Free energy analysis of conductivity and charge selectivity of M2GlyR-derived synthetic channels

Significant progresses have been made in the design, synthesis, modeling and in vitro testing of channel-forming peptides derived from the second transmembrane domain of the α-subunit of the glycine receptor (GlyR). The latest designs, including p22 (KKKKP ARVGL GITTV LTMTT QS), are highly soluble in water with minimal aggregation propensity and insert efficiently into cell membranes to form highly conductive ion channels. The last obstacle to a potential lead sequence for channel replacement treatment of CF patients is achieving adequate chloride selectivity. We have performed free energy simulation to analyze the conductance and charge selectivity of M2GlyR-derived synthetic channels. The results reveal that the pentameric p22 pore is non-selective. Moderate barriers for permeation of both K+ and Cl- are dominated by the desolvation cost. Despite previous evidence suggesting a potential role of threonine side chains in anion selectivity, the hydroxyl group is not a good surrogate of water for coordinating these ions. We have also tested initial ideas of introducing additional rings of positive changes to various positions along the pore to increase anion selectivity. The results support the feasibility of achieving anion selectivity by modifying the electrostatic properties of the pore, but at the same time suggest that the peptide assembly and pore topology may also be dramatically modified, which could abolish the effects of modified electrostatics on anion selectivity. This was confirmed by subsequent two-electrode voltage clamp measurements showing that none of the tested mono-, di- and tri-Dap substituted sequences was selective. The current study thus highlights the importance of controlling channel topology besides modifying pore electrostatics for achieving anion selectivity. Several strategies are now being explored in our continued efforts to design an anion selective peptide channel with suitable biophysical, physiological and pharmacological properties as a potential treatment modality for channel replacement therapy

Características químicas e digestibilidade in vitro de silagens de girassol.

Silages of 13 cultivars (AS243, AS603, Cargill 11, Contiflor 3, Contiflor 7, DK180, M734, M737, M738, M742, Rumbosol 90, Rumbosol 91, V2000 and Media) of sunflower (Helianthus annuus L.) were evaluated for chemical characteristics and in vitro dry matter digestibility (IVDMD). The silages were produced when the plants had >90% ripe grains. The experimental procedure followed an entirely randomized design and the mean values were compared with SNK test (P9.0% crude protein was observed in 5 cultivars. The obtained ether extract (EE; 14.7%) was quite high when compared with the other roughages. The neutral detergent fibre, acid detergent fibre (ADF) and lignin contents were 45.8, 35.7 and 6.4%, respectively, and were negatively correlated to IVDMD (general average of 49.8%). The silages had low concentrations of residual soluble carbohydrates. The high ADF and lignin, and the average of IVDMD (49.8%) can restrict the application of sunflower silage and the high EE content can limit the level of inclusion of silage in bovine rations.

Etnovariedades de mandioca (Manihot esculenta Crantz) cultivadas em assentamentos rurais de Corumbá, MS.

bitstream/CPAP/56599/1/BP78.pdfFormato Eletrônico

Etnovariedades de mandioca (Manihot esculenta Crantz) cultivadas em assentamentos rurais de Corumbá, MS.

bitstream/CPATC-2009-09/20206/1/BP78.pd

Design of a functional calcium channel protein: Inferences about an ion channel‐forming motif derived from the primary structure of voltage‐gated calcium channels

To identify sequence‐specific motifs associated with the formation of an ionic pore, we systematically evaluated the channel‐forming activity of synthetic peptides with sequence of predicted transmembrane segments of the voltage‐gated calcium channel. The amino acid sequence of voltage‐gated, dihydropyridine (DHP)‐sensitive calcium channels suggests the presence in each of four homologous repeats (I–IV) of six segments (S1–S6) predicted to form membrane‐spanning, α‐helical structures. Only peptides representing amphipathic segments S2 or S3 form channels in lipid bilayers. To generate a functional calcium channel based on a four‐helix bundle motif, four‐helix bundle proteins representing IVS2 (T4CaIVS2) or IVS3 (T4CaIVS3) were synthesized. Both proteins form cation‐selective channels, but with distinct characteristics: the single‐channel conductance in 50 mM BaCl2 is 3 pS and 10 pS. For T4CaIVS3, the conductance saturates with increasing concentration of divalent cation. The dissociation constants for Ba2+, Ca2+, and Sr2+ are 13.6 mM, 17.7 mM, and 15.0 mM, respectively. The conductance of T4CaIVS2 does not saturate up to 150 mM salt. Whereas T4CaIVS3 is blocked by μM Ca2+ and Cd2+, T4CaIVS2 is not blocked by divalent cations. Only T4CaIVS3 is modulated by enantiomers of the DHP derivative BayK 8644, demonstrating sequence requirement for specific drug action. Thus, only T4CaIVS3 exhibits pore properties characteristic also of authentic calcium channels. The designed functional calcium channel may provide insights into fundamental mechanisms of ionic permeation and drug action, information that may in turn further our understanding of molecular determinants underlying authentic pore structures. Copyright © 1993 The Protein Societ

Sistema de produção e utilização da mandioca em assentamentos rurais de Corumbá, MS.

Como base para estudos sobre a viabilidade de utilização de resíduos da cultura da mandioca (Manihot esculenta Crantz) para a alimentação de ruminantes na época de seca, foi avaliado o sistema de produção e de utilização dessa cultura em assentamentos rurais localizados no município de Corumbá, MS. A coleta de dados foi realizada por observação participante e por aplicação de questionários dirigidos a 235 produtores de base familiar residentes nos assentamentos Mato Grande, Paiolzinho, Tamarineiro II e Taquaral. Procurou-se identificar a influência de fatores sociais, econômicos e culturais sobre o processo produtivo e de utilização da mandioca nessas comunidades. Os resultados obtidos indicam a bovinocultura como a principal atividade econômica desenvolvida pelas famílias assentadas, seguida pela agricultura. A mandioca destaca-se como uma cultura importante para a região, tanto economicamente como para a segurança alimentar das famílias assentadas, com um grande percentual de famílias produtoras (69,7%). A parte aérea da mandioca vem sendo utilizada apenas como alimento fresco fornecido aos animais no momento da colheita. A necessidade de possuir triturador de forragem foi identificada pela própria comunidade como um dos principais fatores limitantes para a utilização dos resíduos da cultura da mandioca como subproduto da produção agrícola a ser empregado para alimentação de ruminantes.bitstream/CPAP/55940/1/BP70.pdfFormato Eletrônico

Feno da parte aérea da mandioca para a produção de ruminantes em sistemas organicos.

As produtividades de feno observadas neste estudo indicam que o aproveitamento da parte aérea da mandioca pode representar uma estratégia de integração das atividades produtivas em uma mesma propriedade apropriada para a alimentação de ruminantes durante o período de escassez das pastagens, constituindo em um recurso disponível para manter índices adequados de produtividade dos rebanhos da região manejados de acordo com as diretrizes da produção orgânica.bitstream/CPAP-2010/57326/1/CT88.pd

Channel Replacement Therapy for Cystic Fibrosis

No abstract available

Design, synthesis and functional characterization of a pentameric channel protein that mimics the presumed pore structure of the nicotinic cholinergic receptor

AbstractNicotinic cholinergic receptors are membrane proteins composed of five subunits organized around a central aqueous pore. A pentameric channel protein, T5M2δ, that emulates the presumed pore-forming structure of this receptor was generated by assembling five helix-forming peptide modules at the lysine ϵ-amino groups of the 11-residue template [K∗AK∗KK∗PGK∗EK∗G], where ∗ indicates attachment sites. Helical modules represent the sequence of the M2 segment of the Torpedo californica acetylcholine receptor (AChR) δ subunit; M2 segments are considered involved in pore-lining. Purified T5M2δ migrates in SDS-PAGE with an apparent Mr~14,000, concordant with a protein of 126 residues. T5M2δ forms cation-selective channels when reconstituted in planar lipid bilayers. The single channel conductance in symmetric 0.5 M K.C1 is 40 pS. This value approximates the 45 pS single channel conductance characteristic of authentic purified Torpedo AChR, recorded under otherwise identical conditions. These results, together with conformational energy calculations, support the notion that a bundle of five amphipathic a-helices is a plausible structural motif underlying the inner bundle that forms the pore of the pentameric AChR channel

An ABC Transporter Is Required for Secretion of Peptide Sex Pheromones in Enterococcus faecalis

ABSTRACT Enterococci are leading causes of hospital-acquired infection in the United States and continue to develop resistances to new antibiotics. Many Enterococcus faecalis isolates harbor pheromone-responsive plasmids that mediate horizontal transfer of even large blocks of chromosomal genes, resulting in hospital-adapted strains over a quarter of whose genomes consist of mobile elements. Pheromones to which the donor cells respond derive from lipoprotein signal peptides. Using a novel bacterial killing assay dependent on the presence of sex pheromones, we screened a transposon mutant library for functions that relate to the production and/or activity of the effector pheromone. Here we describe a previously uncharacterized, but well-conserved, ABC transporter that contributes to pheromone production. Using three distinct pheromone-dependent mating systems, we show that mutants defective in expressing this transporter display a 5- to 6-order-of-magnitude reduction in conjugation efficiency. In addition, we demonstrate that the ABC transporter mutant displays an altered biofilm architecture, with a significant reduction in biofilm biomass compared to that of its isogenic parent, suggesting that pheromone activity also influences biofilm development. The conservation of this peptide transporter across the Firmicutes suggests that it may also play an important role in cell-cell communication in other species within this important phylum