Synthesis, biochemical evaluation of a range of potent 4-substituted phenyl alkyl imidazole-based inhibitors of the enzyme complex 17alpha-Hydroxylase/17,20-Lyase (P45017alpha)

We report the synthesis, biochemical evaluation and rationalisation of the inhibitory activity of a number of azole-based compounds as inhibitors of the two components of the cytochrome P-450 enzyme 17alpha-hydroxylase/17,20-lyase (P450(17alpha)), i.e. 17alpha-hydroxylase (17alpha-OHase) and 17,20-lyase (lyase). The results suggest that the compounds synthesised are potent inhibitors, with 7-phenyl heptyl imidazole (11) (IC(50)=320 nM against 17alpha-OHase and IC(50)=100 nM against lyase); 1-[7-(4-fluorophenyl) heptyl] imidazole (14) (IC(50)=170 nM against 17alpha-OHase and IC(50)=57 nM against lyase); 1-[5-(4-bromophenyl) pentyl] imidazole (19) (IC(50)=500 nM against 17alpha-OHase and IC(50)=58 nM against lyase) being the most potent inhibitors within the current study, in comparison to ketoconazole (KTZ) (IC(50)=3.76 microM against 17alpha-OHase and IC(50)=1.66 microM against lyase). Furthermore, consideration of the inhibitory activity against the two components shows that all of the compounds tested are less potent towards the 17alpha-OHase in comparison to the lyase component, a desirable property in the development of novel inhibitors of P450(17alpha). From the modelling of these compounds onto the novel substrate heme complex (SHC) for the overall enzyme complex, the length of the compound, along with its ability to undergo interaction with the active site corresponding to the C(3) area of the steroidal backbone, are suggested to play a key role in determining the overall inhibitory activity.     

Nitration as a Mechanism of Na+, K+-ATPase Modification During Hypoxia in the Cerebral Cortex of the Guinea Pig Fetus

Previous studies have shown that hypoxia induces nitric oxide synthase-mediated generation of nitric oxide free radicals leading to peroxynitrite production. The present study tests the hypothesis that hypoxia results in NO-mediated modification of Na⁺, K⁺-ATPase in the fetal brain. Studies were conducted in guinea pig fetuses of 58-days gestation. The mothers were exposed to FiO₂ of 0.07% for 1 hour. Brain tissue hypoxia in the fetus was confirmed biochemically by decreased ATP and phosphocreatine levels. P₂ membrane fractions were prepared from normoxic and hypoxic fetuses and divided into untreated and treated groups. The membranes were treated with 0.5 mM peroxynitrite at pH 7.6. The Na⁺, K⁺-ATPase activity was determined at 37°C for five minutes in a medium containing 100 mM NaCl, 20 mM KCl, 6.0 mM MgCl₂, 50 mM Tris HCl buffer pH 7.4, 3.0 mM ATP with or without 10 mM ouabain. Ouabain sensitive activity was referred to as Na⁺, K⁺-ATPase activity. Following peroxynitrite exposure, the activity of Na⁺, K⁺-ATPase in guinea pig brain was reduced by 36% in normoxic membranes and further 29% in hypoxic membranes. Enzyme kinetics was determined at varying concentrations of ATP (0.5 mM-2.0 mM). The results indicate that peroxynitrite treatment alters the affinity of the active site of Na⁺, K⁺-ATPase for ATP and decreases the Vmax by 35% in hypoxic membranes. When compared to untreated normoxic membranes Vmax decreases by 35.6% in treated normoxic membranes and further to 52% in treated hypoxic membranes. The data show that peroxynitrite treatment induces modification of Na⁺, K⁺-ATPase. The results demonstrate that peroxynitrite decreased activity of Na⁺, K⁺-ATPase enzyme by altering the active sites as well as the microenvironment of the enzyme. We propose that nitric oxide synthase-mediated formation of peroxynitrite during hypoxia is a potential mechanism of hypoxia-induced decrease in Na⁺, K⁺-ATPase activity.     

Reactive centre loop mutants of α-1-antitrypsin reveal position-specific effects on intermediate formation along the polymerization pathway

The common severe Z mutation (E342K) of α₁-antitrypsin forms intracellular polymers that are associated with liver cirrhosis. The native fold of this protein is well-established and models have been proposed from crystallographic and biophysical data for the stable inter-molecular configuration that terminates the polymerization pathway. Despite these molecular ‘snapshots’, the details of the transition between monomer and polymer remain only partially understood. We surveyed the RCL (reactive centre loop) of α₁-antitrypsin to identify sites important for progression, through intermediate states, to polymer. Mutations at P₁₄P₁₂ and P₄, but not P₁₀P₈ or P₂P_1′, resulted in a decrease in detectable polymer in a cell model that recapitulates the intracellular polymerization of the Z variant, consistent with polymerization from a near-native conformation. We have developed a FRET (Förster resonance energy transfer)-based assay to monitor polymerization in small sample volumes. An in vitro assessment revealed the position-specific effects on the unimolecular and multimolecular phases of polymerization: the P₁₄P₁₂ region self-inserts early during activation, while the interaction between P₆P₄ and β-sheet A presents a kinetic barrier late in the polymerization pathway. Correspondingly, mutations at P₆P₄, but not P₁₄P₁₂, yield an increase in the overall apparent activation energy of association from ~360 to 550 kJ mol⁻¹.     

Profiling Deleterious Non-synonymous SNPs of Smoker's Gene CYP1A1

CYP1A1 gene belongs to the cytochrome P450 family and is known better as smokers’ gene due to its hyperactivation as a consequence of long term smoking. The expression of CYP1A1 induces polycyclic aromatic hydrocarbon production in the lungs, which when over expressed, is known to cause smoking related diseases, such as cardiovascular pathologies, cancer, and diabetes. Single nucleotide polymorphisms (SNPs) are the simplest form of genetic variations that occur at a higher frequency, and are denoted as synonymous and non-synonymous SNPs on the basis of their effects on the amino acids. This study adopts a systematic in silico approach to predict the deleterious SNPs that are associated with disease conditions. It is inferred that four SNPs are highly deleterious, among which the SNP with rs17861094 is commonly predicted to be harmful by all tools. Hydrophobic (isoleucine) to hydrophilic (serine) amino acid variation was observed in the candidate gene. Hence, this investigation aims to characterize a candidate gene from 159 SNPs of CYP1A1.     

Development and validation of a loop-mediated isothermal amplification assay for the detection of <Emphasis Type="Italic">Mycoplasma bovis</Emphasis> in mastitic milk

Mycoplasma mastitis is often difficult to control due to a lack of rapid and accurate diagnostic tools. The aim of the current study was to develop a loop-mediated isothermal amplification (LAMP) assay for the detection of Mycoplasma bovis (M. bovis) in mastitic milk. The assay was developed using primers designed for three different target genes: uvrC, 16S rRNA, and gyrB, and validated using mastitic milk samples previously found positive for the target pathogen. Specificity of the developed assay was determined by testing cross-reactivity of LAMP primers against closely related bovine mastitis bacterial pathogens. The sensitivity was found to be higher compared to conventional polymerase chain reaction (PCR). The LAMP assay was also capable of detecting M. bovis in PCR-negative milk samples of cows with clinical mastitis. The uvrC primers were found to be more sensitive, while gyrB primers were more specific; however, 16S rRNA primers were less specific and sensitive compared to either uvrC or gyrB primers. Cohen’s kappa values for uvrC, gyrB, and 16S rRNA primers used in the LAMP assays were 0.940, 0.970, and 0.807, respectively. There was a high level of agreement between the test results and the true-disease status as indicated by the receiver operating characteristic (ROC) curve. Our findings suggest that the newly developed LAMP assays targeting the uvrC and gyrB genes could be a useful tool for rapid and accurate diagnosis of mastitis caused by M. bovis.     

Successful phytoremediation of crude-oil contaminated soil at an oil exploration and production company by plants-bacterial synergism

Phytoremediation is a promising approach for the cleanup of soil contaminated with petroleum hydrocarbons. This study aimed to develop plant-bacterial synergism for the successful remediation of crude oil-contaminated soil. A consortia of three endophytic bacteria was augmented to two grasses, Leptochloa fusca and Brachiaria mutica, grown in oil-contaminated soil (46.8 g oil kg^?1 soil) in the vicinity of an oil exploration and production company. Endophytes augmentation improved plant growth, crude oil degradation, and soil health. Maximum oil degradation (80%) was achieved with B. mutica plants augmented with the endophytes and it was significantly (P < 0.05) higher than the use of plants or bacteria individually. Moreover, endophytes showed more persistence, the abundance and expression of alkB gene in the rhizosphere as well as in the endosphere of the tested plants than in unvegetated soil. A positive relationship (r = 0.70) observed between gene expression and crude oil reduction indicates that catabolic gene expression is important for hydrocarbon mineralization. This investigation showed that the use of endophytes with appropriate plant is an effective strategy for the cleanup of oil-contaminated soil under field conditions.     

Development of intron targeted amplified polymorphic markers of metal homeostasis genes for monitoring their introgression from <Emphasis Type="Italic">Aegilops</Emphasis> species to wheat

The identification of transfers of useful alien genes for metal homeostasis from non-progenitor Aegilops species using the widely available anchored wheat SSR markers is difficult due to their lower polymorphism with the distant related wild species and the lack of locus specificity further restricts their application. The present study deals with the development of intron targeted amplified polymorphic (ITAP) markers for the metal homeostasis genes present on chromosomes of groups 2 and 7 of Triticeae. The mRNA sequences of 27 metal homeostasis genes were retrieved from different plant species using NCBI database and their BLASTn was performed against the wheat draft genome sequences in Ensemblplants to get exonic and intronic sequences of the corresponding metal homeostasis genes in wheat. The ITAP primers were developed in such a way that they would anneal to the conserved flanking exonic regions of the genes and amplify across highly variable introns within the PCR limits. The primers led to the amplification of variable intronic sequences of genes with polymorphism between non-progenitor Aegilops species and the recipient wheat cultivars. Further, the polymorphic ITAP markers were used to characterize the transfers of metal homeostasis genes from the non-progenitor Aegilops species to the BC₂F₅ wheat-Aegilops derivatives, developed through induced homoeologous pairing. The derivatives with significant percent increase in grain Fe and Zn content over the elite cultivar PBW343 LrP showed the introgression of some of the useful Aegilops alleles of the metal homeostasis genes. The use of different metal homeostasis genes using this approach is the first report of the direct contribution of the genes for increasing the grain micronutrient content for developing biofortified wheat lines with reduced linkage drag.     

Phosphorylation of Wat1, human Lst8 homolog is critical for the regulation of TORC2 –Gad8 dependent pathway in fission yeast Schizosacchromyces pombe

Mammalian Lst8 interacts with the kinase domain of mTOR and stabilizes its interaction with Raptor regulating cell growth through the mTOR-S6K1 signalling pathway. Fission yeast Wat1, an ortholog of mammalian Lst8 is also an essential component of TOR complex 1 (TORC1) and TOR Complex 2 (TORC2) that control protein kinases essential for metabolic pathways. Here, we show that in response to osmotic stress, the Wat1 protein undergoes hyper-phosphorylation at S116 position. Wat1 interacts with the C-terminal region of Tor1 that also contain kinase domain. Co-immunoprecipitation and molecular modelling studies suggest that Wat1-Tor1 interaction is stabilized by FATC domain of Tor1 protein present at the C-terminal region. We have also demonstrated a physical interaction of Wat1 with Gad8, an AGC family protein kinase that is dependent on phosphorylation of Wat1 at S116 residue. Wat1 phosphorylation is required for the maintenance of vacuolar integrity and sexual differentiation. Collectively, our study reveals Wat1 phosphorylation regulates Gad8 function in a manner dependent on Tor1 interaction.     

Salt tolerance of two perennial grass <Emphasis Type="Italic">Brachypodium sylvaticum</Emphasis> accessions

Key message

We studied the salt stress tolerance of two accessions isolated from different areas of the world (Norway and Tunisia) and characterized the mechanism(s) regulating salt stress in Brachypodium sylvaticum Osl1 and Ain1.

Abstract

Perennial grasses are widely grown in different parts of the world as an important feedstock for renewable energy. Their perennial nature that reduces management practices and use of energy and agrochemicals give these biomass crops advantages when dealing with modern agriculture challenges such as soil erosion, increase in salinized marginal lands and the runoff of nutrients. Brachypodium sylvaticum is a perennial grass that was recently suggested as a suitable model for the study of biomass plant production and renewable energy. However, its plasticity to abiotic stress is not yet clear. We studied the salt stress tolerance of two accessions isolated from different areas of the world and characterized the mechanism(s) regulating salt stress in B. sylvaticum Osl1, originated from Oslo, Norway and Ain1, originated from Ain-Durham, Tunisia. Osl1 limited sodium transport from root to shoot, maintaining a better K/Na homeostasis and preventing toxicity damage in the shoot. This was accompanied by higher expression of HKT8 and SOS1 transporters in Osl1 as compared to Ain1. In addition, Osl1 salt tolerance was accompanied by higher abundance of the vacuolar proton pump pyrophosphatase and Na⁺/H⁺ antiporters (NHXs) leading to a better vacuolar pH homeostasis, efficient compartmentation of Na⁺ in the root vacuoles and salt tolerance. Although preliminary, our results further support previous results highlighting the role of Na⁺ transport systems in plant salt tolerance. The identification of salt tolerant and sensitive B. sylvaticum accessions can provide an experimental system for the study of the mechanisms and regulatory networks associated with stress tolerance in perennials grass.