Multiple loci are associated with white blood cell phenotypes

White blood cell (WBC) count is a common clinical measure from complete blood count assays, and it varies widely among healthy individuals. Total WBC count and its constituent subtypes have been shown to be moderately heritable, with the heritability estimates varying across cell types. We studied 19,509 subjects from seven cohorts in a discovery analysis, and 11,823 subjects from ten cohorts for replication analyses, to determine genetic factors influencing variability within the normal hematological range for total WBC count and five WBC subtype measures. Cohort specific data was supplied by the CHARGE, HeamGen, and INGI consortia, as well as independent collaborative studies. We identified and replicated ten associations with total WBC count and five WBC subtypes at seven different genomic loci (total WBC count-6p21 in the HLA region, 17q21 near ORMDL3, and CSF3; neutrophil count-17q21; basophil count- 3p21 near RPN1 and C3orf27; lymphocyte count-6p21, 19p13 at EPS15L1; monocyte count-2q31 at ITGA4, 3q21, 8q24 an intergenic region, 9q31 near EDG2), including three previously reported associations and seven novel associations. To investigate functional relationships among variants contributing to variability in the six WBC traits, we utilized gene expression- and pathways-based analyses. We implemented gene-clustering algorithms to evaluate functional connectivity among implicated loci and showed functional relationships across cell types. Gene expression data from whole blood was utilized to show that significant biological consequences can be extracted from our genome-wide analyses, with effect estimates for significant loci from the meta-analyses being highly corellated with the proximal gene expression. In addition, collaborative efforts between the groups contributing to this study and related studies conducted by the COGENT and RIKEN groups allowed for the examination of effect homogeneity for genome-wide significant associations across populations of diverse ancestral backgrounds.     

Unlocking the barley genome by chromosomal and comparative genomics

We used a novel approach that incorporated chromosome sorting, next-generation sequencing, array hybridization, and systematic exploitation of conserved synteny with model grasses to assign ~86% of the estimated ~32,000 barley (Hordeum vulgare) genes to individual chromosome arms. Using a series of bioinformatically constructed genome zippers that integrate gene indices of rice (Oryza sativa), sorghum (Sorghum bicolor), and Brachypodium distachyon in a conserved synteny model, we were able to assemble 21,766 barley genes in a putative linear order. We show that the barley (H) genome displays a mosaic of structural similarity to hexaploid bread wheat (Triticum aestivum) A, B, and D subgenomes and that orthologous genes in different grasses exhibit signatures of positive selection in different lineages. We present an ordered, information-rich scaffold of the barley genome that provides a valuable and robust framework for the development of novel strategies in cereal breeding.     

A new method for the reconstitution of the anion transport system of the human erythrocyte membrane

Summary The anion transport protein of the human erythrocyte membrane, band 3, was solubilized and purified in solutions of the non-ionic detergent Triton X-100. It was incorporated into spherical lipid bilayers by the following procedure: (1) Dry phosphatidylcholine was suspended in the protein solution. Octylglucopyranoside was added until the milky suspension became clear. (2) The sample was dialyzed overnight against detergentfree buffer. (3) Residual Triton X-100 was removed from the opalescent vesicle suspension by sucrose density gradient centrifugation and subsequent dialysis. Sulfate efflux from the vesicles was studied, under exchange conditions, using a filtration method. Three vesicle subpopulations could be distinguished by analyzing the time course of the efflux. One was nearly impermeable to sulfate, and efflux from another was due to leaks. The largest subpopulation, however, showed transport characteristics very similar to those of the anion transport system of the intact erythrocyte membrane: transport numbers (at 30°C) close to 20 sulfate molecules per band 3 and min, an activation energy of approx. 140 kJ/mol, a pH maximum at pH 6.2, saturation of the sulfate flux at sulfate concentrations around 100mm, inhibition of the flux by H₂DIDS and flufenamate (approx.K _l-values at 30°C: 0.1 and 0.7 m, respectively), and right-side-out orientation of the transport protein (as judged from the inhibition of sulfate efflux by up to 98% by externally added H₂DIDS). Thus, the system represents, for the first time, a reconstitution of all the major properties of the sulfate transport across the erythrocyte membrane.     

The Claudin Megatrachea Protein Complex

Claudins are integral transmembrane components of the tight junctions forming trans-epithelial barriers in many organs, such as the nervous system, lung, and epidermis. In Drosophila three claudins have been identified that are required for forming the tight junctions analogous structure, the septate junctions (SJs). The lack of claudins results in a disruption of SJ integrity leading to a breakdown of the trans-epithelial barrier and to disturbed epithelial morphogenesis. However, little is known about claudin partners for transport mechanisms and membrane organization. Here we present a comprehensive analysis of the claudin proteome in Drosophila by combining biochemical and physiological approaches. Using specific antibodies against the claudin Megatrachea for immunoprecipitation and mass spectrometry, we identified 142 proteins associated with Megatrachea in embryos. The Megatrachea interacting proteins were analyzed in vivo by tissue-specific knockdown of the corresponding genes using RNA interference. We identified known and novel putative SJ components, such as the gene product of CG3921. Furthermore, our data suggest that the control of secretion processes specific to SJs and dependent on Sec61p may involve Megatrachea interaction with Sec61 subunits. Also, our findings suggest that clathrin-coated vesicles may regulate Megatrachea turnover at the plasma membrane similar to human claudins. As claudins are conserved both in structure and function, our findings offer novel candidate proteins involved in the claudin interactome of vertebrates and invertebrates.     

‘Further Development’ of Mendel’s legacy? Erich von Tschermak-Seysenegg in the context of Mendelian–biometry controversy, 1901–1906

The contribution of Erich von Tschermak-Seysenegg (1871?C1962) to the beginning of classical genetics is a matter of dispute. The aim of this study is to analyse, based on newly accessible archive materials, the relevance of his positions and theoretical views in a debate between advocates of early Mendelian explanation of heredity and proponents of biometry, which took place in England around 1901?C1906. We challenge not only his role of an ??external consultant??, which at the time de facto confirmed his status of ??rediscoverer?? of Mendel??s work but also analyse his ambivalent positions which are to be seen as a part of ??further development?? (Weiterführung), a development of Mendel??s legacy as he understood it. Second, there is an interesting aspect of establishing connections within an ??experimental culture?? along the Mendel??s lines of thought that was parallel to the first step of institutionalizing the new discipline of Genetics after 1905/06. Part of the study is also the analysis of contribution of his older brother Armin von Tschermak-Seysenegg (1870?C1952) who??much like in the case of ??rediscovery?? of 1900?C1901??was for his younger brother an important source of theoretical knowledge. In this particular case, it regarded Bateson??s ??Defence?? of Mendel from 1902.     

Conformational changes in the rod domain of human keratin 8 following heterotypic association with keratin 18 and its implication for filament stability

Keratin intermediate filaments are heteropolymers of type I and type II polypeptides that constitute the bulk of the epithelial cytoskeleton. We microinjected seven keratin monoclonal antibodies into human epithelial cells, and two of them, only A45-B/B3 and LP3K, caused the formation of keratin aggregates. The keratin filaments in human epithelial cells were also disrupted by a monovalent A45-B/B3 Fab fragment, suggesting that the binding of the antibody, rather than cross-linking, collapses the filaments. Immunoblotting and ELISA experiments suggested that the antibody reacted weakly with recombinant K8 but did not react with recombinant K18 at all. However, the antibody reactivity increased substantially when a mixture of the two keratin polypeptides, either recombinant or derived from MCF-7, was used. The epitopes of 15 monoclonal antibodies recognizing human K8 were characterized by their reactivity with recombinant fragments of K8. Reactivity of antibody A45-B/B3 with fragments of K8 in the presence of K18 revealed that the antibody recognizes an epitope in the rod domain of K8, between residues 313 and 332, on the amino-terminal side of the stutter in helix 2B, which is involved in heterotypic association. The data suggest that this region of K8 undergoes a conformational change following interaction with the complementary K18 either to expose the epitope or to increase its affinity for the antibody. Taken together, the data highlight the role of this epitope in heterotypic association and in filament stabilization.     

Ligand-induced coupling versus receptor pre-association: cellular automaton simulations of FGF-2 binding

The binding of basic fibroblast growth factor (FGF-2) to its cell surface receptor (CSR) and subsequent signal transduction is known to be enhanced by heparan sulfate proteoglycans (HSPGs). HSPGs bind FGF-2 with low affinity and likely impact CSR-mediated signaling via stabilization of FGF-2-CSR complexes via association with both the ligand and the receptor. What is unknown is whether HSPG associates with CSR in the absence of FGF-2. In this paper, we determine conditions by which pre-association would impact CSR-FGF-2-HSPG triad formation assuming diffusion-limited surface reactions. Using mean-field rate equations, we show that (i) when [HSPG] is much higher than [CSR], the presence of pre-formed complexes does not affect the steady state of FGF-2 binding, and (ii) when the concentrations are comparable, the presence of pre-formed complexes substantially increases the steady-state concentration of FGF-2 bound to CSR. These findings are supported by explicit cellular automaton simulations, which justify the mean-field treatment. We discuss the advantages of such a two-receptor system compared to a single-receptor model, when the parameters are comparable. Further, we speculate that the observed high concentration of HSPG in intact cells ([HSPG]-100[CSR]) provides a way to ensure that the binding levels of FGF-2 to its signaling receptor remains high, irrespective of the presence of pre-formed CSR-HSPG complexes on the cell surface, while allowing the cell to finely tune the response to FGF-2 via down-regulation of the signaling receptor.     

Structure and function of vertebrate CMP-sialic acid synthetases

Activation of sugars into nucleotide sugars is critical for their entry into biosynthetic pathways. In eukaryotic cells, the activation of the acidic nine-carbon sugar sialic acid to CMP-sialic acid takes place in the cell nucleus, whereas all other nucleotide sugars are made in the cytoplasm. Molecular cloning of vertebrate CMP-sialic acid synthetases confirmed the nuclear localization and introduced new molecular tools for directly exploring the functional mechanisms of the enzymes, as well as the physiological relevance of their nuclear transport. Although major advances have been made in understanding structure-function relationships and defining elements involved in the nuclear transport, the riddle surrounding the physiological relevance of nuclear localization awaits resolution.