Sodium channel selectivity and conduction: Prokaryotes have devised their own molecular strategy

Striking structural differences between voltage-gated sodium (Nav) channels from prokaryotes (homotetramers) and eukaryotes (asymmetric, four-domain proteins) suggest the likelihood of different molecular mechanisms for common functions. For these two channel families, our data show similar selectivity sequences among alkali cations (relative permeability, Pion/PNa) and asymmetric, bi-ionic reversal potentials when the Na/K gradient is reversed. We performed coordinated experimental and computational studies, respectively, on the prokaryotic Nav channels NaChBac and NavAb. NaChBac shows an “anomalous,” nonmonotonic mole-fraction dependence in the presence of certain sodium–potassium mixtures; to our knowledge, no comparable observation has been reported for eukaryotic Nav channels. NaChBac’s preferential selectivity for sodium is reduced either by partial titration of its highly charged selectivity filter, when extracellular pH is lowered from 7.4 to 5.8, or by perturbation—likely steric—associated with a nominally electro-neutral substitution in the selectivity filter (E191D). Although no single molecular feature or energetic parameter appears to dominate, our atomistic simulations, based on the published NavAb crystal structure, revealed factors that may contribute to the normally observed selectivity for Na over K. These include: (a) a thermodynamic penalty to exchange one K⁺ for one Na⁺ in the wild-type (WT) channel, increasing the relative likelihood of Na⁺ occupying the binding site; (b) a small tendency toward weaker ion binding to the selectivity filter in Na–K mixtures, consistent with the higher conductance observed with both sodium and potassium present; and (c) integrated 1-D potentials of mean force for sodium or potassium movement that show less separation for the less selective E/D mutant than for WT. Overall, tight binding of a single favored ion to the selectivity filter, together with crucial inter-ion interactions within the pore, suggests that prokaryotic Nav channels use a selective strategy more akin to those of eukaryotic calcium and potassium channels than that of eukaryotic Nav channels.     

糖浓度及BMP4对小鼠诱导型多能干细胞成骨作用的研究

诱导型多能干细胞（induced pluripotent stem cells,iPS cells）技术的建立为自体组织工程治疗带来了新的希望。鉴于糖尿病患者常伴有骨再生性障碍,该研究比较了不同葡萄糖浓度下小鼠iPS细胞的成骨能力,并探讨了骨形态蛋4（bone morphogenetic protein4,BMP4）在该过程中的作用。实验结果显示：成骨诱导21d后,低糖组茜素红阳性细胞比例和成骨基INRunx2、Osteocalcin的表达水平显著高于高糖组和自发分化组（P〈0．05）;BMP4的添加提高了高糖组茜素红阳性细胞比例及Osteocalcin的表达水平（P〈0．05）,而对自发分化组细胞的成骨水平无影响。该结果表明：低葡萄糖含量对小鼠iPS细胞的骨向分化有促进作用’尽管BMP4可以提高高糖组小鼠iPS细胞的成骨能力,但仅在成骨分化条件下发挥作用。     

The 9p21 Locus Is Associated with Coronary Artery Disease and Cardiovascular Events in the Presence (but Not in the Absence) of Coronary Calcification

Variants at the 9p21 locus have been associated with coronary artery disease (CAD); coronary artery calcification (CAC) is related to CAD and other cardiovascular events. To determine the association of the 9p21 locus with CAD in the presence and absence of CAC, 4 groups were enrolled in a case-control study, including 527 CAD patients without CAC, 692 CAD patients with CAC, 585 individuals with simple CAC but no CAD, and 725 healthy controls. The rs1333049 representing the locus was associated with CAD in the presence of CAC (odds ratio = 1.38 in allelic analysis, 95%CI, 1.19–1.60, P<0.001), but not in the absence of CAC. Additionally, rs1333049 was not associated with simple CAC or CAC severity/extent in CAD patients with CAC. 849 CAD patients undergoing revascularization (660 with CAC and 189 without CAC) were enrolled in a cohort study to test its association with cardiovascular events in CAD patients with and without CAC in a 3-year follow-up. rs1333049 was significantly associated with the incidence of cardiovascular events in non-target vessels in patients with CAC (hazard ratio = 1.44, 95%CI, 1.08–1.91, P = 0.012), but not in those without CAC. The variants at the 9p21 locus were related to CAD and post-revascularization events only in the presence of CAC, suggesting that they may confer risk of calcification-related coronary atherosclerosis.     

Functional Motor Recovery from Motoneuron Axotomy Is Compromised in Mice with Defective Corticospinal Projections

Brachial plexus injury (BPI) and experimental spinal root avulsion result in loss of motor function in the affected segments. After root avulsion, significant motoneuron function is restored by re-implantation of the avulsed root. How much this functional recovery depends on corticospinal inputs is not known. Here, we studied that question using Celsr3|Emx1 mice, in which the corticospinal tract (CST) is genetically absent. In adult mice, we tore off right C5–C7 motor and sensory roots and re-implanted the right C6 roots. Behavioral studies showed impaired recovery of elbow flexion in Celsr3|Emx1 mice compared to controls. Five months after surgery, a reduced number of small axons, and higher G-ratio of inner to outer diameter of myelin sheaths were observed in mutant versus control mice. At early stages post-surgery, mutant mice displayed lower expression of GAP-43 in spinal cord and of myelin basic protein (MBP) in peripheral nerves than control animals. After five months, mutant animals had atrophy of the right biceps brachii, with less newly formed neuromuscular junctions (NMJs) and reduced peak-to-peak amplitudes in electromyogram (EMG), than controls. However, quite unexpectedly, a higher motoneuron survival rate was found in mutant than in control mice. Thus, following root avulsion/re-implantation, the absence of the CST is probably an important reason to hamper axonal regeneration and remyelination, as well as target re-innervation and formation of new NMJ, resulting in lower functional recovery, while fostering motoneuron survival. These results indicate that manipulation of corticospinal transmission may help improve functional recovery following BPI.     

A Pharmacogenetics-Based Warfarin Maintenance Dosing Algorithm from Northern Chinese Patients

Inconsistent associations with warfarin dose were observed in genetic variants except VKORC1 haplotype and CYP2C9*3 in Chinese people, and few studies on warfarin dose algorithm was performed in a large Chinese Han population lived in Northern China. Of 787 consenting patients with heart-valve replacements who were receiving long-term warfarin maintenance therapy, 20 related Single nucleotide polymorphisms were genotyped. Only VKORC1 and CYP2C9 SNPs were observed to be significantly associated with warfarin dose. In the derivation cohort (n = 551), warfarin dose variability was influenced, in decreasing order, by VKORC1 rs7294 (27.3%), CYP2C9*3(7.0%), body surface area(4.2%), age(2.7%), target INR(1.4%), CYP4F2 rs2108622 (0.7%), amiodarone use(0.6%), diabetes mellitus(0.6%), and digoxin use(0.5%), which account for 45.1% of the warfarin dose variability. In the validation cohort (n = 236), the actual maintenance dose was significantly correlated with predicted dose (r = 0.609, P<0.001). Our algorithm could improve the personalized management of warfarin use in Northern Chinese patients.     

Neutral sphingomyelinase 2 is a key factor for PorB‐dependent invasion of Neisseria gonorrhoeae

Disseminated gonococcal infection (DGI) is a rare but serious complication caused by the spread of Neisseria gonorrhoeae in the human host. Gonococci associated with DGI mainly express the outer membrane protein PorB_IA that binds to the scavenger receptor expressed on endothelial cells (SREC‐I) and mediates bacterial uptake. We recently demonstrated that this interaction relies on intact membrane rafts that acquire SREC‐I upon attachment of gonococci and initiates the signalling cascade that finally leads to the uptake of gonococci in epithelial cells. In this study, we analysed the role of sphingomyelinases and their breakdown product ceramide. Gonococcal infection induced increased levels of ceramide that was enriched at bacterial attachment sites. Interestingly, neutral but not acid sphingomyelinase was mandatory for PorB_IA‐mediated invasion into host cells. Neutral sphingomyelinase was required to recruit the PI3 kinase to caveolin and thereby activates the PI3 kinase‐dependent downstream signalling leading to bacterial uptake. Thus, this study elucidates the initial signalling processes of bacterial invasion during DGI and demonstrates a novel role for neutral sphingomyelinase in the course of bacterial infections.     

Warming and grazing interact to affect root dynamics in an alpine meadow

Plant and Soil - Arbuscular Mycorrhizal Fungi (AMF) are ubiquitous soil microorganisms playing a vital role in the functioning of agricultural ecosystems. Although AMF are generally considered to...     

Cypher/ZASP Is a Novel A-kinase Anchoring Protein

PKA signaling is important for the post-translational modification of proteins, especially those in cardiomyocytes involved in cardiac excitation-contraction coupling. PKA activity is spatially and temporally regulated through compartmentalization by protein kinase A anchoring proteins. Cypher/ZASP, a member of PDZ-LIM domain protein family, is a cytoskeletal protein that forms multiprotein complexes at sarcomeric Z-lines. It has been demonstrated that Cypher/ZASP plays a pivotal structural role in the structural integrity of sarcomeres, and several of its mutations are associated with myopathies including dilated cardiomyopathy. Here we show that Cypher/ZASP, interacting specifically with the type II regulatory subunit RIIα of PKA, acted as a typical protein kinase A anchoring protein in cardiomyocytes. In addition, we show that Cypher/ZASP itself was phosphorylated at Ser²⁶⁵ and Ser²⁹⁶ by PKA. Furthermore, the PDZ domain of Cypher/ZASP interacted with the L-type calcium channel through its C-terminal PDZ binding motif. Expression of Cypher/ZASP facilitated PKA-mediated phosphorylation of the L-type calcium channel in vitro. Additionally, the phosphorylation of the L-type calcium channel at Ser¹⁹²⁸ induced by isoproterenol was impaired in neonatal Cypher/ZASP-null cardiomyocytes. Moreover, Cypher/ZASP interacted with the Ser/Thr phosphatase calcineurin, which is a phosphatase for the L-type calcium channel. Taken together, our data strongly suggest that Cypher/ZASP not only plays a structural role for the sarcomeric integrity, but is also an important sarcomeric signaling scaffold in regulating the phosphorylation of channels or contractile proteins.