Structures of human MAP kinase kinase 1 (MEK1) and MEK2 describe novel noncompetitive kinase inhibition

MEK1 and MEK2 are closely related, dual-specificity tyrosine/threonine protein kinases found in the Ras/Raf/MEK/ERK mitogen-activated protein kinase (MAPK) signaling pathway. Approximately 30% of all human cancers have a constitutively activated MAPK pathway, and constitutive activation of MEK1 results in cellular transformation. Here we present the X-ray structures of human MEK1 and MEK2, each determined as a ternary complex with MgATP and an inhibitor to a resolution of 2.4 A and 3.2 A, respectively. The structures reveal that MEK1 and MEK2 each have a unique inhibitor-binding pocket adjacent to the MgATP-binding site. The presence of the potent inhibitor induces several conformational changes in the unphosphorylated MEK1 and MEK2 enzymes that lock them into a closed but catalytically inactive species. Thus, the structures reported here reveal a novel, noncompetitive mechanism for protein kinase inhibition.     

Pregnancy does not affect HIV incidence test results obtained using the BED capture enzyme immunoassay or an antibody avidity assay

Background

Accurate incidence estimates are needed for surveillance of the HIV epidemic. HIV surveillance occurs at maternal-child health clinics, but it is not known if pregnancy affects HIV incidence testing.

Methods

We used the BED capture immunoassay (BED) and an antibody avidity assay to test longitudinal samples from 51 HIV-infected Ugandan women infected with subtype A, C, D and intersubtype recombinant HIV who were enrolled in the HIVNET 012 trial (37 baseline samples collected near the time of delivery and 135 follow-up samples collected 3, 4 or 5 years later). Nineteen of 51 women were also pregnant at the time of one or more of the follow-up visits. The BED assay was performed according to the manufacturer''s instructions. The avidity assay was performed using a Genetic Systems HIV-1/HIV-2 + O EIA using 0.1M diethylamine as the chaotropic agent.

Results

During the HIVNET 012 follow-up study, there was no difference in normalized optical density values (OD-n) obtained with the BED assay or in the avidity test results (%) when women were pregnant (n = 20 results) compared to those obtained when women were not pregnant (n = 115; for BED: p = 0.9, generalized estimating equations model; for avidity: p = 0.7, Wilcoxon rank sum). In addition, BED and avidity results were almost exactly the same in longitudinal samples from the 18 women who were pregnant at only one study visit during the follow-up study (p = 0.6, paired t-test).

Conclusions

These results from 51 Ugandan women suggest that any changes in the antibody response to HIV infection that occur during pregnancy are not sufficient to alter results obtained with the BED and avidity assays. Confirmation with larger studies and with other HIV subtypes is needed.     

Role of Human Nucleoside Transporters in the Uptake and Cytotoxicity of Azacitidine and Decitabine

The nucleoside analogs 5-azacytidine (azacitidine) and 5-aza-2′-deoxycytidine (decitabine) are active against acute myeloid leukemia and myelodysplastic syndromes. Cellular transport across membranes is crucial for uptake of these highly polar hydrophilic molecules. We assessed the ability of azacitidine, decitabine, and, for comparison, gemcitabine, to interact with human nucleoside transporters (hNTs) in Saccharomyces cerevisiae cells (hENT1/2, hCNT1/2/3) or Xenopus laevis oocytes (hENT3/4). All three drugs inhibited hCNT1/3 potently (K _i values, 3–26 μM), hENT1/2 and hCNT2 weakly (K _i values, 0.5–3.1 mM), and hENT3/4 poorly if at all. Rates of transport of [³H]gemcitabine, [¹⁴C]azacitidine, and [³H]decitabine observed in Xenopus oocytes expressing individual recombinant hNTs differed substantially. Cytotoxicity of azacitidine and decitabine was assessed in hNT-expressing or hNT-deficient cultured human cell lines in the absence or presence of transport inhibitors where available. The rank order of cytotoxic sensitivities (IC ₅₀ values, μM) conferred by hNTs were hCNT1 (0.1) > hENT1 (0.3) ? hCNT2 (8.3), hENT2 (9.0) for azacitidine and hENT1 (0.3) > hCNT1 (0.8) ? hENT2, hCNT2 (>100) for decitabine. Protection against cytotoxicity was observed for both drugs in the presence of inhibitors of nucleoside transport, thus suggesting the importance of hNTs in manifestation of toxicity. In summary, all seven hNTs transported azacitidine, with hCNT3 showing the highest rates, whereas hENT1 and hENT2 showed modest transport and hCNT1 and hCNT3 poor transport of decitabine. Our results show for the first time that azacitidine and decitabine exhibit different human nucleoside transportability profiles and their cytotoxicities are dependent on the presence of hNTs, which could serve as potential biomarkers of clinical response.     

Population structure and phylogeography reveal pathways of colonization by a migratory marine reptile (Chelonia mydas) in the central and eastern Pacific

Climate, behavior, ecology, and oceanography shape patterns of biodiversity in marine faunas in the absence of obvious geographic barriers. Marine turtles are an example of highly migratory creatures with deep evolutionary lineages and complex life histories that span both terrestrial and marine environments. Previous studies have focused on the deep isolation of evolutionary lineages (>3 mya) through vicariance; however, little attention has been given to the pathways of colonization of the eastern Pacific and the processes that have shaped diversity within the most recent evolutionary time. We sequenced 770 bp of the mtDNA control region to examine the stock structure and phylogeography of 545 green turtles from eight different rookeries in the central and eastern Pacific. We found significant differentiation between the geographically separated nesting populations and identified five distinct stocks (F_ST = 0.08–0.44, P < 0.005). Central and eastern Pacific Chelonia mydas form a monophyletic group containing 3 subclades, with Hawaii more closely related to the eastern Pacific than western Pacific populations. The split between sampled central/eastern and western Pacific haplotypes was estimated at around 0.34 mya, suggesting that the Pacific region west of Hawaii has been a more formidable barrier to gene flow in C. mydas than the East Pacific Barrier. Our results suggest that the eastern Pacific was colonized from the western Pacific via the Central North Pacific and that the Revillagigedos Islands provided a stepping‐stone for radiation of green turtles from the Hawaiian Archipelago to the eastern Pacific. Our results fit with a broader paradigm that has been described for marine biodiversity, where oceanic islands, such as Hawaii and Revillagigedo, rather than being peripheral evolutionary “graveyards”, serve as sources and recipients of diversity and provide a mechanism for further radiation.     

Bridging the gap: How the National Core Arts Standards forge the divide between the arts and assessment-based learning

ABSTRACT

This article is a review and thematic analysis of the 2014 National Core Arts Standards (NCAS). Historically, there exists a gap between the arts and assessment-based educational practices. Thematic analysis of the NCAS reveals a policy striving to bridge the gap between the way arts educators envision arts in schools, and the current reality of assessment-based schooling in the United States. This policy could become the foundation for the recognition of the arts as academically rigorous subject matter, capable of existing and thriving in an assessment-oriented world; it remains to be seen as to how and if arts educators will use and adapt NCAS. Is the NCAS merely a symbolic policy or does it have the support behind it to be a material policy that truly creates change in the educational system?     

Contested Citizenship and Social Exclusion: Adolescent Arab American Immigrants' Views of the Social Contract

Drawing from social contract theory, we explore how some adolescent Arab immigrants' (n = 99) sensitivity to the image of their ethnic group as enemies of America colors their interpretation of the social contract. Analyses of data collected in 1997 reveal that those youth who reported that the American media portray Arab people and nations as enemies of the United States are more attuned to personal experiences of prejudice based on their ethnic identity and are more dubious that the tenets of the social contract apply equally across groups. Negative images of Arab Americans were well in place prior to September 11, 2001, a pivotal moment that altered the lives of Arab Americans as well as the discourse on immigration and citizenship.     

Biogeographic context is related to local scale tree demography,co-occurrence and functional differentiation

Identifying the drivers of community structure and dynamics is a major pursuit in ecology. Emphasis is typically placed on the importance of local scale interactions when attempting to explain these fundamental ecological patterns. However, regional scale phenomena are also important predictors. The importance of regional scale context should be more evident in assemblages where multiple species are close to their range margins. Here, we test the importance of regional scale context using data from a temperate forest plot that contains two species groups – one near its northern range limit and one near its southern range limit. We show the proximity of species to their southern or northern range margins is linked to local scale co-occurrence, similarity in gene expression responses to a key environmental driver, demographic performance and inter-specific variation in conspecific negative density dependence. In sum, many of the key local scale patterns and processes of interest to community ecologists are linked to biogeographic context that is frequently ignored.     

Changes in Nkx2.1, Sox2, Bmp4, and Bmp16 expression underlying the lung‐to‐gas bladder evolutionary transition in ray‐finned fishes

The key to understanding the evolutionary origin and modification of phenotypic traits is revealing the responsible underlying developmental genetic mechanisms. An important organismal trait of ray‐finned fishes is the gas bladder, an air‐filled organ that, in most fishes, functions for buoyancy control, and is homologous to the lungs of lobe‐finned fishes. The critical morphological difference between lungs and gas bladders, which otherwise share many characteristics, is the general direction of budding during development. Lungs bud ventrally and the gas bladder buds dorsally from the anterior foregut. We investigated the genetic underpinnings of this ventral‐to‐dorsal shift in budding direction by studying the expression patterns of known lung genes (Nkx2.1, Sox2, and Bmp4) during the development of lungs or gas bladder in three fishes: bichir, bowfin, and zebrafish. Nkx2.1 and Sox2 show reciprocal dorsoventral expression patterns during tetrapod lung development and are important regulators of lung budding; their expression during bichir lung development is conserved. Surprisingly, we find during gas bladder development, Nkx2.1 and Sox2 expression are inconsistent with the hypothesis that they regulate the direction of gas bladder budding. Bmp4 is expressed ventrally during lung development in bichir, akin to the pattern during mouse lung development. During gas bladder development, Bmp4 is not expressed. However, Bmp16, a paralogue of Bmp4, is expressed dorsally in the developing gas bladder of bowfin. Bmp16 is present in the known genomes of Actinopteri (ray‐finned fishes excluding bichir) but absent from mammalian genomes. We hypothesize that Bmp16 was recruited to regulate gas bladder development in the Actinopteri in place of Bmp4.     

Structural insights into the allosteric effects of 4EBP1 on the eukaryotic translation initiation factor eIF4E

The eukaryotic translation initiation factor eIF4E plays key roles in cap-dependent translation and mRNA export. These functions rely on binding the 7-methyl-guanosine moiety (5'cap) on the 5'-end of all mRNAs. eIF4E is regulated by proteins such as eIF4G and eIF4E binding proteins (4EBPs) that bind the dorsal surface of eIF4E, distal to the cap binding site, and modulate cap binding activity. Both proteins increase the affinity of eIF4E for 5'cap. Our understanding of the allosteric effects and structural underpinnings of 4EBP1 or eIF4G binding can be advanced by obtaining structural data on cap-free eIF4E bound to one of these proteins. Here, we report the crystal structure of apo-eIF4E and cap-free eIF4E in complex with a 4EBP1 peptide. We also monitored 4EBP1 binding to cap-free eIF4E in solution using NMR. Together, these studies suggest that 4EBP1 transforms eIF4E into a cap-receptive state. NMR methods were also used to compare the allosteric routes activated by 4EBP1, eIF4G, and the arenavirus Z protein, a negative regulator of cap binding. We observed chemical shift perturbation at the dorsal binding site leading to alterations in the core of the protein, which were ultimately communicated to the unoccupied cap binding site of eIF4E. There were notable similarities between the routes taken by 4EBP1 and eIF4G and differences from the negative regulator Z. Thus, binding of 4EBP1 or eIF4G allosterically drives alterations throughout the protein that increase the affinity of eIF4E for the 5'cap.