A Dual Anchoring Strategy for the Directed Evolution of Improved Artificial Transfer Hydrogenases Based on Carbonic Anhydrase

International audienc

Kavezno izlaganje lubina (Dicentrarchus labrax) u procjeni genotoksičnog utjecaja onečišćenja

Genotoxic effects are often the earliest signs of pollution-related environmental disturbance. In this study, we used the comet assay and micronucleus test to assess DNA damage in the erythrocytes of the European sea bass (Dicentrarchus labrax) exposed to environmental pollution in situ. Fish were collected from a fi sh farm in the Trogir Bay and their cages placed at an unpolluted reference site Šolta (Nečujam Bay) and a polluted site Vranjic (Kaštela Bay) for four weeks. A group of fi sh which remained at the fi sh farm Trogir Bay were used as the second control group. Fish exposed at the Vranjic site showed a signifi cantly higher erythrocyte DNA damage, measured by the comet assay, than either control group. Micronucleus induction showed a similar gradient of DNA damage, but did not reach statistical signifi cance. Our results show that cage exposure of a marine fi sh D. labrax can be useful in environmental biomonitoring and confi rm the comet assay as a suitable tool for detecting pollution-related genotoxicity.Genotoksični učinak često je jedan od najranijih pokazatelja štetnog djelovanja onečišćenja okoliša. U ovom radu procijenjeno je oštećenje DNA u eritrocitima lubina (Dicentrarchus labrax) izloženima okolišnom onečišćenju s pomoću komet-testa i mikronukleus-testa. Lubini su prikupljeni na ribogojilištu i kavezno izloženi u periodu od četiri tjedna na dvije postaje različitog stupnja onečišćenja na jadranskoj obali: na kontrolnoj postaji Šolta (zaljev Nečujam) i na onečišćenoj postaji Vranjic (Kaštelanski zaljev). Zasebna skupina lubina skupljena na ribogojilištu poslužila je kao druga kontrola. Rezultati komet-testa pokazali su statistički značajan porast oštećenja DNA na postaji Vranjic u usporedbi s obje kontrolne postaje. Rezultati mikronukleus-testa pokazali su sličan gradijent onečišćenja, iako nisu dosegli statističku značajnost. Ovi rezultati upućuju na primjenjivost kaveznog izlaganja lubina D. labrax u biomonitoringu vodenog okoliša te potvrđuju korisnost komet-testa kao prikladne metode za detekciju genotoksičnog utjecaja onečišćenja

The Zea mays mutants opaque-2 and opaque-7 disclose extensive changes in endosperm metabolism as revealed by protein, amino acid, and transcriptome-wide analyses

<p>Abstract</p> <p>Background</p> <p>The changes in storage reserve accumulation during maize (<it>Zea mays </it>L.) grain maturation are well established. However, the key molecular determinants controlling carbon flux to the grain and the partitioning of carbon to starch and protein are more elusive. The <it>Opaque-2 </it>(<it>O2</it>) gene, one of the best-characterized plant transcription factors, is a good example of the integration of carbohydrate, amino acid and storage protein metabolisms in maize endosperm development. Evidence also indicates that the <it>Opaque-7 </it>(<it>O7</it>) gene plays a role in affecting endosperm metabolism. The focus of this study was to assess the changes induced by the <it>o2 </it>and <it>o7 </it>mutations on maize endosperm metabolism by evaluating protein and amino acid composition and by transcriptome profiling, in order to investigate the functional interplay between these two genes in single and double mutants.</p> <p>Results</p> <p>We show that the overall amino acid composition of the mutants analyzed appeared similar. Each mutant had a high Lys and reduced Glx and Leu content with respect to wild type. Gene expression profiling, based on a unigene set composed of 7,250 ESTs, allowed us to identify a series of mutant-related down (17.1%) and up-regulated (3.2%) transcripts. Several differentially expressed ESTs homologous to genes encoding enzymes involved in amino acid synthesis, carbon metabolism (TCA cycle and glycolysis), in storage protein and starch metabolism, in gene transcription and translation processes, in signal transduction, and in protein, fatty acid, and lipid synthesis were identified. Our analyses demonstrate that the mutants investigated are pleiotropic and play a critical role in several endosperm-related metabolic processes. Pleiotropic effects were less evident in the <it>o7 </it>mutant, but severe in the <it>o2 </it>and <it>o2o7 </it>backgrounds, with large changes in gene expression patterns, affecting a broad range of kernel-expressed genes.</p> <p>Conclusion</p> <p>Although, by necessity, this paper is descriptive and more work is required to define gene functions and dissect the complex regulation of gene expression, the genes isolated and characterized to date give us an intriguing insight into the mechanisms underlying endosperm metabolism.</p

Estimates of DNA damage by the comet assay in the direct-developing frog Eleutherodactylus johnstonei (Anura, Eleutherodactylidae)

The aim of this study was to use the Comet assay to assess genetic damage in the direct-developing frog Eleutherodactylus johnstonei. A DNA diffusion assay was used to evaluate the effectiveness of alkaline, enzymatic and alkaline/enzymatic treatments for lysing E. johnstonei blood cells and to determine the amount of DNA strand breakage associated with apoptosis and necrosis. Cell sensitivity to the mutagens bleomycin (BLM) and 4-nitro-quinoline-1-oxide (4NQO) was also assessed using the Comet assay, as was the assay reproducibility. Alkaline treatment did not lyse the cytoplasmic and nuclear membranes of E. johnstonei blood cells, whereas enzymatic digestion with proteinase K (40 μg/mL) yielded naked nuclei. The contribution of apoptosis and necrosis (assessed by the DNA diffusion assay) to DNA damage was estimated to range from 0% to 8%. BLM and 4NQO induced DNA damage in E. johnstonei blood cells at different concentrations and exposure times. Dose-effect curves with both mutagens were highly reproducible and showed consistently low coefficients of variation (CV ≤ 10%). The results are discussed with regard to the potential use of the modified Comet assay for assessing the exposure of E. johnstonei to herbicides in ecotoxicological studies

Cytotoxicity and genotoxicity of lanthanides for Vicia faba L. are mediated by their chemical speciation in different exposure media

International audienceA comprehensive study of the toxicity of lanthanides (LN) in relation to the media composition will enhance the prediction of their potential adverse effects for living organisms. Here we examined the effect of different media on the V. faba root elongation and on the cytotoxic (mitotic index) and the genotoxic (micronucleated cell number) effects from toxicity tests with Ce, Gd and Lu (100, 800 and 6400 μg L −1). Three different exposure media were selected: the standard Hoagland media (SH); an alternative SH, without phosphates (SH-P); and distilled water (DW). In the SH no cyto-genotoxic effects were observed and even, for low LN content, potential root elongation stimulation was reported. The absence of toxic effects was explained by a drastic decrease of the total dissolved LN concentration due to the presence of phosphates causing LN precipitation. In SH-P, LN remained largely soluble and inhibition of root elongation was observed mainly for the highest treatments. While in the tests done in DW, toxic effects were obtained for all treatments. Our results showed that in absence of phosphorous, LN appear mainly as free form and complexed in carbonates and sulphates, and can cause toxic effects, whereas toxicity is not expected when phosphorous is available in aquatic media. The highest LN root contents were observed for the tests using distilled water, possibly due to the absence of competition by Ca 2+ for uptake. The present work demonstrated that media composition has a great impact in assessing the ecotoxicology of lanthanides

Development of a series of 3-hydroxyquinolin-2(1H)-ones as selective inhibitors of HIV-1 reverse transcriptase associated RNase H activity

We report herein the synthesis of a series of 3-hydroxyquinolin-2(1H)-one derivatives. Esters and amide groups were introduced at position 4 of the basis scaffold and some modulations of the benzenic moiety were performed. Most compounds presented selective inhibitory properties in the 10–20 μM range against HIV-1 reverse transcriptase associated ribonuclease H activity, without affecting the integrase and reverse transcriptase DNA polymerase activities. Unfortunately all tested compounds exhibited high cellular cytotoxicity in cell culture which limited their applications as antiviral agents

Sphingolipid-induced cell death in Arabidopsis is negatively regulated by the papain-like cysteine protease RD21

It is now well established that sphingoid Long Chain Bases (LCBs) are crucial mediators of programmed cell death. In plants, the mycotoxin fumonisin B1 (FB1) produced by the necrotrophic fungus Fusarium moniliforme disrupts the sphingolipid biosynthesis pathway by inhibiting the ceramide synthase leading to an increase in the amount of phytosphingosine (PHS) and dihydrosphingosine (DHS), the two major LCBs in Arabidopsis thaliana. To date, the signaling pathway involved in FB1-induced cell death remains largely uncharacterized. It is also well acknowledged that plant proteases such as papain-like cysteine protease are largely involved in plant immunity. Here, we show that the papain-like cysteine protease RD21 (responsive-to-desiccation-21) is activated in response to PHS and FB1 in Arabidopsis cultured cells and leaves, respectively. Using two allelic null mutants of RD21, and two different PCD bioassays, we demonstrate that the protein acts as a negative regulator of FB1-induced cell death in Arabidopsis