Two-step Ca2+ intracellular release underlies excitation-contraction coupling in mouse urinary bladder myocytes

The relative contributions of Ca²⁺-induced Ca²⁺ release (CICR) versus Ca²⁺ influx through voltage-dependent Ca²⁺ channels (VDCCs) to excitation-contraction coupling has not been defined in most smooth muscle cells (SMCs). The present study was undertaken to address this issue in mouse urinary bladder (UB) smooth muscle cells (UBSMCs). Confocal Ca²⁺ images were obtained under voltage- or current-clamp conditions. When UBSMCs were activated by a 30-ms depolarization to 0 mV, intracellular Ca²⁺ concentration ([Ca²⁺]_i) increased in several small, discrete areas just beneath the cell membrane. These Ca²⁺ "hot spots" then spread slowly through the myoplasm as Ca²⁺ waves, which continued even after repolarization. Shorter depolarizations (5 ms) elicited only a few Ca²⁺ sparks, which declined quickly. The number of Ca²⁺ sparks, or hot spots, was closely related to the depolarization duration in the range of 5–20 ms. There was an apparent threshold depolarization duration of 10 ms within which to induce enough Ca²⁺ transients to spread globally and then induce a contraction. Application of 100 µM ryanodine to the pipette solution did not change the resting [Ca²⁺]_i or the VDCC current, but it did abolish Ca²⁺ hot spots elicited by depolarization. Application of 3 µM xestospongin C reduced ACh-induced Ca²⁺ release but did not affect depolarization-induced Ca²⁺ events. The addition of 100 µM ryanodine to tissue segments markedly reduced the amplitude of contractions triggered by direct electrical stimulation. In conclusion, global [Ca²⁺]_i rise triggered by a single action potential is not due mainly to Ca²⁺ influx through VDCCs but is attributable to the subsequent two-step CICR. Ca²⁺-induced Ca²⁺ release; Ca²⁺-activated K⁺ current; voltage-dependent Ca²⁺ channel     

Protein kinase C delta (deltaPKC)-annexin V interaction: a required step in deltaPKC translocation and function

Protein kinase C (PKC) plays a critical role in diseases such as cancer, stroke, and cardiac ischemia, and participates in a variety of signal transduction pathways such as apoptosis, cell proliferation, and tumor suppression. Though much is known about PKC downstream signaling events, the mechanisms of regulation of PKC activation and subsequent translocation have not been elucidated. Protein-protein interactions regulate and determine the specificity of many cellular signaling events. Such a specific protein-protein interaction is described here between deltaPKC and annexin V. We demonstrate, at physiologically relevant conditions, that a transient interaction between annexin V and deltaPKC occurs in cells after deltaPKC stimulation, but before deltaPKC translocates to the particulate fraction. Evidence of deltaPKC-annexin V binding is provided also by FRET and by in vitro binding studies. Dissociation of the deltaPKC-annexin V complex requires ATP and microtubule integrity. Furthermore, depletion of endogenous annexin V, but not annexin IV, with siRNA inhibits deltaPKC translocation following PKC stimulation. A rationally designed eight amino acid peptide, corresponding to the interaction site for deltaPKC on annexin V, inhibits deltaPKC translocation and deltaPKC-mediated function as evidenced by its protective effect in a model of myocardial infarction. Our data indicate that translocation of deltaPKC is not simply a diffusion-driven process, but is instead a multi-step event regulated by protein-protein interactions. We show that following cell activation, deltaPKC-annexin V binding is a transient and an essential step in the function of deltaPKC, thus identifying a new role for annexin V in PKC signaling and a new step in PKC activation.     

Characterization and classification of ATP-binding cassette transporter ABCA3 mutants in fatal surfactant deficiency

The ATP-binding cassette transporter ABCA3 is expressed predominantly at the limiting membrane of the lamellar bodies in lung alveolar type II cells. Recent study has shown that mutation of the ABCA3 gene causes fatal surfactant deficiency in newborns. In this study, we investigated in HEK293 cells the intracellular localization and N-glycosylation of the ABCA3 mutants so far identified in fatal surfactant deficiency patients. Green fluorescent protein-tagged L101P, L982P, L1553P, Q1591P, and Ins1518fs/ter1519 mutant proteins remained localized in the endoplasmic reticulum, and processing of oligosaccharide was impaired, whereas wild-type and N568D, G1221S, and L1580P mutant ABCA3 proteins trafficked to the LAMP3-positive intracellular vesicle, accompanied by processing of oligosaccharide from high mannose type to complex type. Vanadate-induced nucleotide trapping and ATP-binding analyses showed that ATP hydrolysis activity was dramatically decreased in the N568D, G1221S, and L1580P mutants, accompanied by a moderate decrease in ATP binding in N568D and L1580P mutants but not in the G1221S mutant, compared with the wild-type ABCA3 protein. In addition, mutational analyses of the Gly-1221 residue in the 11th transmembrane segment and the Leu-1580 residue in the cytoplasmic tail, and homology modeling of nucleotide binding domain 2 demonstrate the significance of these residues for ATP hydrolysis and suggest a mechanism for impaired ATP hydrolysis in G1221S and L1580P mutants. Thus, surfactant deficiency because of ABCA3 gene mutation may be classified into two categories as follows: abnormal intracellular localization (type I) and normal intracellular localization with decreased ATP binding and/or ATP hydrolysis of the ABCA3 protein (type II). These distinct pathophysiologies may reflect both the severity and effective therapy for surfactant deficiency.     

Activation of the superoxide-producing phagocyte NADPH oxidase requires co-operation between the tandem SH3 domains of p47phox in recognition of a polyproline type II helix and an adjacent alpha-helix of p22phox

Activation of the superoxide-producing phagocyte NADPH oxidase, crucial for host defence, requires an SH3 (Src homology 3)-domain-mediated interaction of the regulatory protein p47phox with p22phox, a subunit of the oxidase catalytic core flavocytochrome b558. Although previous analysis of a crystal structure has demonstrated that the tandem SH3 domains of p47phox sandwich a short PRR (proline-rich region) of p22phox (amino acids 151-160), containing a polyproline II helix, it has remained unknown whether this model is indeed functional in activation of the oxidase. In the present paper we show that the co-operativity between the two SH3 domains of p47phox, as expected from the model, is required for oxidase activation. Deletion of the linker between the p47phox SH3 domains results not only in a defective binding to p22phox but also in a loss of the activity to support superoxide production. The present analysis using alanine-scanning mutagenesis identifies Pro152, Pro156 and Arg158 in the p22phox PRR as residues indispensable for the interaction with p47phox. Pro152 and Pro156 are recognized by the N-terminal SH3 domain, whereas Arg158 contacts with the C-terminal SH3 domain. Amino acid substitution for any of the three residues in the p22phox PRR abrogates the superoxide-producing activity of the oxidase reconstituted in intact cells. The bis-SH3-mediated interaction of p47phox with p22phox thus functions to activate the phagocyte oxidase. Furthermore, we provide evidence that a region C-terminal to the PRR of p22phox (amino acids 161-164), adopting an a-helical conformation, participates in full activation of the phagocyte oxidase by fortifying the association with the p47phox SH3 domains.     

Molecular phylogenetic and chemical analyses of the microbial mats in deep-sea cold seep sediments at the northeastern Japan Sea

Microbial communities inhabiting deep-sea cold seep sediments at the northeastern Japan Sea were characterized by molecular phylogenetic and chemical analyses. White patchy microbial mats were observed along the fault offshore the Hokkaido Island and sediment samples were collected from two stations at the southern foot of the Shiribeshi seamount (M1 site at a depth of 2,961 m on the active fault) and off the Motta Cape site (M2 site at a depth of 3,064 m off the active fault). The phylogenetic and terminal-restriction fragment polymorphism analyses of PCR-amplified 16S rRNA genes revealed that microbial community structures were different between two sampling stations. The members of ANME-2 archaea and diverse bacterial components including sulfate reducers within Deltaproteobacteria were detected from M1 site, indicating the occurrence of biologically mediated anaerobic oxidation of methane, while microbial community at M2 site was predominantly composed of members of Marine Crenarchaeota group I, sulfate reducers of Deltaproteobacteria, and sulfur oxidizers of Epsilonproteobacteria. Chemical analyses of seawater above microbial mats suggested that concentrations of sulfate and methane at M1 site were largely decreased relative to those at M2 site and carbon isotopic composition of methane at M1 site shifted heavier (¹³C-enriched), the results of which are consistent with molecular analyses. These results suggest that the mat microbial communities in deep-sea cold seep sediments at the northeastern Japan Sea are significantly responsible for sulfur and carbon circulations and the geological activity associated with plate movements serves unique microbial habitats in deep-sea environments.     

Photon emissions from rice cells elicited by N-acetylchitooligosaccharide are generated through phospholipid signaling in close association with the production of reactive oxygen species

Biophotons are ultraweak light emissions from biochemical reactions in a living body. They increase in suspension-cultured rice (Oryza sativa L.) cells when elicited by N-acetylchitooligosaccharide. Biochemical analyses were undertaken to investigate the relationship between disease response and biophotons in order to clarify the emission mechanism of biophotons caused by this elicitor. Photon emissions induced by N-acetylchitohexaose were suppressed when cells were pretreated with the reactive oxygen species (ROS)-generating inhibitors: pyrocatechol-3,5-disulfonic acid disodium salt (Tiron); diphenylene iodonium (DPI); and salicylhydroxamic acid (SHAM). Conversely, exogenously applied ROS (superoxide and hydrogen peroxide) were able to induce photon emissions. The effects of protein phosphorylation (K-252a) and the Ca(2+) signaling inhibitors, ethylene glycol-bis(beta-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA) and LaCl(3), caused photon emissions to decrease. It is clear that photon emissions from rice cells elicited by N-acetylchitohexaose are closely associated with the ROS-generating system, and are regulated by Ca(2+) signaling and protein phosphorylation. Exogenously applied phosphatidic acid (PA), the second messenger in the signal transduction of disease response, raised photon emissions in rice cells. Comparisons of photon emissions from PA and N-acetylchitohexaose regarding time courses, spectral compositions, and the inhibition ratios of several inhibitors, as well as a loss- and gain-of-function assay using the protein synthesis inhibitor cycloheximide (CHX) and PA, showed the possibility that photon emissions from rice cells elicited by N-acetylchitooligosaccharide were generated through PA, an intermediate of phospholipid signaling.     

Identification of diabetes susceptibility loci in db mice by combined quantitative trait loci analysis and haplotype mapping

To identify the disease-susceptibility genes of type 2 diabetes, we performed quantitative trait loci (QTL) analysis in F(2) populations generated from a BKS.Cg-m+/+Lepr(db) and C3H/HeJ intercross, taking advantage of genetically determined obesity and diabetes traits associated with the db gene. A genome-wide scan in the F(2) populations divided by sex and db genotypes identified 14 QTLs in total and 3 major QTLs on chromosome (Chr) 3 (LOD 5.78) for fat pad weight, Chr 15 (LOD 6.64) for body weight, and Chr 16 (LOD 8.15) for blood glucose concentrations. A linear-model-based genome scan using interactive covariates allowed us to consider sex- or sex-by db-specific effects of each locus. For the most significant QTL on Chr 16, the high-resolution haplotype comparison between BKS and C3H strains reduced the critical QTL interval from 20 to 4.6 Mb by excluding shared haplotype regions and identified 11 nonsynonymous single-nucleotide polymorphisms in six candidate genes.     

Development of a versatile expression plasmid construction system for Aspergillus oryzae and its application to visualization of mitochondria

We report here a development of the MultiSite Gateway(TM)-based versatile plasmid construction system applicable for the rapid and efficient preparation of Aspergillus oryzae expression plasmids. This system allows the simultaneous connection of the three DNA fragments inserted in entry clones along with a destination vector in a defined order and orientation. We prepared a variety of entry clones and destination vectors containing promoters, genes encoding carrier-proteins and fusion tags, and selectable markers, which makes it possible to generate 80 expression plasmids for each target protein. Using this system, plasmids for expression of the EGFP fused with the mitochondrial-targeting signal of citrate synthase (AoCit1) were generated. Tubular structures of mitochondria were visualized in the transformants expressing the AoCit1-EGFP fusion protein. This plasmid construction system allows us to prepare a large number of expression plasmids without laborious DNA manipulations, which would facilitate molecular biological studies on A. oryzae.