Two guard cell mitogen‐activated protein kinases,MPK9 and MPK12, function in methyl jasmonate‐induced stomatal closure in Arabidopsis thaliana

Methyl jasmonate (MeJA) and abscisic acid (ABA) signalling cascades share several signalling components in guard cells. We previously showed that two guard cell‐preferential mitogen‐activated protein kinases (MAPKs), MPK9 and MPK12, positively regulate ABA signalling in Arabidopsis thaliana. In this study, we examined whether these two MAP kinases function in MeJA signalling using genetic mutants for MPK9 and MPK12 combined with a pharmacological approach. MeJA induced stomatal closure in mpk9‐1 and mpk12‐1 single mutants as well as wild‐type plants, but not in mpk9‐1 mpk12‐1 double mutants. Consistently, the MAPKK inhibitor PD98059 inhibited the MeJA‐induced stomatal closure in wild‐type plants. MeJA elicited reactive oxygen species (ROS) production and cytosolic alkalisation in guard cells of the mpk9‐1, mpk12‐1 and mpk9‐1 mpk12‐1 mutants, as well in wild‐type plants. Furthermore, MeJA triggered elevation of cytosolic Ca²⁺ concentration ([Ca²⁺]_cyt) in the mpk9‐1 mpk12‐1 double mutant as well as wild‐type plants. Activation of S‐type anion channels by MeJA was impaired in mpk9‐1 mpk12‐1. Together, these results indicate that MPK9 and MPK12 function upstream of S‐type anion channel activation and downstream of ROS production, cytosolic alkalisation and [Ca²⁺]_cyt elevation in guard cell MeJA signalling, suggesting that MPK9 and MPK12 are key regulators mediating both ABA and MeJA signalling in guard cells.     

Characteristics of DNase activities in excretory/secretory products of infective larvae of Haemonchus contortus

While multiple DNase activities occur in the excretory/secretory products (ESPs) of the adult Haemonchus contortus, the DNase activities in ESPs of the infective larvae (L3) have not been studied. Thus, the DNase activities in ESPs of H. contortus L3 were investigated and compared to those of adults for developmental stage-specific analysis. The DNase activities had relative molecular masses (M rs) of 34 and 36 kDa upon zymographic analysis at pH 5.0 and 7.0 when the larvae were incubated for over 48 h. The 34 and 36 kDa DNases of L3 ESPs were also detected in adult ESPs with similar characteristics. However, the 37 and 38.5 kDa DNases of the adult ESPs were not detected in the L3 ESPs. Since the 37 and 38.5 kDa DNase activities were mainly detected in adult ESPs, these activities appear to be specific to the adult stage whereas the other ESP DNase activities appear to be expressed during multiple stages of the parasite's life cycle. While the difference in DNase activities of L3 and adults remains obscure, the role of DNase in larval development should be further clarified and the identification of stage-specific developmental markers will lead to the discovery of specific factors that stimulate larval development.     

Independent Relationship between Amyloid Precursor Protein (APP) Dimerization and γ-Secretase Processivity

Altered production of β-amyloid (Aβ) from the amyloid precursor protein (APP) is closely associated with Alzheimer’s disease (AD). APP has a number of homo- and hetero-dimerizing domains, and studies have suggested that dimerization of β-secretase derived APP carboxyl terminal fragment (CTFβ, C99) impairs processive cleavage by γ-secretase increasing production of long Aβs (e.g., Aβ1-42, 43). Other studies report that APP CTFβ dimers are not γ-secretase substrates. We revisited this issue due to observations made with an artificial APP mutant referred to as 3xK-APP, which contains three lysine residues at the border of the APP ectodomain and transmembrane domain (TMD). This mutant, which dramatically increases production of long Aβ, was found to form SDS-stable APP dimers, once again suggesting a mechanistic link between dimerization and increased production of long Aβ. To further evaluate how multimerization of substrate affects both initial γ-secretase cleavage and subsequent processivity, we generated recombinant wild type- (WT) and 3xK-C100 substrates, isolated monomeric, dimeric and trimeric forms of these proteins, and evaluated both ε-cleavage site utilization and Aβ production. These show that multimerization significantly impedes γ-secretase cleavage, irrespective of substrate sequence. Further, the monomeric form of the 3xK-C100 mutant increased long Aβ production without altering the initial ε-cleavage utilization. These data confirm and extend previous studies showing that dimeric substrates are not efficient γ-secretase substrates, and demonstrate that primary sequence determinants within APP substrate alter γ-secretase processivity.     

Characterization of HTT Inclusion Size,Location, and Timing in the zQ175 Mouse Model of Huntington′s Disease: An In Vivo High-Content Imaging Study

Huntington’s disease (HD) is an autosomal dominant neurodegenerative disorder caused by a CAG trinucleotide repeat expansion in the huntingtin gene. Major pathological hallmarks of HD include inclusions of mutant huntingtin (mHTT) protein, loss of neurons predominantly in the caudate nucleus, and atrophy of multiple brain regions. However, the early sequence of histological events that manifest in region- and cell-specific manner has not been well characterized. Here we use a high-content histological approach to precisely monitor changes in HTT expression and characterize deposition dynamics of mHTT protein inclusion bodies in the recently characterized zQ175 knock-in mouse line. We carried out an automated multi-parameter quantitative analysis of individual cortical and striatal cells in tissue slices from mice aged 2–12 months and confirmed biochemical reports of an age-associated increase in mHTT inclusions in this model. We also found distinct regional and subregional dynamics for inclusion number, size and distribution with subcellular resolution. We used viral-mediated suppression of total HTT in the striatum of zQ175 mice as an example of a therapeutically-relevant but heterogeneously transducing strategy to demonstrate successful application of this platform to quantitatively assess target engagement and outcome on a cellular basis.     

Phosphorylation is required for alteration of kv1.5 K(+) channel function by the Kvbeta1.3 subunit.

The Kv1.5 K(+) channel is functionally altered by coassembly with the Kvbeta1.3 subunit, which induces fast inactivation and a hyperpolarizing shift in the activation curve. Here we examine kinase regulation of Kv1.5/Kvbeta1.3 interaction after coexpression in human embryonic kidney 293 cells. The protein kinase C inhibitor calphostin C (3 microM) removed the fast inactivation (66 +/- 1.9 versus 11 +/- 0.25%, steady state/peak current) and the beta-induced hyperpolarizing voltage shift in the activation midpoint (V(1/2)) (-21.9 +/- 1.4 versus -4.3 +/- 2.0 mV). Calphostin C had no effect on Kv1.5 alone with respect to inactivation kinetics and V(1/2). Okadaic acid, but not the inactive derivative, blunted both calphostin C effects (V(1/2) = -17.6 +/- 2.2 mV, 38 +/- 1.8% inactivation), consistent with dephosphorylation being required for calphostin C action. Calphostin C also removed the fast inactivation (57 +/- 2.6 versus 16 +/- 0.6%) and the shift in V(1/2) (-22.1 +/- 1.4 versus -2.1 +/- 2.0 mV) conferred onto Kv1.5 by the Kvbeta1.2 subunit, which shares only C terminus sequence identity with Kvbeta1. 3. In contrast, modulation of Kv1.5 by the Kvbeta2.1 subunit was unaffected by calphostin C. These data suggest that Kvbeta1.2 and Kvbeta1.3 subunit modification of Kv1.5 inactivation and voltage sensitivity require phosphorylation by protein kinase C or a related kinase.     

Genome-Wide Analysis of Human Metapneumovirus Evolution

Human metapneumovirus (HMPV) has been described as an important etiologic agent of upper and lower respiratory tract infections, especially in young children and the elderly. Most of school-aged children might be introduced to HMPVs, and exacerbation with other viral or bacterial super-infection is common. However, our understanding of the molecular evolution of HMPVs remains limited. To address the comprehensive evolutionary dynamics of HMPVs, we report a genome-wide analysis of the eight genes (N, P, M, F, M2, SH, G, and L) using 103 complete genome sequences. Phylogenetic reconstruction revealed that the eight genes from one HMPV strain grouped into the same genetic group among the five distinct lineages (A1, A2a, A2b, B1, and B2). A few exceptions of phylogenetic incongruence might suggest past recombination events, and we detected possible recombination breakpoints in the F, SH, and G coding regions. The five genetic lineages of HMPVs shared quite remote common ancestors ranging more than 220 to 470 years of age with the most recent origins for the A2b sublineage. Purifying selection was common, but most protein genes except the F and M2-2 coding regions also appeared to experience episodic diversifying selection. Taken together, these suggest that the five lineages of HMPVs maintain their individual evolutionary dynamics and that recombination and selection forces might work on shaping the genetic diversity of HMPVs.     

Increase of yeast survival under oxidative stress by the expression of the laccase gene from Coprinellus congregatus

Coprinellus congregatus secreted a laccase isozyme when the culture was transferred to an acidic liquid medium (pH 4.1). The laccase cDNA gene (clac2) was used as a probe for cloning of the genomic laccase gene (lac2) including the promoter (Plac2). The open reading frame (ORF) of lac2 had 526 deduced amino acids and four conserved copper binding domains as other fungal laccases. Recombinant plasmid (pRSlac2p-cDNA) of lac2 cDNA with its own promoter was transformed in Saccharomyces cerevisiae. Expression of the transformed lac2 gene was induced by oxidative stress (H2O2) in yeast and the survival rate of the transformed yeast strain was greatly increased when compared with that of the control strain transformed with pRS316 yeast vector.     

Structural and biochemical analysis of mammalian methionine sulfoxide reductase B2

Methionine sulfoxide reductases are antioxidant enzymes that repair oxidatively damaged methionine residues in proteins. Mammals have three members of the methionine-R-sulfoxide reductase family, including cytosolic MsrB1, mitochondrial MsrB2, and endoplasmic reticulum MsrB3. Here, we report the solution structure of reduced Mus musculus MsrB2 using high resolution nuclear magnetic resonance (NMR) spectroscopy. MsrB2 is a β-strand rich globular protein consisting of eight antiparallel β-strands and three N-terminal α-helical segments. The latter secondary structure elements represent the main structural difference between mammalian MsrB2 and MsrB1. Structural comparison of mammalian and bacterial MsrB structures indicates that the general topology of this MsrB family is maintained and that MsrB2 more resembles bacterial MsrBs than MsrB1. Structural and biochemical analysis supports the catalytic mechanism of MsrB2 that, in contrast to MsrB1, does not involve a resolving cysteine (Cys). pH dependence of catalytically relevant residues in MsrB2 was accessed by NMR spectroscopy and the pK(a) of the catalytic Cys162 was determined to be 8.3. In addition, the pH-dependence of MsrB2 activity showed a maximum at pH 9.0, suggesting that deprotonation of the catalytic Cys is a critical step for the reaction. Further mobility analysis showed a well-structured N-terminal region, which contrasted with the high flexibility of this region in MsrB1. Our study highlights important structural and functional aspects of mammalian MsrB2 and provides a unifying picture for structure-function relationships within the MsrB protein family.     

SKI306X, an oriental herbal mixture, suppresses gastric leukotriene B4 synthesis without causing mucosal injury and the diclofenac-induced gastric lesions

SKI306X compound is a herbal mixture. This plant was in oriental medicine and was clinically approved for the treatment of osteoarthritis (OA) in Korea. SKI306X was previously found to have anti-inflammatory, analgesic and cartilage protective effects in several experimental models. In this study, SKI306X was investigated for its gastro-sparing effects on the gastric mucosa comparing with those of diclofenac, a conventional NSAID, and celecoxib, a cyclooxygenase-2 (COX-2) specific inhibitor. To investigate acute gastric damaging properties of SKI306X, the stomach of the animals was histologically and immuno-histochemically examined after single or repeated administration, and SKI306X demonstrated excellent gastric tolerability. SKI306X did not cause significant gastric irritation, erosion, or ulceration up to the orally administered dose of 2 g/kg and the intraperitoneal (i.p.) dose of 125 mg/kg. In contrast, diclofenac caused mucosal erosion, ulceration and bleeding at clinically effective doses. To determine the mode of gastro-sparing action, eicosanoid synthesis was examined in gastric mucosa and blood. SKI306X significantly decreased gastric and blood leukotriene B(4) (LTB(4)) production. However, SKI306X showed either no effect or a slight increase in levels of prostaglandin E(2) (PGE(2)). In addition, gastro-protective effects of SKI306X were exhibited by suppressing diclofenac-induced erosion and ulceration of gastric mucosa in a rat model and the possible mechanism of these effects were investigated. These studies demonstrated that SKI306X did not produce any significant damage up to dose of 2 g/kg and was effective in significantly protecting the damage associated to diclofenac-induced gastric ulcerations. SKI306X could spare the gastric mucosa through significantly suppressing gastric leukotriene (LT) synthesis.