An Interaction between Glutathione and the Capsid Is Required for the Morphogenesis of C-Cluster Enteroviruses

Glutathione (GSH) is the most abundant cellular thiol playing an essential role in preserving a reduced cellular environment. Cellular GSH levels can be efficiently reduced by the GSH biosynthesis inhibitor, L-buthionine sulfoximine (BSO). The aim of our study was to determine the role of GSH in the growth of two C-cluster enteroviruses, poliovirus type 1 (PV1) and coxsackievirus A20 (CAV20). Our results show that the growth of both PV1 and CAV20 is strongly inhibited by BSO and can be partially reversed by the addition of GSH. BSO has no effect on viral protein synthesis or RNA replication but it strikingly reduces the accumulation of 14S pentamers in infected cells. GSH-pull down assays show that GSH directly interacts with capsid precursors and mature virus made in the absence of BSO whereas capsid precursors produced under GSH-depletion do not bind to GSH. In particular, the loss of binding of GSH may debilitate the stability of 14S pentamers, resulting in their failure to assemble into mature virus. Immunofluorescence cell imaging demonstrated that GSH-depletion did not affect the localization of viral capsid proteins to the replication complex. PV1 BSO resistant (BSOr) mutants evolved readily during passaging of the virus in the presence of BSO. Structural analyses revealed that the BSOr mutations, mapping to VP1 and VP3 capsid proteins, are primarily located at protomer/protomer interfaces. BSOr mutations might, in place of GSH, aid the stability of 14S particles that is required for virion maturation. Our observation that BSOr mutants are more heat resistant and need less GSH than wt virus to be protected from heat inactivation suggests that they possess a more stable capsid. We propose that the role of GSH during enterovirus morphogenesis is to stabilize capsid structures by direct interaction with capsid proteins both during and after the formation of mature virus particles.     

Warming and Elevated CO<Subscript>2</Subscript> Interact to Alter Seasonality and Reduce Variability of Soil Water in a Semiarid Grassland

Global changes that alter soil water availability may have profound effects on semiarid ecosystems. Although both elevated CO₂ (eCO₂) and warming can alter water availability, often in opposite ways, few studies have measured their combined influence on the amount, timing, and temporal variability of soil water. Here, we ask how free air CO₂ enrichment (to 600 ppmv) and infrared warming (+?1.5 °C day, +?3 °C night) effects on soil water vary within years and across wet-dry periods in North American mixed-grass prairie. We found that eCO₂ and warming interacted to influence soil water and that those interactions varied by season. In the spring, negative effects of warming on soil water largely offset positive effects of eCO₂. As the growing season progressed, however, warming reduced soil water primarily (summer) or only (autumn) in plots treated with eCO₂. These interactions constrained the combined effect of eCO₂ and warming on soil water, which ranged from neutral in spring to positive in autumn. Within seasons, eCO₂ increased soil water under drier conditions, and warming decreased soil water under wetter conditions. By increasing soil water under dry conditions, eCO₂ also reduced temporal variability in soil water. These temporal patterns explain previously observed plant responses, including reduced leaf area with warming in summer, and delayed senescence with eCO₂ plus warming in autumn. They also suggest that eCO₂ and warming may favor plant species that grow in autumn, including winter annuals and C₃ graminoids, and species able to remain active under the dry conditions moderated by eCO₂.     

Antagonistic roles for CTLA-4 and the mammalian target of rapamycin in the regulation of clonal anergy: enhanced cell cycle progression promotes recall antigen responsiveness.

CD4(+) T cells that undergo multiple rounds of cell division during primary Ag challenge in vivo produce IL-2 on secondary Ag rechallenge, whereas cells that fail to progress through the cell cycle are anergic to restimulation. Anti-CTLA-4 mAb treatment during primary Ag exposure increases cell cycle progression and enhances recall Ag responsiveness; however, simultaneous treatment with rapamycin, an inhibitor of the mammalian target of rapamycin and potent antiproliferative agent, prevents both effects. The data suggest that cell cycle progression plays a primary role in the regulation of recall Ag responsiveness in CD4(+) T cells in vivo. CTLA-4 molecules promote clonal anergy development only indirectly by limiting cell cycle progression during the primary response.     

Addressing chemical pollution in biodiversity research

Climate change, biodiversity loss, and chemical pollution are planetary-scale emergencies requiring urgent mitigation actions. As these “triple crises” are deeply interlinked, they need to be tackled in an integrative manner. However, while climate change and biodiversity are often studied together, chemical pollution as a global change factor contributing to worldwide biodiversity loss has received much less attention in biodiversity research so far. Here, we review evidence showing that the multifaceted effects of anthropogenic chemicals in the environment are posing a growing threat to biodiversity and ecosystems. Therefore, failure to account for pollution effects may significantly undermine the success of biodiversity protection efforts. We argue that progress in understanding and counteracting the negative impact of chemical pollution on biodiversity requires collective efforts of scientists from different disciplines, including but not limited to ecology, ecotoxicology, and environmental chemistry. Importantly, recent developments in these fields have now enabled comprehensive studies that could efficiently address the manifold interactions between chemicals and ecosystems. Based on their experience with intricate studies of biodiversity, ecologists are well equipped to embrace the additional challenge of chemical complexity through interdisciplinary collaborations. This offers a unique opportunity to jointly advance a seminal frontier in pollution ecology and facilitate the development of innovative solutions for environmental protection.     

Characterization of the backbone dynamics of an anti-digoxin antibody VL domain by inverse detected 1H–15N NMR: Comparisons with X-ray data for the Fab

The dynamic behavior of the polypeptide backbone of a recombinant anti-digoxin antibody V_L domain has been characterized by measurements of ¹⁵N T₁ and T₂ relaxation times, ¹H–¹⁵N NOE values, and ¹H–²H exchange rates. These data were acquired with 2D inverse detected heteronuclear ¹H–¹⁵N NMR methods. The relaxation data are interpreted in terms of model free spectral density functions and exchange contributions to transverse relaxation rates R₂ (= 1/T₂). All characterized residues display low-amplitude picosecond timescale librational motions. Fifteen residues undergo conformational changes on the nanosecond timescale, and 24 residues have significant R₂ exchange contributions, which reflect motions on the microsecond to millisecond timescale. For several residues, microsecond to millisecond motions of nearby aromatic rings are postulated to account for some or all of their observed R₂ exchange contributions. The measured ¹H–²H exchange rates are correlated with hydrogen bonding patterns and distances from the solvent accessible surface. The degree of local flexibility indicated by the NMR measurements is compared to crystallographic B-factors derived from X-ray analyses of the native Fab and the Fab/digoxin complex. In general, both the NMR and X-ray data indicate enhanced flexibility in the turns, hypervariable loops, and portions of β-strands A, B, and G. However, on a residue-specific level, correlations among the various NMR data, and between the NMR and X-ray data, are often absent. This is attributed to the different dynamic processes and environments that influence the various observables. The combined data indicate that certain regions of the V_L domain, including the three hypervariable loops, undergo dynamic changes upon V_L:V_H association and/ or complexation with digoxin. Overall, the 26–10 V_L domain exhibits relatively low flexibility on the ps–ns timescale. The possible functional consequences of this result are considered. © 1993 Wiley-Liss, Inc.     

Matching the crystallographic structure of ribosomal protein S7 to a three-dimensional model of the 16S ribosomal RNA.

Two recently published but independently derived structures, namely the X-ray crystallographic structure of ribosomal protein S7 and the "binding pocket" for this protein in a three-dimensional model of the 16S rRNA, have been correlated with one another. The known rRNA-protein interactions for S7 include a minimum binding site, a number of footprint sites, and two RNA-protein crosslink sites on the 16S rRNA, all of which form a compact group in the published 16S rRNA model (despite the fact that these interactions were not used as primary modeling constraints in building that model). The amino acids in protein S7 that are involved in the two crosslinks to 16S rRNA have also been determined in previous studies, and here we have used these sites to orient the crystallographic structure of S7 relative to its rRNA binding pocket. Some minor alterations were made to the rRNA model to improve the fit. In the resulting structure, the principal positively charged surface of the protein is in contact with the 16S rRNA, and all of the RNA-protein interaction data are satisfied. The quality of the fit gives added confidence as to the validity of the 16S rRNA model. Protein S7 is furthermore known to be crosslinked both to P site-bound tRNA and to mRNA at positions upstream of the P site codon; the matched S7-16S rRNA structure makes a prediction as to the location of this crosslink site within the protein molecule.     

Recovery of free ADP, Pi, and free energy of ATP hydrolysis in human skeletal muscle

We measuredsignificant undershoots of the concentrations of free ADP([ADP]) and P_i([P_i]) and the freeenergy of ATP hydrolysis (G_ATP) belowinitial resting levels during recovery from severe ischemic exercisewith ³¹P-nuclear magneticresonance spectroscopy in 11 healthy sports students. Undershoots ofthe rate of oxidative phosphorylation would be predicted if the rate ofoxidative phosphorylation would depend solely on free[ADP],[P_i], orG_ATP. However,undershoots of the rate of oxidative phosphorylation have not beenreported in the literature. Furthermore, undershoots of the rate ofoxidative phosphorylation are unlikely because there is evidence that a balance between ATP production and consumption cannot be achieved if anundershoot of the rate of oxidative phosphorylation actually occurs.Therefore, oxidative phosphorylation seems to depend not only on free[ADP],[P_i], orG_ATP. Anexplanation is that acidosis-related or other factors control oxidativephosphorylation additionally, at least under some conditions.