Characterization of the tonoplast proton pumps in Cucumis sativus L. root cells

Large-scale preparation of highly purified tonoplast from cucumber (Cucumis sativus L.) roots was obtained after centrifugation of microsome pellet (10,000 – 80,000 g) on discontinuous sucrose density gradient (20, 28, 32 and 42 %). Lack of PEP carboxylase (cytosol marker) and cytochrome c oxidase (mitochondrial marker) together with a slight activity of VO₄-ATPase (plasma membrane marker) and NADH-cytochrome c reductase (ER marker) in tonoplast preparation confirmed its high purity. Using latency of nitrate-inhibited ATPase and H⁺ pumping as criteria it was established that the majority of tonoplast vesicles were sealed and oriented right(cytoplasmic)-side-out. Strong acidification of the interior of vesicles observed at the presence of both, ATP and PP_i, confirmed that obtained tonoplast contains two classes of proton pumps: V-ATPase and H⁺PP_iase. To examine and characterise of proton-transport systems in tonoplast, the effect of various inhibitors on H⁺ pumping and hydrolytic activities of ATPase and PP_iase were measured. ATP-dependent activities (H⁺ flux and ATP hydrolysis) were specifically decreased by nitrate and bafilomycin A₁, whereas the PP_iase activities were reduced in the presence of fluoride and Na⁺ ions. Both enzymes showed a similar sensitivity to DCCD and DES. The results of experiments with KCl and NaCl suggested that the vacuolar ATPase was stimulated by Cl⁻, whereas the vacuolar Pp_iase requires K⁺ ions for its activity.     

Starch degradation by glucoamylase Glm from Saccharomycopsis fibuligera IFO 0111 in the presence and absence of a commercial pullulanase

This study describes the course of enzymatic hydrolysis of the native corn starches Maritena 100 and Maritena 300. Hydrolyses were carried out with glucoamylase Glm produced by Saccharomycopsis fibuligera IFO 0111, which degrades also native starch, with the purpose to substitute a two-step hydrolysis (amylase followed by glucoamylase) by a one-step process (glucoamylase only). Hydrolysis generally became more effective by adding the pullulanase Promozyme D, which cleaves alpha-1,6-glycosidic bonds more effectively than glucoamylase Glm does. The time course (kinetics) of hydrolysis was followed by determination of the glucose concentration and calculation of dextrose equivalents.     

2′, 3′-<Emphasis Type="Italic">O</Emphasis>-(4-benzoylbenzoyl)–ATP-mediated calcium signaling in rat glioma C6 cells: role of the P2Y<Subscript>2</Subscript> nucleotide receptor

In this study, we examined the response of glioma C6 cells to 2′,3′-O-(4-benzoylbenzoyl)-ATP (BzATP) and showed that the BzATP-induced calcium signaling does not involve the P2X₇ receptor activity. We show here that in the absence of extracellular Ca²⁺, BzATP-generated increase in [Ca²⁺]_i via Ca²⁺ release from intracellular stores. In the presence of calcium ions, BzATP established a biphasic Ca²⁺ response, in a manner typical for P2Y receptors. Brilliant Blue G, a selective antagonist of the rat P2X₇ receptor, did not reduce any of the two components of the Ca²⁺ response elicited by BzATP. Periodate-oxidized ATP blocked not only BzATP- but also UTP-induced Ca²⁺ elevation. Moreover, BzATP did not open large transmembrane pores. What is more, a cross-desensitization between UTP and BzATP occurred, which clearly shows that in glioma C6 cells BzATP activates most likely the P2Y₂ but not the P2X₇ receptors.     

Subspecies of the Eeuropean beaver<Emphasis Type="Italic">Castor fiber</Emphasis> Linnaeus, 1758

Subspecific relationships of the European beaverCastor fiber have been obscured by failure to follow the rules of the International Code of Zoological Nomenclature and remain an open issue. Inconsequence in the use of subspecies names caused a nomenclatural as well as a taxonomic confusion. We discuss these controversial and incorrect nomenclatural decisions, and recognize 9 nominal subspecies of European beaver, bearing 9 available names:C. f. fiber Linnaeus, 1758,C. f. galliae Geoffroy, 1803, C. f. albicus Matschie, 1907,C. f. vistulanus Matschie, 1907,C. f. pohlei Serebrennikov, 1929,C. f. birulai Serebrennikov, 1929,C. f. tuvinicus Lavrov, 1969,C. f. belorussicus Lavrov, 1981, andC. f. orientoeuropaeus Lavrov, 1981.     

Genetic and nongenetic factors influencing callus induction inMiscanthus sinensis (Anderss.) anther cultures

Miscanthus sinensis is a promising species for biomass production. Influences of genetic and nongenetic factors on androgenesis induction efficiency were investigated. This is the first report on successful induction of pollen-derived callus inM. sinensis. The callus yield was strongly affected by genotype. A beneficial influence of cold pretreatment of spikes on androgenesis induction was observed. The highest yield of calli was obtained in cultures on a modified C17 medium. The results suggest that the high callus yield might be caused by the late culture initiation. The beginning of anther culture at the end of the flowering season caused a 17-fold increase in callus yield, in comparison to culture initiated at the height of the flowering season (August). It is likely, however, that the efficiency of androgenesis induction in the case ofM. sinensis anther culture beginning in October could be related to a positive influence of growing donor plants in conditions of cooler and shorter day, i.e. 11-h day with temperature around 11°C and 13-h night with temperature around 5°C. Results of this study can significantly support the development of effective methods ofM. sinensis haploidization, which could be used in crop improvement by breeding.     

A real-time PCR diagnostic method for detection of Naegleria fowleri

Naegleria fowleri is a free-living amoeba that can cause primary amoebic meningoencephalitis (PAM). While, traditional methods for diagnosing PAM still rely on culture, more current laboratory diagnoses exist based on conventional PCR methods; however, only a few real-time PCR processes have been described as yet. Here, we describe a real-time PCR-based diagnostic method using hybridization fluorescent labelled probes, with a LightCycler instrument and accompanying software (Roche), targeting the Naegleria fowleriMp2Cl5 gene sequence.Using this method, no cross reactivity with other tested epidemiologically relevant prokaryotic and eukaryotic organisms was found. The reaction detection limit was 1 copy of the Mp2Cl5 DNA sequence. This assay could become useful in the rapid laboratory diagnostic assessment of the presence or absence of Naegleria fowleri.     

Intravenous rutin in rat exacerbates isoprenaline-induced cardiotoxicity likely due to intracellular oxidative stress

Objectives: Rutin, quercetin-3-O-rutinoside, a natural flavonol glycoside, has shown various in vitro benefits with potential use treating human diseases, especially cardiovascular system disorders. Antioxidant properties are assumed to underlie the majority of these benefits. Yet rutin pro-oxidant properties have been reported as well. Our research group has recently shown aggravating effects on isoprenaline (ISO)-induced cardiotoxicity in Wistar:Han rats after 24?hours.

Methods: This study was designed to examine in more detail the reasons for the negative effects of rutin (11.5 and 46?mg/kg, i.v.) after administration of ISO (100?mg/kg, s.c.) in rats within 2?hours of continuous experiment and in the H9c2 cardiomyoblast-derived cell line.

Results: Like our previous findings, rutin did not (11.5 or 46?mg/kg, i.v.) reduce the ISO-induced mortality within 2?hours although the lower dose significantly reduced cardiac troponin T (cTnT) and partly improved the histological findings. In contrast, the higher dose increased the mortality in comparison with solvent (1.26% w/v sodium bicarbonate). This was not caused by any specific haemodynamic disturbances. It appears to be associated with oxidative stress as rutin enhanced intracellular reactive oxygen species formation in vitro and had the tendency to increase it in vivo.

Conclusions: Rutin, likely due to its pro-oxidative effects, can exacerbate catecholamine cardiotoxicity depending on the dose used.     

Exogenous abscisic acid increases stability of polysomes in embryos of triticale caryopses during germination

Some posttranslational processes that occur in embryos of germinating triticale caryopses treated with different concentrations of abscisic acid (ABA) were examined. ABA increased the ratio of cytoskeleton-bound polysomes in the total population of polysomes and depressed the share of free and membrane-bound polysomes. Using exogenous RNase, stability of the total polysomal population as well as each polysomal fraction was investigated. The total extractable polysomes isolated from embryonic tissues of germinating triticale caryopses treated with ABA were more stable than the polysomes isolated from the control sample caryopses. The contribution of the polysomes that were not digested by RNase was increased by higher concentrations of ABA applied during germination. At high concentrations of ABA (50, 100 μM), the quantitative contribution of polysomes in the total ribosomal fraction was almost 100% of the amount of polysomes before digestion and the modifications observed consisted mainly of the shift of the so-called heavy polysomes towards light polysomes, containing a few ribosomes. Within each polysomal population, cytoskeleton-bound polysomes (CBP and CMBP) were the most stable, which may imply that the bonds between polysomes and these protein filaments, created in all eukaryotic cells increased their stability. It is assumed that mRNAs are stabilised or destabilised by interaction of proteins with their various sequences. A plant hormone may depress or elevate the quantities of these proteins, thus regulating the stability of different mRNAs. The results confirm the multi-faceted mechanism of ABA-induced response, where one of the constituents is the effect of ABA on the stability of mRNAs molecules. The co-ordinated regulation of mRNAs synthesis and their stability provide plants with improved adaptability.     

Characterization of an aerated submerged hollow fiber ultrafiltration device for efficient microalgae harvesting

The present work characterizes a submerged aerated hollow fiber polyvinylidene fluorid (PVDF) membrane (0.03 μm) device (Harvester) designed for the ultrafiltration (UF) of microalgae suspensions. Commercial baker''s yeast served as model suspension to investigate the influence of the aeration rate of the hollow fibers on the critical flux (CF, J _c) for different cell concentrations. An optimal aeration rate of 1.25 vvm was determined. Moreover, the CF was evaluated using two different Chlorella cultures (axenic and non‐axenic) of various biomass densities (0.8–17.5 g DW/L). Comparably high CFs of 15.57 and 10.08 L/m/²/h were measured for microalgae concentrations of 4.8 and 10.0 g DW/L, respectively, applying very strict CF criteria. Furthermore, the J _c‐values correlated (negative) linearly with the biomass concentration (0.8–10.0 g DW/L). Concentration factors between 2.8 and 12.4 and volumetric reduction factors varying from 3.5 to 11.5 could be achieved in short‐term filtration, whereat a stable filtration handling biomass concentrations up to 40.0 g DW/L was feasible. Measures for fouling control (aeration of membrane fibers, periodic backflushing) have thus been proven to be successful. Estimations on energy consumption revealed very low energy demand of 17.97 kJ/m³ treated microalgae feed suspension (4.99 × 10⁻³ kWh/m³) and 37.83 kJ/kg treated biomass (1.05 × 10⁻² kWh/kg), respectively, for an up‐concentration from 2 to 40 g DW/L of a microalgae suspension.