A surface plasmon resonance approach to monitor toxin interactions with an isolated voltage-gated sodium channel paddle motif

Animal toxins that inhibit voltage-gated sodium (Na_v) channel fast inactivation can do so through an interaction with the S3b–S4 helix-turn-helix region, or paddle motif, located in the domain IV voltage sensor. Here, we used surface plasmon resonance (SPR), an optical approach that uses polarized light to measure the refractive index near a sensor surface to which a molecule of interest is attached, to analyze interactions between the isolated domain IV paddle and Na_v channel–selective α-scorpion toxins. Our SPR analyses showed that the domain IV paddle can be removed from the Na_v channel and immobilized on sensor chips, and suggest that the isolated motif remains susceptible to animal toxins that target the domain IV voltage sensor. As such, our results uncover the inherent pharmacological sensitivities of the isolated domain IV paddle motif, which may be exploited to develop a label-free SPR approach for discovering ligands that target this region.     

The effect of ethylene glycol on the phytovolatilization of 1,4-dioxane

Phytoremediation at contaminated sites is often complicated by the presence of more than one chemical However, the effects of common co-contaminants such as ethylene glycol on the phytoremediation of other chemicals, e.g., 1,4-dioxane, is not well understood. Field studies with DN34 poplar trees revealed a 28% decline in growth rate in response to 10 g/L ethylene glycol in the groundwater, thus indicating a significant and deleterious effect on tree viability, and likely, the plants' utility for phytoremediation. Thorough investigations using Arabidopsis thaliana, with its small size and rapid life cycle, indicated significant growth reduction at 10 g/L and complete inhibition of germination at 40 g/L ethylene glycol Ethylene glycol was almost as severe a stressor as the well characterized osmotic inhibitor, sorbitoL Watering potted trees with 10 g/L ethylene glycol reduced their growth by more than 50%, and similar results were observed in hydroponically grown poplar and willow trees. Under hydroponic conditions, 60 g/L ethylene glycol inhibited the phytovolatilization of l,4-dioxane by more than 80%, and all trees evapo-transpired 1,4-dioxane less efficiently than water. In fact, this efficiency differed between trees and the difference became more pronounced in the presence of ethylene glycol.     

Identification of differentially expressed genes in a porcine in vivo model of adipogenesis using suppression subtractive hybridization

Although they provide valuable information, in vitro models of adipocyte development often require high doses of hormones and growth factors, which may influence gene expression and adipocyte differentiation patterns. To overcome these problems, a novel in vivo model of adipose tissue development was used to characterize genes involved in adipogenesis. The suppression subtractive hybridization technique was used to identify genes showing differential expression between the adipose tissue of a day 90 gestating sow, which is enriched in adipocytes, and day 90 fetal adipose tissue, which is enriched in preadipocytes. A total of 149 expressed sequence tags corresponding to identified genes and tentative consensus sequences emerged. Thirty-seven clones matched expressed sequence tags or genomic DNA sequences and six novel sequences were also identified. Adipogenesis-related genes were identified, many of which have never been reported to be expressed in mammalian adipose tissue, and may play a role in regulation of adipose tissue differentiation. Validation of differentially expressed genes was confirmed for perilipin, monocyte to macrophage differentiation-associated, myocilin, paraoxonase 3, stearoyl-CoA desaturase, angiotensinogen and adiponectin genes using real-time RT-PCR.     

Seasonal variation of infestation by ectoparasitic chigger mite larvae (Acarina: Trombiculidae) on resident and migratory birds in coffee agroecosystems of Chiapas, Mexico

Parasitism is not well documented for birds found in tropical habitats. Long-distance migratory birds may face additional risks to an already hazardous journey when infected. This study explores the ecology of an ectoparasite infestation in Chiapas, Mexico. During a mist-netting project in 2 different coffee management systems, chigger mites (Acarina: Trombiculidae), ectoparasitic during the larval stage, were found on both resident and migratory birds. Using a rapid assessment protocol, it was observed that 17 of 26 species of long-distance migrants and 33 of 71 resident species had at least 1 infested individual. Infestation prevalences were unexpectedly high on some long-distance migrants, as high as 0.73 for Swainson's thrush (Catharus ustulatus), a value on par with heavily infested resident species. Prevalence was highest during winter sampling: 0.18 overall, 0.16 of migrants, and 0.23 of residents. Prevalence was 0.14 for resident birds during the summer breeding season. Mean abundance and mean intensity of infestation are reported for 97 species captured and inspected during the course of this study. In this region, chigger mite larvae are relatively common on birds and their abundance varies seasonally. High prevalence for some migratory birds suggests that more research and monitoring of ectoparasites are needed, especially in light of emerging diseases.     

Annexin 2 promotes the formation of lipid microdomains required for calcium-regulated exocytosis of dense-core vesicles

Annexin 2 is a calcium-dependent phospholipid-binding protein that has been implicated in a number of membrane-related events, including regulated exocytosis. In chromaffin cells, we previously reported that catecholamine secretion requires the translocation and formation of the annexin 2 tetramer near the exocytotic sites. Here, to obtain direct evidence for a role of annexin 2 in exocytosis, we modified its expression level in chromaffin cells by using the Semliki Forest virus expression system. Using a real-time assay for individual cells, we found that the reduction of cytosolic annexin 2, and the consequent decrease of annexin 2 tetramer at the cell periphery, strongly inhibited exocytosis, most likely at an early stage before membrane fusion. Secretion also was severely impaired in cells expressing a chimera that sequestered annexin 2 into cytosolic aggregates. Moreover, we demonstrate that secretagogue-evoked stimulation triggers the formation of lipid rafts in the plasma membrane, essential for exocytosis, and which can be attributed to the annexin 2 tetramer. We propose that annexin 2 acts as a calcium-dependent promoter of lipid microdomains required for structural and spatial organization of the exocytotic machinery.     

An electrostatic steering mechanism of Cdc42 recognition by Wiskott-Aldrich syndrome proteins

The specific and rapid formation of protein complexes is essential for diverse cellular processes such as remodeling of actin filaments in response to the interaction between Rho GTPases and the Wiskott-Aldrich syndrome proteins (WASp and N-WASp). Although Cdc42, TC10, and other members of the Rho family have been implicated in binding to and activating the WAS proteins, the exact nature of such a protein-protein recognition process has remained obscure. Here, we describe a mechanism that ensures rapid and selective long-range Cdc42-WASp recognition. The crystal structure of TC10, together with mutational and bioinformatic analyses, proved that the basic region of WASp and two unique glutamates in Cdc42 generate favorable electrostatic steering forces that control the accelerated WASp-Cdc42 association reaction. This process is a prerequisite for WASp activation and a critical step in temporal regulation and integration of WASp-mediated cellular responses.     

Abi1 regulates the activity of N-WASP and WAVE in distinct actin-based processes

Neural Wiskott-Aldrich syndrome protein (N-WASP) and WAVE are members of a family of proteins that use the Arp2/3 complex to stimulate actin assembly in actin-based motile processes. By entering into distinct macromolecular complexes, they act as convergent nodes of different signalling pathways. The role of WAVE in generating lamellipodial protrusion during cell migration is well established. Conversely, the precise cellular functions of N-WASP have remained elusive. Here, we report that Abi1, an essential component of the WAVE protein complex, also has a critical role in regulating N-WASP-dependent function. Consistently, Abi1 binds to N-WASP with nanomolar affinity and, cooperating with Cdc42, potently induces N-WASP activity in vitro. Molecular genetic approaches demonstrate that Abi1 and WAVE, but not N-WASP, are essential for Rac-dependent membrane protrusion and macropinocytosis. Conversely, Abi1 and N-WASP, but not WAVE, regulate actin-based vesicular transport, epidermal growth factor receptor (EGFR) endocytosis, and EGFR and transferrin receptor (TfR) cell-surface distribution. Thus, Abi1 is a dual regulator of WAVE and N-WASP activities in specific processes that are dependent on actin dynamics.     

An efficient algorithm for the identification of structured motifs in DNA promoter sequences

We propose a new algorithm for identifying cis-regulatory modules in genomic sequences. The proposed algorithm, named RISO, uses a new data structure, called box-link, to store the information about conserved regions that occur in a well-ordered and regularly spaced manner in the data set sequences. This type of conserved regions, called structured motifs, is extremely relevant in the research of gene regulatory mechanisms since it can effectively represent promoter models. The complexity analysis shows a time and space gain over the best known exact algorithms that is exponential in the spacings between binding sites. A full implementation of the algorithm was developed and made available online. Experimental results show that the algorithm is much faster than existing ones, sometimes by more than four orders of magnitude. The application of the method to biological data sets shows its ability to extract relevant consensi.