Vitamin D and melatonin protect the cell’s viability and ameliorate the CCl4 induced cytotoxicity in HepG2 and Hep3B hepatoma cell lines

Carbon tetrachloride (CCl₄) is widely used to induce liver toxicity in in vitro/in vivo models. Lipid peroxidation (LPO) begins with toxicity and affects cell viability. Recently, the beneficial effects of melatonin and Vitamin D on cell proliferation in human normal and cancer cells were found. This study was planned to evaluate antioxidant and cytoprotective activity of melatonin and Vitamin D in CCl₄ induced cytotoxicity in HepG2 and Hep3B hepatoma cell lines. Based on the cytotoxicity assay, melatonin and Vitamin D were evaluated for cytotoprotective potential against CCl₄ induced toxicity in HepG2 and Hep3B liver cell lines by monitoring cell viability, LPO and glutathione (GSH) level. Different dosages of CCl₄ (0.1, 0.2, 0.3 and 0.4 % v/v) were applied to HepG2 and Hep3B cells in order to determine the most toxic dosage of it in a time dependent manner. The same experiments were repeated with exogenously applied melatonin (MEL) and Vitamin D to groups treated with/without CCL₄. Cell viability was determined with MTT measurements at the 2nd, 24th and 48th h. GSH content and Malondialdehyde levels were measured from the cell lysates. As a result, both melatonin and Vitamin D administration during CCl₄ exposure protected liver cells from CCl₄ induced cell damage. Increase in LPO and decrease in GSH were found in the CCl₄ groups of both cells. Contrary to these results administration of MEL and Vitamin D on cells exhibited results similar to the control groups. Therefore, melatonin and Vitamin D might be a promising therapeutic agent in several toxic hepatic diseases.     

Is there any genetic predisposition of MMP-9 gene C1562T and MTHFR gene C677T polymorphisms with essential hypertension?

The current study was conducted to determine whether there is a relation between hypertension and two different polymorphisms, including C1562T of the Matrix metalloproteinase-9 (MMP-9) gene and C677T of the methylenetetrahydrofolate reductase (MTHFR) gene. Genomic DNA obtained from 224 persons (125 patients with hypertension and 99 healthy controls) were used in the study. Polymorphisms were determined by using polymerase chain reaction-restriction fragment length polymorphism and electrophoresis. The results were statistically analyzed and were found to be statistically significant. The frequencies of the C1562T genotypes were found to be, in controls CC 75.8 % and CT 24.2 % and in patients CC 71.2 %, and CT 28.8 %. The frequencies of C677T genotype were found to be, in controls CC 56.6 %, CT 38.4 and TT 5.1 % in controls and in patients CC 52 %, CT 30.4 % and TT 17.6 %. In conclusion, we may suggest that there is no relation between the essential hypertension and C1562T polymorphism of MMP-9 gene; on the other hand C677T polymorphism (genotype TT) of MTHFR gene can be regarded as a genetic indicator for the development of essential hypertension.     

Protective Effects of Caffeic Acid Phenethyl Ester on Cyclophosphamide‐Induced Hemorrhagic Cystitis in Rats

We investigated the protective effect of caffeic acid phenethyl ester (CAPE) on cyclophosphamide‐induced hemorrhagic cystitis in rats in comparison with 2‐mercaptoethane sulfonate (MESNA). Forty male rats were randomized into four groups: group 1 (control), group 2 (cyclophosphamide), group 3 (cyclophosphamide + MESNA), group 4 (cyclophosphamide + CAPE). Cyclophosphamide injection increased malondialdehyde levels indicating oxidative stress, whereas CAPE and MESNA ameliorated malondialdehyde levels in the bladder (p < 0.05). Only catalase activities were decreased significantly in both groups (cyclophosphamide + MESNA and cyclophosphamide + CAPE, p < 0.05). Pretreatment with CAPE (p < 0.01) resulted in a significant decrease in nitric oxide levels when compared with the cyclophosphamide group. When we consider the studies that show the critical importance of increased nitric oxide levels in pathogenesis of cyclophosphamide‐induced hemorrhagic cystitis, we suggest that it would be more beneficial to use MESNA with CAPE to prevent histological damage.     

Fab fragments imprinted SPR biosensor for real-time human immunoglobulin G detection

F(ab) fragments imprinted surface plasmon resonance (SPR) chip was prepared for the real-time detection of human immunoglobulin G (IgG). In order to attach polymerization precursor on SPR chip, the SPR chip surface was modified with allyl mercaptan. F(ab) fragments of the IgG molecules were prepared by papain digestion procedure and collected by fast protein liquid chromatography (FPLC) system using Hi-Trap_r Protein A FF column. The collected F(ab) fragments were complexed with histidine containing specific monomer, N-methacryloyl-l-histidine methyl ester (MAH). Molecular imprinted polymeric nanofilm was prepared on SPR chip in the presence of ethylene glycol dimethacrylate and 2-hydroxyethylmethacrylate. The template molecules, F(ab) fragments, were removed from the polymeric nanofilm using 1M NaCl solution (pH: 7.4, phosphate buffer system). The molecular imprinted SPR chip was characterized by contact angle, atomic force microscopy and Fourier transform infrared spectroscopy. By the real-time IgG detection studies carried out using aqueous IgG solutions in different concentrations, the kinetics and isotherm parameters of the molecular imprinted SPR chip-IgG system were calculated. To show selectivity and specificity of the molecular imprinted SPR chip, competitive kinetic analyses were performed using bovine serum albumin (BSA), IgG, F(ab) and F(c) fragments in singular and competitive manner. As last step, IgG detection studies from human plasma were performed and the measured IgG concentrations were well matched with the results determined by enzyme-linked immunosorbent assay (ELISA). The results obtained with the molecular imprinted SPR chip were well fitted to Langmuir isotherm and the detection limit was found as 56 ng/mL. In the light of the results, we can conclude that the proposed molecular imprinted SPR chip can detect IgG molecules from both aqueous solutions and complex natural samples.     

Effect of feeding and sludge age on acclimated bacterial community and fate of slowly biodegradable substrate

The study investigated the effect of feeding regime and sludge age on starch utilization. For this purpose, parallel sequencing batch reactors were operated with pulse and continuous feeding of soluble starch at sludge ages of 8 and 2 days. Pulse feeding induced almost complete conversion of starch to glycogen, while storage was lowered and accompanied with direct growth under continuous feeding, regardless of sludge age. Low sludge age did not alter simultaneous storage and utilization for direct growth but it slightly favoured direct utilization due to faster growing biomass. Experimental results suggested adsorption of starch onto biomass as a preliminary removal mechanism prior to hydrolysis at sludge age of 8 days. Adsorption was not noticeable as substrate removal, glycogen generation and dissolved oxygen decrease were synchronous at sludge age of 2 days. Bacterial community always included fractions storing glycogen although sludge age only affected the relative magnitude of filamentous growth.     

The use of a white rot fungi (Pleurotus ostreatus) immobilized on Amberlite XAD-4 as a new biosorbent in trace metal determination

The present work proposes the use of Pleurotus ostreatus immobilized on Amberlite XAD-4 as new biosorbent in trace metal determination. The effects of experimental parameters, such as “pH and flow rate of sample solution, amount of solid phase, eluent type, and concentration” on the recovery of the metal ions were investigated. Maximum adsorption of Cr(III), Cd(II) and Cu(II) ions took place in the pH range 4-5. These metal ions can be desorbed with 1 M HCl (recovery 95-100%). 0.2 g adsorbent amount and 2.5 mL min⁻¹ flow rate was found to be optimum of all preconcentration experiments. The sorption capacity after 10 cycles of sorption and desorption does not vary more than 2.0%. The influences of the contaminant ions on the retentions of the analytes were also examined. The results showed that P. ostreatus immobilized on Amberlite XAD-4 can be considered as very promising material in trace metal determination.     

Kinetic and docking studies of phenol-based inhibitors of carbonic anhydrase isoforms I, II, IX and XII evidence a new binding mode within the enzyme active site

Carbonic anhydrases (CAs, EC 4.2.1.1) are inhibited by sulfonamides, inorganic anions, phenols, coumarins (acting as prodrugs) and polyamines. A novel class of CA inhibitors (CAIs), interacting with the CA isozymes I, II (cytosolic) and IX, XII (transmembrane, tumor-associated) in a different manner, is reported here. Kinetic measurements allowed us to identify hydroxy-/methoxy-substituted benzoic acids as well as di-/tri-methoxy benzenes as submicromolar-low micromolar inhibitors of the four CA isozymes. Molecular docking studies of a set of such inhibitors within CA I and II allowed us to understand the inhibition mechanism. This new class of inhibitors binds differently compared to all other classes of inhibitors known to date: they were found between the phenol-binding site and the coumarin-binding site, filling thus the middle of the enzyme cavity. They exploit different interactions with amino acid residues and water molecules from the CA active site compared to other classes of inhibitors, offering the possibility to design CAIs with an interesting inhibition profile compared to the clinically used sulfonamides/sulfamates.     

Microparticulate Based Topical Delivery System of Clobetasol Propionate

Psoriasis is a chronic, autoimmune skin disease affecting approximately 2% of the world's population. Clobetasol propionate which is a superpotent topical corticosteroid is widely used for topical treatment of psoriasis. Conventional dosage forms like creams and ointments are commonly prefered for the therapy. The purpose of this study was to develop a new topical delivery system in order to provide the prolonged release of clobetasol propionate and to reduce systemic absorption and side effects of the drug. Clobetasol propionate loaded-poly(D,L-lactic-co-glycolic acid) (PLGA) microspheres were prepared by oil-in-water emulsion–solvent evaporation technique. Particle size analysis, morphological characterization, DSC and XRD analyses and in vitro drug release studies were performed on the microparticle formulations. Emulgel formulations were prepared as an alternative for topical delivery of clobetasol propionate. In vitro drug release studies were carried out from the emulgel formulations containing pure drug and drug-loaded microspheres. In addition, the same studies were performed to determine the drug release from the commercial cream product of clobetasol propionate. The release of clobetasol propionate from the emulgel formulations was significantly higher than the commercial product. In addition, the encapsulation of clobetasol propionate in the PLGA microspheres significantly delayed the drug release from the emulgel formulation. As a result, the decrease in the side effects of clobetasol propionate by the formulation containing PLGA microspheres is expected.     

Differential hydrolysis of homocysteine thiolactone by purified human serum (192)Q and (192)R PON1 isoenzymes

Human serum paraoxonase 1 (PON1) is a HDL-associated enzyme that catalyzes the hydrolysis of a variety of aromatic carboxylic acid esters and several organophosphates. Recently it has been suggested that a physiological substrate of serum PON1 is homocysteine thiolactone which is a putative risk factor in atherosclerosis. In this study, human (192)Q and (192)R PON1 isoenzymes were purified from the respective phenotype human serum, using a protocol consisting of ammonium sulfate precipitation and four chromatography steps: gel filtration, ion-exchange, non-specific affinity, and a second ion-exchange. Using paraoxon as substrate, overall purification fold was found as 742 for (192)R PON1 and 590 for (192)Q PON1. The final purified enzymes were shown as single protein bands close to 45kDa on SDS-PAGE and confirmed by Western blot. Substrate kinetics were studied with phenyl acetate, paraoxon and homocysteine thiolactone. Both PON1 isoenzymes showed mixed type inhibition with phenyl acetate. K(m) values of (192)Q and (192)R PON1 for homocysteine thiolactone were 23.5mM and 22.6mM respectively. For (192)R PON1, the V(max) was 2.5-fold and k(cat)/K(m) was 2.6-fold higher than those for (192)Q PON1 when homocysteine thiolactone is used as substrate. The present data suggest that defining (192)Q and (192)R PON1 isoforms could be a good predictor and prognostic marker in the cardiovascular risk assessment.