Colorimetric coupled enzyme assay for gamma-glutamyltransferase activity using glutathione as substrate

A colorimetric coupled enzyme assay for the determination of gamma-glutamyltransferase (GGT) activity using glutathione as substrate is described. The cysteine released from glutathione upon sequential action of GGT and leucine aminopeptidase is spectrophotometrically detected through its reaction with ninhydrin at 100 degrees C in acidic conditions. The method was applied to the determination of the activity of both bovine kidney and human serum GGT. In the described assay conditions with final GGT concentrations ranging from 0.18 to 4 mU/ml, a linear relationship between produced cysteine and incubation times up to 90 min was observed. When a standard chromogenic assay for GGT using L-gamma-glutamyl-3-carboxy-4-nitroanilide as substrate and the proposed assay were applied on the same serum sample a linear relationship between the two method was observed. Since the use of GSH as substrate, the proposed method can be usefully adopted for enzymological studies on GGT-related enzymes, a class of enzymes which is still waiting to be characterized.     

Optimization of formulation variables of benzocaine liposomes using experimental design

This study aimed to optimize, by means of an experimental design multivariate strategy, a liposomal formulation for topical delivery of the local anaesthetic agent benzocaine. The formulation variables for the vesicle lipid phase uses potassium glycyrrhizinate (KG) as an alternative to cholesterol and the addition of a cationic (stearylamine) or anionic (dicethylphosphate) surfactant (qualitative factors); the percents of ethanol and the total volume of the hydration phase (quantitative factors) were the variables for the hydrophilic phase. The combined influence of these factors on the considered responses (encapsulation efficiency (EE%) and percent drug permeated at 180 min (P%)) was evaluated by means of a D-optimal design strategy. Graphic analysis of the effects indicated that maximization of the selected responses requested opposite levels of the considered factors: For example, KG and stearylamine were better for increasing EE%, and cholesterol and dicethylphosphate for increasing P%. In the second step, the Doehlert design, applied for the response-surface study of the quantitative factors, pointed out a negative interaction between percent ethanol and volume of the hydration phase and allowed prediction of the best formulation for maximizing drug permeation rate. Experimental P% data of the optimized formulation were inside the confidence interval (P < 0.05) calculated around the predicted value of the response. This proved the suitability of the proposed approach for optimizing the composition of liposomal formulations and predicting the effects of formulation variables on the considered experimental response. Moreover, the optimized formulation enabled a significant improvement (P < 0.05) of the drug anaesthetic effect with respect to the starting reference liposomal formulation, thus demonstrating its actually better therapeutic effectiveness.     

Design,synthesis and anticancer activity of N-(1-(4-(dibenzo[b,f][1,4]thiazepin-11-yl)piperazin-1-yl)-1-oxo-3-phenylpropan-2-yl derivatives

Novel N-(1-(4-(dibenzo[b,f][1,4]thiazepin-11-yl)piperazin-1-yl)-1-oxo-3-phenylpropan-2-yl derivatives were designed, synthesized and their chemical structures were confirmed by ¹H NMR, ¹³C NMR and Mass spectra. The anticancer activities of the newly synthesized compounds were evaluated in vitro against three human cancer cell lines including K562, Colo-205 and MDA-MB 231 by MTT assay. The screening results showed that five compounds (16b, 16d, 16i, 16p and 16q) exhibited potent cytotoxic activities with IC₅₀ values between 20 and 40 μM. Further in vitro studies revealed that inhibition of sirtuins could be the possible mechanism of action of these molecules.     

Evaluation of the viability and germination of tempisque [<i>Sideroxylon capiri</i> (A.DC.) Pittier Sapotaceae]

The tempisque (Sideroxylon capiri) is a tree native to Mexico used by the rural population for housing construction, poles and hedges, as fuel (wood) and also for fodder and ornamental purposes, among others. It is considered an endangered species. In order to contribute to its preservation and sustainable management, it was considered important to determine the proportion of viable seeds, the loss of viability due to storage period and the germination process by applying pregerminative treatments. We found that freshly collected seeds showed 100% viability, which decreased to 0% after 5 months of storage. According to the cumulative germination significant differences between treatments (p≤0.01) were found. It was observed that seeds can accelerate their time of germination with the previous exposure of 24 h in water at room temperature. The soaking treatment in water for 24 h at room temperature obtained final germination of 55%, while with the control 39% was reached. Soaking in hydrogen peroxide and scarification were the treatments with lower germination percentage (33 and 23%, respectively). To get a higher percentage of germinated seeds in a short time, it is necessary to give a soaking treatment in water for 24 h before sowing.     

Influence of aflatoxin B1 on seed germination, seedling growth, chlorophyll and carotenoid contents of mustard (brassica juncea L. var. pusa bold) seeds

Five different concentrations (100, 250, 500, 1000 and 2000 μg/L of aflatoxin B₁ were found to be inhibitory to seed germination and seedling growth (root and shoot lengths) of mustard seeds (variety Pusa bold). These also lowered the levels of chlorophyll and carotenoids in the emerging leaves during seedling growth. The inhibitory effect was correlated with the concentration of applied toxin.     

Use of morphological characters for species separation within the genus Artemia (Crustacea, Branchiopoda)

Doubts have been raised about the use of morphological characters as criteria for species separation within the genus Artemia because of their relative validity. The characters considered to date, i.e. morphology of the frontal knobs of the male and the presence/absence of a spine-like projection on the basal part of the penis, are only partly efficacious, since they allow us to reliably distinguish only the Mediterranean species, Artemia salina, from the other bisexual species. While A. salina is characterized by subconical frontal knobs and the absence of the spine-like projection, the other species present subspherical frontal knobs and the spine-like projections. There is no information about the aspect of the frontal knobs in the Asian bisexual species. The aim of the present study is to clarify the situation by means of SEM observations of characters not considered in detail (ornamentation of the apical part of the spine-like projections) and by examination of the frontal knobs in the Asian species, which were not described at the time of the first studies.     

SEM morphological observations on Artemiopsis stefanssoni Johansen, 1921 (Crustacea, Anostraca)

The morphology of the female ovisac of the fairy shrimp Artemiopsis stefanssoni Johansen, 1921, is analysed by SEM. SEM observations reveal the existence of a genital opening located ventrally, but anterior to the area where the genital pore is normally situated. Ornamented frontal knobs resembling those on the second antennae of males of Artemia were also found in Artemiopsismales, and account for its particular mating behaviour.     

Radioenzymatic determination of adenosine

Adenosine has been measured at the nanomolar level by an enzymatic radioactive assay. The nucleoside is converted into [U-14C]ribose-labeled inosine via the following reactions: adenosine + H2O----adenine + ribose (adenosine nucleosidase); adenine + [U-14C]ribose 1-phosphate in equilibrium with T[U-14C]ribose-adenosine + Pi (adenosine phosphorylase); [U-14C]ribose-adenosine + H2O----[U-14C]ribose-inosine + NH3 (adenosine deaminase). The radioactivity of inosine, separated by thin-layer chromatography, is a measure of the adenosine initially present.     

Activity and Structural Changes of Euphorbia characias Peroxidase in the Presence of Trifluoroethanol

Activity assays, conformational changes and transitional switches between secondary structures of a peroxidase from Euphorbia characias were studied in the presence of trifluoroethanol and in the presence or absence of calcium ions. The addition of trifluoroethanol up to 10–20% first induced a drastic decrease of α-helix content followed by an increase of tryptophan fluorescence emission intensity, a progressive re-induction of the formation of α-helical elements concomitant with loss of enzyme activity. In the presence of calcium ions, the fluorescence of the enzyme almost remained unchanged in the trifluoroethanol concentration range 5–20%. Further increase in trifluoroethanol concentration led to a protein structure characterized by a progressive re-induction of α-helical elements, a remarkable increase of the tryptophan fluorescence and a loss of enzyme activity. These results indicate that calcium ions in Euphorbia peroxidase play an essential role in maintaining the hydrophobic interactions on the protein structure preserving enzymatic activity.