Involvement of autocrine CXCL12/CXCR4 system in the regulation of ovarian carcinoma cell invasion

Ovarian carcinomas are often highly invasive, especially in the peritoneal cavity; however, the mechanism involved in invasion is not yet fully understood. In the present research, we studied the role of NF-κB in the invasiveness of ovarian carcinoma cells by using (−)-DHMEQ, a specific inhibitor of NF-κB. (−)-DHMEQ inhibited invasion in vitro and the expression of CXCL12 and CXCR4. We found that neutralizing antibody against CXCR4 or knockdown of CXCR4 suppressed the invasion. Proteomic analysis revealed that CXCR4-siRNA treatment lowered the secretion of several invasion-related proteins, such as MMP-9 and uPA. These data imply that (−)-DHMEQ suppressed ovarian cell invasion via inhibition of the NF-κB-regulated autocrine system of CXCL12-CXCR4.     

Complete Nucleotide Sequences of Virulence-Resistance Plasmids Carried by Emerging Multidrug-Resistant Salmonella enterica Serovar Typhimurium Isolated from Cattle in Hokkaido,Japan

In the present study, we have shown that virulence-resistance plasmids from emerging multidrug-resistant isolates of Salmonella enterica serovar Typhimurium were derived from a virulence-associated plasmid, essential for systematic invasiveness of S. Typhimurium in mice (pSLT), through acquisition of a large insert containing a resistance island flanked by IS1294 elements. A bla _CMY-2-carrying plasmid from a cefotaxime-resistant isolate comprised a segment of Escherichia coli plasmid pAR060302 and the replication region (IncFIB) of a virulence-resistance plasmid. These results provide insights into the evolution of drug resistance in emerging clones of S. Typhimurium.     

Heterotrimeric G Proteins Serve as a Converging Point in Plant Defense Signaling Activated by Multiple Receptor-Like Kinases

In fungi and metazoans, extracellular signals are often perceived by G-protein-coupled receptors (GPCRs) and transduced through heterotrimeric G-protein complexes to downstream targets. Plant heterotrimeric G proteins are also involved in diverse biological processes, but little is known about their upstream receptors. Moreover, the presence of bona fide GPCRs in plants is yet to be established. In Arabidopsis (Arabidopsis thaliana), heterotrimeric G protein consists of one Gα subunit (G PROTEIN α-SUBUNIT1), one Gβ subunit (ARABIDOPSIS G PROTEIN β-SUBUNIT1 [AGB1]), and three Gγs subunits (ARABIDOPSIS G PROTEIN γ-SUBUNIT1 [AGG1], AGG2, and AGG3). We identified AGB1 from a suppressor screen of BAK1-interacting receptor-like kinase1-1 (bir1-1), a mutant that activates cell death and defense responses mediated by the receptor-like kinase (RLK) SUPPRESSOR OF BIR1-1. Mutations in AGB1 suppress the cell death and defense responses in bir1-1 and transgenic plants overexpressing SUPPRESSOR OF BIR1-1. In addition, agb1 mutant plants were severely compromised in immunity mediated by three other RLKs, FLAGELLIN-SENSITIVE2 (FLS2), Elongation Factor-TU RECEPTOR (EFR), and CHITIN ELICITOR RECEPTOR KINASE1 (CERK1), respectively. By contrast, G PROTEIN α-SUBUNIT1 is not required for either cell death in bir1-1 or pathogen-associated molecular pattern-triggered immunity mediated by FLS2, EFR, and CERK1. Further analysis of agg1 and agg2 mutant plants indicates that AGG1 and AGG2 are also required for pathogen-associated molecular pattern-triggered immune responses mediated by FLS2, EFR, and CERK1, as well as cell death and defense responses in bir1-1. We hypothesize that the Arabidopsis heterotrimeric G proteins function as a converging point of plant defense signaling by mediating responses initiated by multiple RLKs, which may fulfill equivalent roles to GPCRs in fungi and animals.Receptor-like kinases (RLKs) represent one of the largest protein families in plants and play diverse roles in plant development and stress signaling (Morillo and Tax, 2006). In Arabidopsis (Arabidopsis thaliana), there are over 600 RLKs (Shiu and Bleecker, 2001). Most RLKs contain an extracellular domain, a single transmembrane motif, and a cytoplasmic kinase domain. It is believed that the extracellular domains are involved in ligand recognition that subsequently leads to activation of the cytoplasmic kinase domain. Pathogen-associated molecular pattern (PAMP) receptors FLAGELLIN-SENSITIVE2 (FLS2), Elongation Factor (EF)-TU RECEPTOR (EFR), and CHITIN ELICITOR RECEPTOR KINASE1 (CERK1) all belong to the RLK family. FLS2 and EFR function as receptors for bacterial flagellin and EF-Tu, respectively (Gómez-Gómez and Boller, 2000; Zipfel et al., 2006), while CERK1 is involved in the perception of chitin, a common component of the fungal cell wall (Miya et al., 2007; Wan et al., 2008). CERK1 was also shown to play an important role in defense against bacterial pathogens (Gimenez-Ibanez et al., 2009). Another RLK, Brassinosteroid Insensitive1-associated receptor kinase1 (BAK1), functions as a coreceptor for FLS2 and EFR (Chinchilla et al., 2007; Heese et al., 2007). In addition, a rice (Oryza sativa) RLK, Xa21, functions as the receptor for a peptide derived from AvrXa21 (Song et al., 1995; Lee et al., 2009).Activation of different PAMP receptors often leads to rapid downstream responses, such as oxidative burst, calcium influx, mitogen-activated protein kinase (MAPK) activations, and the up-regulation of defense gene expression (Boller and Felix, 2009). However, our knowledge of how defense responses are regulated downstream of the RLKs remains limited. Genetic analysis of mutants defective in EFR-mediated PAMP responses showed that endoplasmic reticulum (ER) quality control plays an important role in the accumulation of EFR (Li et al., 2009; Lu et al., 2009; Nekrasov et al., 2009; Saijo et al., 2009). ER-resident chaperones are also required for the accumulation of the tobacco (Nicotiana tabacum) INDUCED RECEPTOR-LIKE KINASE (Caplan et al., 2009). BOTRYTIS-INDUCED KINASE1 (BIK1) encodes a cytoplasmic RLK that directly interacts with FLS2 and likely EFR and CERK1 (Lu et al., 2010; Zhang et al., 2010). Knocking out BIK1 leads to modest reductions of PAMP-induced callose deposition, H₂O₂ accumulation, and pathogen resistance. Recently, a group of redundant calcium-dependent protein kinases (CDPKs) were identified as critical regulators of MAPK-independent defense pathways downstream of FLS2 (Boudsocq et al., 2010).In yeast (Saccharomyces cerevisiae) and metazoans, heterotrimeric G proteins, composed of α-, β-, and γ-subunits, serve as essential signaling intermediates between cell surface G-protein-coupled receptors (GPCRs) and their downstream targets (Temple and Jones, 2007). Binding of ligands to GPCRs leads to the exchange of GDP for GTP in the α-subunit, resulting in the activation of the G protein. In Arabidopsis, there is one Gα subunit (G PROTEIN α-SUBUNIT1 [GPA1]), one Gβ subunit (ARABIDOPSIS G PROTEIN β-SUBUNIT1 [AGB1]), and three Gγs subunits (ARABIDOPSIS G PROTEIN γ-SUBUNIT1 [AGG1], AGG2, and AGG3; Temple and Jones, 2007; Chakravorty et al., 2011). Whereas AGG1 and AGG2 are closely related, AGG3 only shares very limited homology to AGG1 and AGG2. GPCR-like proteins have been identified in plants, and their interactions with Gα have been experimentally proven, although their status as bona fide GPCRs remains controversial (Liu et al., 2007; Gookin et al., 2008; Pandey et al., 2009).Heterotrimeric G proteins have been shown to be involved in a wide range of biological processes, including plant immunity (Perfus-Barbeoch et al., 2004). Analysis of rice dwarf1 mutants with defects in the Gα subunit indicates that the rice Gα subunit plays an important role in resistance against rice blast (Suharsono et al., 2002). In Arabidopsis, loss of function of the Gβ or Gγ subunits leads to compromised resistance against the necrotrophic pathogen Plectosphaerella cucumerina (Llorente et al., 2005; Delgado-Cerezo et al., 2012). By contrast, loss of function of the Gα subunit GPA1 results in enhanced resistance against the pathogen (Llorente et al., 2005). Resistance against other necrotrophic pathogens, such as Fusarium oxysporum, Alternaria brassicicola, and Botrytis cinerea, was also found to be compromised in AGB1- and AGG1/AGG2-deficient mutants (Trusov et al., 2006, 2007, 2009). In addition, loss of function of the Gβ subunit leads to reduced elf18-induced resistance against Agrobacterium tumefaciens as well as reduced reactive oxygen species (ROS) production triggered by flagellin22 (flg22) and elf18 (an 18-amino acid peptide that represents the N terminus of bacterial EF-Tu; Ishikawa, 2009). Multiple surface residues of AGB1 were recently shown to play important roles in resistance against necrotrophic pathogens and flg22-induced ROS production (Jiang et al., 2012).Arabidopsis GPA1 was also found to play an important role in stomatal defense. In gpa1 mutants, flg22-induced inhibition of stomatal opening and inward K⁺ channels in guard cells are blocked (Zhang et al., 2008b). Growth of the coronatine-deficient Pseudomonas syringae pv tomato DC3118 is dramatically increased in gpa1 mutant plants (Zeng and He, 2010). In Nicotiana benthamiana, silencing of the Gα and Gβ subunits also leads to reduced elicitor-induced stomatal closure as well as hypersensitive responses induced by harpin (Zhang et al., 2012a).Arabidopsis BAK1-INTERACTING RECEPTOR-LIKE KINASE1 (BIR1) encodes a BAK1-associated RLK (Gao et al., 2009). Knocking out BIR1 results in constitutive activation of cell death and defense responses in a manner that is partially dependent on PHYTOALEXIN DEFICIENT4 (PAD4), a positive regulator of resistance mediated by the Toll-Interleukin-1 Receptor-like-Nucleotide-Binding-Leu-Rich Repeat domain class of resistance proteins. To identify signaling components downstream of BIR1, a suppressor screen was performed in the bir1-1 pad4-1 double-mutant background. A number of suppressor of bir1-1 (sobir) mutants suppressing the seedling lethality phenotype of bir1-1 were identified. SOBIR1 encodes another RLK whose overexpression is sufficient to activate cell death and defense responses (Gao et al., 2009). Combining the sobir1-1 and pad4-1 mutations results in complete suppression of cell death and enhanced pathogen resistance in bir1-1, suggesting that SOBIR1 and PAD4 function in parallel to regulate cell death and defense responses. Here, we report the discovery and characterization of SOBIR2, which encodes the Arabidopsis heterotrimeric G-protein β-subunit AGB1 that functions downstream of SOBIR1 to regulate cell death and defense responses.     

PLEIOTROPIC REGULATORY LOCUS 2 exhibits unequal genetic redundancy with its homolog PRL1

In plants, signaling leading to resistance against biotrophic pathogens is complex. Perception of pathogenic microbes by resistance (R) proteins is relayed though successive activities of downstream components, in a network that is not well understood. PLEIOTROPIC REGULATORY LOCUS 1 (PRL1) and >20 other proteins are members of the MOS4-associated complex (MAC), a regulatory node in defense signaling. Of all characterized MAC members, mutations in PRL1 cause the most severe susceptibility towards both virulent and avirulent microbial pathogens. Genetic suppressors of prl1 represent new signaling elements and may aid in further unraveling of defense mechanisms. Our identification and characterization of a dominant suppressor of prl1 revealed a regulatory, gain-of-function mutation in PLEIOTROPIC REGULATORY LOCUS 2 (PRL2), a close homolog of PRL1. Loss-of-function mutants of PRL2 do not exhibit altered phenotypes; however, prl1 prl2 double mutants exhibit enhanced morphological defects consistent with unequal genetic redundancy between the homologs. Up-regulated gene expression mediated by the dominant prl2-1D allele completely suppresses disease susceptibility in the prl1 mutant background and also restores wild-type appearance, further supporting functional equivalence between the two PRL proteins.     

The Arabidopsis TAC Position Viewer: a high‐resolution map of transformation‐competent artificial chromosome (TAC) clones aligned with the Arabidopsis thaliana Columbia‐0 genome

We present a high‐resolution map of genomic transformation‐competent artificial chromosome (TAC) clones extending over all Arabidopsis thaliana (Arabidopsis) chromosomes. The Arabidopsis genomic TAC clones have been valuable genetic tools. Previously, we constructed an Arabidopsis genomic TAC library consisting of more than 10 000 TAC clones harboring large genomic DNA fragments extending over the whole Arabidopsis genome. Here, we determined 13 577 end sequences from 6987 Arabidopsis TAC clones and mapped 5937 TAC clones to precise locations, covering approximately 90% of the Arabidopsis chromosomes. We present the large‐scale data set of TAC clones with high‐resolution mapping information as a Java application tool, the Arabidopsis TAC Position Viewer, which provides ready‐to‐go transformable genomic DNA clones corresponding to certain loci on Arabidopsis chromosomes. The TAC clone resources will accelerate genomic DNA cloning, positional walking, complementation of mutants and DNA transformation for heterologous gene expression.     

The carboxyl-terminal region of the geranylgeranyl diphosphate synthase is indispensable for the stabilization of the region involved in substrate binding and catalysis

Rat geranylgeranyl diphosphate synthase (GGPS) and its deletion mutants from the carboxyl terminus were analysed using Escherichia coli harbouring pACYC-crtIB, which contains crtI and crtB encoding the carotenoid biosynthetic enzymes. Mutants (delta-4, -8, -12 and -16) produced lycopene-derived red colour, but mutants (delta-17, -18, -19, -20, -23, -57 and -70) did not. The histidine-tagged mutants (delta-4, -8, -12 and -16) were overexpressed in E. coli BL21 (DE3) and purified in a stable form by nickel affinity chromatography except for one mutant (delta-16). The farnesyl-transferring activities of wild-type GGPS, delta-4, -8 and -12 mutants were relatively in a ratio of 1.0, 0.84, 0.26 and 0.0015. Each Km value of the four recombinants were estimated to be 0.71, 2.0 2.8 and 55 microM for farnesyl diphosphate and to be 2.9, 5.1, 56 and >100 microM for isopentenyl diphosphate, respectively. Allylic substrate specificities of these recombinants were estimated by quantitative analysis of the products, revealing that delta-8 and -12 mutants lack the ability to accept dimethylallyl and geranyl diphosphates compared to wild-type GGPS and delta-4 mutant. These results suggest that the KMFTEENE residing on the carboxyl-terminal sequence of GGPS stabilizes the active region involved in the substrate binding and catalysis.     

Preferential Accumulation of 14C-N-Glycolylneuraminic Acid over 14C-N-Acetylneuraminic Acid in the Rat Brain after Tail Vein Injection

The two main molecular species of sialic acid existing in nature are N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc). Neu5Ac is abundant in mammalian brains and plays crucial roles in many neural functions. In contrast, Neu5Gc is present only at a trace level in vertebrate brains. The brain-specific suppression of Neu5Gc synthesis, which is a common feature in mammals, suggests that Neu5Gc has toxicity against brain functions. However, in vivo kinetics of Neu5Gc in the whole body, especially in the brain, has not been studied in sufficient detail. To determine the in vivo kinetics of Neu5Gc, ¹⁴C-Neu5Gc was enzymatically synthesized and injected into rat tail veins. Although most of ¹⁴C-Neu5Gc was excreted in urine, a small amount of ¹⁴C-Neu5Gc was detected in the brain. Brain autoradiography indicated that ¹⁴C-Neu5Gc was accumulated predominantly in the hippocampus. ¹⁴C-Neu5Gc transferred into the brain was incorporated into gangliosides including GM1, GD1a, GD1b, GT1b and GQ1b. Reduction of ¹⁴C-Neu5Gc after intracerebroventricular infusion was slower than that of ¹⁴C-Neu5Ac in the brain and hippocampus. The results suggest that Neu5Gc is transferred from blood into the brain across the blood brain barrier and accumulates in the brain more preferentially than does Neu5Ac.     

Terrestrial influences on the Shiraho fringing reef, Ishigaki Island, Japan: high carbon input relative to phosphate

Measurements of CO₂ system parameters and nutrients showed the potential influence of terrestrial inputs on the carbon budget of a fringing coral reef on Ishigaki Island, Japan. Land-derived freshwaters, including river and ground water, make a relatively large contribution (0.5–1.2% by volume) to the water circulation of the reef. These terrestrial waters exhibit extremely high fugacity of CO₂ (fCO₂ up to 6400 μatm), reflecting enrichments in total alkalinity and dissolved inorganic carbon. Since the dissolved inorganic C:P ratio is anomalously high in terrestrial waters compared with the mean C:P ratios of primary production, excess carbon could be released into the atmosphere. Terrestrial inputs deliver a large quantity of C relative to P. The study site was a source of CO₂ at the time of measurement. Such a situation is likely to prevail all year round, and it may occur elsewhere. Accepted: 20 February 2000     

Effect of food residues on norovirus survival on stainless steel surfaces

Background

In households and food processing plants, minute food residues left behind from improper cleaning may influence the survivability of human norovirus on surfaces. In this study, the survivability of norovirus on desiccated food residue-attached stainless steel coupons was investigated.

Methodology/Principal Findings

Using murine norovirus-1 (MNV-1) as a surrogate of human norovirus, the survivability of norovirus was investigated on lettuce, cabbage, or ground pork-attached stainless steel coupons. A 6.2 log MPN/ml of MNV-1 infectivity was completely lost at day 30 in residue-free coupons, whereas only a 1.4 log MPN/ml reduction was observed in coupons with residues. Moreover, the disinfective effect of sodium hypochlorite was reduced when residues were present on the coupons.

Conclusions/Significance

This study revealed that the food residues increased the survivability and the resistance to chemicals of norovirus, indicating the need of thorough cleaning in food processing plants and household settings.     

IL-27 Enhances the Expression of TRAIL and TLR3 in Human Melanomas and Inhibits Their Tumor Growth in Cooperation with a TLR3 Agonist Poly(I:C) Partly in a TRAIL-Dependent Manner

Interleukin (IL)-27 is a member of the IL-6/IL-12 cytokine family and possesses potent antitumor activity, which is mediated by multiple mechanisms. Toll-like receptor (TLR)3 is the critical sensor of the innate immune system that serves to identify viral double-stranded RNA. TLR3 is frequently expressed by various types of malignant cells, and recent studies reported that a synthetic TLR3 agonist, polyinosinic-polycytidylic acid [poly(I:C)], induces antitumor effects on malignant cells. In the present study, we have explored the effect of IL-27 on human melanomas and uncovered a previously unknown mechanism. We found that IL-27 inhibits in vitro tumor growth of human melanomas and greatly enhances the expression of TNF-related apoptosis inducing ligand (TRAIL) in a dose-dependent manner. Neutralizing antibody against TRAIL partly but significantly blocked the IL-27–mediated inhibition of tumor growth. In addition, IL-27 and poly(I:C) cooperatively augmented TRAIL expression and inhibited tumor growth. The cooperative effect could be ascribed to the augmented expression of TLR3, but not retinoic acid-inducible gene-I or anti-melanoma differentiation-associated gene 5, by IL-27. The inhibition of tumor growth by the combination was also significantly abrogated by anti-TRAIL neutralizing antibody. Moreover, IL-27 and poly(I:C) cooperatively suppressed in vivo tumor growth of human melanoma in immunodeficient mice. Taken together, these results suggest that IL-27 enhances the expression of TRAIL and TLR3 in human melanomas and inhibits their tumor growth in cooperation with poly(I:C), partly in a TRAIL-dependent manner. Thus, IL-27 and the combination of IL-27 and poly(I:C) may be attractive candidates for cancer immunotherapy.