l-Lactate generates hydrogen peroxide in purified rat liver mitochondria due to the putative l-lactate oxidase localized in the intermembrane space

AbstractIn order to ascertain whether and how mitochondria can produce hydrogen peroxide (H2O2) as a result of l-lactate addition, we monitored H2O2 generation in rat liver mitochondria and in submitochondrial fractions free of peroxisomal and cytosolic contamination. We found that H2O2 is produced independently on the respiratory chain with 1:1 stoichiometry with pyruvate, due to a putative flavine-dependent l-lactate oxidase restricted to the intermembrane space. The l-lactate oxidase reaction shows a hyperbolic dependence on l-lactate concentration and is inhibited by NAD+ in a competitive manner, being the enzyme different from the l-lactate dehydrogenase isoenzymes as shown by their pH profiles

Riboflavin uptake and FAD synthesis in Saccharomyces cerevisiae mitochondria: Involvement of the Flx1p carrier in FAD export

We have studied the functional steps by which Saccharomyces cerevisiae mitochondria can synthesize FAD from cytosolic riboflavin (Rf). Riboflavin uptake into mitochondria took place via a mechanism that is consistent with the existence of (at least two) carrier systems. FAD was synthesized inside mitochondria by a mitochondrial FAD synthetase (EC 2.7.7.2), and it was exported into the cytosol via an export system that was inhibited by lumiflavin, and which was different from the riboflavin uptake system. To understand the role of the putative mitochondrial FAD carrier, Flx1p, in this pathway, an flx1Δ mutant strain was constructed. Coupled mitochondria isolated from flx1Δ mutant cells were compared with wild-type mitochondria with respect to the capability to take up Rf, to synthesize FAD from it, and to export FAD into the extramitochondrial phase. Mitochondria isolated from flx1Δ mutant cells specifically lost the ability to export FAD, but did not lose the ability to take up Rf, FAD, or FMN and to synthesize FAD from Rf. Hence, Flx1p is proposed to be the mitochondrial FAD export carrier. Moreover, deletion of the FLX1 gene resulted in a specific reduction of the activities of mitochondrial lipoamide dehydrogenase and succinate dehydrogenase, which are FAD-binding enzymes. For the flavoprotein subunit of succinate dehydrogenase we could demonstrate that this was not due to a changed level of mitochondrial FAD or to a change in the degree of flavinylation of the protein. Instead, the amount of the flavoprotein subunit of succinate dehydrogenase was strongly reduced, indicating an additional regulatory role for Flx1p in protein synthesis or degradation

Silver staining (Campbell-Switzer) of neuronal α-synuclein assemblies induced by multiple system atrophy and Parkinson's disease brain extracts in transgenic mice.

Synucleinopathies [Parkinson's disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy (MSA)] share filamentous α-synuclein assemblies in nerve cells and glial cells. We compared the abilities of brain extracts from MSA and PD patients to induce neuronal α-synuclein assembly and neurodegeneration following intracerebral injection in heterozygous mice transgenic for human mutant A53T α-synuclein. MSA extracts were more potent than PD extracts in inducing α-synuclein assembly and in causing neurodegeneration. MSA assemblies were Campbell-Switzer- and Gallyas-silver-positive, whereas PD assemblies were only Campbell-Switzer-positive, in confirmation of previous findings. However, induced α-synuclein inclusions were invariably Campbell-Switzer-positive and Gallyas-negative, irrespective of whether MSA or PD brain extracts were injected. The α-synuclein inclusions of non-injected homozygous mice transgenic for A53T α-synuclein were also Campbell-Switzer-positive and Gallyas-negative. These findings demonstrate that transgene expression and its intracellular environment dominated over the silver staining properties of the conformers of assembled α-synuclein

Bidirectional Control between Cholesterol Shuttle and Purine Signal at the Central Nervous System

: Recent studies have highlighted the mechanisms controlling the formation of cerebral cholesterol, which is synthesized in situ primarily by astrocytes, where it is loaded onto apolipoproteins and delivered to neurons and oligodendrocytes through interactions with specific lipoprotein receptors. The "cholesterol shuttle" is influenced by numerous proteins or carbohydrates, which mainly modulate the lipoprotein receptor activity, function and signaling. These molecules, provided with enzymatic/proteolytic activity leading to the formation of peptide fragments of different sizes and specific sequences, could be also responsible for machinery malfunctions, which are associated with neurological, neurodegenerative and neurodevelopmental disorders. In this context, we have pointed out that purines, ancestral molecules acting as signal molecules and neuromodulators at the central nervous system, can influence the homeostatic machinery of the cerebral cholesterol turnover and vice versa. Evidence gathered so far indicates that purine receptors, mainly the subtypes P2Y2, P2X7 and A2A, are involved in the pathogenesis of neurodegenerative diseases, such as Alzheimer's and Niemann-Pick C diseases, by controlling the brain cholesterol homeostasis; in addition, alterations in cholesterol turnover can hinder the purine receptor function. Although the precise mechanisms of these interactions are currently poorly understood, the results here collected on cholesterol-purine reciprocal control could hopefully promote further research

Proteasome function is required for activation of programmed cell death in heat shocked tobacco Bright-Yellow 2 cells

AbstractTo find out whether and how proteasome is involved in plant programmed cell death (PCD) we measured proteasome function in tobacco cells undergoing PCD as a result of heat shock (HS-PCD). Reactive oxygen species (ROS) production, cytochrome c levels and caspase-3-like protease activation were also measured in the absence or presence of MG132, a proteasome inhibitor. We show that proteasome activation occurs in early phase of HS-PCD upstream of the caspase-like proteases activation; moreover inhibition of proteasome function by MG132 results in prevention of PCD perhaps due to the prevention of ROS production, cytochrome c release and caspase-3-like protease activation

Intercomparison Exercise for Heavy Metals in PM10

The Joint Research Centre (JRC) has carried out an Intercomparison Exercise (IE) for the determination of heavy metals in particulate matter (PM10). The IE focussed on Lead (Pb), Arsenic (As), Nickel (Ni) and Cadmium (Cd), the heavy metals regulated by the 1st and 4th Daughter Directives for Air Pollution. Copper (Cu), Chromium (Cr) and Zinc (Zn), the elements included in the EMEP programme together with Aluminium (Al), Cobalt (Co), Iron (Fe), Manganese (Mn) and Vanadium (V) were also tested. Fourteen Laboratories, generally members of the Network of Air Quality Reference Laboratories (AQUILA), participated in the IE. The participants mainly used microwave digestion with nitric acid and hydrogen peroxide and Inductively Coupled Plasma Mass Spectrometry (ICP-MS) or Graphite Furnace Atomic Absorption Spectrometry (GF-AAS) for analysis as recommended in the reference method (EN 14902). However, a few participants used other methods: Energy Dispersive X-ray Fluorescence (EDXRF), Atomic Emission Spectrometry (ICP-AES) and Voltammetry for analysis and vaporisation on hot plate before microwave digestion, Soxhlet extraction, high pressure or cold Hydrogen Fluoride methods for digestion. Each participant received 5 samples to be analysed: a liquid sample prepared by dilution of a Certified Reference Material (CRM), a solution of a dust CRM sample digested by the JRC13F, a sub-sample of a dust CRM that each participating laboratory had to digest and analyse, a solution prepared by JRC after digestion of an exposed filter and a pair of filters (one blank filter and one exposed filter) to be digested and analysed by each participant. For 89 % of all types of samples, the DQOs of the 1st and 4th European Directives (uncertainty of 25 % for Pb and 40 % for As, Cd and Ni) were met. All together, this is a very good score. The best results were obtained for the liquid CRM, dust CRM digested by JRC, dust CRM and filter digested by JRC with 92, 90, 96 and 93 % of DQOs being met, respectively. It was found that the DQOs were not met if the difference of acidity between test samples and participant calibration standards was high. Conversely, only 76 % of DQOs were met for the filter to be digested by each participant with (about 85 % for Cd and Ni, 73/64 % for Pb and As, the most difficult element to determine). The worst results were associated with special events: explosion in microwave oven during digestion for two participants, a wrong dilution factor used by one participant and a huge contamination in the blank filter for another participant. Among the two explosions, one of them was probably the effect of a lack of temperature control in the digestion vessel. For the other explosion, the microwave digestion and the digestion program advised by EN 14902 is to be questioned. Moreover, satisfactory results were obtained using Soxhlet extraction, high pressure method and cold Hydrogen Fluoride digestion methods which are not presented in EN 14902. The DQOs of As and Cd could not be met with EDXRF whose limit of detection was too high for these two elements and for Cd using Voltammetry which suffered a strong interference for this element. Regarding the methods of analysis, apart the points mentioned just before about EDXRF and Voltammetry, good results were observed using ICP-OES for Cd, Ni and Pb. A few discrepancies were also registered for GF-AAS and ICP-MS but they were created by the special events or acidity problem mentioned before. This shows that even though GF-AAS and ICP-MS are found suitable, the implementation by each participant may be responsible for important mistakes.JRC.H.4-Transport and air qualit

Common and rare variant association analyses in amyotrophic lateral sclerosis identify 15 risk loci with distinct genetic architectures and neuron-specific biology

A cross-ancestry genome-wide association meta-analysis of amyotrophic lateral sclerosis (ALS) including 29,612 patients with ALS and 122,656 controls identifies 15 risk loci with distinct genetic architectures and neuron-specific biology. Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease with a lifetime risk of one in 350 people and an unmet need for disease-modifying therapies. We conducted a cross-ancestry genome-wide association study (GWAS) including 29,612 patients with ALS and 122,656 controls, which identified 15 risk loci. When combined with 8,953 individuals with whole-genome sequencing (6,538 patients, 2,415 controls) and a large cortex-derived expression quantitative trait locus (eQTL) dataset (MetaBrain), analyses revealed locus-specific genetic architectures in which we prioritized genes either through rare variants, short tandem repeats or regulatory effects. ALS-associated risk loci were shared with multiple traits within the neurodegenerative spectrum but with distinct enrichment patterns across brain regions and cell types. Of the environmental and lifestyle risk factors obtained from the literature, Mendelian randomization analyses indicated a causal role for high cholesterol levels. The combination of all ALS-associated signals reveals a role for perturbations in vesicle-mediated transport and autophagy and provides evidence for cell-autonomous disease initiation in glutamatergic neurons