Combined effects of thrombosis pathway gene variants predict cardiovascular events

The genetic background of complex diseases is proposed to consist of several low-penetrance risk loci. Addressing this complexity likely requires both large sample size and simultaneous analysis of different predisposing variants. We investigated the role of four thrombosis genes: coagulation factor V (F5), intercellular adhesion molecule 1 (ICAM1), protein C (PROC), and thrombomodulin (THBD) in cardiovascular diseases. Single allelic gene variants and their pair-wise combinations were analyzed in two independently sampled population cohorts from Finland. From among 14,140 FINRISK participants (FINRISK-92, n = 5,999 and FINRISK-97, n = 8,141), we selected for genotyping a sample of 2,222, including 528 incident cardiovascular disease (CVD) cases and random subcohorts totaling 786. To cover all known common haplotypes (>10%), 54 single nucleotide polymorphisms (SNPs) were genotyped. Classification-tree analysis identified 11 SNPs that were further analyzed in Cox's proportional hazard model as single variants and pair-wise combinations. Multiple testing was controlled by use of two independent cohorts and with false-discovery rate. Several CVD risk variants were identified: In women, the combination of F5 rs7542281 x THBD rs1042580, together with three single F5 SNPs, was associated with CVD events. Among men, PROC rs1041296, when combined with either ICAM1 rs5030341 or F5 rs2269648, was associated with total mortality. As a single variant, PROC rs1401296, together with the F5 Leiden mutation, was associated with ischemic stroke events. Our strategy to combine the classification-tree analysis with more traditional genetic models was successful in identifying SNPs-acting either in combination or as single variants--predisposing to CVD, and produced consistent results in two independent cohorts. These results suggest that variants in these four thrombosis genes contribute to arterial cardiovascular events at population level.     

The Arabidopsis MATE transporter TT12 acts as a vacuolar flavonoid/H+ -antiporter active in proanthocyanidin-accumulating cells of the seed coat

Phenotypic characterization of the Arabidopsis thaliana transparent testa12 (tt12) mutant encoding a membrane protein of the multidrug and toxic efflux transporter family, suggested that TT12 is involved in the vacuolar accumulation of proanthocyanidin precursors in the seed. Metabolite analysis in tt12 seeds reveals an absence of flavan-3-ols and proanthocyanidins together with a reduction of the major flavonol quercetin-3-O-rhamnoside. The TT12 promoter is active in cells synthesizing proanthocyanidins. Using translational fusions between TT12 and green fluorescent protein, it is demonstrated that this transporter localizes to the tonoplast. Yeast vesicles expressing TT12 can transport the anthocyanin cyanidin-3-O-glucoside in the presence of MgATP but not the aglycones cyanidin and epicatechin. Inhibitor studies demonstrate that TT12 acts in vitro as a cyanidin-3-O-glucoside/H(+)-antiporter. TT12 does not transport glycosylated flavonols and procyanidin dimers, and a direct transport activity for catechin-3-O-glucoside, a glucosylated flavan-3-ol, was not detectable. However, catechin-3-O-glucoside inhibited TT12-mediated transport of cyanidin-3-O-glucoside in a dose-dependent manner, while flavan-3-ol aglycones and glycosylated flavonols had no effect on anthocyanin transport. It is proposed that TT12 transports glycosylated flavan-3-ols in vivo. Mutant banyuls (ban) seeds accumulate anthocyanins instead of proanthocyanidins, yet the ban tt12 double mutant exhibits reduced anthocyanin accumulation, which supports the transport data suggesting that TT12 mediates anthocyanin transport in vitro.     

Targeting of alpha(v) integrins interferes with FAK activation and smooth muscle cell migration and invasion

Aberrant migration of smooth muscle cells (SMCs) is a key feature of restenosis. Since extracellular matrix proteins and their receptors of the integrin family play a critical role in this process, it is instrumental to understand their contribution to cell migration and invasive motility of SMC on the molecular level. Therefore, we investigated the role of alpha(v)-containing integrins expressed by primary human coronary artery smooth muscle cells (hCASMCs) in vitronectin (VN)-initiated signaling events and cell migration. In hCASMC plated on VN, alpha(v)-containing integrins were localized at focal adhesion sites. Haptotactic stimulation through VN led to a dose-dependent increase in cell migration and concomitantly to enhanced tyrosine phosphorylation of focal adhesion kinase. Both events were completely blocked by a specific inhibitor of integrin alpha(v). Additionally, the integrin alpha(v) inhibitor abolished PDGF-BB-stimulated chemotactic migration. Confocal microscopy confirmed the increased tyrosine phosphorylation at VN-initiated focal contact sites in hCASMC, that was abolished upon alpha(v) inhibition. In vitro invasion of hCASMC was severely compromised in the presence of the integrin alpha(v) inhibitor paralleled by decreased levels of secreted matrix metalloprotease 2 (MMP-2). Together, integrin alpha(v) inhibition abrogates tyrosine phosphorylation at focal adhesion sites and diminishes MMP-2 secretion leading to reduced migration and invasion of hCASMCs.     

Differential inhibition of inducible T cell cytokine secretion by potent iron chelators

Effector functions and proliferation of T helper (Th) cells are influenced by cytokines in the environment. Th1 cells respond to a synergistic effect of interleukin-12 (IL-12) and interleukin-18 (IL-18) to secrete interferon-gamma (IFN-gamma). In contrast, Th2 cells respond to interleukin-4 (IL-4) to secrete IL-4, interleukin-13 (IL-13), interleukin-5 (IL-5), and interleukin-10 (IL-10). The authors were interested in identifying nonpeptide inhibitors of the Th1 response selective for the IL-12/IL-18-mediated secretion of IFN-gamma while leaving the IL-4-mediated Th2 cytokine secretion relatively intact. The authors established a screening protocol using human peripheral blood mononuclear cells (PBMCs) and identified the hydrazino anthranilate compound 1 as a potent inhibitor of IL-12/IL-18-mediated IFN-gamma secretion from CD3(+) cells with an IC(50) around 200 nM. The inhibitor was specific because it had virtually no effect on IL-4-mediated IL-13 release from the same population of cells. Further work established that compound 1 was a potent intracellular iron chelator that inhibited both IL-12/IL-18- and IL-4-mediated T cell proliferation. Iron chelation affects multiple cellular pathways in T cells. Thus, the IL-12/IL-18-mediated proliferation and IFN-gamma secretion are very sensitive to intracellular iron concentration. However, the IL-4-mediated IL-13 secretion does not correlate with proliferation and is partially resistant to potent iron chelation.     

Time course of plant diversity effects on Centaurea jacea establishment and the role of competition and herbivory

Aims Invasion resistance in experimental plant communities is known to increase with increasing diversity and further to depend on the presence of particular functional groups. To test whether these effects also hold true for the invader establishment phase beyond the seedling stage, we studied survival and performance of Centaurea jacea L. (brown knapweed) planted into experimental grassland communities of varying plant biodiversity over three consecutive years. Moreover, we analysed the role of insect herbivory and biomass of the recipient community for mediating diversity effects.Methods In 2005, seedlings of Centaurea were transplanted into experimental grassland communities (the Jena Experiment) covering a species richness (1–60) and functional group richness (1–4) gradient. Half of these transplants and the community surrounding them in each plot were sprayed with insecticide while the other half served as control. In 2006 and 2007 (during the second and third year after transplantation), we recorded survival, growth-related (e.g. transplant biomass, height) and reproduction-related traits (e.g. number of flower heads). Annual data on community aboveground biomass served as covariate to investigate mediating effects of aboveground competition with the recipient community.Important findings Species richness was the most important factor responsible for Centaurea limitation. Higher levels of diversity decreased survival and all performance traits in both years. These diversity effects were partly driven by community biomass, but not fully explained by that covariate, suggesting the importance also of further processes. The influence of functional group richness was strong in the second year after transplantation and weaker in the third year. Among the particular functional groups, only the presence of legumes showed strong negative effects on Centaurea survival and weak negative effects on growth and reproduction, the latter two being mediated by biomass. Insect herbivore reduction considerably benefited Centaurea in sprayed monocultures, where it grew significantly larger than in all other diversity levels and than in the control subplots. We conclude that effects of plant community properties on invading individuals change in the course of establishment, that plant species richness effects are also important during later stages of establishment, and that biomass (especially at high diversity) and herbivory (especially at low diversity) of the recipient community are important in mediating community effects on invaders.     

Kap120 functions as a nuclear import receptor for ribosome assembly factor Rpf1 in yeast

The nucleocytoplasmic exchange of macromolecules is mediated by receptors specialized in passage through the nuclear pore complex. The majority of these receptors belong to the importin beta protein family, which has 14 members in Saccharomyces cerevisiae. Nine importins carry various cargos from the cytoplasm into the nucleus, whereas four exportins mediate nuclear export. Kap120 is the only receptor whose transport cargo has not been found previously. Here, we characterize Kap120 as an importin for the ribosome maturation factor Rpf1, which was identified in a two-hybrid screen. Kap120 binds directly to Rpf1 in vitro and is released by Ran-GTP. At least three parallel import pathways exist for Rpf1, since nuclear import is defective in strains with the importins Kap120, Kap114, and Nmd5 deleted. Both kap120 and rpf1 mutants accumulate large ribosomal subunits in the nucleus. The nuclear accumulation of 60S ribosomal subunits in kap120 mutants is abolished upon RPF1 overexpression, indicating that Kap120 does not function in the actual ribosomal export step but rather in import of ribosome maturation factors.     

Strong genetic evidence of DCDC2 as a susceptibility gene for dyslexia

We searched for linkage disequilibrium (LD) in 137 triads with dyslexia, using markers that span the most-replicated dyslexia susceptibility region on 6p21-p22, and found association between the disease and markers within the VMP/DCDC2/KAAG1 locus. Detailed refinement of the LD region, involving sequencing and genotyping of additional markers, showed significant association within DCDC2 in single-marker and haplotype analyses. The association appeared to be strongest in severely affected patients. In a second step, the study was extended to include an independent sample of 239 triads with dyslexia, in which the association--in particular, with the severe phenotype of dyslexia--was confirmed. Our expression data showed that DCDC2, which contains a doublecortin homology domain that is possibly involved in cortical neuron migration, is expressed in the fetal and adult CNS, which--together with the hypothesized protein function--is in accordance with findings in dyslexic patients with abnormal neuronal migration and maturation.     

Relationship of hyperthermia-induced hemolysis of human erythrocytes to the thermal denaturation of membrane proteins

Hemolysis of human erythrocytes as a function of time of exposure to 47.4-54.5 degrees C was measured and correlated to thermal transitions in the membranes of intact erythrocytes as determined by differential scanning calorimetry (DSC). Curves of hemoglobin leakage (a measure of hemolysis) as a function of time have a shoulder region exhibiting no leakage, indicative of the ability to accumulate sublethal damage (i.e., damage not sufficient to cause lysis), followed by a region of leakage approximating pseudo-first-order kinetics. Inverse leakage rates (Do) of 330-21 min were obtained from 47.4-54.5 degrees C, respectively. A relatively high activation energy of 304 +/- 22 kJ/mol was obtained for leakage, eliminating the involvement of metabolic processes but implicating a transition as the rate-limiting step. Membrane protein involvement was suggested by the very low rate (10(-2) of the rate from erythrocytes) and low activation energy (50 +/- 49 kJ/mol) of hemoglobin leakage from liposomes containing no membrane protein. A model was developed that predicts a transition temperature (Tm) for the critical target (rate-limiting step) of 60 degrees C when measured at a scan rate of 1 K/min. DSC scans were obtained from intact erythrocytes and a procedure developed to fit and remove the transition for hemoglobin denaturation which dominated the scan. Three transitions remained (transitions A, B, and C) with Tm values of 50.0, 56.8, and 63.8 degrees C, respectively. These correspond to, but occur at slightly different temperatures than, the A, B, and C transitions of isolated erythrocyte membranes in the same salt solution (Tm = 49.5, 53-58, and 65.5 degrees C, respectively). In addition, the relative enthalpies of the three transitions differ between isolated membranes and erythrocytes, suggestive of membrane alterations occurring during isolation. Thus, all analyses were conducted on DSC scans of intact erythrocytes. The B transition is very broad and probably consists of several transitions. An inflection, which is seen as a distinct peak (transition B3) in fourth-derivative curves, occurs at 60.8 degrees C and correlates well with the predicted Tm of the critical target. Ethanol (2.2%) lowers the Tm of B3 by 4.0-4.5 K, close to the shift of 3.3 K predicted from its effect on hemolysis. Glycerol (10%) has very little effect on both hemolysis and the Tm of B3, but it stabilizes spectrin (delta Tm = 1.5 K) against thermal denaturation.(ABSTRACT TRUNCATED AT 400 WORDS)     

The heparan sulfate proteoglycan agrin contributes to barrier properties of mouse brain endothelial cells by stabilizing adherens junctions

Barrier characteristics of brain endothelial cells forming the blood–brain barrier (BBB) are tightly regulated by cellular and acellular components of the neurovascular unit. During embryogenesis, the accumulation of the heparan sulfate proteoglycan agrin in the basement membranes ensheathing brain vessels correlates with BBB maturation. In contrast, loss of agrin deposition in the vasculature of brain tumors is accompanied by the loss of endothelial junctional proteins. We therefore wondered whether agrin had a direct effect on the barrier characteristics of brain endothelial cells. Agrin increased junctional localization of vascular endothelial (VE)-cadherin, β-catenin, and zonula occludens-1 (ZO-1) but not of claudin-5 and occludin in the brain endothelioma cell line bEnd5 without affecting the expression levels of these proteins. This was accompanied by an agrin-induced reduction of the paracellular permeability of bEnd5 monolayers. In vivo, the lack of agrin also led to reduced junctional localization of VE-cadherin in brain microvascular endothelial cells. Taken together, our data support the notion that agrin contributes to barrier characteristics of brain endothelium by stabilizing the adherens junction proteins VE-cadherin and β-catenin and the junctional protein ZO-1 to brain endothelial junctions.