Mechanistic study of the structure–activity relationship for the free radical scavenging activity of baicalein

Density functional theory calculations were performed to evaluate the antioxidant activity of baicalein. The conformational behaviors of both the isolated and the aqueous-solvated species (simulated with the conductor-like polarizable continuum solvation model) were analyzed at the M052X/6-311 + G(d,p) level. The most stable tautomers of various forms of baicalein displayed three IHBs between O4 and OH5, O5 and OH6, and O6 and OH7. The most stable tautomer of the baicalein radical was obtained by dehydrogenating the hydroxyl at C6, while the most stable anion tautomer was obtained by deprotonating the C7 hydroxyl in gaseous and aqueous phases. The expected antioxidant activity of baicalein was explained by its ionization potentials (IPs) and homolytic O–H bond dissociation enthalpies (BDEs), which were obtained via the UM052X optimization level of the corresponding radical species. Heterolytic O–H bond cleavages (proton dissociation enthalpies, PDEs) were also computed. The calculated IP, BDE, and PDE values suggested that one-step H-atom transfer, rather than sequential proton loss–electron transfer or electron transfer–proton transfer, would be the most favorable mechanism for explaining the antioxidant activity of baicalein in the gas phase and in nonpolar solvents. In aqueous solution, the SPLET mechanism was more important.     

Trace elements in aerial parts and rhizosphere of <Emphasis Type="Italic">Thymus pannonicus</Emphasis> All.

This paper brings out the results of the study on the levels of selected trace elements (Cu, Fe, Mn, Zn and Cr) in aerial parts of Thymus pannonicus All. (Lamiaceae) and rhizosphere soil from twelve locations in Serbia. Prior to assays by flame and flameless atomic absorption spectrometry, samples were subjected to microwave-assisted acid digestion. Real and potential acidity of soil samples were also measured. Obtained results for soil samples, although slightly higher for some elements (Cu: 12.38–45.18 mg/kg; Fe: 22102–46193 mg/kg; Mn: 776.95–4901.27 mg/kg; Zn: 62.27–214.02 mg/kg; Cr: 48.86–69.13 mg/kg), were found to fit into biogeochemical background. Element contents in plant samples differed depending on collecting site (Cu: 5.26–14.07 mg/kg; Fe: 25.92–1454.07 mg/kg; Mn: 89.29–278.25 mg/kg; Zn: 1.81–10.64 mg/kg; Cr: 1.11–3.51 mg/kg), which can be partly explainable by different nutrient availability influenced by soil acidity. Zinc levels in T. pannonicus were below expected and seem to be strongly influenced by plant physiological properties.     

Clinical and functional characterization of URAT1 variants

Idiopathic renal hypouricaemia is an inherited form of hypouricaemia, associated with abnormal renal handling of uric acid. There is excessive urinary wasting of uric acid resulting in hypouricaemia. Patients may be asymptomatic, but the persistent urinary abnormalities may manifest as renal stone disease, and hypouricaemia may manifest as exercise induced acute kidney injury. Here we have identified Macedonian and British patients with hypouricaemia, who presented with a variety of renal symptoms and signs including renal stone disease, hematuria, pyelonephritis and nephrocalcinosis. We have identified heterozygous missense mutations in SLC22A12 encoding the urate transporter protein URAT1 and correlate these genetic findings with functional characterization. Urate handling was determined using uptake experiments in HEK293 cells. This data highlights the importance of the URAT1 renal urate transporter in determining serum urate concentrations and the clinical phenotypes, including nephrolithiasis, that should prompt the clinician to suspect an inherited form of renal hypouricaemia.     

Stream section types of the Danube River in Serbia according to the distribution of macroinvertebrates

The aim of this study was to use the data on the distribution of aquatic macroinvertebrates obtained from 14 sites within a 413 km long stretch of the Danube River in Serbia to show the relevance of the bordering zone between the Middle and Lower Danube. A total of 68 macroinvertebrate taxa were observed. Molluscs were the major component with regard to species richness and relative abundance. Lithoglyphus naticoides (C. Pfeiffer, 1828) was the most abundant species and Unio tumidus (Retzius, 1788) was the most frequent species. Product-moment correlation coefficients or Pearson r coefficient was used to analyse the relation between the sites based on macroinvertebrate distribution. The data obtained by product-moment correlation served as input for cluster analyses. According to a cluster analyses Danube River in Serbia could be separated in the free-flowing sector, the stretch with a backwater effect and the area of the Iron Gate.     

Coexistence of Probe Conformations in Lipid Phases—A Polarized Fluorescence Microspectroscopy Study

Several well-established fluorescence methods depend on environment-sensitive probes that report about molecular properties of their local environment. For reliable interpretation of experiments, careful characterization of probes’ behavior is required. In this study, bleaching-corrected polarized fluorescence microspectroscopy with nanometer spectral peak position resolution was applied to characterize conformations of two alkyl chain-labeled 7-nitro-2-1,3-benzoxadiazol-4-yl phospholipids in three model membranes, representing the three main lipid phases. The combination of polarized and spectral detection revealed two main probe conformations with their preferential fluorophore dipole orientations roughly parallel and perpendicular to membrane normal. Their peak positions were separated by 2–6 nm because of different local polarities and depended on lipid environment. The relative populations of conformations, estimated by a numerical model, indicated a specific sensitivity of the two probes to molecular packing with cholesterol. The coexistence of probe conformations could be further exploited to investigate membrane organization below microscopy spatial resolution, such as lipid rafts. With the addition of polarized excitation or detection to any environment-sensitive fluorescence imaging technique, the conformational analysis can be directly applied to explore local membrane complexity.     

Deriving fluorometer-specific values of relative PSI fluorescence intensity from quenching of F 0 fluorescence in leaves of Arabidopsis thaliana and Zea mays

The effect of stepwise increments of red light intensities on pulse-amplitude modulated (PAM) chlorophyll (Chl) fluorescence from leaves of A. thaliana and Z. mays was investigated. Minimum and maximum fluorescence were measured before illumination (F ₀ and F _M, respectively) and at the end of each light step ( $ F^{\prime}_{0} $ and $ F^{\prime}_{\text{M}} $ , respectively). Calculated $ F^{\prime}_{0} $ values derived from F ₀, F _M and $ F^{\prime}_{\text{M}} $ fluorescence according to Oxborough and Baker (1997) were lower than the corresponding measured $ F^{\prime}_{0} $ values. Based on the concept that calculated $ F^{\prime}_{0} $ values are under-estimated because the underlying theory ignores PSI fluorescence, a method was devised to gain relative PSI fluorescence intensities from differences between calculated and measured $ F^{\prime}_{0} $ . This method yields fluorometer-specific PSI data as its input data (F ₀, F _M, $ F^{\prime}_{0} $ and $ F^{\prime}_{\text{M}} $ ) depend solely on the spectral properties of the fluorometer used. Under the present conditions, the PSI contribution to F ₀ fluorescence was 0.24 in A. thaliana and it was independent on the light acclimation status; the corresponding value was 0.50 in Z. mays. Correction for PSI fluorescence affected Z. mays most: the linear relationship between PSI and PSII photochemical yields was clearly shifted toward the one-to-one proportionality line and maximum electron transport was increased by 50 %. Further, correction for PSI fluorescence increased the PSII reaction center-specific parameter, 1/F ₀ ? 1/F _M, up to 50 % in A. thaliana and up to 400 % in Z. mays.     

Evaluation of the DNA damaging effects of amitraz on human lymphocytes in the Comet assay

Amitraz is formamidine pesticide widely used as insecticide and acaricide. In veterinary medicine, amitraz has important uses against ticks, mites and lice on animals. Also, amitraz is used in apiculture to control Varroa destructor. It this study, the alkaline Comet assay was used to evaluate DNA damaging effects of amitraz in human lymphocytes. Isolated human lymphocytes were incubated with varying concentrations of amitraz (0.035, 0.35, 3.5, 35 and 350 μg/mL). The Comet assay demonstrated that all concentrations of amitraz caused statistically significant increase in the level of DNA damage, thus indicating that amitraz possesses genotoxic potential. The concentration of amitraz that produced the highest DNA damage (3.5 μg/mL) was chosen for further analysis with the antioxidant catalase. The obtained results showed that co-treatment with antioxidant catalase (100 IU/mL or 500 IU/mL) significantly reduced the level of DNA damage, indicating the possible involvement of reactive oxygen species in DNA damaging effects of amitraz. Flow cytometric analysis revealed increase of the apoptotic index following treatment with amitraz. However, co-treatment with catalase reduced the apoptotic index, while treatment with catalase alone reduced the percentage of apoptotoc cells even in comparison with the negative control. Therefore, catalase had protective effects against ROS-mediated DNA damage and apoptosis.     

Histopathological indicators: a useful fish health monitoring tool in common carp (Cyprinus carpio Linnaeus, 1758) culture

In order to evaluate the relationship between water quality in ponds and indices of histopathological changes occurring in the vital organs of the common carp (Cyprinus carpio L., 1758), two six-month field experiments were carried out using two different water supplies: from the nearby stream and a tube well. The fish were fed supplemental feed: raw cereals, pelleted and extruded compound feed. Histopathological analysis, alteration frequencies, and semi-quantitative scoring of the changes were used to assess the health status of the fish. Ponds supplied by stream water were characterized by higher water hardness, dissolved oxygen and pH values, while those supplied by the tube well had higher electroconductivity, total ammonium and orthophosphates content. Fish survival rate and habitat suitability index were lower in ponds supplied by stream water, while the weight gain did not differ between the two water supplies. The use of stream water resulted in a higher level of histopathological changes in gills and liver. Among the water quality parameters, pH level had the strongest influence on fish. Differences in water supply produced greater influence on the level of histopathological changes than the type of feed applied. Gills were the most sensitive organ, while the kidney was the least responsive.     

GreenGate - A Novel,Versatile, and Efficient Cloning System for Plant Transgenesis

Building expression constructs for transgenesis is one of the fundamental day-to-day tasks in modern biology. Traditionally it is based on a multitude of type II restriction endonucleases and T4 DNA ligase. Especially in case of long inserts and applications requiring high-throughput, this approach is limited by the number of available unique restriction sites and the need for designing individual cloning strategies for each project. Several alternative cloning systems have been developed in recent years to overcome these issues, including the type IIS enzyme based Golden Gate technique. Here we introduce our GreenGate system for rapidly assembling plant transformation constructs, which is based on the Golden Gate method. GreenGate cloning is simple and efficient since it uses only one type IIS restriction endonuclease, depends on only six types of insert modules (plant promoter, N-terminal tag, coding sequence, C-terminal tag, plant terminator and plant resistance cassette), but at the same time allows assembling several expression cassettes in one binary destination vector from a collection of pre-cloned building blocks. The system is cheap and reliable and when combined with a library of modules considerably speeds up cloning and transgene stacking for plant transformation.