Cryptic purine transporters in Aspergillus nidulans reveal the role of specific residues in the evolution of specificity in the NCS1 family

NCS1 proteins are H⁺ or Na⁺ symporters responsible for the uptake of purines, pyrimidines or related metabolites in bacteria, fungi and some plants. Fungal NCS1 are classified into two evolutionary and structurally distinct subfamilies, known as Fur‐ and Fcy‐like transporters. These subfamilies have expanded and functionally diversified by gene duplications. The Fur subfamily of the model fungus Aspergillus nidulans includes both major and cryptic transporters specific for uracil, 5‐fluorouracil, allantoin or/and uric acid. Here we functionally analyse all four A. nidulans Fcy transporters (FcyA, FcyC, FcyD and FcyE) with previously unknown function. Our analysis shows that FcyD is moderate‐affinity, low‐capacity, highly specific adenine transporter, whereas FcyE contributes to 8‐azaguanine uptake. Mutational analysis of FcyD, supported by homology modelling and substrate docking, shows that two variably conserved residues (Leu356 and Ser359) in transmembrane segment 8 (TMS8) are critical for transport kinetics and specificity differences among Fcy transporters, while two conserved residues (Phe167 and Ser171) in TMS3 are also important for function. Importantly, mutation S359N converts FcyD to a promiscuous nucleobase transporter capable of recognizing adenine, xanthine and several nucleobase analogues. Our results reveal the importance of specific residues in the functional evolution of NCS1 transporters.     

Theories and Major Hypotheses in Ethnobotany

Ethnobotany has evolved from a discipline that largely documented the diversity of plant use by local people to one focused on understanding how and why people select plants for a wide range of uses. This progress has been in response to a repeated call for theory-inspired and hypothesis-driven research to improve the rigor of the discipline. Despite improvements, recent ethnobotanical research has overemphasized the use of quantitative ethnobotany indices and statistical methods borrowed from ecology, yet underemphasized the development and integration of a strong theoretical foundation. To advance the field of ethnobotany as a hypothesis-driven, theoretically inspired discipline, it is important to first synthesize the existing theoretical lines of research. We review and discuss 17 major theories and hypotheses in ethnobotany that can be used as a starting point for developing research questions that advance our understanding of people–plant interactions. For each theory or major hypothesis, we identify its primary predictions and testable hypotheses and then discuss how these predictions have been tested. Developing research to test these predictions will make significant contributions to the field of ethnobotany and create the critical mass of primary literature necessary to develop meta-analyses and to advance new theories in ethnobotany.     

Eliciting and integrating expert knowledge to assess the viability of the critically endangered golden sun‐moth Synemon plana

The critically endangered golden sun‐moth Synemon plana occurs in urban fringe areas of southeastern Australia that are currently experiencing rapid and extensive development. The urban fringe is a complex and uncertain environment in which to manage threatened species with the intersection of fragmented natural habitats, built environments and human populations generating novel, poorly understood interactions. In this context, management frameworks must incorporate ecological processes as well as social considerations. Here, we explore how biodiversity sensitive urban design might improve the fate of the golden sun‐moth, and threatened species generally, in urban fringe environments. We: (i) developed an expert‐informed Bayesian Belief Network model that synthesizes the current understanding of key determinants of golden sun‐moth population viability at sites experiencing urbanizing pressure; (ii) quantified the nature and strength of cause‐effect relationships between these factors using expert knowledge; and (iii) used the model to assess expectations of moth population viability in response to different combinations of management actions. We predict that adult survival, bare ground cover and cover of resource plants are the most important variables affecting the viability of golden sun‐moth populations. We also demonstrate the potential for biodiversity sensitive urban design as a complementary measure to conventional management for this species. Our findings highlight how expert knowledge may be a valuable component of conservation management, especially in addressing uncertainty around conservation decisions when empirical data are lacking, and how structured expert judgements become critical in supporting decisions that may help ameliorate extinction risks faced by threatened species in urban environments.     

Ephrin-as guide the formation of functional maps in the visual cortex

Ephrin-As and their receptors, EphAs, are expressed in the developing cortex where they may act to organize thalamic inputs. Here, we map the visual cortex (V1) in mice deficient for ephrin-A2, -A3, and -A5 functionally, using intrinsic signal optical imaging and microelectrode recording, and structurally, by anatomical tracing of thalamocortical projections. V1 is shifted medially, rotated, and compressed and its internal organization is degraded. Expressing ephrin-A5 ectopically by in utero electroporation in the lateral cortex shifts the map of V1 medially, and expression within V1 disrupts its internal organization. These findings indicate that interactions between gradients of EphA/ephrin-A in the cortex guide map formation, but that factors other than redundant ephrin-As are responsible for the remnant map. Together with earlier work on the retinogeniculate map, the current findings show that the same molecular interactions may operate at successive stages of the visual pathway to organize maps.     

Alopecia scoring: the quantitative assessment of hair loss in captive macaques

Many captive animals show forms of pelage loss that are absent in wild or free-living conspecifics, which result from grooming or plucking behaviours directed at themselves or at other individuals. For instance, dorsal hair loss in primates such as rhesus macaques (Macaca mulatta) in research facilities, results from excessive hair-pulling or over-grooming by cage-mates. This behaviour appears to be associated with stress, and is controllable to some extent with environmental enrichment. Quantifying alopecia in primates (as in many species) is therefore potentially useful for welfare assessment. A simple system for scoring alopecia was developed and its reliability was tested. Study 1 showed high interobserver reliability between two independent scorers in assessing the state of monkeys coats from photographs. Study 2 showed that there were no significant differences between the scores derived from photographs and from direct observations. Thus, where hair loss due to hair pulling exists in captive primates, this scoring system provides an easy, rapid, and validated quantitative method, for use in assessing the success of attempts to reduce it via improved husbandry. In the future, such scoring systems might also prove useful for quantifying barbering in laboratory rodents.     

Electrostatic modeling predicts the activities of orthopoxvirus complement control proteins

Regulation of complement activation by pathogens and the host are critical for survival. Using two highly related orthopoxvirus proteins, the vaccinia and variola (smallpox) virus complement control proteins, which differ by only 11 aa, but differ 1000-fold in their ability to regulate complement activation, we investigated the role of electrostatic potential in predicting functional activity. Electrostatic modeling of the two proteins predicted that altering the vaccinia virus protein to contain the amino acids present in the second short consensus repeat domain of the smallpox protein would result in a vaccinia virus protein with increased complement regulatory activity. Mutagenesis of the vaccinia virus protein confirmed that changing the electrostatic potential of specific regions of the molecule influences its activity and identifies critical residues that result in enhanced function as measured by binding to C3b, inhibition of the alternative pathway of complement activation, and cofactor activity. In addition, we also demonstrate that despite the enhanced activity of the variola virus protein, its cofactor activity in the factor I-mediated degradation of C3b does not result in the cleavage of the alpha' chain of C3b between residues 954-955. Our data have important implications in our understanding of how regulators of complement activation interact with complement, the regulation of the innate immune system, and the rational design of potent complement inhibitors that might be used as therapeutic agents.     

The high-resolution Structure of LeuB (Rv2995c) from Mycobacterium tuberculosis

The crystal structure of the enzyme 3-isopropylmalate dehydrogenase (IPMDH) from Mycobacterium tuberculosis (LeuB, Mtb-IPMDH, Rv2995c) without substrate or co-factor was determined at 1.65 A resolution, which is the highest resolution reported for an IPMDH to date. The crystals contain two functional dimers in the asymmetric unit in an arrangement close to a tetramer of D2 symmetry. Despite the absence of a substrate or inhibitor bound to the protein, the structure of the monomer resembles the previously observed closed form of the enzyme more closely than the open form. A comparison with the substrate complex of IPMDH from Thiobacillus ferrooxidans and the co-factor complex of the Thermus thermophilus enzyme revealed a close relationship of the active-site architecture between the various bacterial enzymes. The inhibitor O-isobutenyl oxalylhydroxamate was found to bind to the active site of IPMDH in a mode similar to the substrate isopropylmalate.     

Expression profiling in squamous carcinoma cells reveals pleiotropic effects of vitamin D3 analog EB1089 signaling on cell proliferation,differentiation, and immune system regulation

The active form of vitamin D3, 1alpha,25-dihydroxyvitamin D3 [1,25-(OH)2D3] is key mediator of calcium homeostasis and is a component of the complex homeostatic system of the skin. 1,25-(OH)2D3 regulates cellular differentiation and proliferation and has broad potential as an anticancer agent. Oligonucleotide microarrays were used to assess profiles of target gene regulation at several points over a 48 h period by the low calcemic 1,25-(OH)2D3 analog EB1089 in human SCC25 head and neck squamous carcinoma cells. One hundred fifty-two targets were identified, composed of 89 up- and 63 down-regulated genes distributed in multiple profiles of regulation. Results are consistent with EB1089 driving SCC25 cells toward a less malignant phenotype, similar to that of basal keratinocytes. Targets identified control inter- and intra-cellular signaling, G protein-coupled receptor function, intracellular redox balance, cell adhesion, and extracellular matrix composition, cell cycle progression, steroid metabolism, and more than 20 genes modulating immune system function. The data indicate that EB1089 performs three key functions of a cancer chemoprevention agent; it is antiproliferative, it induces cellular differentiation, and has potential genoprotective effects. While no evidence was found for gene-specific differences in efficacy of 1,25-(OH)2D3 and EB1089, gene regulation by 1,25-(OH)2D3 was generally more transient. Treatment of cells with 1,25-(OH)2D3 and the cytochrome P450 inhibitor ketoconazole produced profiles of regulation essentially identical to those observed with EB1089 alone, indicating that the more sustained regulation by EB1089 was due to its resistance to inactivation by induced 24-hydroxylase activity. This suggests that differences in action of the two compounds arise more from their relative sensitivities to metabolism than from differing effects on VDR function.     

Interaction of early growth response protein 1 (Egr-1), specificity protein 1 (Sp1), and cyclic adenosine 3'5'-monophosphate response element binding protein (CREB) at a proximal response element is critical for gastrin-dependent activation of the chromogranin A promoter

Recently, binding of specific protein 1 (Sp1) and cAMP response element binding protein (CREB) to a GC-rich element at -92/-62 has been identified as a critical step in gastrin-dependent regulation of the chromogranin A (CgA) gene in gastric epithelial cells. Here we demonstrate that binding of early growth response protein 1 (Egr-1) to the distal part of the -92/-62 site is also required for gastrin-dependent CgA transactivation. Gastrin elevated cellular and nuclear Egr-1 levels in a time-dependent manner and also increased Egr-1 binding to the CgA -92/-73 region. Disruption of this site reduced gastrin responsiveness without influencing basal promoter activity, while loss of Sp1 and/or CREB binding sites diminished basal and gastrin-stimulated CgA promoter activity. Ectopic Egr-1 overexpression potently stimulated the CgA promoter, whereas coexpression of Egr-1 with Sp1 and/or CREB resulted in additive effects. Functional analysis of Sp1-, Egr-1-, or CREB-specific promoter mutations in transfection studies confirmed the tripartite organization of the CgA -92/-62 element. Signaling studies revealed that MAPK kinase 1 (MEK1)/ERK1/2 cascades are critical for gastrin-dependent Egr-1 protein accumulation as well as Egr-1 binding to the CgA promoter. Our studies for the first time identify Egr-1 as a nuclear target of gastrin and show that functional interplay of Egr-1, Sp1, and CREB is indispensable for gastrin-dependent CgA transactivation in gastric epithelial cells.