This broad replication provides unprecedented evidence for association between genetic variants at chromosome 9p21.3 and risk of CAD.

No abstract

We present a three-stage analysis of genome-wide SNP data in 1,222 German individuals with myocardial infarction and 1,298 controls, in silico replication in three additional genome-wide datasets of coronary artery disease (CAD) and subsequent replication in ~25,000 subjects. We identified one new CAD risk locus on 3q22.3 in MRAS (P = 7.44 × 10−13; OR = 1.15, 95% CI = 1.11–1.19), and suggestive association with a locus on 12q24.31 near HNF1A-C12orf43 (P = 4.81 × 10−7; OR = 1.08, 95% CI = 1.05–1.11).

Cell-free expression is emerging as a prime method for the rapid production of preparative quantities of high-quality membrane protein samples. The technology facilitates easy access to large numbers of proteins that have been extremely difficult to obtain. Most frequently used are cell-free systems based on extracts of Escherichia coli cells, and the reaction procedures are reliable and efficient. This protocol describes the preparation of all essential reaction components such as the E. coli cell extract, T7 RNA polymerase, DNA templates as well as the individual stock solutions. The setups of expression reactions in analytical and preparative scales, including a variety of reaction designs, are illustrated. We provide detailed reaction schemes that allow the preparation of milligram amounts of functionally folded membrane proteins of prokaryotic and eukaryotic origin in less than 24 h.

Cell-free expression has become a highly promising tool for the fast and efficient production of integral membrane proteins. The proteins can be produced as precipitates that solubilize in mild detergents usually without any prior denaturation steps. Alternatively, membrane proteins can be synthesized in a soluble form by adding detergents to the cell-free system. However, the effects of a representative variety of detergents on the production, solubility and activity of a wider range of membrane proteins upon cell-free expression are currently unknown. We therefore analyzed the cell-free expression of three structurally very different membrane proteins, namely the bacterial a-helical multidrug transporter, EmrE, the b-barrel nucleoside transporter, Tsx, and the porcine vasopressin receptor of the eukaryotic superfamily of G-protein coupled receptors. All three membrane proteins could be produced in amounts of several mg per one ml of reaction mixture. In general, the detergent 1-myristoyl-2-hydroxy-snglycero-3-[phospho-rac-(1-glycerol)] was found to be most effective for the resolubilization of membrane protein precipitates, while long chain polyoxyethylene-alkyl-ethers proved to be most suitable for the soluble expression of all three types of membrane proteins. The yield of soluble expressed membrane protein remained relatively stable above a certain threshold concentration of the detergents. We report, for the first time, the high-level cell-free expression of a b-barrel type membrane protein in a functional form. Structural and functional variations of the analyzed membrane proteins are evident that correspond with the mode of expression and that depend on the supplied detergent. (20)-oleyl-ether; CE, continuous exchange; CF, cell-free; CMC, critical micellar concentration; C mic , micellar concentration; DDM, N-dodecyl-b-D-maltoside; DHPC, 1,2-diheptanoyl-sn-glycero-3-phosphocholine; diC 6 PC, 1,2-dihexanoyl-sn-glycero-3-phosphocholine; diC 8 PC, 1,2-dioctanoyl-sn-glycero-3-phosphocholine; DM, n-decyl-b-maltoside; DPC, dodecylphosphocholine; FM, feeding mixture; Genapol C-100, polyoxyethylene-(10)-dodecyl-ether; GPCR, G-protein coupled receptor; LMPG, 1-myristoyl-2-hydroxy-sn-glycero-3-[phospho-rac-(1-glycerol)]; LPPG, 1-palmitoyl-2-hydroxy-sn-glycero-3-[phospho-rac-(1-glycerol)]; MP, integral membrane protein; NP40, nonylphenyl polyethylene glycol; RM, reaction mixture; Thesit, polyethylene glycol 400 dedecyl ether; TMS, transmembrane segment; Triton X-100, polyethylene glycol P-1,1,3,3-tetramethyl-butylphenyl ether; Tween 20, polyoxyethylene sorbitan monolaurate 20.

Tail-anchored (TA) proteins are involved in cellular processes including trafficking, degradation, and apoptosis. They contain a C-terminal membrane anchor and are posttranslationally delivered to the endoplasmic reticulum (ER) membrane by the Get3 adenosine triphosphatase interacting with the hetero-oligomeric Get1/2 receptor. We have determined crystal structures of Get3 in complex with the cytosolic domains of Get1 and Get2 in different functional states at 3.0, 3.2, and 4.6 angstrom resolution. The structural data, together with biochemical experiments, show that Get1 and Get2 use adjacent, partially overlapping binding sites and that both can bind simultaneously to Get3. Docking to the Get1/2 complex allows for conformational changes in Get3 that are required for TA protein insertion. These data suggest a molecular mechanism for nucleotide-regulated delivery of TA proteins.

Although physiological data on microcircuits involving a few inhibitory neurons in the mammalian cerebral cortex are available, data on the quantitative relation between inhibition and excitation in cortical circuits involving thousands of neurons are largely missing. Because the distribution of neurons is very inhomogeneous in the cerebral cortex, it is critical to map all neurons in a given volume rather than to rely on sparse sampling methods. Here, we report the comprehensive mapping of interneurons (INs) in cortical columns of rat somatosensory cortex, immunolabeled for neuron-specific nuclear protein and glutamate decarboxylase. We found that a column contains ∼2,200 INs (11.5% of ∼19,000 neurons), almost a factor of 2 less than previously estimated. The density of GABAergic neurons was inhomogeneous between layers, with peaks in the upper third of L2/3 and in L5A. IN density therefore defines a distinct layer 2 in the sensory neocortex. In addition, immunohistochemical markers of IN subtypes were layer-specific. The "hot zones" of inhibition in L2 and L5A match the reported low stimulus-evoked spiking rates of excitatory neurons in these layers, suggesting that these inhibitory hot zones substantially suppress activity in the neocortex.F or a quantitative understanding of the interaction between excitatory and inhibitory synaptic transmission in the neocortex, it is crucial to obtain data on the absolute numbers and the relative distribution of excitatory and inhibitory (mostly GABAergic) neurons [interneurons (INs)] in a cortical column. Only on the basis of such prevalence numbers is it possible to interpret data on single-cell physiology (1-8) and synaptic connections of pairs of neurons (9-12) at the circuit level. The distribution of cortical neurons and INs has therefore been the objective of several studies over the past decades (13-15). Statistical sampling methods were used because mapping tens of thousands of neurons was not feasible. Although the nominal error bounds of such methods are in the range of 10-20%, the reported absolute numbers differed strongly among studies, especially for sparse neuron populations, such as INs and their subtypes. The ratio of INs reported for the somatosensory cortex varied between 15% and 25%, thus by almost a factor of 2 (13,16,17). Data on possible differences in IN density between layers in a cortical column were contradictory (13,15,16,18,19).To resolve these substantial discrepancies, we undertook the complete mapping of INs in entire cortical columns using the cytoarchitectonic barrels in L4 as a reference frame for the thalamocortical innervation volume (20) in postnatal day (P) 25-36 rat vibrissal cortex. Our data show that the overall prevalence of inhibitory neurons was previously overestimated by almost a factor of 2. We find a unique substructure of the distribution of INs in a cortical column that can explain the layer-specific differences in the excitability in neocortex. The distribution of INs defines layer 2 as a distinct neocortical layer.

The RJ software package is freely available at http://www.randomjungle.org