Oxidation of hydrogen sulfide remains a priority in mammalian cells and causes reverse electron transfer in colonocytes

Sulfide (H₂S) is an inhibitor of mitochondrial cytochrome oxidase comparable to cyanide. In this study, poisoning of cells was observed with sulfide concentrations above 20 µM. Sulfide oxidation has been shown to take place in organisms/cells naturally exposed to sulfide. Sulfide is released as a result of metabolism of sulfur containing amino acids. Although in mammals sulfide exposure is not thought to be quantitatively important outside the colonic mucosa, our study shows that a majority of mammalian cells, by means of the mitochondrial sulfide quinone reductase (SQR), avidly consume sulfide as a fuel. The SQR activity was found in mitochondria isolated from mouse kidneys, liver, and heart. We demonstrate the precedence of the SQR over the mitochondrial complex I. This explains why the oxidation of the mineral substrate sulfide takes precedence over the oxidation of other (carbon-based) mitochondrial substrates. Consequently, if sulfide delivery rate remains lower than the SQR activity, cells maintain a non-toxic sulfide concentration (< 1 µM) in their external environment. In the colonocyte cell line HT-29, sulfide oxidation provided the first example of reverse electron transfer in living cells, such a transfer increasing sulfide tolerance. However, SQR activity was not detected in brain mitochondria and neuroblastoma cells. Consequently, the neural tissue would be more sensitive to sulfide poisoning. Our data disclose new constraints concerning the emerging signaling role of sulfide.     

Testing ecological and behavioral correlates of nest predation

Variation in nest predation rates among bird species are assumed to reflect differences in risk that are specific to particular nest sites. Theoretical and empirical studies suggest that parental care behaviors can evolve in response to nest predation risk and thereby differ among ecological conditions that vary in inherent risk. However, parental care also can influence predation risk. Separating the effects of nest predation risk inherent to a nest site from the risk imposed by parental strategies is needed to understand the evolution of parental care. Here we identify correlations between risks inherent to nest sites, and risk associated with parental care behaviors, and use an artificial nest experiment to assess site-specific differences in nest predation risk across nesting guilds and between habitats that differed in nest predator abundance. We found a strong correlation between parental care behaviors and inherent differences in nest predation risk, but despite the absence of parental care at artificial nests, patterns of nest predation risk were similar for real and artificial nests both across nesting guilds and between predator treatments. Thus, we show for the first time that inherent risk of nest predation varies with nesting guild and predator abundance independent of parental care.     

Structural and functional characterization of enantiomeric glutamic acid derivatives as potential transition state analogue inhibitors of MurD ligase

Mur ligases play an essential role in the intracellular biosynthesis of bacterial peptidoglycan, the main component of the bacterial cell wall, and represent attractive targets for the design of novel antibacterials. UDP-N-acetylmuramoyl-L-alanine:D-glutamate ligase (MurD) catalyses the addition of D-glutamic acid to the cytoplasmic intermediate UDP-N-acetylmuramoyl-L-alanine (UMA) and is the second in the series of Mur ligases. MurD ligase is highly stereospecific for its substrate, D-glutamic acid (D-Glu). Here, we report the high resolution crystal structures of MurD in complexes with two novel inhibitors designed to mimic the transition state of the reaction, which contain either the D-Glu or the L-Glu moiety. The binding modes of N-sulfonyl-D-Glu and N-sulfonyl-L-Glu derivatives were also characterised kinetically. The results of this study represent an excellent starting point for further development of novel inhibitors of this enzyme.     

Chronic lithium administration attenuates up-regulated brain arachidonic acid metabolism in a rat model of neuroinflammation

Neuroinflammation, caused by a 6-day intracerebroventricular infusion of lipopolysaccharide (LPS) in rats, is associated with the up-regulation of brain arachidonic acid (AA) metabolism markers. Because chronic LiCl down-regulates markers of brain AA metabolism, we hypothesized that it would attenuate increments of these markers in LPS-infused rats. Incorporation coefficients k* of AA from plasma into brain, and other brain AA metabolic markers, were measured in rats that had been fed a LiCl or control diet for 6 weeks, and subjected in the last 6 days on the diet to intracerebroventricular infusion of artificial CSF or of LPS. In rats on the control diet, LPS compared with CSF infusion increased k* significantly in 28 regions, whereas the LiCl diet prevented k* increments in 18 of these regions. LiCl in CSF infused rats increased k* in 14 regions, largely belonging to auditory and visual systems. Brain cytoplasmic phospholipase A(2) activity, and prostaglandin E(2) and thromboxane B(2) concentrations, were increased significantly by LPS infusion in rats fed the control but not the LiCl diet. Chronic LiCl administration attenuates LPS-induced up-regulation of a number of brain AA metabolism markers. To the extent that this up-regulation has neuropathological consequences, lithium might be considered for treating human brain diseases accompanied by neuroinflammation.     

Functional calcitonin gene-related peptide receptors are formed by the asymmetric assembly of a calcitonin receptor-like receptor homo-oligomer and a monomer of receptor activity-modifying protein-1

In addition to their interactions with hetero-trimeric G proteins, seven-transmembrane domain receptors are now known to form multimeric complexes that can include receptor homo- or hetero-oligomers and/or accessory proteins that modulate their activity. The calcitonin gene-related peptide (CGRP) receptor requires the assembly of the seven-transmembrane domain calcitonin receptor-like receptor with the single-transmembrane domain receptor activity-modifying protein-1 to reach the cell surface and be active. However, the relative stoichiometric arrangement of these two proteins within a receptor complex remains unknown. Despite recent advances in the development of protein-protein interactions assays, determining the composition and stoichiometric arrangements of such signaling complexes in living cells remains a challenging task. In the present study, we combined bimolecular fluorescence complementation (BiFC) with bioluminescence resonance energy transfer (BRET) to probe the stoichiometric arrangement of the CGRP receptor complex. Together with BRET competition assays, co-immunoprecipitation experiments, and BiFC imaging, dual BRET/BiFC revealed that functional CGRP receptors result from the association of a homo-oligomer of the calcitonin receptor-like receptor with a monomer of the accessory protein receptor activity-modifying protein-1. In addition to revealing the existence of an unexpected asymmetric oligomeric organization for a G protein-coupled receptor, our study illustrates the usefulness of dual BRET/BiFC as a powerful tool for analyzing constitutive and dynamically regulated multiprotein complexes.     

Probing the opening of the pancreatic lipase lid using site-directed spin labeling and EPR spectroscopy

Access to the active site of human pancreatic lipase (HPL) is controlled by a surface loop (the lid) that undergoes a conformational change in the presence of amphiphiles and lipid substrate. The question of how and when the lid opens still remains to be elucidated, however. A paramagnetic probe was covalently bound to the lid via the D249C mutation, and electron paramagnetic resonance (EPR) spectroscopy was used to monitor the conformational change in solution. Two EPR spectral components, corresponding to distinct mobilities of the probe, were attributed to the closed and open conformations of the HPL lid, based on experiments performed with the E600 inhibitor. The open conformation of the lid was observed in solution at supramicellar bile salt concentrations. Colipase alone did not induce lid opening but increased the relative proportions of the open conformation in the presence of bile salts. The opening of the lid was found to be a reversible process. Using various colipase to lipase molar ratios, a correlation between the proportion of the open conformation and the catalytic activity of HPL was observed.     

Conversion of methionine to cysteine in Bacillus subtilis and its regulation

Bacillus subtilis can use methionine as the sole sulfur source, indicating an efficient conversion of methionine to cysteine. To characterize this pathway, the enzymatic activities of CysK, YrhA and YrhB purified in Escherichia coli were tested. Both CysK and YrhA have an O-acetylserine-thiol-lyase activity, but YrhA was 75-fold less active than CysK. An atypical cystathionine beta-synthase activity using O-acetylserine and homocysteine as substrates was observed for YrhA but not for CysK. The YrhB protein had both cystathionine lyase and homocysteine gamma-lyase activities in vitro. Due to their activity, we propose that YrhA and YrhB should be renamed MccA and MccB for methionine-to-cysteine conversion. Mutants inactivated for cysK or yrhB grew similarly to the wild-type strain in the presence of methionine. In contrast, the growth of an DeltayrhA mutant or a luxS mutant, inactivated for the S-ribosyl-homocysteinase step of the S-adenosylmethionine recycling pathway, was strongly reduced with methionine, whereas a DeltayrhA DeltacysK or cysE mutant did not grow at all under the same conditions. The yrhB and yrhA genes form an operon together with yrrT, mtnN, and yrhC. The expression of the yrrT operon was repressed in the presence of sulfate or cysteine. Both purified CysK and CymR, the global repressor of cysteine metabolism, were required to observe the formation of a protein-DNA complex with the yrrT promoter region in gel-shift experiments. The addition of O-acetyl-serine prevented the formation of this protein-DNA complex.     

Evolution and Conservation of Central African Biodiversity: Priorities for Future Research and Education in the Congo Basin and Gulf of Guinea

The tropical forests of the Congo Basin and Gulf of Guinea harbor some of the greatest terrestrial and aquatic biological diversity in the world. However, our knowledge of the rich biological diversity of this region and the evolutionary processes that have shaped it remains limited, as is our understanding of the capacity for species to adapt or otherwise respond to current and projected environmental change. In this regard, research efforts are needed to increase current scientific knowledge of this region's biodiversity, identify the drivers of past diversification, evaluate the potential for species to adapt to environmental change and identify key populations for future conservation. Moreover, when evolutionary research is combined with ongoing environmental monitoring efforts, it can also provide an important set of tools for assessing and mitigating the impacts of development activities. Building on a set of recommendations developed at an international workshop held in Gabon in 2011, we highlight major areas for future evolutionary research that could be directly tied to conservation priorities for the region. These research priorities are centered around five disciplinary themes: (1) documenting and discovering biodiversity; (2) identifying drivers of evolutionary diversification; (3) monitoring environmental change; (4) understanding community and ecosystem level processes; (5) investigating the ecology and epidemiology of disease from an evolutionary perspective (evolutionary epidemiology). Furthermore, we also provide an overview of the needs and priorities for biodiversity education and training in Central Africa.