4,627 research outputs found
Cold Induction of EARLI1, a Putative Arabidopsis Lipid Transfer Protein, Is Light and Calcium Dependent
As sessile organisms, plants must adapt to their environment. One approach toward understanding this adaptation is to investigate environmental regulation of gene expression. Our focus is on the environmental regulation of EARLI1, which is activated by cold and long-day photoperiods. Cold activation of EARLI1 in short-day photoperiods is slow, requiring several hours at 4ºC to detect an increase in mRNA abundance. EARLI1 is not efficiently cold-activated in etiolated seedlings, suggesting that photomorphogenesis is necessary for its cold activation. Cold activation of EARLI1 is inhibited in the presence of the calcium channel blocker lanthanum chloride or the calcium chelator EGTA. Addition of the calcium ionophore Bay K8644 results in cold-independent activation of EARLI1. These data suggest that EARLI1 is not an immediate target of the cold response, and that calcium flux affects its expression. EARLI1 is a putative secreted protein and has motifs found in lipid transfer proteins. Over-expression of EARLI1 in transgenic plants results in reduced electrolyte leakage during freezing damage, suggesting that EARLI1 may affect membrane or cell wall stability in response to low temperature stress
Purification and Reconstitution of the Glutamate Carrier GltT of the Thermophilic Bacterium Bacillus stearothermophilus
An affinity tag consisting of six adjacent histidine residues followed by an enterokinase cleavage site was genetically engineered at the N-terminus of the glutamate transport protein GltT of the thermophilic bacterium Bacillus stearothermophilus. The fusion protein was expressed in Escherichia coli and shown to transport glutamate. The highest levels of expression were observed in E. coli strain DH5α grown on rich medium. The protein could be purified in a single step by Ni2+-NTA affinity chromatography after solubilization of the cytoplasmic membranes with the detergent Triton X100. Purified GltT was reconstituted in an active state in liposomes prepared from E. coli phospholipids. The protein was reconstituted in detergent-treated preformed liposomes, followed by removal of the detergent with polystyrene beads. Active reconstitution was realized with a wide range of Triton X100 concentrations. Neither the presence of glycerol, phospholipids, nor substrates of the transporter was necessary during the purification and reconstitution procedure to keep the enzyme in an active state. In B. stearothermophilus, GltT translocates glutamate in symport with protons or sodium ions. In membrane vesicles derived from E. coli cells expressing GltT, the Na+ ion dependency seems to be lost, suggesting a role for the lipid environment in the cation specificity. In agreement with the last observation, glutamate transport catalyzed by purified GltT reconstituted in E. coli phospholipid is driven by an electrochemical gradient of H+ but not of Na+.
The future of journalism: risks, threats and opportunities
Today journalism, as an industry and a profession, is characterised by everincreasing turbulence and change, for better and for worse. Profound transformations affect every aspect of the institution, including the economic health of journalism, the conditions and self-understandings of its practitioners, its ability to serve as a watchdog on concentrations of power, its engagement with and relationship to its audience, and its future prospects. This emerging and dynamic ecology can be viewed as a unique constellation of challenges and opportunities. For these reasons, the fifth Future of Journalism conference, held in Cardiff on 10–11 September 2015, focused on the theme of Risks, Threats and Opportunities. The conference saw over 120 papers from around the world presented across 34 sessions, with keynote speeches from Dan Gillmor, Stephen Reese and Jean Seaton. This introduction briefly outlines some of these key risks, threats and opportunities, drawing on work presented at the conference, as well as insights from the field of journal- ism studies
Increasing galactose consumption by Saccharomyces cerevisiae through metabolic engineering of the GAL gene regulatory network
Inhibition of Fungi and Gram-Negative Bacteria by Bacteriocin BacTN635 Produced by Lactobacillus plantarum sp. TN635
The aim of this study was to evaluate 54 lactic acid bacteria (LAB) strains isolated from meat, fermented vegetables and dairy products for their capacity to produce antimicrobial activities against several bacteria and fungi. The strain designed TN635 has been selected for advanced studies. The supernatant culture of this strain inhibits the growth of all tested pathogenic including the four Gram-negative bacteria (Salmonella enterica ATCC43972, Pseudomonas aeruginosa ATCC 49189, Hafnia sp. and Serratia sp.) and the pathogenic fungus Candida tropicalis R2 CIP203. Based on the nucleotide sequence of the 16S rRNA gene of the strain TN635 (1,540 pb accession no FN252881) and the phylogenetic analysis, we propose the assignment of our new isolate bacterium as Lactobacillus plantarum sp. TN635 strain. Its antimicrobial compound was determined as a proteinaceous substance, stable to heat and to treatment with surfactants and organic solvents. Highest antimicrobial activity was found between pH 3 and 11 with an optimum at pH = 7. The BacTN635 was purified to homogeneity by a four-step protocol involving ammonium sulfate precipitation, centrifugal microconcentrators with a 10-kDa membrane cutoff, gel filtration Sephadex G-25, and C18 reverse-phase HPLC. SDS-PAGE analysis of the purified BacTN635, revealed a single band with an estimated molecular mass of approximately 4 kDa. The maximum bacteriocin production (5,000 AU/ml) was recorded after a 16-h incubation in Man, Rogosa, and Sharpe (MRS) medium at 30 °C. The mode of action of the partial purified BacTN635 was identified as bactericidal against Listeria ivanovii BUG 496 and as fungistatic against C. tropicalis R2 CIP203
Identification of hepatitis a virus mimotopes by phage display, antigenicity and immunogenicity
A phage-displayed peptide approach was used to identify ligands mimicking antigenic determinants of hepatitis A virus (HAV) for the first time. Bacteriophages displaying HAV mimotopes were isolated from a phage-display peptide library by affinity selection on serum antibodies from hepatitis A patients. Selected phage-peptides were screened for reactivity with sera from HAV infected patients and healthy controls. Four cloned peptides with different sequences were identified as mimotopes of HAV; three of them showed similarity in their amino acid sequences with at least one of the VP3 and VP1 antigenic proteins of HAV. One clone was recognised by 92% of the positive sera. The phagotopes competed effectively with HAV for absorption of anti-HAV-specific antibodies in human sera, as determined by ELISA. The four phage clones induced neutralising anti-HAV antibodies in immunised mice. These results demonstrate the potential of this method to elucidate the disease related epitopes of HAV and to use these mimotopes in diagnostic applications or in the development of a mimotope-based hepatitis A vaccine without the necessity of manipulation of the virus
Tertiary-Treated Municipal Wastewater is a Significant Point Source of Antibiotic Resistance Genes Into Duluth-Superior Harbor
In this study, the impact of tertiary-treated municipal wastewater on the quantity of several antibiotic resistance determinants in Duluth-Superior Harbor was investigated by collecting surface water and sediment samples from 13 locations in Duluth-Superior Harbor, the St. Louis River, and Lake Superior. Quantitative PCR (qPCR) was used to target three different genes encoding resistance to tetracycline (tet(A), tet(X), and tet(W)), the gene encoding the integrase of class 1 integrons (intI1), and total bacterial abundance (16S rRNA genes) as well as total and human fecal contamination levels (16S rRNA genes specific to the genus Bacteroides). The quantities of tet(A), tet(X), tet(W), intI1, total Bacteroides, and human-specific Bacteroides were typically 20-fold higher in the tertiary-treated wastewater than in nearby surface water samples. In contrast, the quantities of these genes in the St. Louis River and Lake Superior were typically below detection. Analysis of sequences of tet(W) gene fragments from four different samples collected throughout the study site supported the conclusion that tertiary-treated municipal wastewater is a point source of resistance genes into Duluth-Superior Harbor. This study demonstrates that the discharge of exceptionally treated municipal wastewater can have a statistically significant effect on the quantities of antibiotic resistance genes in otherwise pristine surface waters
Gyrase inhibitors induce an oxidative damage cellular death pathway in Escherichia coli
Modulation of bacterial chromosomal supercoiling is a function of DNA gyrase-catalyzed strand breakage and rejoining. This reaction is exploited by both antibiotic and proteic gyrase inhibitors, which trap the gyrase molecule at the DNA cleavage stage. Owing to this interaction, double-stranded DNA breaks are introduced and replication machinery is arrested at blocked replication forks. This immediately results in bacteriostasis and ultimately induces cell death. Here we demonstrate, through a series of phenotypic and gene expression analyses, that superoxide and hydroxyl radical oxidative species are generated following gyrase poisoning and play an important role in cell killing by gyrase inhibitors. We show that superoxide-mediated oxidation of iron–sulfur clusters promotes a breakdown of iron regulatory dynamics; in turn, iron misregulation drives the generation of highly destructive hydroxyl radicals via the Fenton reaction. Importantly, our data reveal that blockage of hydroxyl radical formation increases the survival of gyrase-poisoned cells. Together, this series of biochemical reactions appears to compose a maladaptive response, that serves to amplify the primary effect of gyrase inhibition by oxidatively damaging DNA, proteins and lipids
COMPARATIVE EFFECTS OF 4 LEGUME SPECIES ON PLASMA-LIPIDS AND FECAL STEROID-EXCRETION IN HYPERCHOLESTEROLEMIC PIGS
The effect of four species of legume seeds on plasma cholesterol levels and faecal steroid excretion was studied in pigs. Thirty-six growing boars were randomly allocated in groups of six to six diets which they ate continuously for 42 d. The diets fed were: 1, a semi-purified (SP; control group 1) diet; 2, SP 10 g cholesterol/kg (control group 2); 3, 4, 5, 6, SP + cooked legumes (70:30, w/w; respectively baked beans (Phaseolus vulgaris), peas (Pisum sativum), lentils (Lens culinaris Medik.), butter beans (Phaseolus lunatus)) + 10 g cholesterol/kg. Fasting blood samples were taken on days 0, 14, 28, and 42 for the determination of total plasma cholesterol, high-density-lipoprotein (HDL)-cholesterol and triacylglycerols. Between days 7 and 11 and days 28 and 32 complete 5 d faecal collections were made for the measurement of neutral, acidic and conjugated steroids. After 42 d total cholesterol and HDL-cholesterol levels were raised significantly in all groups, but to different extents. In comparison with control group 2, diet-induced hypercholesterolaemia was significantly inhibited in the groups consuming baked beans, peas and butter beans, although HDL-cholesterol levels were maintained. Faecal steroid excretion by the legume groups was not significantly different from that of control group 2. The results suggest that the mechanism for the hypocholesterolaemic effect does not involve increased hepatic bile acid synthesis and thereby increased cholesterol clearance via the intestinal route
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