Rational design toward developing a more efficient laccase: Catalytic efficiency and selectivity.

Laccases are multicopper oxidases that catalyze the oxidation of variety of substrates. The specificity and efficiency of laccases are clearly the important components leading to their remarkable uses. To develop an improved biocatalysts, site directed mutagenesis of laccase from Bacillus HR03 was carried out in the current study. Based on the ABTS-bound crystal structure of CotA from B. subtilis and alignment with closely related enzymes, T415 and T418 at the vicinity of the type 1 copper site were chosen and several mutants (T415I, T418I, T415G, T415G/T418I) were made. Kinetic parameters of the constructs were then determined using ABTS and SGZ as substrates. In comparison with the wild-type, catalytic efficiency toward ABTS was improved by 4 fold in T415I and 1.5 fold in T418I and T415G. T415I and T418I variants were identified to be almost 11 and 27 times more specific for ABTS than for SGZ compared with the wild type. T415I was also found to acquire enhanced thermal stability with the half-life of 60min at 80°C. Secondary and tertiary structure of mutants were analyzed by CD and fluorescence spectroscopy. Our result illustrated that replacement of residues in the substrate-binding pocket would change the specificity and efficiency of variants.

Evaluation of The Number of CD4(+) CD25(+) FoxP3(+) Treg Cells in Normal Mice Exposed to AFB1 and Treated with Aged Garlic Extract.

OBJECTIVE: Aflatoxin B1 (AFB1) suppresses the immune system. To decrease such suppressive effects on the immune system, a wide range of herbal medicines like garlic are utilized. Biological activities of garlic in vitro and in vivo have also been verified. Our previous studies demonstrated that aged garlic (dry garlic bulbs preserved in the freezer for six months at -20˚C) have increased immunostimulator fractions and reduced immunosuppressor fractions. This study focuses on the immunosuppressor activity of AFB1 and immunostimulator activity of aged garlic extract (AGE) through the evaluation of CD4(+) CD25(+) FoxP(+) regulator cell (Treg) counts and the pattern of cytokine production in Balb/c normal mice. MATERIALS AND METHODS: In this experimental research, AFB1 was separated from Aspergillus flavus (PTCC 5004) by HPLC and AGE prepared using the Mantis method. The Delayed-Type Hypersensitivity (DTH) test was carried out to determinate the effectiveness of different doses of AGE and AFB1, which can both have an effect on the immune system. Subsequent experiments were carried out on 20 Balb/c mice to estimate the effects of AGE and AFB1 on the number of Treg cell in 4 groups: 10 µl/kg/day of AFB1 and AGE diluents were administered for 4 consecutive days to group 1. AFB1, 2. control, 3. AGE + AFB1 and 4. AGE via intraperitoneal (IP) route, respectively. Mice were sacrificed and splenocytes harvested and the percentage of splenic Treg cells was measured by flow cytometry analysis. The ELISA method was utilized to measure Cytokine production. RESULTS: The findings reveal that AGE increased the level of IFN-λ and IL-4 cytokines produced by splenocytes stimulated by specific tumor antigen and decreased the number of Treg cells in the spleen (p<0.05). AFB1 increased the number Treg cells in the spleen and decreased cytokine production (p<0.05). In groups 2 (control) and 4 (AGE) the number of Treg cells decreased (p value<0.05) whereas in groups 1 and 3 the number of Treg cells increased (p<0.05). CONCLUSION: This study indicated that AGE is able to alter the cytokine production in normal mice into a Th1 protective pattern which is beneficial to the immune system in general and anti-tumor immunity in particular. AFB1 is able to alter the cytokine production into a Th2 protective pattern. Therefore, AGE might be used as herbal medicine with few side effects as compared to chemotherapy in treating cancers caused by substances like AFB1.

Assessment of salivary and serum antioxidant status in patients with recurrent aphthous stomatitis

Objective: The aim of this study was to determine the possible association of oxidant/antioxidant status and recurrentaphthous stomatitis (RAS).Study design: The study consis ted of thirty-one patients with RAS and thirty-two healthy controls from whom salivaand blood samples were collected. Superoxide dismutase (SOD), glutathione peroxidase (GSHPx) and catalase(CAT) were measured in erythrocytes and total antioxidant status (TAS) was measured in plasma and saliva.Results: Erythrocyte SOD activity was significantly lower in RAS patients in comparison to healthy controls(P=0.012). No significant differences were found in erythrocyte GSHPx, CAT activities, and salivary and plasmaTAS between RAS patients and control subjects (p>0.1).Conclusion: Changes in SOD activity may be important in the inflammatory reactions observed in RAS, but othertested defense systems such as CAT and GSHPx do not seem to play a primary role in the aetiopathogenesis ofRAS. Moreover, the antioxidant system in saliva and plasma is not as affected as in erythrocytes in RAS patients,and therefore it may not be considered an appropriate indicator of the body’s total antioxidant status (AU)

Assessment of salivary and serum antioxidant status in patients with recurrent aphthous stomatitis.

OBJECTIVE: The aim of this study was to determine the possible association of oxidant/antioxidant status and recurrent aphthous stomatitis (RAS). STUDY DESIGN: The study consisted of thirty-one patients with RAS and thirty-two healthy controls from whom saliva and blood samples were collected. Superoxide dismutase (SOD), glutathione peroxidase (GSHPx) and catalase (CAT) were measured in erythrocytes and total antioxidant status (TAS) was measured in plasma and saliva. RESULTS: Erythrocyte SOD activity was significantly lower in RAS patients in comparison to healthy controls (P=0.012). No significant differences were found in erythrocyte GSHPx, CAT activities, and salivary and plasma TAS between RAS patients and control subjects (p>0.1). CONCLUSION: Changes in SOD activity may be important in the inflammatory reactions observed in RAS, but other tested defense systems such as CAT and GSHPx do not seem to play a primary role in the aetiopathogenesis of RAS. Moreover, the antioxidant system in saliva and plasma is not as affected as in erythrocytes in RAS patients, and therefore it may not be considered an appropriate indicator of the body's total antioxidant status.

Synthesis and dopaminergic activity of some E-3-(piperidin-1-yl)-1-(4-substituted phenyl)prop-2-en-1-one derivatives.

A convenient route for the synthesis of some 2-propen-1-one derivatives with E isomeric configuration is described. The activity of the synthesized compounds was evaluated through behavioral studies of apomorphine-induced licking in animal models. It was demonstrated that most of the synthesized compounds showed moderate activity in inhibition of lickings, among which 6a, was the most active compound at 30 mg/kg.

Binding properties of adenosine deaminase interacted with theophylline.

Thermodynamic studies were carried out to evaluate the binding of theophylline on adenosine deaminase (ADA) in 50 mM sodium phosphate buffer pH 7.5, at 300 K, using isothermal titration calorimetry (ITC). A simple method for determination of binding isotherm in the drug--ADA interaction was applied using ITC data. ADA has two binding sites for theophylline, which show positive cooperativity in its sites. The intrinsic association equilibrium constants are 6 and 52 mM(-1) in the first and second binding sites, respectively. Hence, occupation of the first site has produced an appreciable enhancement by 8.7 of the binding affinity of the second site. The molar enthalpies of binding are -12.2 and -14.9 kJ/mol in the first and second binding sites, respectively.