Immobilized Pseudomonas sp. lipase: A powerful biocatalyst for asymmetric acylation of (±)-2-amino-1-phenylethanols with vinyl acetate

Pseudomonas sp. lipase was immobilized onto glutaraldehyde-activated Florisil^® support via Schiff base formation and stabilized by reducing Schiff base with sodium cyanoborohydride. The immobilization performance was evaluated in terms of bound protein per gram of support (%) and recovered activity (%). A 4-factor and 3-level Box–Behnken design was applied for the acylation of (±)-2-(propylamino)-1-phenylethanol, a model substrate, with vinyl acetate and the asymmetric acylations of other (±)-2-amino-1-phenylethanols with different alkyl substituents onto nitrogen atom such as (±)-2-(methylamino)-1-phenylethanol, (±)-2-(ethylamino)-1-phenylethanol, (±)-2-(butylamino)-1-phenylethanol and (±)-2-(hexylamino)-1-phenylethanol were performed under the optimized conditions. The optimal conditions were bulk water content of 1.8%, reaction temperature of 51.5 °C, initial molar ratio of vinyl acetate to amino alcohol of 1.92, and immobilized lipase loading of 47 mg mL^?1. (R)-enantiomers of tested amino alcohols were preferentially acylated and the reaction purely took place on the hydroxyl group of 2-amino-1-phenylethanols. The increase of alkyl chain length substituted onto nitrogen atom caused an increase in the acylation yield and ee values of (S)-enantiomers. Enantiomeric ratio values were >200 for all the reactions. Our results demonstrate that the immobilized lipase is a promising biocatalyst for the preparation of (S)-2-amino-1-phenylethanols and their corresponding (R)-esters via O-selective acylation of (±)-2-amino-1-phenylethanols with vinyl acetate.     

Purification and characterization of carbonic anhydrase from sheep kidney and effects of sulfonamides on enzyme activity

Carbonic anhydrase (CA, EC: 4.2.1.1) was purified from sheep kidney by affinity chromatography on a Sepharose 4B-tyrosine-sulfanilamide column. By means of two consecutive procedures, the enzyme (sCA) was purified 227.61-fold with a yield of 60.75%, and a specific activity of 838.89 U/mg proteins. The optimum temperature, ionic strength and pH were determined to be 35 °C, 20 mM and 8.5, respectively. The molecular weight determined by SDS–PAGE was found to be 29 kDa. The kinetic parameters, K_M and V_max values were determined for the 4-nitrophenyl acetate (p-NpA) hydrolysis reaction. Some sulfonamides were tested as inhibitors against the purified CAs enzyme. The K_i constants for benzenesulfonamide (1), sulfanilamide (2), mafenide (3), 4-(2-aminoethyl) benzenesulfonamide (4), 4-methyl-benzenesulfonamide (5), 2-bromo-benzenesulfonamide (6), naphthalene-2-sulfonamide (7), 4-amino-6-chlorobenzene-1,3-disulfonamide (8) and saccharin (9) were in the range 1.348–69.31 μM.     

Regulatory Networks and Complex Interactions between the Insulin and Angiotensin II Signalling Systems: Models and Implications for Hypertension and Diabetes

The cross-talk between insulin and angiotensin II signalling pathways plays a significant role in the co-occurrence of diabetes and hypertension. We developed a mathematical model of the system of interactions among the biomolecules that are involved in the cross-talk between the insulin and angiotensin II signalling pathways. We have identified several feedback structures that regulate the dynamic behavior of the individual signalling pathways and their interactions. Different scenarios are simulated and dominant steady-state, dynamic and stability characteristics are revealed. The proposed mechanistic model describes how angiotensin II inhibits the actions of insulin and impairs the insulin-mediated vasodilation. The model also predicts that poor glycaemic control induced by diabetes contributes to hypertension by activating the renin angiotensin aystem.     

Transmission electron microscopy study of the effects of cadmium and copper on fetal rat liver tissue

During the entire period of their pregnancies, three groups of adult pregnant Wistar albino rats were provided with tap water (control; group I) or with tap water containing 10 mg/kg CdCl₂ (group II) or 10 mg/kg CdCl₂ plus 10 mg/kg CuSO₄ (group III). At term, the animals were sacrificed and the fetal livers were removed and examined under electron microscopy. The liver tissue of the fetuses in maternal groups II and III showed degenerative changes to their hepatocytes. In group II, the smooth endoplasmic reticulum tubules showed dilatation, and the mitochondria showed a dense matrix. In group III, some mitochondrial degeneration was also seen, with a diluted matrix and mitochondrial dilatation. There were also more heterochromatic nuclei and an increased number of ribosomes. None of these histopathological changes were present in the fetal liver samples from the maternal group I control animals.     

Reciprocal Activating Crosstalk between c-Met and Caveolin 1 Promotes Invasive Phenotype in Hepatocellular Carcinoma

c-Met, the receptor for Hepatocyte Growth Factor (HGF), overexpressed and deregulated in Hepatocellular Carcinoma (HCC). Caveolin 1 (CAV1), a plasma membrane protein that modulates signal transduction molecules, is also overexpressed in HCC. The aim of this study was to investigate biological and clinical significance of co-expression and activation of c-Met and CAV1 in HCC. We showed that c-Met and CAV1 were co-localized in HCC cells and HGF treatment increased this association. HGF-triggered c-Met activation caused a concurrent rise in both phosphorylation and expression of CAV1. Ectopic expression of CAV1 accelerated c-Met signaling, resulted in enhanced migration, invasion, and branching-morphogenesis. Silencing of CAV1 downregulated c-Met signaling, and decreased migratory/invasive capability of cells and attenuated branching morphogenesis. In addition, activation and co-localization of c-Met and CAV1 were elevated during hepatocarcinogenesis. In conclusion reciprocal activating crosstalk between c-Met and CAV1 promoted oncogenic signaling of c-Met contributed to the initiation and progression of HCC.     

Crosslinked enzyme aggregates of hydroxynitrile lyase partially purified from Prunus dulcis seeds and its application for the synthesis of enantiopure cyanohydrins

Hydroxynitrile lyases are powerful catalysts in the synthesis of enantiopure cyanohydrins which are key synthons in the preparations of a variety of important chemicals. The response surface methodology including three‐factor and three‐level Box–Behnken design was applied to optimize immobilization of hydroxynitrile lyase purified partially from Prunus dulcis seeds as crosslinked enzyme aggregates (PdHNL‐CLEAs). The quadratic model was developed for predicting the response and its adequacy was validated with the analysis of variance test. The optimized immobilization parameters were initial glutaraldehyde concentration, ammonium sulfate saturation concentration, and crosslinking time, and the response was relative activity of PdHNL‐CLEA. The optimal conditions were determined as initial glutaraldehyde concentration of 25% w/v, ammonium sulfate saturation concentration of 43% w/v, and crosslinking time of 18 h. The preparations of PdHNL‐CLEA were examined for the synthesis of (R)‐mandelonitrile, (R)‐2‐chloromandelonitrile, (R)‐3,4‐dihydroxymandelonitrile, (R)‐2‐hydroxy‐4‐phenyl butyronitrile, (R)‐4‐bromomandelonitrile, (R)‐4‐fluoromandelonitrile, and (R)‐4‐nitromandelonitrile from their corresponding aldehydes and hydrocyanic acid. After 96‐h reaction time, the yield–enantiomeric excess values (%) were 100?99, 100?21, 100?99, 83?91, 100?99, 100?72, and 100?14%, respectively, for (R)‐mandelonitrile, (R)‐2‐chloromandelonitrile, (R)‐3,4‐dihydroxymandelonitrile, (R)‐2‐hydroxy‐4‐phenyl butyronitrile, (R)‐4‐bromomandelonitrile, (R)‐4‐fluoromandelonitrile, and (R)‐4‐nitromandelonitrile. The results show that PdHNL‐CLEA offers a promising potential for the preparation of enantiopure (R)‐mandelonitrile, (R)‐3,4‐dihydroxymandelonitrile, (R)‐2‐hydroxy‐4‐phenyl butyronitrile, and (R)‐4‐bromomandelonitrile with a high yield and enantiopurity. © 2014 American Institute of Chemical Engineers Biotechnol. Prog, 30:818–827, 2014