Ngila: global pairwise alignments with logarithmic and affine gap costs

Ngila is an application that will find the best alignment of a pair of sequences using log-affine gap costs, which are the most biologically realistic gap costs. AVAILABILITY: Portable source code for Ngila can be downloaded from its development website, http://scit.us/projects/ngila/. It compiles on most operating systems.     

Seipin: from human disease to molecular mechanism

The most-severe form of congenital generalized lipodystrophy (CGL) is caused by mutations in BSCL2/seipin. Seipin is a homo-oligomeric integral membrane protein in the endoplasmic reticulum that concentrates at junctions with cytoplasmic lipid droplets (LDs). While null mutations in seipin are responsible for lipodystrophy, dominant mutations cause peripheral neuropathy and other nervous system pathologies. We first review the clinical aspects of CGL and the discovery of the responsible genetic loci. The structure of seipin, its normal isoforms, and mutations found in patients are then presented. While the function of seipin is not clear, seipin gene manipulation in yeast, flies, mice, and human cells has recently yielded a trove of information that suggests roles in lipid metabolism and LD assembly and maintenance. A model is presented that attempts to bridge these new data to understand the role of this fascinating protein.     

Patterns of change over time in darter (Teleostei: Percidae) assemblages of the Arkansas River basin,northeastern Oklahoma,USA

Rivers and streams are among the most threatened ecosystems worldwide, and their fish assemblages have been modified by anthropogenic habitat alteration and introductions of non‐native species. Consequently, two frequently observed patterns of assemblage change over time are species loss and biotic homogenization. In the present study, we compared contemporary (2006–2007) and historical (1948–1955) assemblages of darters, a group of small benthic fishes of the family Percidae, in the Arkansas River drainage of northeastern Oklahoma, USA. Results showed species loss between the two sampling periods, with historical estimates of overall species diversity across the study area exceeding contemporary estimates by five to eight species. Assemblages showed a low degree of darter similarity based on species presence and absence, with pairwise site comparisons (Jaccard's similarity index) between historical and contemporary samples averaging < 0.35. No significant homogenization or differentiation of assemblages occurred. Range expansion of widespread species, one of the primary mechanisms of biotic homogenization, was not observed; rather, all species occurred at a smaller proportion of sites in contemporary samples. Our results highlight the threat posed by anthropogenic habitat alteration to taxonomic groups such as darters, most of which are habitat specialists. However, our results suggest that biotic homogenization is unlikely to occur in the absence of immigration, especially if assemblages are subjected to ‘novel disturbances’ such as dam construction and watershed‐scale habitat degradation which negatively affect all components of the assemblage.     

A superfamily of actin-binding proteins at the actin-membrane nexus of higher plants

Complex animals use a wide variety of adaptor proteins to produce specialized sites of interaction between actin and membranes. Plants do not have these protein families, yet actin-membrane interactions within plant cells are critical for the positioning of subcellular compartments, for coordinating intercellular communication, and for membrane deformation [1]. Novel factors are therefore likely to provide interfaces at actin-membrane contacts in plants, but their identity has remained obscure. Here we identify the plant-specific Networked (NET) superfamily of actin-binding proteins, members of which localize to the actin cytoskeleton and specify different membrane compartments. The founding member of the NET superfamily, NET1A, is anchored at the plasma membrane and predominates at cell junctions, the plasmodesmata. NET1A binds directly to actin filaments via a novel actin-binding domain that defines?a superfamily of thirteen Arabidopsis proteins divided into four distinct phylogenetic clades. Members of other clades identify interactions at the tonoplast, nuclear membrane, and pollen tube plasma membrane, emphasizing the role of this superfamily in mediating actin-membrane interactions.     

Switch 1 Mutation S217A Converts Myosin V into a Low Duty Ratio Motor

We have determined the kinetic mechanism and motile properties of the switch 1 mutant S217A of myosin Va. Phosphate dissociation from myosin V-ADP-P_i (inorganic phosphate) and actomyosin V-ADP-P_i and the rate of the hydrolysis step (myosin V-ATP → myosin V-ADP-P_i) were all ∼10-fold slower in the S217A mutant than in wild type (WT) myosin V, resulting in a slower steady-state rate of basal and filamentous actin (actin)-activated ATP hydrolysis. Substrate binding and ADP dissociation kinetics were all similar to or slightly faster in S217A than in WT myosin V and mechanochemical gating of the rates of dissociation of ADP between trail and lead heads is maintained. The reduction in the rate constants of the hydrolysis and phosphate dissociation steps reduces the duty ratio from ∼0.85 in WT myosin V to ∼0.25 in S217A and produces a motor in which the average run length on actin at physiological concentrations of ATP is reduced 10-fold. Thus we demonstrate that, by mutational perturbation of the switch 1 structure, myosin V can be converted into a low duty ratio motor that is processive only at low substrate concentrations.During the past 2 decades a considerable number of different myosins have been discovered (1). Myosin V is the best characterized among the so-called unconventional myosins (i.e. those not belonging to class II), and it serves as an important model molecule for studying actomyosin interactions and single molecule processive motility (2). Myosin V is a highly processive motor whose role is to transport cargo along actin filaments or bundles inside the cell (3–5). The kinetic mechanism of myosin V is significantly different from that of conventional myosins such as muscle myosin II, as it remains bound to actin (filamentous actin) through a number of ATPase cycles (6–8). Myosin V has a high duty ratio: a single-headed myosin V-S1 (myosin V, subfragment 1) is in the strongly bound AM-ADP state 80–90% of the time during ATP hydrolysis. An additional mechanism for promoting highly processive runs is the preferential release of ADP from the trail head because of mechanochemical gating, which causes a drastic reduction of the rate constant of ADP release from the lead head (9–11). Although there are significant differences between the ATPase mechanisms of the different myosins, the structure of the nucleotide binding pocket (composed of the switch 1 and 2 regions and the P-loop) is highly conserved. The position of the Ser²¹⁷ (Ser²³⁶ in Dictyostelium myosin II) residue of the switch 1 loop (the first serine in the NDNSSRFG sequence) is shown in Fig. 1. It had been shown previously by mutagenesis in Dictyostelium (12) and in smooth muscle myosin II (13) that the substitution of serine 236 to alanine retains at least partial enzymatic and motile function in these mutant myosins. Therefore, the OH group is not an essential part of the catalytic mechanism, but the rate of steady-state ATP hydrolysis is reduced several fold. However, neither of these studies includes a detailed kinetic analysis to determine which steps of the catalytic mechanism were altered by the mutation. Here we have exploited the higher affinity of myosin V-ADP-P_i for actin to determine the effect of the mutation on the rate constants of the product dissociation steps following the power stroke, which could not be determined using either Dictyostelium or smooth muscle myosin. We also conducted single molecule motility studies using total internal reflectance fluorescence (TIRF)⁵ microscopy to determine how the changes in the kinetic mechanism affect the motile properties of the molecule.Open in a separate windowFIGURE 1.Schematic representation of the critical residues in the ATP binding site of myosin based on the MgADP·VO₄ crystal of the Dictyostelium motor domain (Smith and Rayment (30)). The serine in position 217 was mutated to alanine for these kinetic studies. The small spheres are at the position of the oxygen of the water molecules.     

Atypical sialylated N-glycan structures are attached to neuronal voltage-gated potassium channels

Mammalian brains contain relatively high amounts of common and uncommon sialylated N-glycan structures. Sialic acid linkages were identified for voltage-gated potassium channels, Kv3.1, 3.3, 3.4, 1.1, 1.2 and 1.4, by evaluating their electrophoretic migration patterns in adult rat brain membranes digested with various glycosidases. Additionally, their electrophoretic migration patterns were compared with those of NCAM (neural cell adhesion molecule), transferrin and the Kv3.1 protein heterologously expressed in B35 neuroblastoma cells. Metabolic labelling of the carbohydrates combined with glycosidase digestion reactions were utilized to show that the N-glycan of recombinant Kv3.1 protein was capped with an oligo/poly-sialyl unit. All three brain Kv3 glycoproteins, like NCAM, were terminated with alpha2,3-linked sialyl residues, as well as atypical alpha2,8-linked sialyl residues. Additionally, at least one of their antennae was terminated with an oligo/poly-sialyl unit, similar to recombinant Kv3.1 and NCAM. In contrast, brain Kv1 glycoproteins consisted of sialyl residues with alpha2,8-linkage, as well as sialyl residues linked to internal carbohydrate residues of the carbohydrate chains of the N-glycans. This type of linkage was also supported for Kv3 glycoproteins. To date, such a sialyl linkage has only been identified in gangliosides, not N-linked glycoproteins. We conclude that all six Kv channels (voltage-gated K+ channels) contribute to the alpha2,8-linked sialylated N-glycan pool in mammalian brain and furthermore that their N-glycan structures contain branched sialyl residues. Identification of these novel and unique sialylated N-glycan structures implicate a connection between potassium channel activity and atypical sialylated N-glycans in modulating and fine-tuning the excitable properties of neurons in the nervous system.     

Test–retest reliability of the soleus H-reflex is affected by joint positions and muscle force levels

The purpose of this study was to determine the test–retest reliability of the soleus (SOL) H-reflex during rest and isometric contractions at 10%, 30%, and 50% of the maximal voluntary force (MVC) at the ankle joint angles of neutral (0°), plantarflexion (20°), and dorsiflexion (?20°) respectively, in a sitting position. Ten healthy participants, with mean age of 24.9 ± 5.0 (SD) years, height 168.3 ± 8.8 cm, weight 62.7 ± 12.3 kg, were tested for the SOL H-reflex (H_max) on two separate occasions within 7 days. The intraclass correlation coefficient (ICC) for the test–retest of the SOL H-reflex during rest was found to be high at ankle joint angle of neutral (ICC = 0.92) and plantarflexion (0.96), and moderate at dorsiflexion (0.75). Inconsistent ICC values (range from 0.62 to 0.97) were found during the submaximal voluntary contractions at the three ankle joint positions. High ICCs were also found in H_max/M_max ratio at neutral (0.86), plantarflexion (0.96), and dorsiflexion (0.84) positions. It was concluded that the test–retest reliability of the SOL H-reflex was affected by the intensity of voluntary contraction and ankle joint position. The H-reflex demonstrated a higher reliability at the neutral and plantarflexion positions than that at the dorsiflexion position during rest, and a higher reliability at 10% MVC than that at 30% and 50% MVC.     

Separation of the intracellular secretory compartment of rat liver and isolated rat hepatocytes in a single step using self-generating gradients of iodixanol

An automated sampling device coupled to a stirred tank reactor was developed for monitoring intracellular metabolite dynamics. Sample flasks fixed in transport magazines were moved by a step engine in a way that each sample flask was filled within 220 ms, resulting in a sampling rate of 4.5 s-1. Rapid inactivation of the metabolism was achieved by spraying the samples into 60% methanol at -50 degrees C. After centrifugation of the quenched cells at -20 degrees C the metabolites were extracted with perchloric acid and analyzed biochemically or with HPLC. The automated sampling device was applied for investigation of the intracellular metabolite dynamics of glycolysis in Escherichia coli after rapid glucose addition to a glucose-limited steady-state culture. For the first time oscillations of intracellular metabolite concentrations like glucose-6-phosphate, phosphoenolpyruvate, glyceraldehyde 3-phosphate, dihydroxyacetonphosphate, 3-phosphoglycerate, and pyruvate were quantified on a subseconds to seconds scale in E. coli. As an example, the kinetics of the decomposition of fructose 1, 6-bisphosphate to glyceraldehyde 3-phosphate and dihydroxyacetonphosphate were investigated by use of a well-known mechanistic kinetic model and the measured in vivo metabolite dynamics.     

Nitric oxide protects human extravillous trophoblast cells from apoptosis by a cyclic GMP-dependent mechanism and independently of caspase 3 nitrosylation

Apoptosis is thought to play an important regulatory role in placental development and inappropriate trophoblast apoptosis has been implicated in complications of pregnancy such as pre-eclampsia. Here we show that apoptosis of a human extravillous trophoblast-derived cell line (SGHPL-4) can be regulated by nitric oxide (NO). Nitric oxide produced exogenously by the addition of NO donors was able to delay or inhibit apoptosis induced by a combination of tumour necrosis factor alpha and actinomycin D and to suppress the activity of caspase 3. Treatment with hepatocyte growth factor (HGF) stimulated expression of the inducible isoform of NO synthase and was also able to protect SGHPL-4 cells from caspase 3 activation and apoptosis. The inhibition of basal NO production with NO synthase inhibitors was shown to sensitise cells to apoptotic stimuli and to reduce the level of endogenous caspase 3 nitrosylation. The anti-apoptotic effects of NO in these extravillous trophoblast cells appear to be mediated through the production of cyclic GMP as inhibitors of soluble guanylate cyclase inhibited the protective effect of both HGF and NO donors.     

Three Brick genes have distinct functions in a common pathway promoting polarized cell division and cell morphogenesis in the maize leaf epidermis

We have taken a genetic approach to investigating cytoskeleton-dependent mechanisms governing cell morphogenesis in the maize leaf epidermis. Previously, we showed that the Brick1 (Brk1) gene is required for the formation of epidermal cell lobes as well as for properly polarized divisions of stomatal subsidiary mother cells, and encodes an 8 kDa protein highly conserved in plants and animals. Here, we show that two additional Brick genes, Brk2 and Brk3, are involved in the same aspects of epidermal cell morphogenesis and division. As shown previously for Brk1, analysis of the cytoskeleton shows that Brk2 and Brk3 are required for the formation of local F-actin enrichments associated with lobe outgrowth in wild-type cells. Analysis of brk1;brk2, brk1;brk3 and brk2;brk3 double mutants shows that their phenotypes are the same as those of brk single mutants. Mosaic analysis shows that Brk1 acts non cell-autonomously over a short distance. By contrast, Brk2 and Brk3 act cell-autonomously to promote pavement cell lobe formation, but Brk3 acts non cell-autonomously, and Brk2 partially non cell-autonomously, to promote polarized subsidiary mother cell divisions. Together, these observations indicate that all three Brk genes act in a common pathway in which each Brk gene has a distinct function. Recent work demonstrating a function for the mammalian homolog of BRK1 (HSPC300) in activation of Arp2/3-dependent actin polymerization implicates the Brk pathway in local regulation of actin polymerization in plant cells.