Nickel(II) complexes bearing a pincer ligand containing thioamide units: Comparison between SNS- and SCS-pincer ligands

Nickel(II) complexes bearing a κ³SNS pincer ligand, 2,5-bis(benzylaminothiocarbonyl)pyrrolyl (L1) and a κ³SCS-pincer ligand, 2,6-bis(benzylaminothiocarbonyl)phenyl (L2), were synthesized, and their structures and electrochemical properties were elucidated. The crystal structures of [Ni(SNS)Br] (2) and [Ni(SCS)Br] (5) were determined by X-ray crystallography. The electrochemical and crystallographic data obtained from the complexes revealed that the κ³SCS ligand has a stronger electron-donating ability than the κ³SNS ligand.     

Regulation of Microbe-Associated Molecular Pattern-Induced Hypersensitive Cell Death,Phytoalexin Production,and Defense Gene Expression by Calcineurin B-Like Protein-Interacting Protein Kinases,OsCIPK14/15, in Rice Cultured Cells

Although cytosolic free Ca²⁺ mobilization induced by microbe/pathogen-associated molecular patterns is postulated to play a pivotal role in innate immunity in plants, the molecular links between Ca²⁺ and downstream defense responses still remain largely unknown. Calcineurin B-like proteins (CBLs) act as Ca²⁺ sensors to activate specific protein kinases, CBL-interacting protein kinases (CIPKs). We here identified two CIPKs, OsCIPK14 and OsCIPK15, rapidly induced by microbe-associated molecular patterns, including chitooligosaccharides and xylanase (Trichoderma viride/ethylene-inducing xylanase [TvX/EIX]), in rice (Oryza sativa). Although they are located on different chromosomes, they have over 95% nucleotide sequence identity, including the surrounding genomic region, suggesting that they are duplicated genes. OsCIPK14/15 interacted with several OsCBLs through the FISL/NAF motif in yeast cells and showed the strongest interaction with OsCBL4. The recombinant OsCIPK14/15 proteins showed Mn²⁺-dependent protein kinase activity, which was enhanced both by deletion of their FISL/NAF motifs and by combination with OsCBL4. OsCIPK14/15-RNAi transgenic cell lines showed reduced sensitivity to TvX/EIX for the induction of a wide range of defense responses, including hypersensitive cell death, mitochondrial dysfunction, phytoalexin biosynthesis, and pathogenesis-related gene expression. On the other hand, TvX/EIX-induced cell death was enhanced in OsCIPK15-overexpressing lines. Our results suggest that OsCIPK14/15 play a crucial role in the microbe-associated molecular pattern-induced defense signaling pathway in rice cultured cells.Calcium ions regulate diverse cellular processes in plants as a ubiquitous internal second messenger, conveying signals received at the cell surface to the inside of the cell through spatial and temporal concentration changes that are decoded by an array of Ca²⁺ sensors (Reddy, 2001; Sanders et al., 2002; Yang and Poovaiah, 2003). Several families of Ca²⁺ sensors have been identified in higher plants. The best known are calmodulins (CaMs) and CaM-related proteins, which typically contain four EF-hand domains for Ca²⁺ binding (Zielinski, 1998). Unlike mammals, which possess single molecular species of CaM, plants have at least three distinct molecular species of CaM playing diverse physiological functions and whose expression is differently regulated (Yamakawa et al., 2001; Luan et al., 2002; Karita et al., 2004; Takabatake et al., 2007). The second major class is exemplified by the Ca²⁺-dependent protein kinases, which contain CaM-like Ca²⁺-binding domains and a kinase domain in a single protein (Harmon et al., 2000). In addition, a new family of Ca²⁺ sensors was identified as calcineurin B-like (CBL) proteins, which consists of proteins similar to both the regulatory β-subunit of calcineurin and the neuronal Ca²⁺ sensor in animals (Liu and Zhu, 1998; Kudla et al., 1999).Unlike CaMs, which interact with a large variety of target proteins, CBLs specifically target a family of protein kinases referred to as CBL-interacting protein kinases (CIPKs) or SnRK3s (for sucrose nonfermenting 1-related protein kinases type 3), which are most similar to the SNF family protein kinases in yeast (Luan et al., 2002). A database search of the Arabidopsis (Arabidopsis thaliana) genome sequence revealed 10 CBL and 25 CIPK homologues (Luan et al., 2002). Expression patterns of these Ca²⁺ sensors and protein kinases suggest their diverse functions in different signaling processes, including light, hormone, sugar, and stress responses (Batistic and Kudla, 2004). AtCBL4/Salt Overly Sensitive3 (SOS3) and AtCIPK24/SOS2 have been shown to play a key role in Ca²⁺-mediated salt stress adaptation (Zhu, 2002). The CBL-CIPK system has been shown to be involved in signaling pathways of abscisic acid (Kim et al., 2003a), sugar (Gong et al., 2002a), gibberellins (Hwang et al., 2005), salicylic acid (Mahajan et al., 2006), and K⁺ channel regulation (Li et al., 2006; Lee et al., 2007; for review, see Luan, 2009; Batistic and Kudla, 2009). However, physiological functions of most of the family members still remain largely unknown.Plants respond to pathogen attack by activating a variety of defense responses, including the generation of reactive oxygen species (ROS), synthesis of phytoalexins, expression of pathogenesis-related (PR) genes, cell cycle arrest, and mitochondrial dysfunction followed by a form of hypersensitive cell death known as the hypersensitive response (Nürnberger and Scheel, 2001; Greenberg and Yao, 2004; Kadota et al., 2004b). Transient membrane potential changes and Ca²⁺ influx are involved at the initial stage of defense responses (Kuchitsu et al., 1993; Pugin et al., 1997; Blume et al., 2000; Kadota et al., 2004a). Many kinds of defense responses are prevented when Ca²⁺ influx is compromised by Ca²⁺ chelators (Nürnberger and Scheel, 2001; Lecourieux et al., 2002). Since complex spatiotemporal patterns of cytosolic free Ca²⁺ concentration have been suggested to play pivotal roles in defense signaling (Nürnberger and Scheel, 2001; Sanders et al., 2002), multiple Ca²⁺ sensor proteins and their effectors should function in the defense signaling pathways. Although possible involvement of some CaM isoforms (Heo et al., 1999; Yamakawa et al., 2001), Ca²⁺-dependent protein kinases (Romeis et al., 2000, 2001; Ludwig et al., 2005; Kobayashi et al., 2007; Yoshioka et al., 2009), as well as Ca²⁺ regulation of EF-hand-containing enzymes such as ROS-generating NADPH oxidase (Ogasawara et al., 2008) have been suggested, other Ca²⁺-regulated signaling components still remain to be identified. No CBLs or CIPKs have so far been implicated as signaling components in defense signaling.N-Acetylchitooligosaccharides, chitin fragments, are microbe-associated molecular patterns (MAMPs) that are recognized by plasma membrane receptors (Kaku et al., 2006; Miya et al., 2007) and induce a variety of defense responses, such as membrane depolarization (Kuchitsu et al., 1993; Kikuyama et al., 1997), ion fluxes (Kuchitsu et al., 1997), ROS production (Kuchitsu et al., 1995), phytoalexin biosynthesis (Yamada et al., 1993), and induction of PR genes (Nishizawa et al., 1999), without hypersensitive cell death in rice (Oryza sativa) cells. In contrast, a fungal proteinaceous elicitor, xylanase from Trichoderma viride (TvX)/ethylene-inducing xylanase (EIX), which is recognized by two putative plasma membrane receptors, LeEix1 and LeEix2 (Ron and Avni, 2004), triggers hypersensitive cell death along with different kinetics of ROS production and activation of a mitogen-activated protein kinase, OsMPK6, previously named as OsMPK2 or OsMAPK6, in rice cells (Kurusu et al., 2005). These two fungal MAMPs thus provide excellent model systems to study innate immunity in rice cells.This study identified two CIPKs involved in various MAMP-induced layers of defense responses, including PR gene expression, phytoalexin biosynthesis, mitochondrial dysfunction, and cell death, in rice. Molecular characterization of these CIPKs, including interaction with the putative Ca²⁺ sensors as well as their physiological functions, is discussed.     

Mechanism of ER stress-induced brain damage by IP(3) receptor

Deranged Ca(2+) signaling and an accumulation of aberrant proteins cause endoplasmic reticulum (ER) stress, which is a hallmark of cell death implicated in many neurodegenerative diseases. However, the underlying mechanisms are elusive. Here, we report that dysfunction of an ER-resident Ca(2+) channel, inositol 1,4,5-trisphosphate receptor (IP(3)R), promotes cell death during ER stress. Heterozygous knockout of brain-dominant type1 IP(3)R (IP(3)R1) resulted in neuronal vulnerability to ER stress in?vivo, and IP(3)R1 knockdown enhanced ER stress-induced apoptosis via mitochondria in cultured cells. The IP(3)R1 tetrameric assembly was positively regulated by the ER chaperone GRP78 in an energy-dependent manner. ER stress induced IP(3)R1 dysfunction through an impaired IP(3)R1-GRP78 interaction, which has also been observed in the brain of Huntington's disease model mice. These results suggest that IP(3)R1 senses ER stress through GRP78 to alter the Ca(2+) signal to promote neuronal cell death implicated in neurodegenerative diseases.     

Glycosylation of capsaicinoids with Panax ginseng stimulated by salicylic acid

The efficient production of β-glycosides of capsaicin and 8-nordihydrocapsaicin by cultured cells of Panax ginseng is reported. Capsaicin 4-O-(6-O-β-D-xylopyranosyl)-β-D-glucopyranoside (β-primeveroside, 12%) together with capsaicin 4-O-β-D-glucoside (6%) was isolated from the cell suspension of P. ginseng after one week of incubation with capsaicin. On the other hand, 8-nordihydrocapsaicin was glycosylated to 8-nordihydrocapsaicin 4-O-β-D-glucoside (5%) and 8-nordihydrocapsaicin 4-O-β-primeveroside (9%) by P. ginseng. Pretreatment of the cultured cells with salicylic acid greatly enhanced the glucosylation activity toward capsaicinoids. When 500 μM of salicylic acid was added to the cultures prior to the addition of substrate, capsaicin was converted into capsaicin 4-O-β-D-glucoside (17%) and capsaicin β-primeveroside (21%) and 8-nordihydrocapsaicin was glycosylated to 8-nordihydrocapsaicin 4-O-β-D-glucoside (16%) and 8-nordihydrocapsaicin β-primeveroside (15%).     

Structure and biosynthesis of lecanopyrone, a naphtho[1,8-cd]pyran-3-one derivative from cultured lichen mycobionts of Lecanora leprosa

From the cultures of spore-derived mycobionts of the lichen Lecanora leprosa a novel naphtho[1,8-cd]pyran-3-one derivative, lecanopyrone, was isolated. Its structure was determined by spectroscopic methods. The assembly pattern of acetate units in its biosynthesis was verified using sodium [1-13C]-acetate and sodium [1,2-13C2]-acetate.     

Reactive oxygen species induced by Streptococcus pyogenes invasion trigger apoptotic cell death in infected epithelial cells

Streptococcus pyogenes (group A streptococcus, GAS), one of the most common pathogens of humans, attaches and invades into human pharyngeal or skin epithelial cells. We have previously reported that induction of apoptosis is associated with GAS invasion, which induces mitochondrial dysfunction and apoptotic cell death. We demonstrate here that GAS‐induced apoptosis is mediated by reactive oxygen species (ROS) production. Both the induction of apoptosis and ROS production markedly increased upon invasion of wild‐type GAS strain JRS4 into HeLa cells; however, the apoptotic response was not observed in fibronectin‐binding protein F1‐disrupted mutant SAM1‐infected cells. In Bcl‐2‐overexpressing HeLa cells (HBD98‐2‐4), the induction of apoptosis, ROS production and mitochondrial dysfunction were significantly suppressed, whereas the numbers of invaded GAS was not different between HeLa (mock cells) and the HeLa HBD98‐2‐4 cells. Whereas Rac1 activation occurred during GAS invasion, ROS production in GAS‐infected cells was clearly inhibited by transfection with the Rac1 mutants (L37 or V12L37), but not by the dominant active mutant (V12L61) or by the dominant negative mutant (N17). These observations indicate that GAS invasion triggers ROS production through Rac1 activation and generated ROS induced mitochondrial dysfunction leading to cellular apoptosis.     

Roles of calcineurin B-like protein-interacting protein kinases in innate immunity in rice

Cytosolic free Ca²⁺ mobilization induced by microbe/pathogen-asssociated molecular patterns (MAMPs/PAMPs) plays key roles in plant innate immunity. However, components involved in Ca²⁺ signaling pathways still remain to be identified and possible involvement of the CBL (calcineurin B-like proteins)-CIPK (CBL-interacting protein kinases) system in biotic defense signaling have yet to be clarified. Recently we identified two CIPKs, OsCIPK14 and OsCIPK15, which are rapidly induced by MAMPs, involved in various MAMP-induced immune responses including defense-related gene expression, phytoalexin biosynthesis and hypersensitive cell death. MAMP-induced production of reactive oxygen species as well as cell browning were also suppressed in OsCIPK14/15-RNAi transgenic cell lines. Possible molecular mechanisms and physiological functions of the CIPKs in plant innate immunity are discussed.Key words: PAMPs/MAMPs, calcium signaling, CBL-CIPK, hypersensitive cell death, reactive oxygen speciesCa²⁺ plays an essential role as an intracellular second messenger in plants as well as in animals. Several families of Ca²⁺ sensor proteins have been identified in higher plants, which decode spatiotemporal patterns of intracellular Ca²⁺ concentration.^1,2 Calcineurin B-Like Proteins (CBLs) comprise a family of Ca²⁺ sensor proteins similar to both the regulatory β-subunit of calcineurin and neuronal Ca²⁺ sensors of animals.^3,4 Unlike calcineurin B that regulates protein phosphatases, CBLs specifically target a family of protein kinases referred to as CIPKs (CBL-Interacting Protein Kinases).⁵ The CBL-CIPK system has been shown to be involved in a wide range of signaling pathways, including abiotic stress responses such as drought and salt, plant hormone responses and K+ channel regulation.^6,7Following the recognition of pathogenic signals, plant cells initiate the activation of a widespread signal transduction network that trigger inducible defense responses, including the production of reactive oxygen species (ROS), biosynthesis of phytoalexins, expression of pathogenesis-related (PR) genes and reorganization of cytoskeletons and the vacuole,⁸ followed by a form of programmed cell death known as hypersensitive response (HR).^9,10 Because complexed spatiotemporal patterns of cytosolic free Ca²⁺ concentration ([Ca²⁺]_cyt) have been suggested to play pivotal roles in defense signaling,^1,9 multiple Ca²⁺ sensor proteins and their effectors should function in defense signaling pathways. Although possible involvement of some calmodulin isoforms^11–13 and the calmodulin-domain/calcium-dependent protein kinases (CDPKs)^14–19 has been suggested, other Ca²⁺-regulated signaling components still remain to be identified. No CBLs or CIPKs had so far been implicated as signaling components in innate immunity.     

Mobilicoccus pelagius gen. nov., sp. nov. and Piscicoccus intestinalis gen. nov., sp. nov., two new members of the family Dermatophilaceae, and reclassification of Dermatophilus chelonae (Masters et al. 1995) as Austwickia chelonae gen. nov., comb. nov

Two Gram-positive bacteria, designated strains Aji5-31(T) and Ngc37-23(T), were isolated from the intestinal tracts of fishes. 16S rRNA gene sequence analysis indicated that both strains were related to the members of the family Dermatophilaceae, with 95.6-96.9% 16S rRNA gene sequence similarities. The family Dermatophilaceae contains 2 genera and 3 species: Dermatophilus congolensis, Dermatophilus chelonae and Kineosphaera limosa. However, it has been suggested that the taxonomic position of D. chelonae should be reinvestigated using a polyphasic approach, because the chemotaxonomic characteristics are not known (Stackebrandt, 2006; Stackebrandt and Schumann, 2000). Our present study revealed that strains Aji5-31(T), Ngc37-23(T) and D. chelonae NBRC 105200(T) should be separated from the other members of the family Dermatophilaceae on the basis of the following characteristics: the predominant menaquinone of strain Aji5-31(T) is MK-8(H(2)), strain Ngc37-23(T) possesses iso- branched fatty acids as major components, and the menaquinone composition of D. chelonae is MK-8(H(4)), MK-8 and MK-8(H(2)) (5 : 3 : 2, respectively). On the basis of these distinctive phenotypic characteristics and phylogenetic analysis results, it is proposed that strains Aji5-31(T) and Ngc37-23(T) be classified as two novel genera and species of the family Dermatophilaceae. The names are Mobilicoccus pelagius gen. nov., sp. nov. and Piscicoccus intestinalis gen. nov., sp. nov., and the type strains are Aji5-31(T) (=NBRC 104925(T) =DSM 22762(T)) and Ngc37-23(T) (=NBRC 104926(T) =DSM 22761(T)), respectively. In addition, D. chelonae should be reassigned to a new genus of the family Dermatophilaceae with the name Austwickia chelonae gen. nov., comb. nov.     

Mining frequent stem patterns from unaligned RNA sequences

MOTIVATION: In detection of non-coding RNAs, it is often necessary to identify the secondary structure motifs from a set of putative RNA sequences. Most of the existing algorithms aim to provide the best motif or few good motifs, but biologists often need to inspect all the possible motifs thoroughly. RESULTS: Our method RNAmine employs a graph theoretic representation of RNA sequences and detects all the possible motifs exhaustively using a graph mining algorithm. The motif detection problem boils down to finding frequently appearing patterns in a set of directed and labeled graphs. In the tasks of common secondary structure prediction and local motif detection from long sequences, our method performed favorably both in accuracy and in efficiency with the state-of-the-art methods such as CMFinder. AVAILABILITY: The software is available upon request.