Purification of lactoperoxidase from creek-water buffalo milk and investigation of kinetic and antibacterial properties

Water buffalo lactoperoxidase (WBLP) was purified with Amberlite CG 50 H+ resin, CM Sephadex C-50 ion-exchange chromatography, and Sephadex G-100 gel filtration chromatography from skim milk. All purification steps of the WBLP were shown with SDS-PAGE and Rz (A412/A280) controlled the purification degree of the enzyme. Rz value for the purified WBLP was 0.8. To determine purification steps and kinetic properties, the activity of enzyme was measured by using 2,2-azino-bis-(3-ethylbenzthiazoline-6 sulfonic acid) diammonium salt (ABTS) as a choromogenic substrate at pH=6. Km, Vmax, optimum pH, and optimum temperature for the WBLP were found by means of graphics for ABTS as substrates. Optimum pH and optimum temperature of the WBLP were 6 and 60 degrees C, respectively. Km value at optimum pH and optimum temperature for the WBLP was 0.82 mM. Vmax value at optimum pH and optimum temperature was 13.7 micromol/mL x min. Km value at optimum pH and 25 degrees C for the WBLP was 0.77 mM. Vmax value at optimum pH and 25 degrees C was 4.83 micromol/mL x min. The purified WBLP was found to have high antibacterial activity in a thiocynate-H2O2 medium for some pathogenic bacteria, such as Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginose, Shigella sonnei, Staphylococcus saphrophyticus, Staphylococcus epidermidis, and Shigella dysenteriae and compared with well known antibacterial substances such as tetracycline, penicillin, and netilmicine.     

Synthesis and Cytotoxicity Screening of Some Synthesized Coumarin and Aza-Coumarin Derivatives as Anticancer Agents

Russian Journal of Bioorganic Chemistry - A series of coumarin and aza-coumarin derivatives (II–IX) were synthesized and identified using IR, 1H NMR, and 13C NMR spectral data. All the...     

Rule-based modeling and simulations of the inner kinetochore structure

Background

Combinatorial complexity is a central problem when modeling biochemical reaction networks, since the association of a few components can give rise to a large variation of protein complexes. Available classical modeling approaches are often insufficient for the analysis of very large and complex networks in detail. Recently, we developed a new rule-based modeling approach that facilitates the analysis of spatial and combinatorially complex problems. Here, we explore for the first time how this approach can be applied to a specific biological system, the human kinetochore, which is a multi-protein complex involving over 100 proteins.

Results

Applying our freely available SRSim software to a large data set on kinetochore proteins in human cells, we construct a spatial rule-based simulation model of the human inner kinetochore. The model generates an estimation of the probability distribution of the inner kinetochore 3D architecture and we show how to analyze this distribution using information theory. In our model, the formation of a bridge between CenpA and an H3 containing nucleosome only occurs efficiently for higher protein concentration realized during S-phase but may be not in G1. Above a certain nucleosome distance the protein bridge barely formed pointing towards the importance of chromatin structure for kinetochore complex formation. We define a metric for the distance between structures that allow us to identify structural clusters. Using this modeling technique, we explore different hypothetical chromatin layouts.

Conclusions

Applying a rule-based network analysis to the spatial kinetochore complex geometry allowed us to integrate experimental data on kinetochore proteins, suggesting a 3D model of the human inner kinetochore architecture that is governed by a combinatorial algebraic reaction network. This reaction network can serve as bridge between multiple scales of modeling. Our approach can be applied to other systems beyond kinetochores.     

Strain-induced inflammation in pulmonary alveolar tissue due to mechanical ventilation

Inflammation is the body’s attempt at self-protection to remove harmful stimuli, including damaged cells, irritants, or pathogens and begin the healing process. In this study, strain-induced inflammation in pulmonary alveolar tissue under high tidal volume is investigated through a combination of an inflammation model and fluid structure interaction (FSI) analysis. A realistic three-dimensional organ model for alveolar sacs is built, and FSI is employed to evaluate strain distribution in alveolar tissue for different tidal volume (TV) values under the mechanical ventilation (MV) condition. The alveolar tissue is treated as a hyperelastic solid and provides the environment for the tissue constituents. The influence of different strain distributions resulting from different tidal volumes is investigated. It is observed that strain is highly distributed in the inlet area. In addition, strain versus time curves in different locations through the alveolar model reveals that middle layers in the alveolar region would undergo higher levels of strain during breathing under the MV condition. Three different types of strain distributions in the alveolar region from the FSI simulation are transferred to the CA model to study population dynamics of cell constituents under MV for different TVs; 200, 500 and 1000 mL, respectively. The CA model results suggests that strain distribution plays a significant role in population dynamics. An interplay between strain magnitude and distribution appears to influence healing capability. Results suggest that increasing TV leads to an exponential rise in tissue damage by inflammation.     

Supramolecular interaction of 18‐crown‐6 ether with mesalazine and spectrofluorimetric determination of mesalazine in pharmaceutical formulations

The supramolecular interaction of protonated mesalazine (MSZ) and 18‐crown‐6 ether (18C6) has been examined by Ultraviolet–visible, FT‐IR and fluorescence spectroscopy. The formation of the inclusion complex has been confirmed based on the changes of the spectral properties. The MSZ–18C6 host–guest complex formed in (1:1) stoichiometry and the inclusion constant (K = 1.411 × 10² L mol^–1) was ascertained by the typical double reciprocal plots. Furthermore, the thermodynamic parameters (ΔG°, ΔH° and ΔS°) of (MSZ‐18C6) were obtained. Based on the remarkable enhancement of the fluorescence intensity of MSZ produced through complex formation, a simple, accurate, rapid and highly sensitive spectrofluorometric method for the determination of MSZ in aqueous solution in the presence of 18C6 was developed. The measurement of relative fluorescence intensity was carried with excitation at 298 nm, emission 410 nm. All variables affecting the reactions were studied and optimized. Beer's law was obeyed in the concentration range of 0.1–0.9 µg/mL. The absorbance was found to increase linearly with increasing concentration of MSZ. The molar absorptivity, Sandell sensitivity, limit of detection (LOD) and limit of quantification (LOQ) were calculated. The validity of the described method was assessed, and the method was successfully applied to the determination of MSZ in its pharmaceutical formulation. In addition, a solid inclusion complex was synthesized by the coprecipitation method. Copyright © 2015 John Wiley & Sons, Ltd.     

Antioxidant Properties of Selected <Emphasis Type="Italic">Boletus</Emphasis> Mushrooms

Considering the growing interest for mushrooms and the demand search of natural antioxidants sources, the aim of this study was to investigate the antioxidant properties of two edible widely used Boletus species, Boletus edulis, and Boletus auranticus, collected from Istra region in Croatia in late summer 2007. To evaluate the antioxidant properties and content of antioxidant compounds, scavenging capacity on DPPH˙, OH˙, and O₂˙⁻ radicals, reducing power and capacity to inhibit lipid peroxidation has been investigated. It is determined that content of total phenols (41.82 ± 0.08 mg gallic acid equivalent per gram of dry extract) was higher for B. edulis. Using high performance liquid chromatography/diode array detector analysis, the main antioxidant compound, variegatic acid, has been detected and quantified. 1,1-Diphenyl-2-picryl-hydrazyl-hydrate assay was used as a preliminary free radical–scavenging evaluation. By this assay, it has been found that B. edulis dry mushroom extract exhibits 50% of inhibition value at the extract concentration of 0.016 ± 0.0003 mg/ml. The extracts were capable of reducing iron(III) and, thus, are capable of donating electrons. Using electron paramagnetic resonance spin-trapping and spin-probing techniques, activity against relevant reactive species, ˙OH and O₂˙⁻ radical, was analyzed for both mushroom extracts. Both investigated extracts are determined as good inhibitors for ˙OH radical reduction, and both exhibited significant capacity for scavenging O₂˙⁻ radical and for that could help to prevent or meliorate oxidative damage. Only B. edulis extract prevents lipid peroxidation. Investigated mushroom extracts could represent easily accessible natural antioxidant resource.     

Allelic Variation of Cytochrome P450s Drives Resistance to Bednet Insecticides in a Major Malaria Vector

Scale up of Long Lasting Insecticide Nets (LLINs) has massively contributed to reduce malaria mortality across Africa. However, resistance to pyrethroid insecticides in malaria vectors threatens its continued effectiveness. Deciphering the detailed molecular basis of such resistance and designing diagnostic tools is critical to implement suitable resistance management strategies. Here, we demonstrated that allelic variation in two cytochrome P450 genes is the most important driver of pyrethroid resistance in the major African malaria vector Anopheles funestus and detected key mutations controlling this resistance. An Africa-wide polymorphism analysis of the duplicated genes CYP6P9a and CYP6P9b revealed that both genes are directionally selected with alleles segregating according to resistance phenotypes. Modelling and docking simulations predicted that resistant alleles were better metabolizers of pyrethroids than susceptible alleles. Metabolism assays performed with recombinant enzymes of various alleles confirmed that alleles from resistant mosquitoes had significantly higher activities toward pyrethroids. Additionally, transgenic expression in Drosophila showed that flies expressing resistant alleles of both genes were significantly more resistant to pyrethroids compared with those expressing the susceptible alleles, indicating that allelic variation is the key resistance mechanism. Furthermore, site-directed mutagenesis and functional analyses demonstrated that three amino acid changes (Val¹⁰⁹Ile, Asp³³⁵Glu and Asn³⁸⁴Ser) from the resistant allele of CYP6P9b were key pyrethroid resistance mutations inducing high metabolic efficiency. The detection of these first DNA markers of metabolic resistance to pyrethroids allows the design of DNA-based diagnostic tools to detect and track resistance associated with bednets scale up, which will improve the design of evidence-based resistance management strategies.     

Significance of serum trace element status in patients with rheumatic heart disease: a prospective study

It is known that certain trace elements can affect various heart diseases. In this study, we aimed to evaluate the changes in concentrations of certain serum trace elements in patients with chronic rheumatic heart disease (RHD). Serum analysis of selenium (Se), zinc (Zn), and copper (Cu) trace elements was assayed by atomic absorption spectrophotometry. RHD patients had significantly lower serum concentrations of Se and Zn than control subjects (p < 0.05 and p < 0.001, respectively). However, the serum Cu concentration was significantly higher in RHD patients than in controls (1.93 +/- 0.59 microg/L vs 1.06 +/- 0.29 microg/L; p < 0.001). Similarly, the Cu/Zn ratio in RHD patients was higher than in control subjects (4.70 +/- 0.92 vs 1.68 +/- 0.45; p < 0.001). Additionally, no significant correlation was found among these trace element concentrations and the functional capacity classes (p > 0.05). RHD patients had decreased serum Se and Zn element concentrations and increased serum Cu element concentration. We suggest that Se and Zn deficiency might be contributory factors in the development of rheumatic heart disease, and a high Cu concentration and a high Cu/Zn ratio might reflect an ongoing inflammatory process in this disease.     

Bioavailability to Fish of Sediment PAH as an Indicator of the Success of In Situ Remediation Treatments at an Experimental Oil Spill

A weathered medium crude oil was applied to experimental plots of Scirpus pungens (Three-square Bulrush) in a freshwater wetland to determine the efficacy of strategies for shoreline oil spill bioremediation based on nutrient enrichment (bioremediation) and plant growth (phytoremediation). Plots were unoiled, oiled with no added nutrients, or oiled with repeated applications of phosphate and nitrate fertilizers. Following initial treatments, the experimental plots were raked to simulate the activity of wave action on oil penetration, and plants in one fertilized plot were cut repeatedly. The sediments were sampled at regular intervals for 15 months after oiling, and the loss of oil was assessed by 4-day laboratory tests of polynuclear aromatic hydrocarbon (PAH) bioaccumulation by trout, as demonstrated by increases in activity of liver cytochrome P450 (CYP1A) enzymes. Oil alone, oil mixed with sediments in the lab, and oiled sediments from treated plots all induced CYP1A activity relative to untreated controls, indicating the presence and bioavailability of PAH. Induction did not vary with nutrient treatments, but declined by 80% within 15 months of oiling, and chemical analyses indicated equivalent losses of hydrocarbons in sediment. These results demonstrate that bioavailable PAHs persisted in measurable quantities for at least 1.25 years following oiling, and that stimulation of plant growth did not affect the rate of oil disappearance. The controlling factors were likely weathering and sediment movement.