Potassium channel-oxidative phosphorylation relationship in durum wheat mitochondria from control and hyperosmotic-stressed seedlings

Durum wheat mitochondria (DWM) possess an ATP-inhibited K(+) channel, the plant mitoK(ATP) (PmitoK(ATP) ), which is activated under environmental stress to control mitochondrial ROS production. To do this, PmitoK(ATP) collapses membrane potential (ΔΨ), thus suggesting mitochondrial uncoupling. We tested this point by studying oxidative phosphorylation (OXPHOS) in DWM purified from control seedlings and from seedlings subjected both to severe mannitol and NaCl stress. In severely-stressed DWM, the ATP synthesis via OXPHOS, continuously monitored by a spectrophotometric assay, was about 90% inhibited when the PmitoK(ATP) was activated by KCl. Contrarily, in control DWM, although PmitoK(ATP) collapsed ΔΨ, ATP synthesis, as well as coupling [respiratory control (RC) ratio and ratio between phosphorylated ADP and reduced oxygen (ADP/O)] checked by oxygen uptake experiments, were unaffected. We suggest that PmitoK(ATP) may play an important defensive role at the onset of the environmental/oxidative stress by preserving energy in a crucial moment for cell and mitochondrial bioenergetics. Consistently, under moderate mannitol stress, miming an early stress condition, the channel may efficiently control reactive oxygen species (ROS) generation (about 35-fold from fully open to closed state) without impairing ATP synthesis. Anyway, if the stress significantly proceeds, the PmitoK(ATP) becomes fully activated by decrease of ATP concentration (25-40%) and increase of activators [free fatty acids (FFAs) and superoxide anion], thus impairing ATP synthesis.     

Functional magnetic resonance imaging reveals different neural substrates for the effects of orexin-1 and orexin-2 receptor antagonists

Orexins are neuro-modulatory peptides involved in the control of diverse physiological functions through interaction with two receptors, orexin-1 (OX1R) and orexin-2 (OX2R). Recent evidence in pre-clinical models points toward a putative dichotomic role of the two receptors, with OX2R predominantly involved in the regulation of the sleep/wake cycle and arousal, and the OX1R being more specifically involved in reward processing and motivated behaviour. However, the specific neural substrates underlying these distinct processes in the rat brain remain to be elucidated. Here we used functional magnetic resonance imaging (fMRI) in the rat to map the modulatory effect of selective OXR blockade on the functional response produced by D-amphetamine, a psychostimulant and arousing drug that stimulates orexigenic activity. OXR blockade was produced by GSK1059865 and JNJ1037049, two novel OX1R and OX2R antagonists with unprecedented selectivity at the counter receptor type. Both drugs inhibited the functional response to D-amphetamine albeit with distinct neuroanatomical patterns: GSK1059865 focally modulated functional responses in striatal terminals, whereas JNJ1037049 induced a widespread pattern of attenuation characterised by a prominent cortical involvement. At the same doses tested in the fMRI study, JNJ1037049 exhibited robust hypnotic properties, while GSK1059865 failed to display significant sleep-promoting effects, but significantly reduced drug-seeking behaviour in cocaine-induced conditioned place preference. Collectively, these findings highlight an essential contribution of the OX2R in modulating cortical activity and arousal, an effect that is consistent with the robust hypnotic effect exhibited by JNJ1037049. The subcortical and striatal pattern observed with GSK1059865 represent a possible neurofunctional correlate for the modulatory role of OX1R in controlling reward-processing and goal-oriented behaviours in the rat.     

Ligand binding to truncated hemoglobin N from Mycobacterium tuberculosis is strongly modulated by the interplay between the distal heme pocket residues and internal water

The survival of Mycobacterium tuberculosis requires detoxification of host *NO. Oxygenated Mycobacterium tuberculosis truncated hemoglobin N catalyzes the rapid oxidation of nitric oxide to innocuous nitrate with a second-order rate constant (k'(NOD) approximately 745 x 10(6) m(-1) x s(-1)), which is approximately 15-fold faster than the reaction of horse heart myoglobin. We ask what aspects of structure and/or dynamics give rise to this enhanced reactivity. A first step is to expose what controls ligand/substrate binding to the heme. We present evidence that the main barrier to ligand binding to deoxy-truncated hemoglobin N (deoxy-trHbN) is the displacement of a distal cavity water molecule, which is mainly stabilized by residue Tyr(B10) but not coordinated to the heme iron. As observed in the Tyr(B10)/Gln(E11) apolar mutants, once this kinetic barrier is lowered, CO and O(2) binding is very rapid with rates approaching 1-2 x 10(9) m(-1) x s(-1). These large values almost certainly represent the upper limit for ligand binding to a heme protein and also indicate that the iron atom in trHbN is highly reactive. Kinetic measurements on the photoproduct of the *NO derivative of met-trHbN, where both the *NO and water can be directly followed, revealed that water rebinding is quite fast (approximately 1.49 x 10(8) s(-1)) and is responsible for the low geminate yield in trHbN. Molecular dynamics simulations, performed with trHbN and its distal mutants, indicated that in the absence of a distal water molecule, ligand access to the heme iron is not hindered. They also showed that a water molecule is stabilized next to the heme iron through hydrogen-bonding with Tyr(B10) and Gln(E11).     

Pyridoxal phosphate-sensitized photoinactivation of glutamate decarboxylase from Clostridium perfringens

1. l-Glutamate decarboxylase (EC 4.1.1.15) from Clostridium perfringens was inactivated by exposure to visible light at pH6.2. 2. Inactivation does not occur at pH4.6 or in the absence of bound pyridoxal phosphate. 3. On prolonged photo-oxidation six histidine residues per molecule of enzyme were destroyed. 4. The loss of six cysteine residues per molecule occurred both in irradiated samples and in controls oxygenated in the dark. 5. This dark-oxidation of cysteine residues is apparently required before the photo-oxidation process. 6. The absorbance, fluorescence and circular-dichroism properties of the enzyme as well as its elution volume during Sephadex gel-filtration were unaffected by prolonged irradiation. 7. However, an apparently homogeneous product of photo-oxidation could be separated from the control enzyme by ion-exchange chromatography. 8. The K(m) for l-glutamate was unchanged in an irradiated sample retaining 22% of control activity. 9. These data and the catalytic role of imidazole residues at the active sites of amino acid decarboxylases are discussed.     

Establishment of mammalian cell lines containing multiple nonsense mutations and functional suppressor tRNA genes

We describe the generation of mammalian cell lines carrying amber suppressor genes. Nonsense mutants in the herpes simplex virus thymidine kinase (HSV tk) gene, the Escherichia coli xanthine-guanine phosphoribosyl transferase (Eco-gpt) gene and the aminoglycoside 3′ phosphotransferase gene of the Tn5 transposon (NPT-II) were isolated and characterized. Each gene was engineered with the appropriate control signals to allow expression in both E. coli and mammalian cells. Expression in E. coli made possible the use of well developed bacterial and phage genetic manipulations to isolate and characterize the nonsense mutants. Once characterized, the nonsense mutants were transferred into mammalian cells by microinjection and used, in turn, to select for amber suppressor genes. Xenopus laevis amber suppressor genes, prepared by site-specific mutagenesis of a normal X. laevis tRNA gene, were microinjected into the above cell lines and selected for the expression of one or more of the amber mutant gene products. The resulting cell lines, containing functional amber suppressor genes, are stable and exhibit normal growth rates.     

NLRP3 inflammasome suppression improves longevity and prevents cardiac aging in male mice

While NLRP3‐inflammasome has been implicated in cardiovascular diseases, its role in physiological cardiac aging is largely unknown. During aging, many alterations occur in the organism, which are associated with progressive impairment of metabolic pathways related to insulin resistance, autophagy dysfunction, and inflammation. Here, we investigated the molecular mechanisms through which NLRP3 inhibition may attenuate cardiac aging. Ablation of NLRP3‐inflammasome protected mice from age‐related increased insulin sensitivity, reduced IGF‐1 and leptin/adiponectin ratio levels, and reduced cardiac damage with protection of the prolongation of the age‐dependent PR interval, which is associated with atrial fibrillation by cardiovascular aging and reduced telomere shortening. Furthermore, old NLRP3 KO mice showed an inhibition of the PI3K/AKT/mTOR pathway and autophagy improvement, compared with old wild mice and preserved Nampt‐mediated NAD⁺ levels with increased SIRT1 protein expression. These findings suggest that suppression of NLRP3 prevented many age‐associated changes in the heart, preserved cardiac function of aged mice and increased lifespan.     

Neuron–Astroglial Interactions in Cell-Fate Commitment and Maturation in the Central Nervous System

Neuron–astroglia interactions play a key role in several events of brain development, such as neuronal generation, migration, survival, and differentiation; axonal growth; and synapse formation and function. While there is compelling evidence of the effects of astrocyte factors on neurons, their effects on astrocytes have not been fully determined. In this review, we will focus on the role of neurons in astrocyte generation and maturation. Further, we highlight the great heterogeneity and diversity of astroglial and neural progenitors such as radial glia cells, and discuss the importance of the variety of cellular interactions in controlling the structural and functional organization of the brain. Finally, we present recent data on a new role of astrocytes in neuronal maturation, as mediators of the action of biolipids in the cerebral cortex. We will argue that the functional architecture of the brain depends on an intimate neuron-glia partnership, by briefly discussing the emerging view of how neuron-astrocyte dysfunctions might be associated with neurodegenerative diseases and neurological disorders.     

Cats under cover: Habitat models indicate a high dependency on woodlands by Atlantic Forest felids

Four Neotropical small and medium felids—the ocelot, jaguarundi, margay, and southern tiger cat—have overlapping geographic distributions in the endangered Atlantic Forest. Local studies show that these felids avoid areas with high human impact, but the three smaller ones use human‐modified areas more frequently than do ocelots. To understand how landscape changes affect the habitat distribution of these four felids in the Atlantic Forest of Argentina, we used maximum entropy models to analyze the effect of environmental and anthropogenic factors. We estimated niche breadth and overlap among these felids. The conversion of the native forest to land uses without trees was the most important variable that determined the habitat distribution of the four species. For all four species, the optimal habitat covered about 1/3 of the study area and corresponds mainly to the native forest areas. Nearly 50% of these areas had some level of protection. The niche width was higher for the small felids than for ocelots. Niche overlap was high for all species pairs, but higher among the small felids and lower for each of these with the ocelot. The four felids were negatively affected by native forest loss, with ocelots being more sensitive than the smaller felids. The conversion of unprotected forest areas to other types of land uses would imply a greater habitat loss for these felids. The protection of current remnants of Atlantic Forest in Argentina is important for the long‐term conservation of the four felids. Abstract in Spanish is available with online material.