The Drosophila peptidoglycan recognition protein PGRP-LF blocks PGRP-LC and IMD/JNK pathway activation

Eukaryotic peptidoglycan recognition proteins (PGRPs) are related to bacterial amidases. In Drosophila, PGRPs bind peptidoglycan and function as central sensors and regulators of the innate immune response. PGRP-LC/PGRP-LE constitute the receptor complex in the immune deficiency (IMD) pathway, which is an innate immune cascade triggered upon Gram-negative bacterial infection. Here, we present the functional analysis of the nonamidase, membrane-associated PGRP-LF. We show that PGRP-LF acts as a specific negative regulator of the IMD pathway. Reduction of PGRP-LF levels, in the absence of infection, is sufficient to trigger IMD pathway activation. Furthermore, normal development is impaired in the absence of functional PGRP-LF, a phenotype mediated by the JNK pathway. Thus, PGRP-LF prevents constitutive activation of both the JNK and the IMD pathways. We propose a model in which PGRP-LF keeps the Drosophila IMD pathway silent by sequestering circulating peptidoglycan.     

Selenium addition alters mercury uptake,bioavailability in the rhizosphere and root anatomy of rice (Oryza sativa)

Background and Aims

Mercury (Hg) is an extremely toxic pollutant, especially in the form of methylmercury (MeHg), whereas selenium (Se) is an essential trace element in the human diet. This study aimed to ascertain whether addition of Se can produce rice with enriched Se and lowered Hg content when growing in Hg-contaminated paddy fields and, if so, to determine the possible mechanisms behind these effects.

Methods

Two cultivars of rice (Oryza sativa, japonica and indica) were grown in either hydroponic solutions or soil rhizobags with different Se and Hg treatments. Concentrations of total Hg, MeHg and Se were determined in the roots, shoots and brown rice, together with Hg uptake kinetics and Hg bioavailability in the soil. Root anatonmy was also studied.

Key Results

The high Se treatment (5 μg g^–1) significantly increased brown rice yield by 48 % and total Se content by 2·8-fold, and decreased total Hg and MeHg by 47 and 55 %, respectively, compared with the control treatments. The high Se treatment also markedly reduced ‘water-soluble’ Hg and MeHg concentrations in the rhizosphere soil, decreased the uptake capacity of Hg by roots and enhanced the development of apoplastic barriers in the root endodermis.

Conclusions

Addition of Se to Hg-contaminated soil can help produce brown rice that is simultaneously enriched in Se and contains less total Hg and MeHg. The lowered accumulation of total Hg and MeHg appears to be the result of reduced bioavailability of Hg and production of MeHg in the rhizosphere, suppression of uptake of Hg into the root cells and an enhancement of the development of apoplastic barriers in the endodermis of the roots.     

Quantitative Characterization of Local Protein Solvation To Predict Solvent Effects on Protein Structure

Characterization of solvent preferences of proteins is essential to the understanding of solvent effects on protein structure and stability. Although it is generally believed that solvent preferences at distinct loci of a protein surface may differ, quantitative characterization of local protein solvation has remained elusive. In this study, we show that local solvation preferences can be quantified over the entire protein surface from extended molecular dynamics simulations. By subjecting microsecond trajectories of two proteins (lysozyme and antibody fragment D1.3) in 4 M glycerol to rigorous statistical analyses, solvent preferences of individual protein residues are quantified by local preferential interaction coefficients. Local solvent preferences for glycerol vary widely from residue to residue and may change as a result of protein side-chain motions that are slower than the longest intrinsic solvation timescale of ～10 ns. Differences of local solvent preferences between distinct protein side-chain conformations predict solvent effects on local protein structure in good agreement with experiment. This study extends the application scope of preferential interaction theory and enables molecular understanding of solvent effects on protein structure through comprehensive characterization of local protein solvation.     

The latitudinal position of peak marine diversity in living and fossil biotas

Aim Peak marine taxonomic diversity has only rarely occurred at or near the equator during the Phanerozoic Eon, in contrast to the present‐day pattern. This fundamental difference is difficult to reconcile because the latitude at which peak diversity occurs for living marine taxa has not yet been explicitly determined at a broad taxonomic and spatial scale. Here, we attempt to determine this value in order to compare the contemporary and fossil patterns directly. Location Our data are global in coverage. Methods We used a literature compilation of 149 present‐day marine latitudinal diversity gradients. We summed the number of marine taxa that exhibited peak diversity within 10° latitudinal bins. In addition, we recorded locality data, general habitat (benthic/pelagic), and the taxonomic level of the study organisms. Results We found that peak diversity for most sampled marine taxa currently occurs between 10° and 20° N, even after correcting for a Northern Hemisphere sampling bias. Moreover, this peak position is a global phenomenon: it is found across habitats and higher taxa, within all sampled ocean basins, and on both sides of the Atlantic and Pacific oceans. Benthic taxa, which dominate our data, exhibit one peak at 10°–20° N, while pelagic taxa exhibit a peak at 10°–20° N and an additional peak at 10°–20° S, producing a distinct trough at the equator. Main conclusions Our data indicate that peak marine diversity for many taxa is currently within 10°–20° N rather than at the equator, and that this is not likely to result from either undersampling at lower latitudes or the pattern being dominated by a particular taxon. Possible explanations may include a coincidence with the intertropical convergence zone, a mid‐domain effect, abundant shallow marine habitat, or high ocean temperatures at latitudes nearest the equator. Regardless of its exact cause, the position of peak diversity should be considered a fundamental feature of the latitudinal diversity gradient that must be accounted for within attempts to explain the latter’s existence.     

Stimulation by glycerate 2,3-bisphosphate: a common property of cytosolic IMP-GMP 5'-nucleotidase in rat and human tissues

Glycerate 2,3-bisphosphate, a potent stimulator of the cytosolic 5'-nucleotidase which preferentially hydrolyzes IMP and GMP in human erythrocytes (Bontemps et al., 1988, Biochem. J. 250, 687-696), also stimulates the dephosphorylation of IMP in cytosol fractions of rat heart, liver, brain, kidney, spleen and erythrocytes, and of human polymorphonuclear leucocytes, mixed peripheral blood lymphocytes, platelets and fibroblasts. Depending on the cell type, stimulation by 5 mM glycerate 2,3-bisphosphate varied from 1.5- to 12-fold. Where investigated, glycerate 2,3-bisphosphate had an approx. 5-fold higher affinity for the enzyme than its other stimulator, ATP. These observations provide a useful tool to distinguish IMP-GMP 5'-nucleotidase from other 5'-nucleotidases, and suggest a common origin of the cytosolic IMP-GMP 5'-nucleotidase in various tissues.     

Repeated high doses of avermectins cause prolonged sterilisation, but do not kill, Onchocerca ochengi adult worms in African cattle

Background

Ivermectin (Mectizan?, Merck and CO. Inc.) is being widely used in the control of human onchocerciasis (Onchoverca volvulus) because of its potent effect on microfilariae. Human studies have suggested that, at the standard dose of 150 μg/kg an annual treatment schedule of ivermectin reversibly interferes with female worm fertility but is not macrofilaricidal. Because of the importance of determining whether ivermectin could be macrofilaricidal, the efficacy of high and prolonged doses of ivermectin and a related avermectin, doramectin, were investigated in cattle infected with O. ochengi.

Methods

Drugs with potential macrofilaricidal activity, were screened for the treatment of human onchocerciasis, using natural infections of O. ochengi in African cattle. Three groups of 3 cows were either treated at monthly intervals (7 treatments) with ivermectin (Ivomec^®, Merck and Co. Inc.) at 500 μg/kg or doramectin (Dectamax^®, Pfizer) at 500 μg/kg or not treated as controls. Intradermal nodules were removed at 6 monthly intervals and adult worms were examined for signs of drug activity.

Results

There was no significant decline in nodule diameter, the motility of male and female worms, nor in male and female viability as determined by the ability to reduce tetrazolium, compared with controls, at any time up to 24 months from the start of treatments (mpt). Embryogenesis, however, was abrogated by treatment, which was seen as an accumulation of dead and dying intra-uterine microfilariae (mf) persisting for up to 18 mpt. Skin mf densities in treated animals had fallen to zero by <3 mpt, but by 18 mpt small numbers of mf were found in the skin of some treated animals and a few female worms were starting to produce multi-cellular embryonic stages. Follow-up of the doramectin treated group at 36 mpt showed that mf densities had still only regained a small proportion of their pre-treatment levels.

Conclusion

These results have important implications for onchocerciasis control in the field. They suggest that ivermectin given at repeated high does may sterilise O. volvulus female worms for prolonged periods but is unlikely to kill them. This supports the view that control programmes may need to continue treatments with ivermectin for a period of decades and highlights the need to urgently identify new marcofiliaricidal compounds.     

Microsecond simulations of mdm2 and its complex with p53 yield insight into force field accuracy and conformational dynamics

The p53‐MDM2 complex is both a major target for cancer drug development and a valuable model system for computational predictions of protein‐ligand binding. To investigate the accuracy of molecular simulations of MDM2 and its complex with p53, we performed a number of long (200 ns to 1 µs) explicit‐solvent simulations using a range of force fields. We systematically compared nine popular force fields (AMBER ff03, ff12sb, ff14sb, ff99sb, ff99sb‐ildn, ff99sb‐ildn‐nmr, ff99sb‐ildn‐phi, CHARMM22*, and CHARMM36) against experimental chemical shift data, and found similarly accurate results, with microsecond simulations achieving better agreement compared to 200‐ns trajectories. Although the experimentally determined apo structure has a closed binding cleft, simulations in all force fields suggest the apo state of MDM2 is highly flexible, and able to sample holo‐like conformations, consistent with a conformational selection model. Initial structuring of the MDM2 lid region, known to competitively bind the binding cleft, is also observed in long simulations. Taken together, these results show molecular simulations can accurately sample conformations relevant for ligand binding. We expect this study to inform future computational work on folding and binding of MDM2 ligands. Proteins 2015; 83:1665–1676. © 2015 Wiley Periodicals, Inc.     

The predicted transmembrane fragment 17 of the human multidrug resistance protein 1 (MRP1) behaves as an interfacial helix in membrane mimics

The human multidrug resistance protein MRP1 (or ABCC1) is one of the most important members of the large ABC transporter family, in terms of both its biological (tissue defense) and pharmacological functions. Many studies have investigated the function of MRP1, but structural data remain scarce for this protein. We investigated the structure and dynamics of predicted transmembrane fragment 17 (TM17, from Ala(1227) to Ser(1251)), which contains a single Trp residue (W(1246)) involved in MRP1 substrate specificity and transport function. We synthesized TM17 and a modified peptide in which Ala(1227) was replaced by a charged Lys residue. Both peptides were readily solubilized in dodecylmaltoside (DM) or dodecylphosphocholine (DPC) micelles, as membrane mimics. The interaction of these peptides with DM or DPC micelles was studied by steady-state and time-resolved Trp fluorescence spectroscopy, including experiments in which Trp was quenched by acrylamide or by two brominated analogs of DM. The secondary structure of these peptides was determined by circular dichroism. Overall, the results obtained indicated significant structuring ( approximately 50% alpha-helix) of TM17 in the presence of either DM or DPC micelles as compared to buffer. A main interfacial location of TM17 is proposed, based on significant accessibility of Trp(1246) to brominated alkyl chains of DM and/or acrylamide. The comparison of various fluorescence parameters including lambda(max), lifetime distributions and Trp rotational mobility with those determined for model fluorescent transmembrane helices in the same detergents is also consistent with the interfacial location of TM17. We therefore suggest that TM17 intrinsic properties may be insufficient for its transmembrane insertion as proposed by the MRP1 consensus topological model. This insertion may also be controlled by additional constraints such as interactions with other TM domains and its position in the protein sequence. The particular pattern of behavior of this predicted transmembrane peptide may be the hallmark of a fragment involved in substrate transport.