups and no clear lineal increase with age could be observed. Twenty-seven percent of adults and 55.4% of children and adolescents do not meet international PA recommendations. Identified target groups should be addressed to increase PA in the Spanish populationCoca-Cola Iberia through Spanish Nutrition Foundation (FEN)Coca-Cola Iberi

Argonaute2 Suppresses Drosophila Fragile X Expression Preventing Neurogenesis and Oogenesis Defects

Fragile X Syndrome is caused by the silencing of the Fragile X Mental Retardation gene (FMR1). Regulating dosage of FMR1 levels is critical for proper development and function of the nervous system and germ line, but the pathways responsible for maintaining normal expression levels are less clearly defined. Loss of Drosophila Fragile X protein (dFMR1) causes several behavioral and developmental defects in the fly, many of which are analogous to those seen in Fragile X patients. Over-expression of dFMR1 also causes specific neuronal and behavioral abnormalities. We have found that Argonaute2 (Ago2), the core component of the small interfering RNA (siRNA) pathway, regulates dfmr1 expression. Previously, the relationship between dFMR1 and Ago2 was defined by their physical interaction and co-regulation of downstream targets. We have found that Ago2 and dFMR1 are also connected through a regulatory relationship. Ago2 mediated repression of dFMR1 prevents axon growth and branching defects of the Drosophila neuromuscular junction (NMJ). Consequently, the neurogenesis defects in larvae mutant for both dfmr1 and Ago2 mirror those in dfmr1 null mutants. The Ago2 null phenotype at the NMJ is rescued in animals carrying an Ago2 genomic rescue construct. However, animals carrying a mutant Ago2 allele that produces Ago2 with significantly reduced endoribonuclease catalytic activity are normal with respect to the NMJ phenotypes examined. dFMR1 regulation by Ago2 is also observed in the germ line causing a multiple oocyte in a single egg chamber mutant phenotype. We have identified Ago2 as a regulator of dfmr1 expression and have clarified an important developmental role for Ago2 in the nervous system and germ line that requires dfmr1 function

Research into the Health Benefits of Sprint Interval Training Should Focus on Protocols with Fewer and Shorter Sprints

Over the past decade, it has been convincingly shown that regularly performing repeated brief supramaximal cycle sprints (sprint interval training [SIT]) is associated with aerobic adaptations and health benefits similar to or greater than with moderate-intensity continuous training (MICT). SIT is often promoted as a time-efficient exercise strategy, but the most commonly studied SIT protocol (4–6 repeated 30-s Wingate sprints with 4 min recovery, here referred to as ‘classic’ SIT) takes up to approximately 30 min per session. Combined with high associated perceived exertion, this makes classic SIT unsuitable as an alternative/adjunct to current exercise recommendations involving MICT. However, there are no indications that the design of the classic SIT protocol has been based on considerations regarding the lowest number or shortest duration of sprints to optimise time efficiency while retaining the associated health benefits. In recent years, studies have shown that novel SIT protocols with both fewer and shorter sprints are efficacious at improving important risk factors of noncommunicable diseases in sedentary individuals, and provide health benefits that are no worse than those associated with classic SIT. These shorter/easier protocols have the potential to remove many of the common barriers to exercise in the general population. Thus, based on the evidence summarised in this current opinion paper, we propose that there is a need for a fundamental change in focus in SIT research in order to move away from further characterising the classic SIT protocol and towards establishing acceptable and effective protocols that involve minimal sprint durations and repetitions

Astrocyte activation and reactive gliosis : A new target in stroke?

Stroke is an acute insult to the central nervous system (CNS) that triggers a sequence of responses in the acute, subacute as well as later stages, with prominent involvement of astrocytes. Astrocyte activation and reactive gliosis in the acute stage of stroke limit the tissue damage and contribute to the restoration of homeostasis. Astrocytes also control many aspects of neural plasticity that is the basis for functional recovery. Here, we discuss the concept of intermediate filaments (nanofilaments) and the complement system as two handles on the astrocyte responses to injury that both present attractive opportunities for novel treatment strategies modulating astrocyte functions and reactive gliosis.Peer reviewe