Multiscale Approach to the Determination of the Photoactive Yellow Protein Signaling State Ensemble

The nature of the optical cycle of photoactive yellow protein (PYP) makes its elucidation challenging for both experiment and theory. The long transition times render conventional simulation methods ineffective, and yet the short signaling-state lifetime makes experimental data difficult to obtain and interpret. Here, through an innovative combination of computational methods, a prediction and analysis of the biological signaling state of PYP is presented. Coarse-grained modeling and locally scaled diffusion map are first used to obtain a rough bird''s-eye view of the free energy landscape of photo-activated PYP. Then all-atom reconstruction, followed by an enhanced sampling scheme; diffusion map-directed-molecular dynamics are used to focus in on the signaling-state region of configuration space and obtain an ensemble of signaling state structures. To the best of our knowledge, this is the first time an all-atom reconstruction from a coarse grained model has been performed in a relatively unexplored region of molecular configuration space. We compare our signaling state prediction with previous computational and more recent experimental results, and the comparison is favorable, which validates the method presented. This approach provides additional insight to understand the PYP photo cycle, and can be applied to other systems for which more direct methods are impractical.     

Why do chimpanzees have diverse behavioral repertoires yet lack more complex cultures? Invention and social information use in a cumulative task

Humans are distinctive in their dependence upon products of culture for survival, products that have evolved cumulatively over generations such that many cannot now be created by a single individual. Why the cultural capacity of humans appears unrivalled in the animal kingdom is a topic of ongoing debate. Here we explore whether innovation and/or social learning propensities may constrain the ability of one of our closest living relatives, chimpanzees (Pan troglodytes), to master an extractive foraging and tool-use task designed to afford opportunities for cumulative culture to develop. We further explore the potential demographic characteristics associated with novel task solutions. Chimpanzees (N = 53) were inventive, flexibly exploring the novel task, albeit complex inventions were rare and shaped by prior individual experience with similar tool-use tasks. However, they displayed no evidence of cumulative cultural learning. Communities displayed richer behavioral repertoires and had greater task success than chimpanzees tested in an asocial control condition, but their solution complexity did not surpass what individuals invented. The lack of social transmission of complex and beneficial solutions in contexts like those we studied provides one explanation for the limited cumulative culture observed in this species.     

Nox5 forms a functional oligomer mediated by self-association of its dehydrogenase domain

Nox5 belongs to the calcium-regulated subfamily of NADPH oxidases (Nox). Like other calcium-regulated Noxes, Nox5 has an EF-hand-containing calcium-binding domain at its N-terminus, a transmembrane heme-containing region, and a C-terminal dehydrogenase (DH) domain that binds FAD and NADPH. While Nox1-4 require regulatory subunits, including p22phox, Nox5 activity does not depend on any subunits. We found that inactive point mutants and truncated forms of Nox5 (including the naturally expressed splice form, Nox5S) inhibit full-length Nox5, consistent with formation of a dominant negative complex. Oligomerization of full-length Nox5 was demonstrated using co-immunoprecipitation of coexpressed, differentially tagged forms of Nox5 and occurred in a manner independent of calcium ion. Several approaches were used to show that the DH domain mediates oligomerization: Nox5 could be isolated as a multimer when the calcium-binding domain and/or the N-terminal polybasic region (PBR-N) was deleted, but deletion of the DH domain eliminated oligomerization. Further, a chimera containing the transmembrane domain of Ciona intestinalis voltage sensor-containing phosphatase (CiVSP) fused to the Nox5 DH domain formed a co-immunoprecipitating complex with, and functioned as a dominant inhibitor of, full-length Nox5. Radiation inactivation of Nox5 overexpressed in HEK293 cells and endogenously expressed in human aortic smooth muscle cells indicated molecular masses of ～350 and ～300 kDa, respectively, consistent with a tetramer being the functionally active unit. Thus, Nox5 forms a catalytically active oligomer in the membrane that is mediated by its dehydrogenase domain. As a result of oligomerization, the short, calcium-independent splice form, Nox5S, may function as an endogenous inhibitor of calcium-stimulated ROS generation by full-length Nox5.     

Male chimpanzees' grooming rates vary by female age, parity, and fertility status

Copulation preferences in our closest living relative, the chimpanzee, suggest that males prefer older females who have had previous offspring. However, this finding is counter to some behavioral models, which predict that chimpanzee males, as promiscuous breeders with minimal costs to mating, should show little or no preference when choosing mating partners (e.g. should mate indiscriminately). To determine if the preferences indicated by copulations appear in other contexts as well as how they interact, we examined how male chimpanzees' grooming patterns varied amongst females. We found that males' preferences were based on interactions among females' fertility status, age, and parity. First, grooming increased with increasing female parity. We further found an effect of the estrous cycle on grooming; when females were at the lowest point of their cycle, males preferentially groomed parous females at peak reproductive age, but during maximal tumescence, males preferred the oldest multiparous females. Nulliparous females received relatively little grooming regardless of age or fertility. Thus, male chimpanzees apparently chose grooming partners based on both female's experience and fertility, possibly indicating a two-pronged social investment strategy. Male selectivity seems to have evolved to effectively distribute costly social resources in a pattern which may increase their overall reproductive success.     

The E-loop is involved in hydrogen peroxide formation by the NADPH oxidase Nox4

In contrast to the NADPH oxidases Nox1 and Nox2, which generate superoxide (O(2)(·-)), Nox4 produces hydrogen peroxide (H(2)O(2)). We constructed chimeric proteins and mutants to address the protein region that specifies which reactive oxygen species is produced. Reactive oxygen species were measured with luminol/horseradish peroxidase and Amplex Red for H(2)O(2) versus L-012 and cytochrome c for O(2)(·-). The third extracytosolic loop (E-loop) of Nox4 is 28 amino acids longer than that of Nox1 or Nox2. Deletion of E-loop amino acids only present in Nox4 or exchange of the two cysteines in these stretches switched Nox4 from H(2)O(2) to O(2)(·-) generation while preserving expression and intracellular localization. In the presence of an NO donor, the O(2)()-producing Nox4 mutants, but not wild-type Nox4, generated peroxynitrite, excluding artifacts of the detection system as the apparent origin of O(2)(·-). In Cos7 cells, in which Nox4 partially localizes to the plasma membrane, an antibody directed against the E-loop decreased H(2)O(2) but increased O(2)(·-) formation by Nox4 without affecting Nox1-dependent O(2)(·-) formation. The E-loop of Nox4 but not Nox1 and Nox2 contains a highly conserved histidine that could serve as a source for protons to accelerate spontaneous dismutation of superoxide to form H(2)O(2). Mutation of this but not of four other conserved histidines also switched Nox4 from H(2)O(2) to O(2)(·-) formation. Thus, H(2)O(2) formation is an intrinsic property of Nox4 that involves its E-loop. The structure of the E-loop may hinder O(2)(·-) egress and/or provide a source for protons, allowing dismutation to form H(2)O(2).     

High-throughput Assays for Superoxide and Hydrogen Peroxide: DESIGN OF A SCREENING WORKFLOW TO IDENTIFY INHIBITORS OF NADPH OXIDASES*

Recent progress characterizing the reaction mechanism(s) of fluorescent probes with reactive oxygen species has made it possible to rigorously analyze these reactive species in biological systems. We have developed rapid high throughput-compatible assays for monitoring cellular production of superoxide radical anion and hydrogen peroxide using hydropropidine and coumarin boronic acid probes, respectively. Coupling plate reader-based fluorescence measurements with HPLC-based simultaneous monitoring of superoxide radical anion and hydrogen peroxide provides the basis for the screening protocol for NADPH oxidase (Nox) inhibitors. Using this newly developed approach along with the medium-throughput plate reader-based oximetry and EPR spin trapping as confirmatory assays, it is now eminently feasible to rapidly and reliably identify Nox enzyme inhibitors with a markedly lower rate of false positives. These methodological advances provide an opportunity to discover selective inhibitors of Nox isozymes, through enhanced conceptual understanding of their basic mechanisms of action.     

Task Design Influences Prosociality in Captive Chimpanzees (Pan troglodytes)

Chimpanzees confer benefits on group members, both in the wild and in captive populations. Experimental studies of how animals allocate resources can provide useful insights about the motivations underlying prosocial behavior, and understanding the relationship between task design and prosocial behavior provides an important foundation for future research exploring these animals'' social preferences. A number of studies have been designed to assess chimpanzees'' preferences for outcomes that benefit others (prosocial preferences), but these studies vary greatly in both the results obtained and the methods used, and in most cases employ procedures that reduce critical features of naturalistic social interactions, such as partner choice. The focus of the current study is on understanding the link between experimental methodology and prosocial behavior in captive chimpanzees, rather than on describing these animals'' social motivations themselves. We introduce a task design that avoids isolating subjects and allows them to freely decide whether to participate in the experiment. We explore key elements of the methods utilized in previous experiments in an effort to evaluate two possibilities that have been offered to explain why different experimental designs produce different results: (a) chimpanzees are less likely to deliver food to others when they obtain food for themselves, and (b) evidence of prosociality may be obscured by more “complex” experimental apparatuses (e.g., those including more components or alternative choices). Our results suggest that the complexity of laboratory tasks may generate observed variation in prosocial behavior in laboratory experiments, and highlights the need for more naturalistic research designs while also providing one example of such a paradigm.     

Pro-oxidant and antioxidant effects of N-acetylcysteine regulate doxorubicin-induced NF-kappa B activity in leukemic cells

Clinical debate has arisen over the consequences of antioxidant supplementation during cancer chemotherapy. While antioxidants may impede the efficacy of chemotherapy by scavenging reactive oxygen species and free radicals, it is also possible that antioxidants alleviate unwanted chemotherapy-induced toxicity, thus allowing for increased chemotherapy doses. These contradictory assertions suggest that antioxidant supplementation during chemotherapy treatment can have varied outcomes depending on the cellular context. To gain a more robust understanding of the role that antioxidants play in chemotherapy, we investigated the dose-dependent effects of the antioxidant, N-acetylcysteine (NAC), on the redox-mediated regulation of intracellular signaling. In this study, we systematically evaluated the effect of Dox-induced ROS on the NF-κB pathway in a pediatric acute lymphoblastic leukemia (ALL) cell line by measuring the thiol-based oxidative modifications of redox-sensitive proteins within the pathway. We report a functional consequence of NAC supplementation during doxorubicin (Dox) chemotherapy administration via the NF-kappa B (NF-κB) signal transduction pathway. The ability of NAC to alter Dox-induced NF-κB activity is contingent on the ROS-mediated S-glutathionylation of IKK-β. Moreover, the NAC-dependent alteration of intracellular glutathione redox balance, through pro-oxidant and antioxidant mechanisms, can be exploited to either promote or inhibit Dox-induced NF-κB activity in an NAC-concentration-dependent manner. We developed an electron-transfer-based computational model that predicts the effect of NAC pretreatment on Dox-induced NF-κB signaling for a range of NAC and Dox treatment combinations.     

STAT5 mediates PAF-induced NADPH oxidase NOX5-S expression in Barrett's esophageal adenocarcinoma cells

We have shown that NADPH oxidase NOX5-S is overexpressed in Barrett's esophageal adenocarcinoma (EA) cells and may contribute to the progression from Barrett's esophagus (BE) to EA presumably by increasing cell proliferation and decreasing apoptosis (Fu X, Beer DG, Behar J, Wands J, Lambeth D, Cao W. J Biol Chem 281: 20368-20382, 2006). The mechanism(s) of NOX5-S overexpression in EA, however, is not fully understood. In SEG1 EA cells we found that acid treatment significantly increased platelet-activating factor (PAF) production, which in turn markedly increased NOX5-S expression and hydrogen peroxide (H(2)O(2)) production. Knockdown of NOX5-S by NOX5-S small interfering RNA (siRNA) blocked PAF-dependent H(2)O(2) production. PAF-dependent induction of NOX5-S expression and H(2)O(2) production were significantly decreased by the MAPK kinase 1 inhibitor PD-98059, by the cytosolic phospholipase A(2) (cPLA(2)) inhibitor AACOCF3, and by STAT5 downregulation with STAT5 siRNA. PAF significantly increased the phosphorylation of ERK1/2 MAPK, cPLA(2), and STAT5. Using inhibitors, we demonstrated that PAF-induced STAT5 phosphorylation depends on activation of ERK1/2 MAPK and cPLA(2), whereas PAF-induced cPLA(2) phosphorylation was associated with activation of ERK1/2 MAPK. Given that STAT5 bound to the c-sis-inducible element (TTCTGGTAA) of the NOX5-S promoter, overexpression of STAT5 significantly increased NOX5-S promoter activity. We conclude that acid-induced NOX5-S expression and H(2)O(2) production is mediated in part by production of PAF in SEG1 EA cells, and that PAF-induced increase in NOX5-S expression depends on sequential activation of ERK MAP kinases, cPLA(2), and STAT5 in these cells.