136 research outputs found
Bench-to-bedside review : targeting antioxidants to mitochondria in sepsis
Peer reviewedPublisher PD
Analysis of the Aspergillus fumigatus Proteome Reveals Metabolic Changes and the Activation of the Pseurotin A Biosynthesis Gene Cluster in Response to Hypoxia
The mold Aspergillus fumigatus is the most important airborne fungal pathogen. Adaptation to hypoxia represents an important virulence attribute for A. fumigatus. Therefore, we aimed at obtaining a comprehensive overview about this process on the proteome level. To ensure highly reproducible growth conditions, an oxygen-controlled, glucose-limited chemostat cultivation was established. Two-dimensional gel electrophoresis analysis of mycelial and mitochondrial proteins as well as two-dimensional Blue Native/SDS-gel separation of mitochondrial membrane proteins led to the identification of 117 proteins with an altered abundance under hypoxic in comparison to normoxic conditions. Hypoxia induced an increased activity of glycolysis, the TCA-cycle, respiration, and amino acid metabolism. Consistently, the cellular contents in heme, iron, copper, and zinc increased. Furthermore, hypoxia induced biosynthesis of the secondary metabolite pseurotin A as demonstrated at proteomic, transcriptional, and metabolite levels. The observed and so far not reported stimulation of the biosynthesis of a secondary metabolite by oxygen depletion may also affect the survival of A. fumigatus in hypoxic niches of the human host. Among the proteins so far not implicated in hypoxia adaptation, an NO-detoxifying flavohemoprotein was one of the most highly up-regulated proteins which indicates a link between hypoxia and the generation of nitrosative stress in A. fumigatus
Prediction of Muscle Energy States at Low Metabolic Rates Requires Feedback Control of Mitochondrial Respiratory Chain Activity by Inorganic Phosphate
The regulation of the 100-fold dynamic range of mitochondrial ATP synthesis flux in skeletal muscle was investigated. Hypotheses of key control mechanisms were included in a biophysical model of oxidative phosphorylation and tested against metabolite dynamics recorded by 31P nuclear magnetic resonance spectroscopy (31P MRS). Simulations of the initial model featuring only ADP and Pi feedback control of flux failed in reproducing the experimentally sampled relation between myoplasmic free energy of ATP hydrolysis (ΔGp = ΔGpo′+RT ln ([ADP][Pi]/[ATP]) and the rate of mitochondrial ATP synthesis at low fluxes (<0.2 mM/s). Model analyses including Monte Carlo simulation approaches and metabolic control analysis (MCA) showed that this problem could not be amended by model re-parameterization, but instead required reformulation of ADP and Pi feedback control or introduction of additional control mechanisms (feed forward activation), specifically at respiratory Complex III. Both hypotheses were implemented and tested against time course data of phosphocreatine (PCr), Pi and ATP dynamics during post-exercise recovery and validation data obtained by 31P MRS of sedentary subjects and track athletes. The results rejected the hypothesis of regulation by feed forward activation. Instead, it was concluded that feedback control of respiratory chain complexes by inorganic phosphate is essential to explain the regulation of mitochondrial ATP synthesis flux in skeletal muscle throughout its full dynamic range
Study of a region on yeast chromosome XIII that complements pet G199 mutants (COX7) and carries a new non-essential gene
Reactive oxygen species contribute to dysfunction of bone marrow hematopoietic stem cells in aged C57BL/6 J mice
Cu<sup>2+</sup> Binds to Phosphatidylethanolamine and Increases Oxidation in Lipid Membranes
Herein,
we demonstrate that Cu<sup>2+</sup> binds bivalently to
phosphatidylethanolamine (PE), the second most abundant lipid in mammalian
cells. The apparent equilibrium dissociation constant, <i>K</i><sub>DApp</sub>, for the Cu<sup>2+</sup>–PE complex at physiological
pH is approximately 2 μM and is insensitive to the concentration
of PE in the membrane. By contrast, at pH 10.0, where PE lipids bear
a negative charge, <i>K</i><sub>DApp</sub> decreases with
increasing PE content and has a value of 150 nM for bilayers containing
70 mol % PE. The oxidation of double bonds in PE-containing bilayers
can be monitored in the presence of Cu<sup>2+</sup>. Strikingly, it
was found that the oxidation rate is 8.2 times faster at pH 7.4 for
bilayers containing 70 mol % PE than for pure phosphatidylcholine
(PC) bilayers upon exposure of both to 70 μM Cu<sup>2+</sup> and 10 mM hydrogen peroxide. The rate of oxidation increases linearly
with the PE content in the membrane. These results may help explain
the high level of lipid oxidation in PE-containing membranes for neurodegenerative
diseases and autism where the Cu<sup>2+</sup> concentration in the
body is abnormally high
Supported Lipid Bilayers with Phosphatidylethanolamine as the Major Component
Phosphatidylethanolamine
(PE) is notoriously difficult to incorporate
into model membrane systems, such as fluid supported lipid bilayers
(SLBs), at high concentrations because of its intrinsic negative curvature.
Using fluorescence-based techniques, we demonstrate that having fewer
sites of unsaturation in the lipid tails leads to high-quality SLBs
because these lipids help to minimize the curvature. Moreover, shorter
saturated chains can help maintain the membranes in the fluid phase.
Using these two guidelines, we find that up to 70 mol % PE can be
incorporated into SLBs at room temperature and up to 90 mol % PE can
be incorporated at 37 °C. Curiously, conditions under which three-dimensional
tubules project outward from the planar surface as well as conditions
under which domain formation occurs can be found. We have employed
these model membrane systems to explore the ability of Ni<sup>2+</sup> to bind to PE. It was found that this transition metal ion binds
1000-fold tighter to PE than to phosphatidylcholine lipids. In the
future, this platform could be exploited to monitor the binding of
other transition metal ions or the binding of antimicrobial peptides.
It could also be employed to explore the physical properties of PE-containing
membranes, such as phase domain behavior and intermolecular hydrogen
bonding
Supported Lipid Bilayers with Phosphatidylethanolamine as the Major Component
Phosphatidylethanolamine
(PE) is notoriously difficult to incorporate
into model membrane systems, such as fluid supported lipid bilayers
(SLBs), at high concentrations because of its intrinsic negative curvature.
Using fluorescence-based techniques, we demonstrate that having fewer
sites of unsaturation in the lipid tails leads to high-quality SLBs
because these lipids help to minimize the curvature. Moreover, shorter
saturated chains can help maintain the membranes in the fluid phase.
Using these two guidelines, we find that up to 70 mol % PE can be
incorporated into SLBs at room temperature and up to 90 mol % PE can
be incorporated at 37 °C. Curiously, conditions under which three-dimensional
tubules project outward from the planar surface as well as conditions
under which domain formation occurs can be found. We have employed
these model membrane systems to explore the ability of Ni<sup>2+</sup> to bind to PE. It was found that this transition metal ion binds
1000-fold tighter to PE than to phosphatidylcholine lipids. In the
future, this platform could be exploited to monitor the binding of
other transition metal ions or the binding of antimicrobial peptides.
It could also be employed to explore the physical properties of PE-containing
membranes, such as phase domain behavior and intermolecular hydrogen
bonding
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