Purification and properties of an enantioselective and thermoactive amidase from the thermophilic actinomycete <Emphasis Type="Italic">Pseudonocardia thermophila</Emphasis>

A constitutively expressed thermoactive amidase from the thermophilic actinomycete Pseudonocardia thermophila was purified to homogeneity by applying hydrophobic interaction, anion exchange and gel filtration chromatography, giving a yield of 26% and a specific activity of 19.5 units mg^–1. The purified enzyme has an estimated molecular mass of 108 kDa and an isoelectric point of 4.2. The amidase is active at a broad pH range (pH 4–9) and temperature range (40–80°C) and has a half-life of 1.2 h at 70°C. Inhibition of enzyme activity was observed in the presence of metal ions, such as Co²⁺, Hg²⁺, Cu²⁺, Ni²⁺, and thiol reagents. The amidase has a broad substrate spectrum, including aliphatic, aromatic and amino acid amides. The presence of a double bond or a methyl group near the carboxamide group of aliphatic and amino acid amides enhances the enzymatic activity. Among aromatic amides with substitutions at the o-, m-, or p-position, the p-substituted amides are the preferred substrates. The highest acyl transferase activity was detected with hexanoamide, isobutyramide and propionamide. The K_m values for propionamide, methacrylamide, benzamide and 2-phenylpropionamide are 7.4, 9.2, 4.9 and 0.9 mM, respectively. The amidase is highly S-stereoselective for 2-phenylpropionamide; and the racemic amide was converted to the corresponding S-acid with an enantiomeric excess of >95% at 50% conversion of the substrate. In contrast, the d,l-tryptophanamide and d,l-methioninamide were converted to the corresponding d,l-acids at the same rate. This thermostable enzyme represents the first reported amidase from a thermophilic actinomycete.     

Recognition of 3′ nucleotide context and stop codon readthrough are determined during mRNA translation elongation

The nucleotide context surrounding stop codons significantly affects the efficiency of translation termination. In eukaryotes, various 3′ contexts that are unfavorable for translation termination have been described; however, the exact molecular mechanism that mediates their effects remains unknown. In this study, we used a reconstituted mammalian translation system to examine the efficiency of stop codons in different contexts, including several previously described weak 3′ stop codon contexts. We developed an approach to estimate the level of stop codon readthrough in the absence of eukaryotic release factors (eRFs). In this system, the stop codon is recognized by the suppressor or near-cognate tRNAs. We observed that in the absence of eRFs, readthrough occurs in a 3′ nucleotide context-dependent manner, and the main factors determining readthrough efficiency were the type of stop codon and the sequence of the 3′ nucleotides. Moreover, the efficiency of translation termination in weak 3′ contexts was almost equal to that in the tested standard context. Therefore, the ability of eRFs to recognize stop codons and induce peptide release is not affected by mRNA context. We propose that ribosomes or other participants of the elongation cycle can independently recognize certain contexts and increase the readthrough of stop codons. Thus, the efficiency of translation termination is regulated by the 3′ nucleotide context following the stop codon and depends on the concentrations of eRFs and suppressor/near-cognate tRNAs.     

Ionic liquids: prospects for nucleic acid handling and delivery

Operations with nucleic acids are among the main means of studying the mechanisms of gene function and developing novel methods of molecular medicine and gene therapy. These endeavours usually imply the necessity of nucleic acid storage and delivery into eukaryotic cells. In spite of diversity of the existing dedicated techniques, all of them have their limitations. Thus, a recent notion of using ionic liquids in manipulations of nucleic acids has been attracting significant attention lately. Due to their unique physicochemical properties, in particular, their micro-structuring impact and tunability, ionic liquids are currently applied as solvents and stabilizing media in chemical synthesis, electrochemistry, biotechnology, and other areas. Here, we review the current knowledge on interactions between nucleic acids and ionic liquids and discuss potential advantages of applying the latter in delivery of the former into eukaryotic cells.     

Modern methods of laboratory diagnostics of Chlamydia infections

Need for further improvement of methods for verification of etiological agent of urogenital and respiratory chlamydiosis on the basis of increased biotechnological requirements to antigens for serological reactions, primers for PCR assay (refinement of connection of primers with microorganism's zones of genome most significant for its life activity or formation of most diagnostically significant complexes of primers), and selection of cultivating conditions considering the predicted features of clinical strains of the agent was substantiated.     

Effect of the Pool Size of Stromal Reductants on the Alternative Pathway of Electron Transfer to Photosystem I in Chloroplasts of Intact Leaves

The effect of elevated temperature on electron flow to plastoquinone pool and to PSI from sources alternative to PSII was studied in barley (Hordeum vulgare L.) and maize (Zea mays L.) leaves. Alternative electron flow was characterized by measuring variable fluorescence of chlorophyll and absorption changes at 830 nm that reflect redox changes of P700, the primary electron donor of PSI. The treatment of leaves with elevated temperature resulted in a transient increase in variable fluorescence after cessation of actinic light. This increase was absent in leaves treated with methyl viologen (MV). The kinetics of P700⁺ reduction in barley and maize leaves treated with DCMU and MV exhibited two exponential components. The rate of both components markedly increased with temperature of the heat pretreatment of leaves when the reduction of P700⁺ was measured after short (1 s) illumination of leaves. The acceleration of both kinetic components of P700⁺ reduction by high-temperature treatment was much less pronounced when P700⁺ reduction rate was measured after illumination of leaves for 1 min. Since the treatment of leaves with DCMU and MV inhibited both the electron flow to PSI from PSII and ferredoxin-dependent cycling of electrons around PSI, the accelerated reduction of P700⁺ indicated that high temperature treatment activated electron flow to PSII from reductants localized in the chloroplast stroma. We conclude that the lesser extent of activation of this process by elevated temperature after prolonged illumination of heat-inhibited leaves is caused by depletion of the pool stromal reductants in light due to photoinduced electron transfer from these reductants to oxygen.     

Glutathione system of erythrocyte and plasma in peptic ulcer

A complex study of the blood glutathione system has been carried out for the first time in patients with peptic (gastric and duodenal) ulcer. In erythrocytes and blood plasma of patients with the complicated peptic ulcer and postgastroresection syndromes there was the increase of conjugated dienes (and in the second group the increase in antioxidant activity). Under these conditions the main change was the sharp and identical decrease in glutathione peroxidase activity. In patients with uncomplicated peptic ulcer there was sharp increase in erythrocite and plasma glutathione reductase activity and plasma GSH. In operated but basically healthy patients plasma glutathione peroxidase remained decreased but plasma GSH sharply increased. Evidently complicated peptic ulcer is characterized by decreased functioning of the glutathione system. Activation of this system and the decrease or disappearance of manifestations of oxidative stress are associated with a favorable course of this disease, especially at uncomplicated peptic ulcer. The revealed changes significantly differ from those observed in patients with viral hepatitis, blle excretory diseases and strokes.     

Value of magnetic resonance imaging in the diagnosis of sequels of shoulder joint injuries

The paper presents the results of examining 45 persons with sequels of shoulder joint injuries, by applying magnetic resonance imaging (MRI) (100%) and arthroscopy (95.6%). The data of arthroscopy were compared with those of MRI; thereafter the sensitivity, specificity, and accuracy of MRI were calculated. The findings suggested that there was no statistically significant difference in the capacities of the two comparable techniques MRI and arthroscopy to diagnose labral tears with degenerative changes, synovitis, bursitis and tendinitis (McNemar's test; p > 0.05). The capacities of MRI are greater than those of arthroscopy only to detect the structural disintegrity of the joint shoulder.     

Clinical and radiation diagnosis of circumscribed peritonitis complicating duodenal perforation

This paper deals with the results of ultrasound (US) and multislice computed tomography (MSCT) in patients with perforated duodenal ulcer in different periods after the onset of the disease, with varying clinical manifestations, and in the postoperative period. A comparative analysis of the results of radiation studies has indicated that X-ray study is a basic technique that defines treatment policy in the early development periods of perforation; US study (USS) reveals a change in the duodenum, fluid and infiltration outside the duodenum. As circumscribed peritonitis progresses, USS allows the diagnosis of abdominal abscesses and infiltrations. Out of the radiation techniques, MSCT is of most importance in the diagnosis of interintestinal abscesses and in the determination of syntopy of purulent cavities.     

Macromolecular systems and bactericidal films based on chitin derivatives and silver nanoparticles

A new polymer composite based on carboxymethylchitin and silver nanoparticles was obtained in order to produce biodegradable wound coating films. The number of metal nanoparticles in the composite may be easily regulated as was verified by UV-VIS-spectroscopy data. A comparative evaluation of silver nanoparticle size in the initial system and in the polymer composition was performed by means of photon correlation spectroscopy. Composite films revealed a pronounced concentration-dependent antibacterial activity towards strains Salmonella typhimurium and Staphilococcus aureus.