New aspects of the molecular evolution of legumains, Asn-specific cysteine proteinases

The molecular evolution of asparagine-specific cysteine proteinases, called legumains, from plants and animals was analyzed using newly available related amino acid sequences from lower eukaryotes, bacteria and Archaea. The results suggest that genuine legumains originate from prokaryote pro-legumains. The evolutionary roots of genuine legumains from plants and animals descend from Parabasalia and Alveolata before developing into their respective separate branches headed by Chlorophyta and Placozoa. The branch of legumain-like plant/animal glycosylphosphatidyl inositol transamidases separated from the general evolutionary stem of legumains at the level of lower eukaryotes. Modeling of the 3D structure of a plant genuine legumain underlined the previously suggested similarity of the active site geometry of legumains with caspases, which are Asp-specific bacterial and eukaryote proteinases.     

Legumains - a family of asparagine-specific cysteine endopeptidases involved in propolypeptide processing and protein breakdown in plants

Legumains are a recently discovered family of plant and animal cysteine endopeptidases with a cleavage specificity for Asn in the P1 position of peptide bonds. Asp-flanked peptide bonds also are cleaved, but with a much lower efficiency. Legumains evolved from GPI transamidase-like progenitors. Sequence analysis revealed three major groups of plant legumains corresponding to differences in the developmental and organ-specific gene expression. With the exception of a single cell wall specific representative, all legumains occur in the vacuolar compartment. Legumains are either involved in protein degradation or play a role in the processing of precursor proteins by Asn/Asp-specific limited proteolysis. Which function legumains perform depends on the conformational state of the substrate protein. A legumain acts as a vacuolar processing enzyme when it only has access to the regular processing sites of a precursor polypeptide, but it acts as a degradative enzyme when an altered conformation opens the substrate for unlimited proteolysis. The specificity of these interactions seems to be the result of a co-evolution of enzyme and substrate. The double function of legumains is particularly evident in the events of deposition and mobilisation of storage globulins during seed maturation and germination/seedling growth and in senescing and dying cells.     

A cellular test system expressing hERG K+ channels for assaying drug cardiotoxicity: Elaboration and analysis

Many drugs are cardiotoxic because they inhibit hERG K⁺ channels, thus prolonging the repolarization phase of the cardiomyocyte action potential giving rise to cardiac arrhythmias. Early detection of inhibiting effects of candidate drugs on the activity of K⁺ channels in cardiomyocytes is one of the main challenges in preclinical drug screening. The aim of this study was to obtain a cell line expressing recombinant hERG channels at a stable and reproducible level as a prerequisite to its further application as a test system.     

Bioconversion of C19- and C21-steroids with parent and mutant strains of Curvularia lunata

Regio- and stereospecificity of microbial hydroxylation was studied at the transformation of 3-keto-4-ene steroids of androstane and pregnane series by the filamentous fungus of Curvularia lunata VKM F-644. The products of the transformations were isolated by column chromatography and identified using HPLC, massspectrometry (MS) and proton nuclear magnetic resonance (¹H NMR) analyses. Androst-4-ene-3,17-dione (AD) and its 1(2)-dehydro- and 9α-hydroxylated (9-OH-AD) derivatives were hydroxylated by the fungus mainly in position 14α, while 6α-, 6β- and 7α-hydroxylated products were revealed in minor amounts. At the transformation of C₂₁-steroids (cortexolone and its acetylated derivatives) the presence of 17-acetyl group was shown to facilitate further selectivity of 11β-hydroxylation. Original procedures for protoplasts obtaining, mutagenesis and mutant strain selection have been developed. A stable mutant (M4) of C. lunata with high 11β-hydroxylase activity towards 21-acetate and 17α,21-diacetate of cortexolone was obtained. Yield of 11β-hydroxylated products reached about 90% at the transformation of 17α, 21-diacetate of cortexolone (1 g/l) using mutant strain M4.     

Doxorubicin and menadione decrease cell proliferation of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> by different mechanisms

The aim of this study was to examine the effect of doxorubicin and menadione on cell proliferation, cell cycle, glutathione concentration, the expression of ribonucleotide reductase, and the Yap1 dependent redox-sensitive pathway in Saccharomyces cerevisiae as a eukaryote cell model. Our data showed that menadione induced cell-cycle arrest in the G₁ phase, decreased intracellular GSH and GSSG concentrations, dose-dependently increased expression of ribonucleotide reductase and the activity of Yap1 pathway. Doxorubicin induced the cell-cycle arrest in G₁ and S phases, increased the GSH and GSSG concentration and the expression of ribonucleotide reductase, and modulated the Yap-dependent pathway activity.     

The Effect of Nimodipine on Calcium Homeostasis and Pain Sensitivity in Diabetic Rats

Summary 1. The pathogenesis of diabetic neuropathy is a complex phenomenon, the mechanisms of which are not fully understood. Our previous studies have shown that the intracellular calcium signaling is impaired in primary and secondary nociceptive neurons in rats with streptozotocin (STZ)-induced diabetes. Here, we investigated the effect of prolonged treatment with the L-type calcium channel blocker nimodipine on diabetes-induced changes in neuronal calcium signaling and pain sensitivity.2. Diabetes was induced in young rats (21 p.d.) by a streptozotocin injection. After 3 weeks of diabetes development, the rats were treated with nimodipine for another 3 weeks. The effect of nimodipine treatment on calcium homeostasis in nociceptive dorsal root ganglion neurons (DRG) and substantia gelatinosa (SG) neurons of the spinal cord slices was examined with fluorescent imaging technique.3. Nimodipine treatment was not able to normalize elevated resting intracellular calcium ([Ca²⁺] _i ) levels in small DRG neurons. However, it was able to restore impaired Ca²⁺ release from the ER, induced by either activation of ryanodine receptors or by receptor-independent mechanism in both DRG and SG neurons.4. The beneficiary effects of nimodipine treatment on [Ca²⁺] _i signaling were paralleled with the reversal of diabetes-induced thermal hypoalgesia and normalization of the acute phase of the response to formalin injection. Nimodipine treatment was also able to shorten the duration of the tonic phase of formalin response to the control values.5. To separate vasodilating effect of nimodipine Biessels et al., (Brain Res. 1035:86–93) from its effect on neuronal Ca²⁺ channels, a group of STZ-diabetic rats was treated with vasodilator – enalapril. Enalapril treatment also have some beneficial effect on normalizing Ca²⁺ release from the ER, however, it was far less explicit than the normalizing effect of nimodipine. Effect of enalapril treatment on nociceptive behavioral responses was also much less pronounced. It partially reversed diabetes-induced thermal hypoalgesia, but did not change the characteristics of the response to formalin injection.6. The results of this study suggest that chronic nimodipine treatment may be effective in restoring diabetes-impaired neuronal calcium homeostasis as well as reduction of diabetes-induced thermal hypoalgesia and noxious stimuli responses. The nimodipine effect is mediated through a direct neuronal action combined with some vascular mechanism.     

Behavioural responses to various chemical incentives in hybrid beluga x Russian sturgeon (Huso huso x Acipenser gueldenstaedtii) fry

Behavioural contact feeding responses to various chemical incentives were studied in hybrid beluga x Russian sturgeon ( Huso huso x Acipenser gueldenstaedtii ) fry. Solutions of 29 amino-acids (10–2 M); aqueous extracts of various vegetable, animal and microbiological components of commercial fish diets (30 min infusion of 100 mg in 100 ml), and various artificial and natural substances were tested. The most attractive amino-acids were lysine, methionine and alanine. Most of the dietary extracts tested did not stimulate feeding behaviour, indicating that widespread commercial sturgeon diets (such as ST-07 or ST-04Az) consist of components with little attractiveness for sturgeon fry. In most cases extruded diet components stimulated more bites than granulated ones. Among the diet components, only fish protein concentrate (FPC), dry milk and krill meal stimulated feeding behaviour. Flavour enhancers such as concentrated low molecular weight nitrogenous substancs (CLMNS), sodium glutamate, and "Finnstim" all stimulated feeding behaviour of fry. Some of these substances are recommended as flavour enhancers in sturgeon diets.     

X-ray irradiation stimulates the mitochondrial-dependent generation of reactive oxygen and nitrogen species in K562 leukemia cells

The dynamics of radiation-induced oxidative and nitrative stress, the source of oxygen and nitrogen reactive species in cancer cell line K562 and the role of mitochondria in these processes have been studied. The study was performed using K562 leukemia cell cultures. Intracellular concentration of reactive oxygen species (ROS), nitrogen oxide, and the mitochondrial potential were analyzed after 15, 30 min, 1, 4, 8, 12, 24, and 48 h after irradiation by X-rays at a dose of 4 and 12 Gy. Radiation-induced generation of ROS in K562 cells has two time peaks, the first peak was recorded after 30 min and the second 24 h after exposure to X-rays. Mitochondria are responsible for the increase of the ROS concentration in the period of 12-48 h after irradiation. The increase in ROS concentrations is accompanied by the increase of the mitochondrial potential. The intracellular concentration of nitric oxide begins to grow 8 h after exposure. The increase in the mitochondria-dependent ROS production is accompanied by the increase in the intracellular concentration of nitric oxide.