Salt tolerance in <Emphasis Type="Italic">Solanum pennellii</Emphasis>: antioxidant response and related QTL

Background  

Excessive soil salinity is an important problem for agriculture, however, salt tolerance is a complex trait that is not easily bred into plants. Exposure of cultivated tomato to salt stress has been reported to result in increased antioxidant content and activity. Salt tolerance of the related wild species, Solanum pennellii, has also been associated with similar changes in antioxidants. In this work, S. lycopersicum M82, S. pennellii LA716 and a S. pennellii introgression line (IL) population were evaluated for growth and their levels of antioxidant activity (total water-soluble antioxidant activity), major antioxidant compounds (phenolic and flavonoid contents) and antioxidant enzyme activities (superoxide dismutase, catalase, ascorbate peroxidase and peroxidase) under both control and salt stress (150 mM NaCl) conditions. These data were then used to identify quantitative trait loci (QTL) responsible for controlling the antioxidant parameters under both stress and nonstress conditions.     

Antioxidative response patterns of Norway spruce bark to low-density Ceratocystis polonica inoculation

Key message

Based on time courses of individual antioxidant compounds, bark phenolic metabolism has been recognised to integrate ascorbate–glutathione system as a redox hub in Norway spruce defence against Ceratocystis polonica infection.

Abstract

Temporal courses of individual phenolics, thiols and ascorbate were studied in Norway spruce phloem over a 5-month period after inoculation at low density with Ceratocystis polonica. The initial reaction of Norway spruce 3 days after inoculation was characterised by significantly increased isorhapontin and taxifolin concentrations, accompanied by significantly lowered catechin contents. On later sampling dates, catechin concentrations within infected bark increased until September. The slightly accumulated astringin contents in April and May diminished at later sampling dates in response to infection. The isorhapontin levels strongly raised in April and were slightly lowered from June onwards. Compared to the controls, taxifolin concentrations were higher in the infected samples showing a double peak with maxima in April and June. The taxifolin values eased later but remained above the control levels. The initial response of the ascorbate–glutathione system to fungal infection was characterised by a significantly more oxidised glutathione pool, slightly more reduced ascorbate system and by higher glutathione reductase activity. Three weeks later an accumulation of thiols was observed, whereas total ascorbate was significantly lowered and the ascorbate redox state shifted towards more oxidised values. Until the middle of July a gradual increase of total glutathione was determined within the infected bark, which was accompanied by significantly increased cysteine contents, higher glutathione reductase activity, but significantly lowered total ascorbate contents. The increased pressure on the ascorbate system reflects its interaction with phenolics, as ascorbate is needed for reducing the phenoxyl radicals formed during pathogen defence.     

Receptor-mediated enhancement of beta adrenergic drug activity by ascorbate in vitro and in vivo

Rationale

Previous in vitro research demonstrated that ascorbate enhances potency and duration of activity of agonists binding to alpha 1 adrenergic and histamine receptors.

Objectives

Extending this work to beta 2 adrenergic systems in vitro and in vivo.

Methods

Ultraviolet spectroscopy was used to study ascorbate binding to adrenergic receptor preparations and peptides. Force transduction studies on acetylcholine-contracted trachealis preparations from pigs and guinea pigs measured the effect of ascorbate on relaxation due to submaximal doses of beta adrenergic agonists. The effect of inhaled albuterol with and without ascorbate was tested on horses with heaves and sheep with carbachol-induced bronchoconstriction.

Measurements

Binding constants for ascorbate binding to beta adrenergic receptor were derived from concentration-dependent spectral shifts. Dose- dependence curves were obtained for the relaxation of pre-contracted trachealis preparations due to beta agonists in the presence and absence of varied ascorbate. Tachyphylaxis and fade were also measured. Dose response curves were determined for the effect of albuterol plus-and-minus ascorbate on airway resistance in horses and sheep.

Main Results

Ascorbate binds to the beta 2 adrenergic receptor at physiological concentrations. The receptor recycles dehydroascorbate. Physiological and supra-physiological concentrations of ascorbate enhance submaximal epinephrine and isoproterenol relaxation of trachealis, producing a 3–10-fold increase in sensitivity, preventing tachyphylaxis, and reversing fade. In vivo, ascorbate improves albuterol''s effect on heaves and produces a 10-fold enhancement of albuterol activity in “asthmatic” sheep.

Conclusions

Ascorbate enhances beta-adrenergic activity via a novel receptor-mediated mechanism; increases potency and duration of beta adrenergic agonists effective in asthma and COPD; prevents tachyphylaxis; and reverses fade. These novel effects are probably caused by a novel mechanism involving phosphorylation of aminergic receptors and have clinical and drug-development applications.     

Sinapinic acid can replace ascorbate in the biotin switch assay

Background

Protein S-nitrosation is an important post-translational modification altering protein function. Interaction of nitric oxide with thiols is an active area of research, and is one of the mechanisms by which NO exerts its biological effects. Biotin switch assay is the method, which has been developed to identify S-nitrosated proteins. The major concern with biotin switch assay includes reducing disulfide which may lead to false positives. We report a modification of the biotin switch assay where sinapinic acid is utilized instead of ascorbate to eliminate potential artifacts in the detection of S-nitrosated proteins.

Methods

The denitrosation ability of sinapinic acid was assessed by monitoring either the NO or NO₂^- released by chemiluminescent NO detection or by the griess assay, respectively. DTNB assay was used to compare disulfide reduction by ascorbate and sinapinic acid. Sinapinic acid and ascorbate were compared in the biotin switch detection of S-nitrosoproteins in RAW 264.7 cells ± S-nitrosocysteine (CysNO) exposure.

Results

We show that sinapinic acid has the ability to denitrosate S-nitrosothiols at pH 7.0 and denitrate plus denitrosate at pHs 8 and 8.5. Unlike ascorbate, sinapinic acid degrades S-nitrosothiols, but it does not reduce disulfide bridges.

Conclusions

Sinapinic acid denitrosate RSNO and does not reduce disulfides. Thus can readily replace ascorbate in detection of S-nitrosated proteins in biotin switch assay.

General significance

The work described is important in view of protein S-nitrosation. In this study we provide an important modification that eliminates artifacts in widely used technique for detecting the S-nitrosoproteome, the biotin switch assay.     

Ascorbic acid attenuates endothelial permeability triggered by cell-free hemoglobin

Background

Increased endothelial permeability is central to shock and organ dysfunction in sepsis but therapeutics targeted to known mediators of increased endothelial permeability have been unsuccessful in patient studies. We previously reported that cell-free hemoglobin (CFH) is elevated in the majority of patients with sepsis and is associated with organ dysfunction, poor clinical outcomes and elevated markers of oxidant injury. Others have shown that Vitamin C (ascorbate) may have endothelial protective effects in sepsis. In this study, we tested the hypothesis that high levels of CFH, as seen in the circulation of patients with sepsis, disrupt endothelial barrier integrity.

The evolution of TEP1, an exceptionally polymorphic immunity gene in <Emphasis Type="Italic">Anopheles gambiae</Emphasis>

Background  

Host-parasite coevolution can result in balancing selection, which maintains genetic variation in the susceptibility of hosts to parasites. It has been suggested that variation in a thioester-containing protein called TEP1 (AGAP010815) may alter the ability of Anopheles mosquitoes to transmit Plasmodium parasites, and high divergence between alleles of this gene suggests the possible action of long-term balancing selection. We studied whether TEP1 is a case of an ancient balanced polymorphism in an animal immune system.     

The twisted pharynx phenotype in <Emphasis Type="Italic">C. elegans</Emphasis>

Background  

The pharynx of C. elegans is an epithelial tube whose development has been compared to that of the embryonic heart and the kidney and hence serves as an interesting model for organ development. Several C. elegans mutants have been reported to exhibit a twisted pharynx phenotype but no careful studies have been made to directly address this phenomenon. In this study, the twisting mutants dig-1, mig-4, mnm-4 and unc-61 are examined in detail and the nature of the twist is investigated.     

Functionality of the GAL4/UAS system in <Emphasis Type="Italic">Tribolium</Emphasis> requires the use of endogenous core promoters

Background  

The red flour beetle Tribolium castaneum has developed into an insect model system second only to Drosophila. Moreover, as a coleopteran it represents the most species-rich metazoan taxon which also includes many pest species. The genetic toolbox for Tribolium research has expanded in the past years but spatio-temporally controlled misexpression of genes has not been possible so far.     

A kinetic study of the coupled iron-ceruloplasmin catalyzed oxidation of ascorbate in the presence of albumin

Ascorbate is catalytically oxidized by a coupled iron-ceruloplasmin system, the iron ions functioning as a red/ox cycling intermediate between ceruloplasmin and ascorbate. Serum albumin, an iron binding compound, was found to stimulate the ascorbate oxidation rate. It is proposed that ferrous ions react more rapidly with ceruloplasmin when they are bound to albumin. A K _m value of 39 m was estimated for Fe²⁺-albumin. Citrate and urate inhibit the iron-ceruloplasmin-dependent ascorbate oxidation by chelating ferric ions. In the presence of albumin only citrate reduced the oxidation rate, the observation suggesting the following order of iron binding ability: citrate > albumin > urate. Physiological aspects of the results have been discussed.     

Polyploid genome of <Emphasis Type="Italic">Camelina sativa</Emphasis>revealed by isolation of fatty acid synthesis genes

Background  

Camelina sativa, an oilseed crop in the Brassicaceae family, has inspired renewed interest due to its potential for biofuels applications. Little is understood of the nature of the C. sativa genome, however. A study was undertaken to characterize two genes in the fatty acid biosynthesis pathway, fatty acid desaturase (FAD) 2 and fatty acid elongase (FAE) 1, which revealed unexpected complexity in the C. sativa genome.     

Regions important for the adhesin activity of <Emphasis Type="Italic">Moraxella catarrhalis</Emphasis> Hag

Background  

The Moraxella catarrhalis Hag protein, an Oca autotransporter adhesin, has previously been shown to be important for adherence of this respiratory tract pathogen to human middle ear and A549 lung cells.     

<Emphasis Type="Italic">Brucella melitensis</Emphasis> VjbR and C<Subscript>12</Subscript>-HSL regulons: contributions of the <Emphasis Type="Italic">N</Emphasis>-dodecanoyl homoserine lactone signaling molecule and LuxR homologue VjbR to gene expression

Background  

Quorum sensing is a communication system that regulates gene expression in response to population density and often regulates virulence determinants. Deletion of the luxR homologue vjbR highly attenuates intracellular survival of Brucella melitensis and has been interpreted to be an indication of a role for QS in Brucella infection. Confirmation for such a role was suggested, but not confirmed, by the demonstrated in vitro synthesis of an auto-inducer (AI) by Brucella cultures. In an effort to further delineate the role of VjbR to virulence and survival, gene expression under the control of VjbR and AI was characterized using microarray analysis.     

Role of type 1 and type 3 fimbriae in <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> biofilm formation

Background  

Klebsiella pneumoniae is an important gram-negative opportunistic pathogen causing primarily urinary tract infections, respiratory infections, and bacteraemia. The ability of bacteria to form biofilms on medical devices, e.g. catheters, has a major role in development of many nosocomial infections. Most clinical K. pneumoniae isolates express two types of fimbrial adhesins, type 1 fimbriae and type 3 fimbriae. In this study, we characterized the role of type 1 and type 3 fimbriae in K. pneumoniae biofilm formation.     

An integrative expression vector for <Emphasis Type="Italic">Actinosynnema pretiosum</Emphasis>

Background  

The Actinomycete Actinosynnema pretiosum ssp. auranticum has commercial importance due to its production of ansamitocin P-3 (AP-3), a potent antitumor agent. One way to increase AP-3 production would be to constitutively express selected genes so as to relieve bottlenecks in the biosynthetic pathway; however, an integrative expression vector for A. pretiosum is lacking. The aim of this study was to construct a vector for heterologous gene expression in A. pretiosum.     

Immunoreactivity of the <Emphasis Type="Italic">Mycobacterium avium</Emphasis> subsp. <Emphasis Type="Italic">paratuberculosis</Emphasis> 19-kDa lipoprotein

Background  

The Mycobacterium tuberculosis 19-kDa lipoprotein has been reported to stimulate both T and B cell responses as well as induce a number of Th1 cytokines. In order to evaluate the Mycobacterium avium subsp. paratuberculosis (M. avium subsp. paratuberculosis) 19-kDa lipoprotein as an immunomodulator in cattle with Johne's disease, the gene encoding the 19-kDa protein (MAP0261c) was analyzed.     

Parasexual genetics of <Emphasis Type="Italic">Dictyostelium</Emphasis> gene disruptions: identification of a ras pathway using diploids

Background  

The relative ease of targeted gene disruption in the social amoeba Dictyostelium has stimulated its widespread use as an experimental organism for cell and developmental biology. However, the field has been hamstrung by the lack of techniques to recombine disrupted genes.     

Identification and characterization of NAGNAG alternative splicing in the moss <Emphasis Type="Italic">Physcomitrella patens</Emphasis>

Background  

Alternative splicing (AS) involving tandem acceptors that are separated by three nucleotides (NAGNAG) is an evolutionarily widespread class of AS, which is well studied in Homo sapiens (human) and Mus musculus (mouse). It has also been shown to be common in the model seed plants Arabidopsis thaliana and Oryza sativa (rice). In one of the first studies involving sequence-based prediction of AS in plants, we performed a genome-wide identification and characterization of NAGNAG AS in the model plant Physcomitrella patens, a moss.     

Contribution of SecDF to <Emphasis Type="Italic">Staphylococcus aureus</Emphasis>resistance and expression of virulence factors

Background  

SecDF is an accessory factor of the conserved Sec protein translocation machinery and belongs to the resistance-nodulation-cell division (RND) family of multidrug exporters. SecDF has been shown in Escherichia coli and Bacillus subtilis to be involved in the export of proteins. RND proteins can mediate resistance against various substances and might be of relevance in antimicrobial therapy. The role of RND proteins in Staphylococcus aureus has not yet been determined.