Sarcoplasmic reticulum lumenal Ca2+ has access to cytosolic activation and inactivation sites of skeletal muscle Ca2+ release channel

The effects of sarcoplasmic reticulum lumenal (trans) Ca2+ on cytosolic (cis) ATP-activated rabbit skeletal muscle Ca2+ release channels (ryanodine receptors) were examined using the planar lipid bilayer method. Single channels were recorded in symmetric 0.25 M KCl media with K+ as the major current carrier. With nanomolar [Ca2+] in both bilayer chambers, the addition of 2 mM cytosolic ATP greatly increased the number of short channel openings. As lumenal [Ca2+] was increased from < 0.1 microM to approximately 250 microM, increasing channel activities and events with long open time constants were seen at negative holding potentials. Channel activity remained low at positive holding potentials. Further increase in lumenal [Ca2+] to 1, 5, and 10 mM resulted in a decrease in channel activities at negative holding potentials and increased activities at positive holding potentials. A voltage-dependent activation by 50 microM lumenal Ca2+ was also observed when the channel was minimally activated by < 1 microM cytosolic Ca2+ in the absence of ATP. With microM cytosolic Ca2+ in the presence or absence of 2 mM ATP, single-channel activities showed no or only a weak voltage dependence. Other divalent cations (Mg2+, Ba2+) could not replace lumenal Ca2+. On the contrary, cytosolic ATP-activated channel activities were decreased as lumenal Ca2+ fluxes were reduced by the addition of 1-5 mM BaCl2 or MgCl2 to the lumenal side, which contained 50 microM Ca2+. An increase in [KCl] from 0.25 M to 1 M also reduced single-channel activities. Addition of the "fast" Ca2+ buffer 1,2-bis(2-aminophenoxy)ethanetetraacetic acid (BAPTA) to the cls chamber increased cytosolic ATP-, lumenal Ca(2+)-activated channel activities to a nearly maximum level. These results suggested that lumenal Ca2+ flowing through the skeletal muscle Ca2+ release channel may regulate channel activity by having access to cytosolic Ca2+ activation and Ca2+ inactivation sites that are located in "BAPTA-inaccessible" and "BAPTA-accessible" spaces, respectively

BIOCHEMICAL & ANTIOXIDANT PROPERTIES OF WILD EDIBLE MUSHROOMS USED FOR FOOD BY TRIBAL OF EASTERN INDIA

Objective: The main objective of this research was to analyze some selected indigenous wild edible mushrooms in Eastern India for their novel antioxidant components and their properties specifically used by primitive tribal groups of Eastern India.Methods: The antioxidant components were analyzed by standardized spectrophotometric methods. The antioxidant properties were analyzed by DPPH Free radical scavenging &amp; Reducing power ability assay.Results: The TPC (phenolic content) in the studied edible mushroom varied from 4.55 mg/g (Russula nigricans) to 0.9 mg/g GAE (Lentinus tuberigium). Measured in term of antioxidants Termitomyces group ranked higher than Russula and Volvariella sp. The scavenging effect of studied mushrooms on 1,1 DPPH varied from 61% to as high as 94%. On the other hand, reducing power (RPA) in methanolic extracts were in the order of T. clypeatus (4.21) T. heimi (2.20) ~R. breviceps (1.73) ~ Termitomyces eurrhizus (1.11) ~ T. rufum (1.07). Antioxidant potential inedible wild mushrooms are found to be on account of combinations of biochemicals, rather than any such significant individual components as TPC, AA, or alkaloid. Conclusion: This is for the first time wild edibles such as Termitomyces clypeatus, Termitomyces eurrhizus, Termitomyces heimii, Russula brevipes, Tuber rufum, Russula nigricans, Volvariella volvaceae, Lentinus fusipes, Lentinus tuberigium and R. lepida from eastern India were observed, collected and subjected to nutritional and biochemical analysis. Of significance is the identification of Tuber rufum and Volvariella volvaceae growing wild as edible mushrooms which have not been profiled in the Indian context. The analysed mushroom especially Lentinus fusipes and Lentinus tuberigium was found valuable in terms of iron and calcium, besides having useful phytochemicals such as phenolics, ascorbic acid, carotenoids. Keywords: Deciduous forests, Orissa, Wild food, Phenolics, Ergosterol, Termitomyce

Artificial Neural Network based Body Posture Classification from EMG signal analysis

 This paper deals with the body posture Classification from EMG signal analysis using artificial neural network (ANN). The various statistical features extracted from each EMG signal corresponding to different muscles associated with the different body postures are framed using LABVIEW software. Further-more, these features are taken as the input towards the ANN classifier and thus the corresponding output for the respective classifier predicts the postures like Bowing, Handshaking, and Hugging. The performance of the classifier is determined by the classification rate (CR). The outcome of result indicates that the CR of Multilayer Feed Forward Neural Network (MFNN) type of ANN is rounded up to a percentage of 71.02%

Selectivity and Permeation in Calcium Release Channel of Cardiac Muscle: Alkali Metal Ions

Current was measured from single open channels of the calcium release channel (CRC) of cardiac sarcoplasmic reticulum (over the range +/-180 mV) in pure and mixed solutions (e.g., biionic conditions) of the alkali metal ions Li+, K+, Na+, Rb+, Cs+, ranging in concentration from 25 mM to 2 M. The current-voltage (I-V) relations were analyzed by an extension of the Poisson-Nernst-Planck (PNP) formulation of electrodiffusion, which includes local chemical interaction described by an offset in chemical potential, which likely reflects the difference in dehydration/solvation/rehydration energies in the entry/exit steps of permeation. The theory fits all of the data with few adjustable parameters: the diffusion coefficient of each ion species, the average effective charge distribution on the wall of the pore, and an offset in chemical potential for lithium and sodium ions. In particular, the theory explains the discrepancy between "selectivities" defined by conductance sequence and "selectivities" determined by the permeability ratios (i.e., reversal potentials) in biionic conditions. The extended PNP formulation seems to offer a successful combined treatment of selectivity and permeation. Conductance selectivity in this channel arises mostly from friction: different species of ions have different diffusion coefficients in the channel. Permeability selectivity of an ion is determined by its electrochemical potential gradient and local chemical interaction with the channel. Neither selectivity (in CRC) seems to involve different electrostatic interaction of different ions with the channel protein, even though the ions have widely varying diameters

Two Domains in Dihydropyridine Receptor Activate the Skeletal Muscle Ca 2+ Release Channel

The II-III cytoplasmic loop of the skeletal muscle dihydropyridine receptor (DHPR) alpha(1)-subunit is essential for skeletal-type excitation-contraction coupling. Single channel and [(3)H]ryanodine binding studies with a full-length recombinant peptide (p(666-791)) confirmed that this region specifically activates skeletal muscle Ca2+ release channels (CRCs). However, attempts to identify shorter domains of the II-III loop specific for skeletal CRC activation have yielded contradictory results. We assessed the specificity of the interaction of five truncated II-III loop peptides by comparing their effects on skeletal and cardiac CRCs in lipid bilayer experiments; p(671-680) and p(720-765) specifically activated the submaximally Ca2+-activated skeletal CRC in experiments using both mono and divalent ions as current carriers. A third peptide, p(671-690), showed a bimodal activation/inactivation behavior indicating a high-affinity activating and low-affinity inactivating binding site. Two other peptides (p(681-690) and p(681-685)) that contained an RKRRK-motif and have previously been suggested in in vitro studies to be important for skeletal-type E-C coupling, failed to specifically stimulate skeletal CRCs. Noteworthy, p(671-690), p(681-690), and p(681-685) induced similar subconductances and long-lasting channel closings in skeletal and cardiac CRCs, indicating that these peptides interact in an isoform-independent manner with the CRCs

Short Palate, Lung, and Nasal Epithelial Clone 1 Has Antimicrobial and Antibiofilm Activities against the Burkholderia cepacia Complex

ABSTRACT The opportunistic bacteria of the Burkholderia cepacia complex (Bcc) are extremely pathogenic to cystic fibrosis (CF) patients, and acquisition of Bcc bacteria is associated with a significant increase in mortality. Treatment of Bcc infections is difficult because the bacteria are multidrug resistant and able to survive in biofilms. Short palate, lung, and nasal epithelial clone 1 (SPLUNC1) is an innate defense protein that is secreted by the upper airways and pharynx. While SPLUNC1 is known to have antimicrobial functions, its effects on Bcc strains are unclear. We therefore tested the hypothesis that SPLUNC1 is able to impair Bcc growth and biofilm formation. We found that SPLUNC1 exerted bacteriostatic effects against several Bcc clinical isolates, including B. cenocepacia strain J2315 (50% inhibitory concentration [IC 50 ] = 0.28 μM), and reduced biofilm formation and attachment (IC 50 = 0.11 μM). We then determined which domains of SPLUNC1 are responsible for its antimicrobial activity. Deletions of SPLUNC1's N terminus and α6 helix did not affect its function. However, deletion of the α4 helix attenuated antimicrobial activity, while the corresponding α4 peptide displayed antimicrobial activity. Chronic neutrophilia is a hallmark of CF lung disease, and neutrophil elastase (NE) cleaves SPLUNC1. However, we found that the ability of SPLUNC1 to disrupt biofilm formation was significantly potentiated by NE pretreatment. While the impact of CF on SPLUNC1-Bcc interactions is not currently known, our data suggest that understanding this interaction may have important implications for CF lung disease

Structure of a Yeast Dyn2-Nup159 Complex and Molecular Basis for Dynein Light Chain-Nuclear Pore Interaction

The nuclear pore complex gates nucleocytoplasmic transport through a massive, eight-fold symmetric channel capped by a nucleoplasmic basket and structurally unique, cytoplasmic fibrils whose tentacles bind and regulate asymmetric traffic. The conserved Nup82 complex, composed of Nsp1, Nup82, and Nup159, forms the unique cytoplasmic fibrils that regulate mRNA nuclear export. Although the nuclear pore complex plays a fundamental, conserved role in nuclear trafficking, structural information about the cytoplasmic fibrils is limited. Here, we investigate the structural and biochemical interactions between Saccharomyces cerevisiae Nup159 and the nucleoporin, Dyn2. We find that Dyn2 is predominantly a homodimer and binds arrayed sites on Nup159, promoting the Nup159 parallel homodimerization. We present the first structure of Dyn2, determined at 1.85 Å resolution, complexed with a Nup159 target peptide. Dyn2 resembles homologous metazoan dynein light chains, forming homodimeric composite substrate binding sites that engage two independent 10-residue target motifs, imparting a β-strand structure to each peptide via antiparallel extension of the Dyn2 core β-sandwich. Dyn2 recognizes a highly conserved QT motif while allowing sequence plasticity in the flanking residues of the peptide. Isothermal titration calorimetric analysis of the comparative binding of Dyn2 to two Nup159 target sites shows similar affinities (18 and 13 μm), but divergent thermal binding modes. Dyn2 homodimers are arrayed in the crystal lattice, likely mimicking the arrayed architecture of Dyn2 on the Nup159 multivalent binding sites. Crystallographic interdimer interactions potentially reflect a cooperative basis for Dyn2-Nup159 complex formation. Our data highlight the determinants that mediate oligomerization of the Nup82 complex and promote a directed, elongated cytoplasmic fibril architecture

Regulation of Skeletal Muscle Ca 2+ Release Channel (Ryanodine Receptor) by Ca 2+ and Monovalent Cations and Anions

The effects of ionic composition and strength on rabbit skeletal muscle Ca2+ release channel (ryanodine receptor) activity were investigated in vesicle-45Ca2+ flux, single channel and [3H]ryanodine binding measurements. In <0.01 microM Ca2+ media, the highest 45Ca2+ efflux rate was measured in 0.25 M choline-Cl medium followed by 0.25 M KCl, choline 4-morpholineethanesulfonic acid (Mes), potassium 1,4-piperazinediethanesulfonic acid (Pipes), and K-Mes medium. In all five media, the 45Ca2+ efflux rates were increased when the free [Ca2+] was raised from <0.01 microM to 20 microM and decreased as the free [Ca2+] was further increased to 1 mM. An increase in [KCl] augmented Ca2+-gated single channel activity and [3H]ryanodine binding. In [3H]ryanodine binding measurements, bell-shaped Ca2+ activation/inactivation curves were obtained in media containing different monovalent cations (Li+, Na+, K+, Cs+, and choline+) and anions (Cl-, Mes-, and Pipes-). In choline-Cl medium, substantial levels of [3H]ryanodine binding were observed at [Ca2+] <0.01 microM. Replacement of Cl- by Mes- or Pipes- reduced [3H]ryanodine binding levels at all [Ca2+]. In all media, the Ca2+-dependence of [3H]ryanodine binding could be well described assuming that the skeletal muscle ryanodine receptor possesses cooperatively interacting high-affinity Ca2+ activation and low-affinity Ca2+ inactivation sites. AMP primarily affected [3H]ryanodine binding by decreasing the apparent affinity of the Ca2+ inactivation site(s) for Ca2+, while caffeine increased the apparent affinity of the Ca2+ activation site for Ca2+. Competition studies indicated that ionic composition affected Ca2+-dependent receptor activity by at least three different mechanisms: (i) competitive binding of Mg2+ and monovalent cations to the Ca2+ activation sites, (ii) binding of divalent cations to the Ca2+ inactivation sites, and (iii) binding of anions to specific anion regulatory sites

Mutations in the Procaspase-3 Dimer Interface Affect the Activity of the Zymogen †

The interface of the procaspase-3 dimer plays a critical role in zymogen maturation. We show that replacement of valine 266, the residue at the center of the procaspase-3 dimer interface, with glutamate resulted in an increase in enzyme activity of ~60-fold, representing a pseudoactivation of the procaspase. In contrast, substitution of V266 with histidine abolished the activity of the procaspase-3 as well as that of the mature caspase. While the mutations do not affect the dimeric properties of the procaspase, we show that the V266E mutation may affect the formation of a loop bundle that is important for stabilizing the active site. In contrast, the V266H mutation affects the positioning of loop L3, the loop that forms the bulk of the substrate binding pocket. In some cases, the amino acids affected by the mutations are >20 Å from the interface. Overall, the results demonstrate that the integrity of the dimer interface is important for maintaining the proper active site conformation

Ruthenium Red Modifies the Cardiac and Skeletal Muscle Ca 2+ Release Channels (Ryanodine Receptors) by Multiple Mechanisms

The effects of ruthenium red (RR) on the skeletal and cardiac muscle ryanodine receptors (RyRs) were studied in vesicle-Ca(2+) flux, [(3)H]ryanodine binding, and single channel measurements. In vesicle-Ca(2+) flux measurements, RR was more effective in inhibiting RyRs at 0.2 microM than 20 microM free Ca(2+). [(3)H]Ryanodine binding measurements suggested noncompetitive interactions between RR inhibition and Ca(2+) regulatory sites of RyRs. In symmetric 0.25 M KCl with 10-20 microM cytosolic Ca(2+), cytosolic RR decreased single channel activities at positive and negative holding potentials. In close to fully activated skeletal (20 microM Ca(2+) + 2 mM ATP) and cardiac (200 microM Ca(2+)) RyRs, cytosolic RR induced a predominant subconductance at a positive but not negative holding potential. Lumenal RR induced a major subconductance in cardiac RyR at negative but not positive holding potentials and several subconductances in skeletal RyR. The RR-related subconductances of cardiac RyR showed a nonlinear voltage dependence, and more than one RR molecule appeared to be involved in their formation. Cytosolic and lumenal RR also induced subconductances in Ca(2+)-conducting skeletal and cardiac RyRs recorded at 0 mV holding potential. These results suggest that RR inhibits RyRs and induces subconductances by binding to cytosolic and lumenal sites of skeletal and cardiac RyRs