General mechanism of spider toxin family I acting on sodium channel Nav1.7.

Various peptide toxins in animal venom inhibit voltage-gated sodium ion channel Nav1.7, including Nav-targeting spider toxin (NaSpTx) Family I. Toxins in NaSpTx Family I share a similar structure, i.e., N-terminal, loops 1-4, and C-terminal. Here, we used Mu-theraphotoxin-Ca2a (Ca2a), a peptide isolated from Cyriopagopus albostriatus, as a template to investigate the general properties of toxins in NaSpTx Family I. The toxins interacted with the cell membrane prior to binding to Nav1.7 via similar hydrophobic residues. Residues in loop 1, loop 4, and the C-terminal primarily interacted with the S3-S4 linker of domain II, especially basic amino acids binding to E818. We also identified the critical role of loop 2 in Ca2a regarding its affinity to Nav1.7. Our results provide further evidence that NaSpTx Family I toxins share similar structures and mechanisms of binding to Nav1.7.

Molecular simulation assisted identification of Ca(2+) binding residues in TMEM16A.

Calcium-activated chloride channels (CaCCs) play vital roles in a variety of physiological processes. Transmembrane protein 16A (TMEM16A) has been confirmed as the molecular counterpart of CaCCs which greatly pushes the molecular insights of CaCCs forward. However, the detailed mechanism of Ca(2+) binding and activating the channel is still obscure. Here, we utilized a combination of computational and electrophysiological approaches to discern the molecular mechanism by which Ca(2+) regulates the gating of TMEM16A channels. The simulation results show that the first intracellular loop serves as a Ca(2+) binding site including D439, E444 and E447. The experimental results indicate that a novel residue, E447, plays key role in Ca(2+) binding. Compared with WT TMEM16A, E447Y produces a 30-fold increase in EC50 of Ca(2+) activation and leads to a 100-fold increase in Ca(2+) concentrations that is needed to fully activate the channel. The following steered molecular dynamic (SMD) simulation data suggests that the mutations at 447 reduce the Ca(2+) dissociation energy. Our results indicated that both the electrical property and the size of the side-chain at residue 447 have significant effects on Ca(2+) dependent gating of TMEM16A.

Conformational Motions and Functionally Key Residues for Vitamin B12 Transporter BtuCD-BtuF Revealed by Elastic Network Model with a Function-Related Internal Coordinate.

BtuCD-BtuF from Escherichia coli is a binding protein-dependent adenosine triphosphate (ATP)-binding cassette (ABC) transporter system that uses the energy of ATP hydrolysis to transmit vitamin B12 across cellular membranes. Experimental studies have showed that during the transport cycle, the transporter undergoes conformational transitions between the "inward-facing" and "outward-facing" states, which results in the open-closed motions of the cytoplasmic gate of the transport channel. The opening-closing of the channel gate play critical roles for the function of the transporter, which enables the substrate vitamin B12 to be translocated into the cell. In the present work, the extent of opening of the cytoplasmic gate was chosen as a function-related internal coordinate. Then the mean-square fluctuation of the internal coordinate, as well as the cross-correlation between the displacement of the internal coordinate and the movement of each residue in the protein, were calculated based on the normal mode analysis of the elastic network model to analyze the function-related motions encoded in the structure of the system. In addition, the key residues important for the functional motions of the transporter were predicted by using a perturbation method. In order to facilitate the calculations, the internal coordinate was introduced as one of the axes of the coordinate space and the conventional Cartesian coordinate space was transformed into the internal/Cartesian space with linear approximation. All the calculations were carried out in this internal/Cartesian space. Our method can successfully identify the functional motions and key residues for the transporter BtuCD-BtuF, which are well consistent with the experimental observations.

Statistical properties and error threshold of quasispecies on single-peak Gaussian-distributed fitness landscapes.

The stochastic Eigen model proposed by Feng et al. (2007) (Journal of Theoretical Biology, 246, 28) showed that error threshold is no longer a phase transition point but a crossover region whose width depends on the strength of the random fluctuation in an environment. The underlying cause of this phenomenon has not yet been well examined. In this article, we adopt a single peak Gaussian distributed fitness landscape instead of a constant one to investigate and analyze the change of the error threshold and the statistical property of the quasi-species population. We find a roughly linear relation between the width of the error threshold and the fitness fluctuation strength. For a given quasi-species, the fluctuation of the relative concentration has a minimum with a normal distribution of the relative concentration at the maximum of the averaged relative concentration, it has however a largest value with a bimodal distribution of the relative concentration near the error threshold. The above results deepen our understanding of the quasispecies and error threshold and are heuristic for exploring practicable antiviral strategies.

Neuropeptide proenkephalin A is associated with in-hospital mortality in patients with acute intracerebral hemorrhage.

High plasma proenkephalin A (PENK-A) levels are associated with poor clinical outcome after ischemic stroke. However, not much is known regarding the change of its level in acute intracerebral hemorrhage. Thus, we sought to determine PENK-A in plasma of patients with acute spontaneous basal ganglia hemorrhage and evaluate its relation with disease severity and in-hospital mortality. One hundred and two patients and 100 healthy controls were recruited. Plasma samples were obtained on admission for patients and at study entry for controls. Its concentration was measured by chemoluminescence sandwich immunoassay. Plasma PENK-A levels were substantially higher in patients than in healthy controls (235.5±85.4 pmol/L vs. 90.1±31.3 pmol/L; P<0.0001). A forward stepwise logistic regression selected plasma PENK-A as an independent predictor for in-hospital mortality of patients (odds ratio 1.080, 95% confidence interval 1.018-1.147, P<0.001). A multivariate linear regression demonstrated that plasma PENK-A level was positively associated with National Institutes of Health Stroke Scale (NIHSS) score (t=6.189, P<0.001) and hematoma volume (t=5.388, P<0.001). A receiver operating characteristic curve identified a plasma PENK-A level>267.1 pmol/L predicted in-hospital mortality of patients with 80.0% sensitivity and 74.7% specificity (area under curve, 0.836; 95% confidence interval, 0.750-0.902). Its predictive value was similar to NIHSS score's and hematoma volume's (both P>0.05). However, it did not statistically significantly improve the predictive values of NIHSS score and hematoma volume (both P>0.05). Thus, increased plasma PENK-A levels are associated with disease severity and in-hospital mortality after acute intracerebral hemorrhage.

The cytosolic GH loop regulates the phosphatidylinositol 4,5-bisphosphate-induced gating kinetics of Kir2 channels.

Inwardly rectifying K(+) (Kir) channels set the resting membrane potential and regulate cellular excitability. The activity of Kir channels depends critically on the phospholipid PIP(2). The molecular mechanism by which PIP(2) regulates Kir channel gating is poorly understood. Here, we utilized a combination of computational and electrophysiological approaches to discern structural elements involved in regulating the PIP(2)-induced gating kinetics of Kir2 channels. We identify a novel role for the cytosolic GH loop. Mutations that directly or indirectly affect GH loop flexibility (e.g. V223L, E272G, D292G) increase both the on- and especially the off-gating kinetics. These effects are consistent with a model in which competing interactions between the CD and GH loops for the N terminus regulate the gating of the intracellular G loop gate.

[Polycomb group protein complex involved in plant vernalization].

The polycomb group (PcG) proteins repress transcription of the target genes and control development by chromatin modification in plants and animals. During vernalization, the Polycomb Repressive Complex 2, containing VERNALIZATION 2 (VRN2), CLF/SWN, FIE, and MSI1, can trimethylate the lysine 27 of histone H3 in the chromtin loci of FLOWER LOCUS C (FLC), the main flowering repressor in Arabidopsis. Therefore, the transcription of FLC is repressed during vernalization and the plant was induced to flowering. Here, we review the machanism of flowering regulation mediated by PcG proteins, which is expected to be valuable to research in vernalization mechanism in cereal.

GhMPK7, a novel multiple stress-responsive cotton group C MAPK gene, has a role in broad spectrum disease resistance and plant development.

Mitogen-activated protein kinase (MAPK) cascades play a pivotal role in environmental responses and developmental processes in plants. Previous researches mainly focus on the MAPKs in groups A and B, and little is known on group C. In this study, we isolated and characterized GhMPK7, which is a novel gene from cotton belonging to the group C MAPK. RNA blot analysis indicated that GhMPK7 transcript was induced by pathogen infection and multiple defense-related signal molecules. Transgenic Nicotina benthamiana overexpressing GhMPK7 displayed significant resistance to fungus Colletotrichum nicotianae and virus PVY, and the transcript levels of SA pathway genes were more rapidly and strongly induced. Furthermore, the transgenic N. benthamiana showed reduced ROS-mediated injuries by upregulating expression of oxidative stress-related genes. Interestingly, the transgenic plants germinated earlier and grew faster in comparison to wild-type plants. beta-glucuronidase activity driven by the GhMPK7 promoter was detected in the apical meristem at the vegetative stage, and it was enhanced by treatments with signal molecules and phytohormones. These results suggest that GhMPK7 might play an important role in SA-regulated broad-spectrum resistance to pathogen infection, and that it is also involved in regulation of plant growth and development.

Bacterially expressed double-stranded RNAs against hot-spot sequences of tobacco mosaic virus or potato virus Y genome have different ability to protect tobacco from viral infection.

Posttranscriptional gene silencing, also known as RNA interference, involves degradation of homologous mRNA sequences in organisms. In plants, posttranscriptional gene silencing is part of a defense mechanism against virus infection, and double-stranded RNA is the pivotal factor that induces gene silencing. In this paper, we got seven hairpin RNAs (hpRNAs) constructs against different hot-spot sequences of Tobacco mosaic virus (TMV) or Potato virus Y (PVY) genome. After expression in Escherichia coli HT115, we extracted the seven hpRNAs for the test in tobacco against TMV or PVY infection. The data suggest that different hpRNAs against different hot-spot sequences of TMV or PVY genome had different ability to protect tobacco plants from viral infection. The resistance to TMV conferred by the hpRNA against the TMV movement protein was stronger than other TMV hpRNAs; the resistance to PVY conferred by the hpRNA against the PVY nuclear inclusion b was better than that induced by any other PVY hpRNAs. Northern blotting of siRNA showed that the resistance was indeed an RNA-mediated virus resistance.

The use of an "effective potential" to describe the directed motion of a two-state molecular motor.

Force generation and directed motion of molecular motors using a simple two-state model are studied in the paper. Here we consider the asymmetric and periodic potential in the model. The symmetric and periodic potential is adopted to describe the interactions between motor proteins and filaments that are periodic and polar. The flux and the slope of the effective potential as functions of the temperature and transition rates are calculated in the two-state model. The ratio of the slope of the effective potential to the flux is also calculated. It is concluded that the directed motion of motor proteins is relevant to the effective potential. The slope of the effective potential corresponds to an average force. The non-vanishing force therefore implies that detailed balance is broken in the process of transition between different states. Moreover, we compare the theoretical relationship of load force and velocity with the experimental data. It is shown that they are consistent.