Oxidative stress disrupts nitric oxide synthase activation in liver endothelial cells

Oxidative stress may mediate vascular disruption associated with a loss of endothelial nitric oxide synthase (eNOS) activity and a hypersensitivity to the constrictor effects of endothelin-1 (ET-1). We hypothesize that this is due, in part, to uncoupling of ET(B) receptors from eNOS activation. Thus, we tested whether oxidative stress (OS) affects liver vascular relaxation by reducing basal and ET-1-induced NO production. Primary sinusoidal endothelial cell cultures were pretreated with H(2)O(2) (25 microM) for 1 or 6 h before a 10-min ET-1 stimulation. OS resulted in a significant basal and ET-1-induced decrease in NO production. Acute OS increased the monomeric form of the inhibitory protein caveolin-1 (1.2 +/- 0.05 vs 0.9 +/- 0.02, p < 0.01) and increased the eNOS-caveolin association as determined by coimmunoprecipitation (1.24 +/- 0.04 vs 0.97 +/- 0.04, p < 0.05). ET-1 stimulation further exacerbated these effects. Subacute OS inhibited ET-1-induced eNOS phosphorylation of serine 1177 (activation residue) (1 +/- 0.07 vs 1.6 +/- 0.04, p < 0.05) and dephosphorylation of the inhibitory residue threonine 495 (1.5 +/- 0.08 vs 0.7 +/- 0.02, p < 0.01). Additionally subacute OS resulted in dissociation of eNOS from ET(B) (0.8 +/- 0.09 vs 1.2 +/- 0.06, p < 0.05). Our findings indicate that acute and subacute oxidative stress result in the inhibition of induced nitric oxide synthase activity through distinct mechanisms dependent on caveolin-1 inhibition, ET(B) dissociation, and eNOS phosphorylation.     

Involvement of IL17A,IL17F and IL23R Polymorphisms in Colorectal Cancer Therapy

IL23/IL17 pathway plays an important role in the development of inflammatory bowel diseases (IBD). In general, the genes encoding the cytokines are genetically polymorphic and polymorphisms in genes IL23R and IL17 have been proved to be associated with its susceptibility to inflammatory diseases as well as cancer including colorectal cancer. Moreover, it has been shown that these interleukins are involved in anti-tumor or pro-tumor effects of various cancers. Previously, we showed that there is a significant association between IL17A, IL17F and IL23R polymorphisms as well as the occurrence of colorectal cancer and the clinical features of the disease. The purpose of the present work is to investigate an association between IL17A, IL17F and IL23R polymorphisms in 102 Tunisian patients with colorectal cancer treatment. The association was analyzed by statistical tools. We found that patients with mutated genotypes of IL17A G197A SNP could be a risk factor for the inefficiency of chemotherapy and radiotherapy. Unlike IL17F variant, patients with wild type genotypes require surgery and adjuvant chemotherapy. On the one hand, we found no evidence that supports a significant association between IL23R polymorphism and the combined genotypes of these three genes and the colorectal cancer treatment. On the other hand, we showed that there is an important interaction between IL17A/IL17F polymorphisms and the stage of the disease as well as its treatment. Finally, patients with IL17F wild type genotype highlighted that there is a valid longer OS without all treatments and with radiotherapy and a neoadjuvant chemotherapy. In contrast, we observed that there are no relationships between IL17A, IL23R and the survival of these patients neither with nor without the treatment. Our results suggest that polymorphisms in IL17A and IL17F genes may be a predictive source of colorectal cancer therapy type. Therefore, IL17F may serve as an independent prognostic factor for overall survival in patients with colorectal cancer.     

Occurrence of Diadegma semiclausum,a parasitoid of diamondback moth in lowlands of Syria

The parasitoid, Diadegma semiclausum (Hymenoptera: Ichneumonidae) is one of the most effective parasitoids of diamondback moth (DBM) in the highlands (> 1600 m above sea level) of Asia. A Diadegma population from the lowland areas of Homs, Syria (about 203–487 m above sea level) was examined to determine if it differs at the species-level from the D. semiclausum and other Diadegma populations present in different countries using molecular diagnostic tools. Phylogenetic analysis based on the neighbor-joining method using the mitochondrial cytochrome oxidase I (COI) and the nuclear internal transcribed spacer 2 (ITS2) sequences grouped the Homs (Syria) Diadegma population with D. semiclausum populations from other countries. The results suggest that D. semiclausum occurs in the lowland conditions in Homs (Syria), where the temperature is higher. The Homs (Syria) strain did not show any variations in the parasitism when the parasitized host (DBM) larvae were exposed to varying temperatures for 24 h. It could not survive when the parasitized DBM larvae were continuously reared at 35 °C; however it inflicted significantly higher parasitism when the parasitized DBM larvae were reared at day and night temperatures of 35 °C and 20 °C, respectively. Preliminary results indicate that the D. semiclausum strain from Homs (Syria) possesses some level of heat tolerance, which could be exploited for successful management of DBM in the tropical lowlands.     

Universal stress proteins are important for oxidative and acid stress resistance and growth of Listeria monocytogenes EGD-e in vitro and in vivo

Background

Pathogenic bacteria maintain a multifaceted apparatus to resist damage caused by external stimuli. As part of this, the universal stress protein A (UspA) and its homologues, initially discovered in Escherichia coli K-12 were shown to possess an important role in stress resistance and growth in several bacterial species.

Methods and Findings

We conducted a study to assess the role of three homologous proteins containing the UspA domain in the facultative intracellular human pathogen Listeria monocytogenes under different stress conditions. The growth properties of three UspA deletion mutants (Δlmo0515, Δlmo1580 and Δlmo2673) were examined either following challenge with a sublethal concentration of hydrogen peroxide or under acidic conditions. We also examined their ability for intracellular survival within murine macrophages. Virulence and growth of usp mutants were further characterized in invertebrate and vertebrate infection models.Tolerance to acidic stress was clearly reduced in Δlmo1580 and Δlmo0515, while oxidative stress dramatically diminished growth in all mutants. Survival within macrophages was significantly decreased in Δlmo1580 and Δlmo2673 as compared to the wild-type strain. Viability of infected Galleria mellonella larvae was markedly higher when injected with Δlmo1580 or Δlmo2673 as compared to wild-type strain inoculation, indicating impaired virulence of bacteria lacking these usp genes. Finally, we observed severely restricted growth of all chromosomal deletion mutants in mice livers and spleens as compared to the load of wild-type bacteria following infection.

Conclusion

This work provides distinct evidence that universal stress proteins are strongly involved in listerial stress response and survival under both in vitro and in vivo growth conditions.     

Functional Characterization of the Plastidial 3-Phosphoglycerate Dehydrogenase Family in Arabidopsis

This work contributes to unraveling the role of the phosphorylated pathway of serine (Ser) biosynthesis in Arabidopsis (Arabidopsis thaliana) by functionally characterizing genes coding for the first enzyme of this pathway, 3-phosphoglycerate dehydrogenase (PGDH). We identified two Arabidopsis plastid-localized PGDH genes (3-PGDH and EMBRYO SAC DEVELOPMENT ARREST9 [EDA9]) with a high percentage of amino acid identity with a previously identified PGDH. All three genes displayed a different expression pattern indicating that they are not functionally redundant. pgdh and 3-pgdh mutants presented no drastic visual phenotypes, but eda9 displayed delayed embryo development, leading to aborted embryos that could be classified as early curled cotyledons. The embryo-lethal phenotype of eda9 was complemented with an EDA9 complementary DNA under the control of a 35S promoter (Pro-35S:EDA9). However, this construct, which is poorly expressed in the anther tapetum, did not complement mutant fertility. Microspore development in eda9.1eda9.1 Pro-35S:EDA9 was arrested at the polarized stage. Pollen from these lines lacked tryphine in the interstices of the exine layer, displayed shrunken and collapsed forms, and were unable to germinate when cultured in vitro. A metabolomic analysis of PGDH mutant and overexpressing plants revealed that all three PGDH family genes can regulate Ser homeostasis, with PGDH being quantitatively the most important in the process of Ser biosynthesis at the whole-plant level. By contrast, the essential role of EDA9 could be related to its expression in very specific cell types. We demonstrate the crucial role of EDA9 in embryo and pollen development, suggesting that the phosphorylated pathway of Ser biosynthesis is an important link connecting primary metabolism with development.Plant primary metabolism is a complex process where many interacting pathways must be finely coordinated and integrated in order to achieve proper plant development and acclimation to the environment. An example of such complexity is the biosynthesis of the amino acid l-Ser, which takes place in at least two different organelles and by different pathways. This amino acid is essential for the synthesis of proteins and other biomolecules needed for cell proliferation, including nucleotides and Ser-derived lipids, such as phosphatidylserine and sphingolipids. Additionally, d-Ser has been attributed a signaling function in male gametophyte-pistil communication (Michard et al., 2011).Despite the important role played by Ser in plants, the biological significance of the coexistence of several Ser biosynthetic pathways and how they interact to maintain amino acid homeostasis in cells is not yet understood. Three different Ser biosynthesis pathways have been described in plants (Kleczkowski and Givan, 1988; Ros et al., 2013; Fig. 1). One is the glycolate pathway, which takes place in mitochondria and is associated with photorespiration (Tolbert, 1980, 1997; Douce et al., 2001; Bauwe et al., 2010; Maurino and Peterhansel, 2010). In this pathway, two molecules of Gly are converted to one molecule of Ser in a reaction catalyzed by the Gly decarboxylase complex and Ser hydroxymethyltransferase (Fig. 1). Ser synthesis through the glycolate pathway is obtained in green tissues during daylight hours (Tolbert, 1980, 1985; Douce et al., 2001), suggesting that alternative Ser biosynthesis pathways may be required in the dark and/or in nonphotosynthetic organs. In this respect, Ser can be synthesized through two nonphotorespiratory pathways (Kleczkowski and Givan, 1988), the plastidial phosphorylated pathway (Ho et al., 1998, 1999a, 1999b; Ho and Saito, 2001) and the so-called glycerate pathway, which synthesizes Ser by the dephosphorylation of 3-phosphoglycerate (3-PGA; Kleczkowski and Givan, 1988; Fig. 1). This latter pathway includes the reverse sequence of the section of the oxidative photosynthetic carbon cycle linking 3-PGA to Ser (3-PGA-glycerate-hydroxypyruvate-Ser), these reactions being catalyzed by putative enzymes such as 3-PGA phosphatase, glycerate dehydrogenase, Ala-hydroxypyruvate aminotransferase, and Gly hydroxypyruvate aminotransferase. Although the existence of enzymatic activities of this pathway has been demonstrated (Kleczkowski and Givan, 1988), its functional significance is unknown and genes coding for the specific enzymes of the pathway have not been characterized to date.Open in a separate windowFigure 1.Schematic representation of Ser biosynthesis in plants. The enzymes participating in each Ser biosynthetic pathway are listed separately. Photorespiratory pathway (glycolate pathway): GDC, Gly decarboxylase; SHMT, Ser hydroxymethyltransferase. Glycerate pathway: PGAP, 3-phosphoglycerate phosphatase; GDH, glycerate dehydrogenase; AH-AT, Ala-hydroxypyruvate aminotransferase. Phosphorylated pathway: PSAT, 3-phosphoserine aminotransferase; PSP, 3-phosphoserine phosphatase. Abbreviations used for metabolites are as follows: 3-PHP, 3-phosphohydroxypyruvate; 3-PS, 3-phosphoserine; THF, tetrahydrofolate; 5,10-CH₂-THF, 5,10-methylene-tetrahydrofolate. This figure is adapted from Cascales-Miñana et al. (2013).The plastidial phosphorylated pathway of serine biosynthesis (PPSB; Fig. 1), which is conserved in mammals and plants, synthesizes Ser via 3-phosphoserine utilizing 3-PGA as a precursor (Kleczkowski and Givan, 1988). Evidence for the existence of this pathway in plants stems from the isolation and characterization of its enzyme activities (Handford and Davies, 1958; Slaughter and Davies, 1968; Larsson and Albertsson, 1979; Walton and Woolhouse, 1986). The PPSB involves three enzymes catalyzing sequential reactions: 3-phosphoglycerate dehydrogenase (PGDH), 3-phosphoserine aminotransferase, and 3-phosphoserine phosphatase (PSP; Fig. 1). Genes coding for some isoforms of these enzymes have been cloned and biochemically characterized in Arabidopsis (Arabidopsis thaliana; Ho et al., 1998, 1999a, 1999b; Ho and Saito, 2001).In humans, the PPSB plays a crucial role in cell proliferation control and oncogenesis (Bachelor et al., 2011; Locasale et al., 2011; Pollari et al., 2011; Possemato et al., 2011). The functional significance of the PPSB in plants has recently been unraveled by providing evidence for the crucial role of PSP1, the last enzyme of the pathway in embryo, pollen, and root development (Cascales-Miñana et al., 2013). However, the PPSB still requires further characterization. In order to gain a complete understanding of the PPSB function in plants, precise molecular, metabolic, and genetic knowledge of all the enzymes and genes of the pathway is needed. In this work, we follow a gain- and loss-of-function approach in Arabidopsis to characterize a family of genes coding for putative isoforms of PGDH, the first enzyme of the PPSB. Here, we identify the essential gene of this family and provide evidence for its crucial function in embryo and pollen development.     

Arabidopsis MSI1 connects LHP1 to PRC2 complexes

Polycomb group (PcG) proteins form essential epigenetic memory systems for controlling gene expression during development in plants and animals. However, the mechanism of plant PcG protein functions remains poorly understood. Here, we probed the composition and function of plant Polycomb repressive complex 2 (PRC2). This work established the fact that all known plant PRC2 complexes contain MSI1, a homologue of Drosophila p55. While p55 is not essential for the in vitro enzymatic activity of PRC2, plant MSI1 was required for the functions of the EMBRYONIC FLOWER and the VERNALIZATION PRC2 complexes including trimethylation of histone H3 Lys27 (H3K27) at the target chromatin, as well as gene repression and establishment of competence to flower. We found that MSI1 serves to link PRC2 to LIKE HETEROCHROMATIN PROTEIN 1 (LHP1), a protein that binds H3K27me3 in vitro and in vivo and is required for a functional plant PcG system. The LHP1–MSI1 interaction forms a positive feedback loop to recruit PRC2 to chromatin that carries H3K27me3. Consequently, this can provide a mechanism for the faithful inheritance of local epigenetic information through replication.