Craniofacial malformation in R-spondin2 knockout mice

In vertebrates, craniofacial formation is accomplished by synergistic interaction of many small elements which are generated independently from distinct germ layers. Because of its complexity, the imbalance of one signaling cascade such as Wnt/β-catenin pathway easily leads to craniofacial malformation, which is the most frequent birth defect in humans. To investigate the developmental role of a newly identified activator of Wnt/β-catenin signaling, Rspo2, we generated and characterized Rspo2^−/− mice. We found CLP with mild facial skeletal defects in Rspo2^−/− mice. Additionally, Rspo2^−/− mice also exhibited distal limb loss and lung hypoplasia, and died immediately after birth with respiratory failure. We showed the apparent reduction of Wnt/β-catenin signaling activity at the branchial arch and the apical ectodermal ridge in Rspo2^−/− mice. These findings indicate that Rspo2 regulates midfacial, limb, and lung morphogenesis during development through the Wnt/β-catenin signaling.     

Alpha-ketoglutarate oxidoreductase, an essential salvage enzyme of energy metabolism, in coccoid form of Helicobacter pylori

In the Krebs cycle of Helicobacter pylori, the absence of alpha-ketoglutarate dehydrogenase and succinyl CoA synthetase are shown. Instead, alpha-ketoglutarate is converted to succinyl CoA and succinate by alpha-ketoglutarate oxidoreductase (KOR) and CoA transferase (CoAT). In the present study, when H. pylori transformed to the coccoid form, a viable but non-culturable form of H. pylori with reduced metabolic activity, the KOR activity was enhanced while the CoAT activity was reduced. Direct inactivation of KOR could potently kill the bacteria without allowing conversion to the coccoid form, suggesting a novel treatment strategy for the eradication of H. pylori, especially in cases infected with multiple antibiotic-resistant strains.     

Focus Issue on Calcium Signaling: Calcium-Dependent Protein Kinase CPK6 Positively Functions in Induction by Yeast Elicitor of Stomatal Closure and Inhibition by Yeast Elicitor of Light-Induced Stomatal Opening in Arabidopsis

Yeast elicitor (YEL) induces stomatal closure that is mediated by a Ca²⁺-dependent signaling pathway. A Ca²⁺-dependent protein kinase, CPK6, positively regulates activation of ion channels in abscisic acid and methyl jasmonate signaling, leading to stomatal closure in Arabidopsis (Arabidopsis thaliana). YEL also inhibits light-induced stomatal opening. However, it remains unknown whether CPK6 is involved in induction by YEL of stomatal closure or in inhibition by YEL of light-induced stomatal opening. In this study, we investigated the roles of CPK6 in induction by YEL of stomatal closure and inhibition by YEL of light-induced stomatal opening in Arabidopsis. Disruption of CPK6 gene impaired induction by YEL of stomatal closure and inhibition by YEL of light-induced stomatal opening. Activation by YEL of nonselective Ca²⁺-permeable cation channels was impaired in cpk6-2 guard cells, and transient elevations elicited by YEL in cytosolic-free Ca²⁺ concentration were suppressed in cpk6-2 and cpk6-1 guard cells. YEL activated slow anion channels in wild-type guard cells but not in cpk6-2 or cpk6-1 and inhibited inward-rectifying K⁺ channels in wild-type guard cells but not in cpk6-2 or cpk6-1. The cpk6-2 and cpk6-1 mutations inhibited YEL-induced hydrogen peroxide accumulation in guard cells and apoplast of rosette leaves but did not affect YEL-induced hydrogen peroxide production in the apoplast of rosette leaves. These results suggest that CPK6 positively functions in induction by YEL of stomatal closure and inhibition by YEL of light-induced stomatal opening in Arabidopsis and is a convergent point of signaling pathways for stomatal closure in response to abiotic and biotic stress.Stomata, formed by pairs of guard cells, play a critical role in regulation of plant CO₂ uptake and water loss, thus critically influencing plant growth and water stress responsiveness. Guard cells respond to a variety of abiotic and biotic stimuli, such as light, drought, and pathogen attack (Israelsson et al., 2006; Shimazaki et al., 2007; Melotto et al., 2008).Elicitors derived from microbial surface mimic pathogen attack and induce stomatal closure in various plant species such as Solanum lycopersicum (Lee et al., 1999), Commelina communis (Lee et al., 1999), Hordeum vulgare (Koers et al., 2011), and Arabidopsis (Arabidopsis thaliana; Melotto et al., 2006; Khokon et al., 2010). Yeast elicitor (YEL) induces stomatal closure in Arabidopsis (Klüsener et al., 2002; Khokon et al., 2010; Salam et al., 2013). Our recent studies showed that YEL inhibits light-induced stomatal opening and that protein phosphorylation is involved in induction by YEL of stomatal closure and inhibition by YEL of light-induced stomatal opening (Salam et al., 2013).Cytosolic Ca²⁺ has long been recognized as a conserved second messenger in stomatal movement (Shimazaki et al., 2007; Roelfsema and Hedrich 2010; Hubbard et al., 2012). Elevation of cytosolic free Ca²⁺ concentration ([Ca2+]_cyt) is triggered by influx of Ca²⁺ from apoplast and release of Ca²⁺ from intracellular stores in guard cell signaling (Leckie et al., 1998; Hamilton et al., 2000; Pei et al., 2000; Garcia-Mata et al., 2003; Lemtiri-Chlieh et al., 2003). The influx of Ca²⁺ is carried by nonselective Ca²⁺-permeable cation (I_Ca) channels that are activated by plasma membrane hyperpolarization and H₂O₂ (Pei et al., 2000; Murata et al., 2001; Kwak et al., 2003). Elevation of [Ca2+]_cyt activates slow anion (S-type) channels and down-regulates inward-rectifying potassium (K_in) channels in guard cells (Schroeder and Hagiwara, 1989; Grabov and Blatt, 1999). The activation of S-type channels is a hallmark of stomatal closure, and the suppression of K_in channels is favorable to stomatal closure but not to stomatal opening (Pei et al., 1997; Kwak et al., 2001; Xue et al., 2011; Uraji et al., 2012).YEL induces stomatal closure with extracellular H₂O₂ production, intracellular H₂O₂ accumulation, activation of I_Ca channels, and transient [Ca2+]_cyt elevations (Klüsener et al., 2002; Khokon et al., 2010). However, it remains to be clarified whether YEL activates S-type channels and inhibits K_in channels in guard cells.Calcium-dependent protein kinases (CDPKs) are regulators in Ca²⁺-dependent guard cell signaling (Mori et al., 2006; Zhu et al., 2007; Geiger et al., 2010, 2011; Zou et al., 2010; Munemasa et al., 2011; Brandt et al., 2012; Scherzer et al., 2012). In guard cells, CDPKs regulate activation of S-type and I_Ca channels and inhibition of K_in channels (Mori et al., 2006; Zou et al., 2010; Munemasa et al., 2011). A CDPK, CPK6, positively regulates activation of S-type channels and I_Ca channels without affecting H₂O₂ production in abscisic acid (ABA)- and methyl jasmonate (MeJA)-induced stomatal closure (Mori et al., 2006; Munemasa et al., 2011). CPK6 phosphorylates and activates SLOW ANION CHANNEL-ASSOCIATED1 expressed in Xenopus spp. oocyte (Brandt et al., 2012; Scherzer et al., 2012). These findings underline the role of CPK6 in regulation of ion channel activation and stomatal movement, leading us to test whether CPK6 regulates the induction by YEL of stomatal closure and inhibition by YEL of light-induced stomatal opening.In this study, we investigated activation of S-type channels and inhibition of K_in channels by YEL and roles of CPK6 in induction by YEL of stomatal closure and inhibition by YEL of light-induced stomatal opening. For this purpose, we examined the effects of mutation of CPK6 on induction by YEL of stomatal closure and inhibition by YEL of light-induced stomatal opening, activation of I_Ca channels, transient [Ca2+]_cyt elevations, activation of S-type channels, inhibition of K_in channels, H₂O₂ production in leaves, and H₂O₂ accumulation in leaves and guard cells.     

Methylglyoxal inhibition of cytosolic ascorbate peroxidase from Nicotiana tabacum

Methylglyoxal (MG) is one of the aldehydes accumulated in plants under environmental stress. Cytosolic ascorbate peroxidase (cAPX) plays a key role in the protection of cells from oxidative damage by scavenging reactive oxygen species in higher plants. A cDNA encoding cAPX, named NtcAPX, was isolated from Nicotiana tabacum. We characterized recombinant NtcAPX (rNtcAPX) as a fusion protein with glutathione S‐transferase to investigate the effects of MG on APX. NtcAPX consists of 250 amino acids and has a deduced molecular mass of 27.5 kDa. The rNtcAPX showed a higher APX activity. MG treatments resulted in a reduction of APX activity and modifications of amino groups in rNtcAPX with increasing K_m for ascorbate. On the contrary, neither NaCl nor cadmium reduced the activity of APX. The present study suggests that inhibition of APX is in part due to the modification of amino acids by MG. © 2012 Wiley Periodicals, Inc. J Biochem Mol Toxicol 26:315–321, 2012; View this article online at wileyonlinelibrary.com . DOI 10.1002/jbt.21423     

Stereospecific assignment of H5′ and H5″ in a (5′R)-/(5′S)-deuterium- labeled DNA decamer for3 JHH determination and unambiguous NOE assignments

The stereoselective deuterium labeling at the 5' methylene protons of the ribose ring recently developed by Kawashima et al. [1995, Tetrahedron Lett., 36, 6699–6700] enabled the assignment of pro-R and pro-S protons at the 5' position. The deuterium-labeled nucleotides, [(5'S)-²H]- and [(5'R)-²H]-diastereomers, in an approximate ratio of 2:1, were incorporated in the decamer 5'-d(GCATTAATGC)-3'. Thus, both pro-R and pro-S methylene proton signals without geminal coupling appeared in the NOESY and DQF-COSY spectra. Complete stereospecific assignments and simplified spin systems enabled the determination of 15 ³J coupling constants between H4' and H5'/H5", and the unambiguous assignment of 135 NOESY cross peaks originating from H4'/H5'/H5" resonances.     

A new species of the genusamblyeleotris (pisces: Gobiidae) from Japan

A new shrimp-associated goby,Amblyeleotris melanocephala, is described on the basis of specimens from Okinoshima Island. Kochi Prefecture, and Okinawa Island, Okinawa Prefecture, Japan. The species is distinguished from other members of the genus by the following combination of characters: head dark brown, a few yellow spots on pectoral fin base and opercular margin, 13 second dorsal and 13 anal fin soft rays, 20 pectoral fin rays, longitudinal scales 92–101, proportional length of interpelvic connecting membrane relative to longest pelvic fin ray (CM-value) 0.46–0.55, presence of a ventral frenum, midline of nape naked, sides scaled above midpoint between preopercle and opercle.     

Synthesis and pharmacological evaluation of 1,1,3-substituted urea derivatives as potent TNF-α production inhibitors

A three substituted urea derivative, SA13353 (compound 1a), exhibited potent inhibitory activity against lipopolysaccharide (LPS)-induced TNF-α production. We focused on the 1,1-substituted moiety (R¹ and R²) of SA13353 and investigated substituent effects of this moiety on LPS-induced TNF-α production by oral administration in rats. The synthesis of the urea derivatives was performed rapidly in a one-pot manner using a manual synthesizer. Several compounds containing hydrophobic substituents at this moiety showed more potent inhibitory activities than SA13353.     

Roles of RCN1, regulatory A subunit of protein phosphatase 2A, in methyl jasmonate signaling and signal crosstalk between methyl jasmonate and abscisic acid

Methyl jasmonate (MeJA) as well as abscisic acid (ABA) induces stomatal closure with their signal crosstalk. We investigated the function of a regulatory A subunit of protein phosphatase 2A, RCN1, in MeJA signaling. Both MeJA and ABA failed to induce stomatal closure in Arabidopsis rcn1 knockout mutants unlike in wild-type plants. Neither MeJA nor ABA induced reactive oxygen species (ROS) production and suppressed inward-rectifying potassium channel activities in rcn1 mutants but not in wild-type plants. These results suggest that RCN1 functions upstream of ROS production and downstream of the branch point of MeJA signaling and ABA signaling in Arabidopsis guard cells.