Arsenate reduction mediated by the plasmid-encoded ArsC protein is coupled to glutathione

Resistance to arsenate conferred on Escherichia coli by the ars operon of plasmid R773 requires both the product of the arsC gene and reduction of arsenate to arsenate. A genetic analysis was performed to identify the source of reducing potential in vivo. in addition to the ars genes, arsenate resistance required the products of the gor gene for glutathione reductase and the gshA and gshB genes for glutathione synthesis. Mutations in the trx and grx genes for thioredoxin and glutaredoxin, respectively, had no effect on arsenate resistance. Although resistance required the arsC gene, the rate of reduction of arsenate to arsenate was nearly the same in cells lacking the ars operon. In strains deficient in glutathione biosynthesis this endogenous reduction was greatly diminished, and cells exhibited increased sensitivity to arsenate. When glutathione was supplied exogenously to such mutants, resistance was restored only to cells expressing the ars operon, and only such cells had detectable arsenate reduction after addition of glutathione. Since ArsC-catalysed reduction of arsenate provides high level resistance, physical coupling of the ArsC reaction to efflux of the resulting arsenite is hypothesised.     

Deciphering the enzymatic target of a new family of antischistosomal agents bearing a quinazoline scaffold using complementary computational tools

Abstract

A previous phenotypic screening campaign led to the identification of a quinazoline derivative with promising in vitro activity against Schistosoma mansoni. Follow-up studies of the antischistosomal potential of this candidate are presented here. The in vivo studies in a S. mansoni mouse model show a significant reduction of total worms and a complete disappearance of immature eggs when administered concomitantly with praziquantel in comparison with the administration of praziquantel alone. This fact is of utmost importance because eggs are responsible for the pathology and transmission of the disease. Subsequently, the chemical optimisation of the structure in order to improve the metabolic stability of the parent compound was carried out leading to derivatives with improved drug-like properties. Additionally, the putative target of this new class of antischistosomal compounds was envisaged by using computational tools and the binding mode to the target enzyme, aldose reductase, was proposed.     

In vitro and ex vivo biofilms of dermatophytes: a new panorama for the study of antifungal drugs

Abstract

This study describes an ex vivo model that creates an environment for dermatophyte biofilm growth, with features that resemble those of in vivo conditions, designing a new panorama for the study of antifungal susceptibility. Regarding planktonic susceptibility, MIC ranges were 0.125-1?µg ml^?1 for griseofulvin and 0.000097-0.25?µg ml^?1 for itraconazole and terbinafine. sMIC50 ranges were 2->512?µg ml^?1 for griseofulvin and 0.25->64?µg ml^?1 for itraconazole and terbinafine. CLSM images demonstrated a reduction in the amount of cells within the biofilm, but hyphae and conidia were still observed and biofilm biomass was maintained. SEM analysis demonstrated a retraction in the biofilm matrix, but fungal structures and water channels were preserved. These results show that ex vivo biofilms are more tolerant to antifungal drugs than in vitro biofilms, suggesting that environmental and nutritional conditions created by this ex vivo model favor biofilm growth and robustness, and hence drug tolerance.     

Mechanisms of mutagenesis induced by DNA lesions: multiple factors affect mutations in translesion DNA synthesis

Abstract

Environmental mutagens lead to mutagenesis. However, the mechanisms are very complicated and not fully understood. Environmental mutagens produce various DNA lesions, including base-damaged or sugar-modified DNA lesions, as well as epigenetically modified DNA. DNA polymerases produce mutation spectra in translesion DNA synthesis (TLS) through misincorporation of incorrect nucleotides, frameshift deletions, blockage of DNA replication, imbalance of leading- and lagging-strand DNA synthesis, and genome instability. Motif or subunit in DNA polymerases further affects the mutations in TLS. Moreover, protein interactions and accessory proteins in DNA replisome also alter mutations in TLS, demonstrated by several representative DNA replisomes. Finally, in cells, multiple DNA polymerases or cellular proteins collaborate in TLS and reduce in vivo mutagenesis. Summaries and perspectives were listed. This review shows mechanisms of mutagenesis induced by DNA lesions and the effects of multiple factors on mutations in TLS in vitro and in vivo.     

Effect of arginine on microorganisms involved in dental caries: a systematic literature review of in vitro studies

Abstract

This systematic review aimed to discuss the effects of arginine on caries-related microorganisms in different in vitro biofilm models. The eligibility criteria were in vitro studies that evaluated the effect of arginine at different concentrations on caries-related microorganisms using biofilm models. Eighteen studies published between 2012 and 2019 were included. Different bacterial species were studied. Seventeen studies (94.4%) achieved a low risk of bias and only one showed a medium risk of bias. The studies showed that arginine is a promising approach for the ecological management of dental caries. The focus of this review was to evaluate the effects of arginine on microorganisms involved in the mechanism of dental caries.     

An ex-situ and in vitro approach towards the bioremediation of carcinogenic hexavalent chromium

Abstract

Chromium, ranking the second most among toxic heavy metal pollutants in the world, causing respiratory, cardiovascular and renal problems in human beings is under study herein. We examined the biological remediation of the carcinogenic Cr (VI) polluted soils by indigenous yeast isolates. The total element analysis of the treated sample was determined by Energy Dispersion X-ray Micro Analysis (EDXMA). The sample under study was observed to have a high concentration of 458.29 mgKg^?1 Cr (VI), determined by Atomic Absorption Spectroscopy (AAS) and DPC analysis. The most tolerant isolate designated as CSR was used for in vitro and ex-situ bioremediation studies of Cr (VI). The isolate achieved significant bioremediation of 86% in vitro and 75.12% in ex-situ method. The optimal conditions for in vitro bioremediation were found to be 28?°C and a pH of 6. The ITS1, 5.8S rRNA and D1, D2 domain of LSU rRNA gene characterization of the isolate CSR illustrated that it belongs to Ustilago genera. The isolate was deposited in NCBI GenBank as Ustilago sp. CSR (KY284846). Although, Ustilago is generally a pathogenic fungus, our study opens up the scope of using Ustilago spp. for bioremediation of the carcinogenic heavy metal Chromium.     

1H, 13C and 15N resonance assignments of the arsenate reductase from Synechocystis sp. strain PCC 6803

Arsenate reductases (ArsC) are a group of enzymes that play essential roles in biological arsenic detoxification pathways by catalyzing the intracellular reduction of arsenate to arsenite, which is subsequently extruded from the cells by specific transport systems. The ArsC protein from cyanobacterium Synechocystis sp. strain PCC 6803 (SynArsC) is related to the thioredoxin-dependent ArsC family, but uses the glutathione/glutaredoxin system for arsenate reduction. Therefore, it is classified to a novel thioredoxin/glutaredoxin hybrid arsenate reductase family. Herein we report the chemical shift assignments of ¹H, ¹³C and ¹⁵N atoms for the reduced form of SynArsC, which provides a starting point for further structural analysis and elucidation of its enzymatic mechanism.     

Design,synthesis and biological evaluation of carbohydrate-based sulphonamide derivatives as topical antiglaucoma agents through selective inhibition of carbonic anhydrase II

Abstract

A series of new carbohydrate-based sulphonamide derivatives were designed, synthesised by employing the so-call ‘sugar-tail’ approach. The compounds were evaluated in vitro against a panel of CAs. Compared to their parent compound p-sulfamoylbenzoic acid, these compounds showed nearly 100-fold improvement in their binding affinities against hCA II in vitro. All of compounds showed great water solubility and the pH value of their water solutions of compounds is 7.0. Such properties are advantageous to make them much less irritating to the eye when applied topical glaucomatous drugs, compared to the relatively highly acidic dorzolamide preparations (pH 5.5). Notably, compounds 7d, 7?g, 7?h demonstrated to topically lower intraocular pressure (IOP) in glaucomatous animals better than brinzolamide when applied as a 1% solution directly into the eye. Low cytotoxicity on human cornea epithelial cell was observed in the tested concentrations by the MTT assay.     

Synthesis,anti-inflammatory,cytotoxic, and COX-1/2 inhibitory activities of cyclic imides bearing 3-benzenesulfonamide,oxime, and β-phenylalanine scaffolds: a molecular docking study

Abstract

Cyclic imides containing 3-benzenesulfonamide, oxime, and β-phenylalanine derivatives were synthesised and evaluated to elucidate their in vivo anti-inflammatory and ulcerogenic activity and in vitro cytotoxic effects. Most active anti-inflammatory agents were subjected to in vitro COX-1/2 inhibition assay. 3-Benzenesulfonamides (2–4, and 9), oximes (11–13), and β-phenylalanine derivative (18) showed potential anti-inflammatory activities with 71.2–82.9% oedema inhibition relative to celecoxib and diclofenac (85.6 and 83.4%, respectively). Most active cyclic imides 4, 9, 12, 13, and 18 possessed ED₅₀ of 35.4–45.3?mg kg^?1 relative to that of celecoxib (34.1?mg kg^?1). For the cytotoxic evaluation, the selected derivatives 2–6 and 8 exhibited weak positive cytotoxic effects (PCE = 2/59–5/59) at 10?μM compared to the standard drug, imatinib (PCE = 20/59). Cyclic imides bearing 3-benzenesulfonamide (2–5, and 9), acetophenone oxime (11–14, 18, and 19) exhibited high selectivity against COX-2 with SI > 55.6–333.3 relative to that for celecoxib [SI > 387.6]. β-Phenylalanine derivatives 21–24 and 28 were non-selective towards COX-1/2 isozymes as indicated by their SI of 0.46–0.68.     

In vitro asymbiotic germination and seedling development of Limodorum abortivum (Orchidaceae)

Abstract

Germination tests were carried out using immature seeds of Limodorum abortivum and applying in vitro techniques. The results proved that BM₁ culture medium is suitable to promote both germination and further growth stages. Details of the developmental pattern, and some micromorphological features, are described.     

Influence of Arsenate on Lipid Peroxidation Levels and Antioxidant Enzyme Activities in Bacillus cereus Strain XZM002 Isolated from High Arsenic Aquifer Sediments

Bacillus cereus strain XZM002 isolated from high arsenic aquifer sediments of Datong Basin was applied to examine the effects of arsenate stress on antioxidant enzyme activities, lipid peroxidation levels and cell growth inhibition rate. After 2 d exposure, the cell growth inhibition rate enhanced with an increase of As(V) concentrations (0, 800, 1600 μg/l). Reactive oxygen species and glutathione contents, lipid peroxidation levels, and antioxidant enzymes (glutathione peroxidase, and other three) activities of the treated cells were significantly higher than those of the controls during 3 d exposure (p < 0.05). Besides, the levels of nine parameters reached maximum after 2 d exposure and increased significantly with increasing arsenate stress (p < 0.05). However, they returned to levels similar to those of the control on the fourth day of exposure. The results suggested that the antioxidant defense system in B. cereus strain XZM002 could protect the cells from oxidative damage induced by arsenate.     

Arsenic resistance in Pteris vittata L.: identification of a cytosolic triosephosphate isomerase based on cDNA expression cloning in Escherichia coli

Arsenic hyperaccumulator Pteris vittata L. (Chinese brake fern) grows well in arsenic-contaminated media, with an extraordinary ability to tolerate high levels of arsenic. An expression cloning strategy was employed to identify cDNAs for the genes involved in arsenic resistance in P. vittata. Excised plasmids from the cDNA library of P. vittata fronds were introduced into Escherichia coli XL-1 Blue and plated on medium containing 4 mM of arsenate, a common form of arsenic in the environment. The deduced amino acid sequence of an arsenate-resistant clone, PV4-8, had cDNA highly homologous to plant cytosolic triosephosphate isomerases (cTPI). Cell-free extracts of PV4-8 had 3-fold higher level of triosephosphate isomerase (TPI) specific activities than that found in E. coli XL-1 Blue and had a 42 kD fusion protein immunoreactive to polyclonal antibodies raised against recombinant Solanum chacoense cTPI. The PV4-8 cDNA complemented a TPI-deficient E. coli mutant. PV4-8 expression improved arsenate resistance in E. coli WC3110, a strain deficient in arsenate reductase but not in AW3110 deficient for the whole ars operon. This is consistent with the hypothesis that PV4-8 TPI increased arsenate resistance in E. coli by directly or indirectly functioning as an arsenate reductase. When E. coli tpi gene was expressed in the same vector, bacterial arsenate resistance was not altered, indicating that arsenate tolerance was specific to P. vittata TPI. Paradoxically, P. vittata TPI activity was not more resistant to inhibition by arsenate in vitro than its bacterial counterpart suggesting that arsenate resistance of conventional TPI reaction was not the basis for the cellular arsenate resistance. P. vittata TPI activity was inhibited by incubation with reduced glutathione while bacterial TPI was unaffected. Consistent with cTPI’s role in arsenate reduction, bacterial cells expressing fern TPI had significantly greater per cent of cellular arsenic as arsenite compared to cells expressing E. coli TPI. Excised frond tissue infiltrated with arsenate reduced arsenate significantly more under light than dark. This research highlights a novel role for P. vittata cTPI in arsenate reduction.     

Mutational and gene expression analysis of mtrDEF,omcA and mtrCAB during arsenate and iron reduction in Shewanella sp. ANA‐3

Arsenate respiration and Fe(III) reduction are important processes that influence the fate and transport of arsenic in the environment. The goal of this study was to investigate the impact of arsenate on Fe(III) reduction using arsenate and Fe(III) reduction deficient mutants of Shewanella sp. strain ANA‐3. Ferrihydrite reduction in the absence of arsenate was similar for an arsenate reduction mutant (arrA and arsC deletion strain of ANA‐3) compared with wild‐type ANA‐3. However, the presence of arsenate adsorbed onto ferrihydrite impeded Fe(III) reduction for the arsenate reduction mutant but not in the wild‐type. In an Fe(III) reduction mutant (mtrDEF, omcA, mtrCAB null mutant of ANA‐3), arsenate was reduced similarly to wild‐type ANA‐3 indicating the Fe(III) reduction pathway is not required for ferrihydrite‐associated arsenate reduction. Expression analysis of the mtr/omc gene cluster of ANA‐3 showed that omcA and mtrCAB were expressed under soluble Fe(III), ferrihydrite and arsenate growth conditions and not in aerobically grown cells. Expression of arrA was greater with ferrihydrite pre‐adsorbed with arsenate relative to ferrihydrite only. Lastly, arrA and mtrA were simultaneously induced in cells shifted to anaerobic conditions and exposed to soluble Fe(III) and arsenate. These observations suggest that, unlike Fe(III), arsenate can co‐induce operons (arr and mtr) implicated in arsenic mobilization.     

Ultrastructural effects of trace elements and environmental pollution in Italian “Triangle of Death” on Pseudevernia furfuracea (L.) Zopf

Abstract

The lichen Pseudevernia furfuracea was exposed to environmental trace elements in the district of Acerra (province of Naples, southern Italy), one of the points forming Italy's “Triangle of Death”. P. furfuracea thalli were exposed in bags at different sites for 6 months, periodically collected and examined by transmission electron microscopy (TEM) to assess ultrastructural changes. An our earlier study demonstrated that these exposed lichens were strongly contaminated by trace elements (Sorbo S, Aprile G, Strumia S, Castaldo Cobianchi R, Leone A, et al. 2008. Trace element accumulation in P. furfuracea (L.) Zopf exposed in Italy's so-called Triangle of Death. Sci Total Environ 407: 647–654.). The algal cells were more affected than the fungal symbiont. Exposition at urban sites gave the most frequent changes. Four trace elements (Cd, Pb, Cu and Zn) were used to study the effects of ultrastructural trace element both in field and in vitro treatments. The lichen developed comparable ultrastructural changes when exposed to different trace elements and the changes were not specific to the treatment used (lichen bag exposition, in field and in vitro treatments). The in vitro treatment gave the highest frequency of damage at all time points. X-ray TEM microanalysis revealed trace elements inside the cell walls and the cytoplasmic vesicles of the lichens cultured with the trace elements; this localization is probably related to tolerance mechanisms.     

Arsenate V induced glutathione efflux from human erythrocytes

ObjectiveThe objective of the present study was to investigate if arsenate V exposure results in glutathione efflux from human erythrocytes.ProcedureThe changes in intracellular and extracellular nonprotein sulfhydryl and glutathione levels were determined in arsenate (V) exposed erythrocytes. Presence of any cellular membrane damage was assessed by lactate dehydrogenase activity measurement in the supernatant.ResultsWhen erythrocytes were exposed to 10 mM of arsenate (V) for 4 h, the intracellular NPSH level decreased to 0.28 ± 0025 μmol/ml erythrocyte. In contrast, extracellular nonprotein thiol level was increased to 0.180 ± 0.010 μmol/ml erythrocyte in 4 h. Extracellular glutathione levels reached to 0.028 ± 0.001, 0.052 ± 0.002, and 0.054 ± 0.004 μmol/ml erythrocyte with 1, 5, and 10 mM of arsenate (V), respectively. Utilization of MK571 a multi drug resistance-associated protein 1 inhibitor decreased the rate of glutathione efflux from erythrocytes suggesting a role for this membrane transporter in the process.ConclusionThe results of the present study indicate that erythrocytes efflux glutathione when exposed to arsenate (V).     

Characterization of polymicrobial biofilms obtained from saliva or carious lesions in dentin

Abstract

This study aimed to compare the formation of polymicrobial biofilms using carious dentin or saliva as inoculum for application in in vitro microbiological studies on caries research. For biofilm growth, combined samples of infected dentin or saliva from three donors were used. The biofilms were grown on glass coverslips, under a regimen of intermittent exposure (6?h day^?1) to 1% sucrose for 4?days. Total bacterial loads, as well as specific aciduric bacteria and mutans streptococci loads were quantified and correlated with biofilm acidogenicity and susceptibility to chlorhexidine. The data were evaluated using the Student’s-t, Mann Whitney and Kruskal-Wallis tests. The two biofilms showed similar microbial loads (total bacteria, aciduric bacteria and mutans streptococci) on day 4, and high acidogenicity after 48?h and were susceptible to chlorhexidine at different time intervals. In conclusion, both dentin and saliva can be used as an inoculum in in vitro studies of processes related to biofilm formation.     

Establishment of Aster sedifolius and Aster caucasicus callus cultures as a potential source of antioxidants

Abstract

Callus cultures were established for Aster sedifolius and Aster caucasicus, two Aster species used in natural medicine for their anticancer, antibacterial and antiviral activities attributed to the high content of antioxidant compounds such as polyphenols and ascorbate. The effects of growth medium and light condition on the induction and growth rate of callus from leaf, petiole and root explants are reported. Callus induction and proliferation depended on the genotype and the experimental conditions. In particular, a profuse callus culture was obtained from leaf explants grown in the light on medium supplemented with 2,4-D (0.1 mg l^?1) for A. caucasicus and on medium supplemented with 2,4-D (0.44 mg l^?1) plus 6-benzil-ammino-purine (BAP) (0.22 mg l^?1) for A. sedifolius. The content of total polyphenol and ascorbic acid was estimated in leaf and petiole explants of in vivo plants and in the relative derived calli. In calli, polyphenol content was lower than in the corresponding in vivo organs. Furthermore, the total ascorbic acid content decreased in calli while the reduced ascorbic acid pool increased. These findings demonstrate that Aster callus cultures produce antioxidant compounds and as such might be a model system to investigate the regulation and production of these important metabolites.     

Isolation and Molecular Characterization of Arsenite-Tolerant Alishewanella sp. GIDC-5 Originated from Industrial Effluents

An As-hypertolerant Alishewanella sp. GIDC-5 (Accession no. HQ659190) was isolated from an effluent treatment plant of the industrial area near Sachin, Gujarat (India). In vitro studies revealed that GIDC-5 can tolerate 18 mM of arsenite [As(III)] and 220 mM of arsenate [As(V)]. PCR analysis confirmed the presence of arsenite transporters [arsB and ACR3(1)] and arsenite oxidase gene [aioB]. Specific activities of arsenite oxidase and arsenate reductase, located on membrane and cytoplasmic fractions respectively, increased in dose dependent manner with arsenite concentration. Also, specific activities of antioxidant enzymes viz., catalase, ascorbate peroxidase, superoxide dismutase and glutathione S-transferase increased in presence of arsenite. Increased exposure to arsenite changes enzyme activities of the glycolysis, Krebs and glyoxylate branches dramatically. These results reveal that along with ars operon, metabolic adaptation and antioxidant activities participate in As(III) tolerance in Alishewanella sp. GIDC-5.     

Integrated management of foliar blast through ecofriendly formulated product,Oscext-e developed from Ocimum sanctum ethanolic extract

Ocimum sanctum ethanolic extract was combined with a formulating agent (coded B+) and named Oscext-e, and the product was bioassayed under in vitro conditions against Pyricularia grisea Sacc. that causes blast disease of rice. Fungitoxic patterns such as complete inhibition, granulation in cytoplasm, reduction in germ tube length, production of thinner or coiled germ tube in conidial germination and restriction in mycelial growth registered as evident through the present studies by various experimentation confirm the fungitoxic strength of importance in impairing the successful infection of the pathogen compared with normal infection. The formulated product, Oscext-e retained its fungitoxicity until 24 months storage period in all treatments. In a separate test of the product in greenhouse and field conditions, it was not only found to effectively reduce the foliar blast of rice crop but also found comparable with a standard fungicide carbendazim.