Assessment of the toxicity and antiproliferative activity of hemocyanins from Helix lucorum, Helix aspersa and Rapana venosa

Hemocyanins (Hcs) are respiratory, oxygen-carrying metalloproteins that are freely dissolved in the hemolymph of many molluscs and arthropods. The interest in hemocyanins has grown significantly since it was found that they can be successfully used in immunotherapy of neoplastic diseases as non-specific or active stimulators of the immune system. The present study aims to assess the cytotoxicity, in vivo toxicity and antiproliferative activity of hemocyanins isolated from marine snail Rapana venosa (RvH), garden snails Helix lucorum (HlH) and Helix aspersa (HaH). For in vitro safety testing, 3T3 Neutral Red Uptake (NRU) test was used. The experiments for antiproliferative activity of the hemocyanins were performed by MTT assay on a panel of cell lines - a model of breast cancer. The in vivo toxicological assessment was performed by regular clinical examinations of hemocyanin-treated laboratory mice and histopathological analysis of hematoxylin/eosin stained preparations of parenchymal organs. The evaluation of the in vitro cytotoxicity showed that the tested hemocyanins does not induce toxic effects in nontumorigenic epithelial cell lines. In contrast, significant reduction of the viability of human breast carcinoma cell lines was found after treatment with high concentrations of hemocyanins. The in vivo experiments showed no signs of organ and systemic toxicity in the hemocyanin-treated animals. The presented data indicate that Hcs show a potential for development of novel anticancer therapeutics due to their beneficial properties, biosafety and lack of toxicity or side effects. Key words: hemocyanins (Hcs); cytotoxicity; antitumor activity; in vivo biosafety testing.

FISH investigation of the bacterial groups anammox and Azoarcus-Thauera at treatment of landfill leachate

The landfill leachate is heavily polluted wastewater produced in the landfills. The management of the purification of the leachate is especially challenging and that is why new approaches and indicators are needed. The quantity, localization, interaction, clustering of the key microbial groups, responsible for the critical transformation processes can be used as indication leading to better performance of the technology. This study is focused on two bacterial groups (Anammox and Azoarcus-Thauera cluster) which have potential to serve as indicators for the landfill leachate treatment. Their quantity and activity were studied by FISH during lab-scale treatment of leachate from the Municipal Enterprise for Waste Treatment (MEWT), Sofia, Bulgaria. Two activated sludges (AS) were used – one from the MEWT and another form the WWTP (wastewater treatment plant) of Sofia. The obtained results showed that 74% of the COD was eliminated when leachate was diluted 50 and 25 times and 31% - when undiluted leachate was used. At the end of the process (21 day) the Azoarcus-Thauera group formed large aggregations in the AS from MEWT. They were 17.50% of the bacteria there while in the AS from the WWTP of Sofia they represented only 2.61%. The quantity of the anammox bacteria remained almost unchanged during the process and was 10.75% of the community from MEWT which eliminated 98 mg/L more ammonium ions at the end of the process and 6% from the community from the WWTP of Sofia. The two studied groups gave more complex information about the processes in the AS related to the elimination of the nitrogen and carbon containing pollutants. They could be used for better management of the biological processes during landfill leachate treatment. Key words: landfill leachate; anammox, Azoarcus-Thauera; activated sludge; fluorescence in-situ hybridization

Influence of storage time and temperature on the activity of urease

Enzymes are made of protein, that is why they are sensitive molecules and are affected by storage conditions. A small change in enzyme activity during storage may cause a big error in analysis results. The aim of the study was to evaluate the effects of storage time and temperature on urease activity. Urease solutions were prepared at different activities (from 100 to 2000 U/mL) and stored at room temperature, in the refrigerator (4°C), and in the deep freezer (-18°C and -80°C). Activity measurements were made at regular intervals until 28 days by the modified Weatherburn method. The relative activities of 100-1000 U/mL urease solutions stored at room temperature, 4, -18 or -80°C were 75% and below after 4 days. Twenty-eight days later, for 2000 U/mL urease solutions, only at room temperature, the relative activity was reduced to 37%, while at 4, -18 or -80°C, the relative activities were above 80%. Since urease can be maintained at 4°C for 28 days without significant loss of activity, it has practical importance. Low-activity urease solutions (such as 100-1000 U/mL) should not be stored at -18 or -80°C for short or long term storage, they should be stored at 4°C only for one day. Keywords: Urease activity, storage time, storage temperature

Synthesis, antimicrobial and antimalarial activityof1, 4-benzothiazepine and pyrazolinederivatives incorporating carbazolemoiety

A series of carbazole-based 1,4-benzothiazepine and pyrazoline derivatives were synthesized and the structures of the newly synthesized compounds were confirmed by FT-IR, 1H NMR, 13C NMR and mass spectral studies. All new derivatives 4(a-f) and 5(a-e) were screened for their in vitro antimicrobial activity, and also for their antimalarial activity. Compounds 4a, 4b, 4d, 5a, 5b and 5c exhibited promising antimicrobial and antimalarial activities as compared to positive control. Notably, compounds 4a, 4b and 4d showed excellent antifungal activity against Penicillium sp. comparable to that of a standard drug.

Theoretical insight to intermolecular hydrogen bond interactions between methyl N-(2-pyridyl) carbamate and acetic acid: substituent effects, cooperativity and energy decomposition analysis

In the present work, the hydrogen bond (HB) interactions between substituted syn and anti rotamers of methyl N-(2-pyridyl) carbamate and acetic acid were investigated using quantum mechanical (QM) calculations. The rotamers have two typical active sites to form hydrogen bonds with acetic acid, such that four stable complexes are found on the potential energy surface. The complexes in which the oxygen atom of carbamate acts as proton acceptor are stabilized by EWSs and are destabilized by EDSs. The trend in the effects of substituents is reversed in the other two complexes, in which the nitrogen atom of ring is involved in the interaction. According to energy data, the substituent effects on the interaction energy can be expressed by Hammett constants. The natural resonance theory (NRT) model was used to investigate the charge distribution on the carbamate group and to discuss the interaction energies. The individual HB energies were estimated to evaluate their cooperative contributions on the interaction energies of the complexes. In addition, the localized molecular orbital energy decomposition analyses (LMO-EDA) demonstrate that the electrostatic interactions are the most important stabilizing components of interactions.

Friedel-Crafts acylation of 2-methoxynaphthalene with acetic anhydride catalyzed by phosphotungstic acid in ionic liquid

The Friedel-Crafts acylation of 2-methoxynaphthalene (2-MN) with acetic anhydride (AA) was carried out in the ionic liquid (IL) butylpyridinium tetrafluoroborate ([BPy]BF4) using phosphotungstic acid (H3PW12O40) as the catalyst. The [BPy]BF4-mediated 2-MN acylation displays good conversion and selectivity towards 1-acyl-2-methoxynaphthalene (1-AC-2-MN), with 70.4% conversion of 2-MN and 96.4% selectivity to 1-AC-2-MN obtained under the optimal conditions. Owing to the rearrangement of 1-AC-2-MN, 6-acyl-2-methoxynaphthalene (6-AC-2-MN) can be detected after 1 h of reaction time, with the highest 6-AC-2-MN yield of 11.3% obtained under the examined reaction conditions. The system can be recycled and reused at least 6 times without significant loss of activity, indicating the good stability of the H3PW12O40/[BPy]BF4 catalytic system.

Theory of staircase cyclic voltammetry of two electrode reactions coupled by a chemical reaction

Two reversible electrode reactions that are connected by either reversible or totally irreversible chemical reactions are theoretically analysed by staircase cyclic voltammetry. The dependence of peak potentials on the thermodynamic and kinetic parameters is calculated. If the mechanism is permanently in equilibrium, the stability constant of the reversible chemical reaction can be determined. Furthermore, the critical kinetic parameter is determined and its application to the measurement of the forward rate constant of the chemical reaction is demonstrated. Also, the influence of the kinetics of electrode reactions is discussed. Keywords: ECE mechanism; Cyclic voltammetry; Theory

Voltammetric detection of hydrochlorothiazide at molybdenum oxide modified screen-printed electrodes

Electrochemical oxidation of hydrochlorothiazide (HCT) was investigated both experimentally and theoretically to explore the connection between thermodynamics and the oxidation potential. In this work, screen printed carbon electrodes were modified with molybdenum oxide by means of voltammetry. The modified surfaces demonstrated significant sensitivity toward hydrochlorothiazide detection in pH 2.00–buffered aqueous solutions. The employed method was validated and used for hydrochlorothiazide quantification in commercial drug products. In this work, we presented results of density functional calculations for the standard reduction potential of the HCT (H)2|HCT couple in aqueous solution. After consideration of 20 density functionals with 4 different basis sets, it was found that the hybrid meta functionals provide the most accurate prediction of the reduction potential in comparison with the available experimental data. The reported reduction potential is underestimated with GGA and MGGA and is overestimated with hybrid and meta hybrid functionals. Our results highlighted the importance of including solute-solvent hydrogen bonding effects in the theoretical modeling of redox processes. Keywords: Molybdenum oxide, Hydrochlorothiazide, Screen-printed electrodes, Electrochemical deposition, Density functional theory (DFT).