Endothelial coordination of cerebral vasomotion via myoendothelial gap junctions containing connexins 37 and 40

Control of cerebral vasculature differs from that of systemic vessels outside the blood-brain barrier. The hypothesis that the endothelium modulates vasomotion via direct myoendothelial coupling was investigated in a small vessel of the cerebral circulation. In the primary branch of the rat basilar artery, membrane potential, diameter, and calcium dynamics associated with vasomotion were examined using selective inhibitors of endothelial function in intact and endothelium-denuded arteries. Vessel anatomy, protein, and mRNA expression were studied using conventional electron microscopy high-resolution ultrastructural and confocal immunohistochemistry and quantitative PCR. Membrane potential oscillations were present in both endothelial cells and smooth muscle cells (SMCs), and these preceded rhythmical contractions during which adjacent SMC intracellular calcium concentration ([Ca(2+)](i)) waves were synchronized. Endothelium removal abolished vasomotion and desynchronized adjacent smooth muscle cell [Ca(2+)](i) waves. N(G)-nitro-l-arginine methyl ester (10 microM) did not mimic this effect, and dibutyryl cGMP (300 muM) failed to resynchronize [Ca(2+)](i) waves in endothelium-denuded arteries. Combined charybdotoxin and apamin abolished vasomotion and depolarized and constricted vessels, even in absence of endothelium. Separately, (37,43)Gap27 and (40)Gap27 abolished vasomotion. Extensive myoendothelial gap junctions (3 per endothelial cell) composed of connexins 37 and 40 connected the endothelial cell and SMC layers. Synchronized vasomotion in rat basilar artery is endothelium dependent, with [Ca(2+)](i) waves generated within SMCs being coordinated by electrical coupling via myoendothelial gap junctions.     

Genetics of symbiosis in Lotus japonicus: recombinant inbred lines, comparative genetic maps, and map position of 35 symbiotic loci

Development of molecular tools for the analysis of the plant genetic contribution to rhizobial and mycorrhizal symbiosis has provided major advances in our understanding of plant-microbe interactions, and several key symbiotic genes have been identified and characterized. In order to increase the efficiency of genetic analysis in the model legume Lotus japonicus, we present here a selection of improved genetic tools. The two genetic linkage maps previously developed from an interspecific cross between L. japonicus Gifu and L. filicaulis, and an intraspecific cross between the two ecotypes L. japonicus Gifu and L. japonicus MG-20, were aligned through a set of anchor markers. Regions of linkage groups, where genetic resolution is obtained preferentially using one or the other parental combination, are highlighted. Additional genetic resolution and stabilized mapping populations were obtained in recombinant inbred lines derived by a single seed descent from the two populations. For faster mapping of new loci, a selection of reliable markers spread over the chromosome arms provides a common framework for more efficient identification of new alleles and new symbiotic loci among uncharacterized mutant lines. Combining resources from the Lotus community, map positions of a large collection of symbiotic loci are provided together with alleles and closely linked molecular markers. Altogether, this establishes a common genetic resource for Lotus spp. A web-based version will enable this resource to be curated and updated regularly.     

Extracellular nucleotide signaling in adult neural stem cells: synergism with growth factor-mediated cellular proliferation

We have previously shown that the extracellular nucleoside triphosphate-hydrolyzing enzyme NTPDase2 is highly expressed in situ by stem/progenitor cells of the two neurogenic regions of the adult murine brain: the subventricular zone (type B cells) and the dentate gyrus of the hippocampus (residual radial glia). We explored the possibility that adult multipotent neural stem cells express nucleotide receptors and investigated their functional properties in vitro. Neurospheres cultured from the adult mouse SVZ in the presence of epidermal growth factor and fibroblast growth factor 2 expressed the ecto-nucleotidases NTPDase2 and the tissue non-specific isoform of alkaline phosphatase, hydrolyzing extracellular ATP to adenosine. ATP, ADP and, to a lesser extent, UTP evoked rapid Ca(2+) transients in neurospheres that were exclusively mediated by the metabotropic P2Y(1) and P2Y(2) nucleotide receptors. In addition, agonists of these receptors and low concentrations of adenosine augmented cell proliferation in the presence of growth factors. Neurosphere cell proliferation was attenuated after application of the P2Y(1)-receptor antagonist MRS2179 and in neurospheres from P2Y(1)-receptor knockout mice. In situ hybridization identified P2Y(1)-receptor mRNA in clusters of SVZ cells. Our results infer nucleotide receptor-mediated synergism that augments growth factor-mediated cell proliferation. Together with the in situ data, this supports the notion that extracellular nucleotides contribute to the control of adult neurogenesis.     

Specific sequences within arginine–glycine-rich domains affect mRNA-binding protein function

The discovery of roles for arginine methylation in intracellular transport and mRNA splicing has focused attention on the methylated arginine–glycine (RG)-rich domains found in many eukaryotic RNA-binding proteins. Sequence similarity among these highly repetitive RG domains, combined with interactions between RG-rich proteins, raises the question of whether these regions are general interaction motifs or whether there is specificity within these domains. Using the essential Saccharomyces cerevisiae mRNA-binding protein Npl3 (ScNpl3) as a model system, we first tested the importance of the RG domain for protein function. While Npl3 lacking the RG domain could not support growth of cells lacking Npl3, surprisingly, expression of the RG domain alone supported partial growth of these cells. To address the specificity of this domain, we created chimeric forms of ScNpl3 with RG-rich domains of S. cerevisiae nucleolar proteins, Gar1 and Nop1 (ScGar1, ScNop1), or of the Candida albicans Npl3 ortholog (CaNpl3). Whereas the CaNpl3 RG chimeric protein retained nearly wild-type function in S. cerevisiae, the ScGar1 and ScNop1 RG domains significantly reduced Npl3 function and self-association, indicating RG domain specificity. Nuclear localization of Npl3 also requires specific RG sequences, yet heterologous RG domains allow similar modulation of Npl3 transport by arginine methylation.     

Form–meaning links in the development of visual word recognition

Learning to read takes time and it requires explicit instruction. Three decades of research has taught us a good deal about how children learn about the links between orthography and phonology during word reading development. However, we have learned less about the links that children build between orthographic form and meaning. This is surprising given that the goal of reading development must be for children to develop an orthographic system that allows meanings to be accessed quickly, reliably and efficiently from orthography. This review considers whether meaning-related information is used when children read words aloud, and asks what we know about how and when children make connections between form and meaning during the course of reading development.     

Perception and apperception in autism: rejecting the inverse assumption

In addition to those with savant skills, many individuals with autism spectrum conditions (ASCs) show superior perceptual and attentional skills relative to the general population. These superior skills and savant abilities raise important theoretical questions, including whether they develop as compensations for other underdeveloped cognitive mechanisms, and whether one skill is inversely related to another weakness via a common underlying neurocognitive mechanism. We discuss studies of perception and visual processing that show that this inverse hypothesis rarely holds true. Instead, they suggest that enhanced performance is not always accompanied by a complementary deficit and that there are undeniable difficulties in some aspects of perception that are not related to compensating strengths. Our discussion emphasizes the qualitative differences in perceptual processing revealed in these studies between individuals with and without ASCs. We argue that this research is important not only in furthering our understanding of the nature of the qualitative differences in perceptual processing in ASCs, but can also be used to highlight to society at large the exceptional skills and talent that individuals with ASCs are able to contribute in domains such as engineering, computing and mathematics that are highly valued in industry.     

Telomerase-associated Protein 1, HSP90, and Topoisomerase II�� Associate Directly with the BLM Helicase in Immortalized Cells Using ALT and Modulate Its Helicase Activity Using Telomeric DNA Substrates

The BLM helicase associates with the telomere structural proteins TRF1 and TRF2 in immortalized cells using the alternative lengthening of telomere (ALT) pathways. This work focuses on identifying protein partners of BLM in cells using ALT. Mass spectrometry and immunoprecipitation techniques have identified three proteins that bind directly to BLM and TRF2 in ALT cells: telomerase-associated protein 1 (TEP1), heat shock protein 90 (HSP90), and topoisomerase IIα (TOPOIIα). BLM predominantly co-localizes with these proteins in foci actively synthesizing DNA during late S and G₂/M phases of the cell cycle when ALT is thought to occur. Immunoprecipitation studies also indicate that only HSP90 and TOPOIIα are components of a specific complex containing BLM, TRF1, and TRF2 but that this complex does not include TEP1. TEP1, TOPOIIα, and HSP90 interact directly with BLM in vitro and modulate its helicase activity on telomere-like DNA substrates but not on non-telomeric substrates. Initial studies suggest that knockdown of BLM in ALT cells reduces average telomere length but does not do so in cells using telomerase.Bloom syndrome (BS)⁴ is a genetic disease caused by mutation of both copies of the human BLM gene. It is characterized by sun sensitivity, small stature, immunodeficiency, male infertility, and an increased susceptibility to cancer of all sites and types. The high incidence of spontaneous chromosome breakage and other unique chromosomal anomalies in cells from BS patients indicate an increase in homologous recombination in somatic cells (1). Another notable feature of non-immortalized and immortalized cells from BS individuals is the presence of telomeric associations (TAs) between homologous chromosomes (2). Work from our group and others have suggested a role for BLM in recombination-mediated mechanisms of telomere elongation or ALT (alternative lengthening of telomeres), processes that maintain/elongate telomeres in the absence of telomerase (3–5). However, the exact mechanism by which BLM contributes to telomere stability is unknown.Several proteins interact with and regulate BLM helicase activity, including two telomere-specific proteins, TRF1 and TRF2 (6, 7). Although TRF2 stimulates BLM unwinding of telomeric and non-telomeric 3′-overhang substrates, TRF1 inhibits BLM unwinding of telomeric substrates. TRF2-mediated stimulation of BLM helicase activity on a telomeric substrate is observed when TRF2 is present in excess or with equimolar amount of TRF1 but not when TRF1 is present in molar excess. Both proteins associate with BLM specifically in ALT cells in vivo, suggesting their involvement in the ALT pathways. In addition to TRF1 and TRF2, the telomere single-strand DNA-binding protein POT1 strongly stimulates BLM helicase activity on long telomeric forked duplexes and D-loop structures (8). Other proteins also play an important role in telomere maintenance in telomerase-negative cells, including RAD50, NBS1, and MRE11, which co-localize with TRF1 and TRF2 in specialized ALT-associated promyelocytic leukemia (PML) nuclear bodies (APBs) (9–11). Thus, we hypothesize that BLM complex formation may be essential for the ALT mechanism, and its modification may occur dynamically during the specific nucleic acid transactions required to protect the telomere in cells using the ALT pathways.This study has identified previously unknown protein partners of BLM and TRF2 in ALT cells using double immunoprecipitation and mass spectrometry (MS). These include telomerase-associated protein 1 (TEP1), heat shock protein 90 (HSP90), and topoisomerase IIα (TOPOIIα). These proteins associate with BLM and TRF2 in cells using ALT but not in cells using telomerase and directly interact with BLM in vitro. This complex of proteins localizes to sites of new DNA synthesis in vivo in ALT cells, suggesting a role in telomere maintenance. We also identified HSP90 and TOPOIIα in another ALT-specific complex consisting of BLM, TRF1, and TRF2 but not TEP1. In vitro analyses demonstrate that HSP90 inhibits BLM helicase activity using both telomeric and non-telomeric substrates, whereas TEP1 and TOPOIIα initially slow the kinetics of BLM unwinding only using telomeric substrates. These findings suggest the presence of dynamic BLM-associated ALT complexes that include previously unidentified interacting proteins. The function of TEP1 in the BLM·TRF2 complex remains unclear, although its previously described interaction with the RNA subunit of telomerase (12) suggests an interesting hypothesis of cross-talk between mechanisms of telomere elongation.