Purification and partial characterization of two lectin isoforms fromCratylia mollis mart. (camaratu bean)

Two additional electrophoretically distinct molecular forms, isoforms (iso) 2 and 3, with lectin properties were isolated fromCratylia mollis Mart, seeds (FABACEAE), by extraction with 0.15M NaCl and ammonium sulfate fractionation, followed by chromatography on Sephadex G-75 and Bio-Gel P-200 (iso 2), as well as CM-Cellulose and Sephadex G-75 (iso 3). Both isoforms were human group nonspecific and showed distinct specificity. Polyacrylamide gel electrophoresis resolved iso 2 and 3 in polypeptides of apparent mol wts 60 and 31 kDa, respectively; a distinct isoelectric focusing pattern was obtained for iso 2 and 3, under denaturing and reducing conditions.     

High-performance liquid chromatographic assay for amiodarone N-deethylation in microsomes of rat liver.

A reversed-phase high-performance liquid chromatographic assay using ultraviolet detection is described for determining the production of the major N-dealkylated metabolite of amiodarone in rat liver microsomes. The principal advantages of this method are its simple sample preparation (protein precipitation by acetonitrile), low detection limit for N-desethylamiodarone (0.05 mumol/l) and relatively short analysis time (16 min). Its analytical applicability is demonstrated by the comparison of the kinetic parameters (maximum velocity and Michaelis-Menten constant) between Sprague-Dawley and Dark-Agouti rats.     

Binding evaluation of isoform 1 fromCratylia mollis lectin to human mammary tissues

The phenomenon of altered carbohydrates in transformed cell surfaces has been studied through histochemical techniques using lectins. Specific binding patterns to normal and transformed mammary tissues were evaluated by Isoform 1 fromCratylia mollis lectin (Cra Iso 1). Protocols using a direct method, incubation of Cra Iso I conjugated to peroxidase (Cra Iso 1-Per) with mammary tissues, followed by diaminobenzidine and hydrogen peroxidase interaction, were performed. Neoplastic tissues, marked by Cra Iso 1, showed a higher intensity of staining than normal ones, in comparison withCanavalia ensiformis lectin, Concanavalin A (Con A), conjugated to peroxidase (Con A-Per). The assay with Cra Iso 1 also indicated a possible utilization of this lectin to characterize normal and transformed mammary cells.     

Solid-phase microextraction gas chromatography/mass spectrometry: a new method for species identification of truffles

This study describes a rapid method to identify different truffle species by analysis of their volatile compound fraction using static headspace solid-phase microextraction gas chromatography/mass spectrometry. The volatile organic compounds (VOCs) were extracted using a new 2-cm 50/30 microm DVB/CAR/PDMS fiber placed for 10 min in the headspace of the truffle sample with the vial maintained at 20 degrees C (in a thermostatically controlled analysis room). The mass spectra of the VOC chromatograms were represented as 'fingerprints' of the analysed samples. Next, stepwise factorial discriminant analysis afforded a limited number of characteristic fragment ions that allowed a classification of the truffle species studied. This new method provides an effective approach to rapid quality control and identification of truffle species by analysis of their volatile fraction. Moreover, this method offers the advantage of minimizing thermal, mechanical, and chemical modifications of the truffles, thereby reducing the risk of analytical artifacts.     

Characterization of Two Light-Harvesting Subunits Isolated from the Brown Alga Pelvetia canaliculata: Heterogeneity of Xanthophyll Distribution

The main light-harvesting fraction from Pelvetia canaliculata was isolated on a sucrose density gradient from digitonin-solubilized chloroplasts. After further solubilization by dodecyl maltoside, the bulk fraction was separated into two subunits by preparative isoelectric focusing. The more acidic brown fraction was mainly composed of 22 kDa polypeptides having an apparent pI of 4.55. Its pigment composition was very simple, containing chlorophyll (Chi) a, Chi c and fucoxanthin. The in vivo spectral properties of fucoxanthin, namely a shift in light absorption to the green and efficient energy transmission to Chi a, were conserved in this subunit. No xanthophyll associated with photoprotection was found in this band, even when obtained from photoinhibited thalli. The less acidic green band contained predominantly 22 kDa polypeptides that were resolved into numerous components by denaturing isoelectric focusing. Its pigment composition was more complex, containing, in addition, pigments of the so-called xanthophyll cycle. In photoinhibited thalli, about half of the violaxanthin was converted into antheraxanthin and zeaxanthin. All the pigments of the xanthophyll cycle were specifically associated with this subunit, and it may thus have a central role in the thermal dissipation of the absorbed light energy as postulated for light-harvesting complex II isolated from green plants.     

Partial purification and some properties of a hemolymph lectin from panstrongylus megistus (Hemiptera,Reduviidae)

Hemagglutinating activity was studied in homogenates of three embryonic stages, and in the hemolymph of most instar larvae and in adult insects of Panstrongylus megistus, an important Chagas' disease vector in Brazil. A hemolymph lectin from the 5th instar larvae of P. megistus was purified through a biospecific adsorption by using formaldehyde-treated erythrocytes. The lectin fraction was desorbed with 0.2M D-galactose in 0.15M NaCl. The lectin fraction activity was inhibited by L-rhamnose, D-lactose, raffinose, D-galactose, and D-fucose. The electrophoretic pattern to native and acidic proteins resolved lectin fraction in two main bands with lectin activity. These bands were considered as multiple molecular forms or isoforms of P. megistus lectin. Under denaturating conditions, isoform 1 showed one band with apparent mol wt (MW) of 64 kDa while isoform 2 was resolved in two bands with MW of 64 and 33 kDa.     

Plasma antioxidant capacity determination: comparative evaluation of chemiluminescent and spectrophotometric assays

All aerobic organisms have developed different mechanisms for neutralising the free radicals, mostly produced by the monoelectronic reduction of O(2), and preventing the severe damages these can provoke. The efficiency of these mechanisms can be assessed, in different matrices, by a simple and direct chemiluminescent assay (CL) based on luminol oxidation catalysed by horseradish peroxidase. Light emission is mediated by the production of free radicals and it is inhibited after a sample addition in a way that is directly proportional to the sample total content of molecules displaying antioxidant activity. The performances of this chemiluminescent assay were compared with those of two spectrophotometric methods already applied in clinical practice. First spectrophotometric method measures, like CL assay, the total antioxidant capacity, whereas the second one determines free thiol groups content. The chemiluminescent assay has a linearity interval between 0.60 and 9.46 mumol l(-1) of Trolox (y=34.91x+3.10; r=0.999; n=5) with an imprecision, expressed as CV, of 3.8% and an inaccuracy, expressed as percentage recovery, of 109%. The first spectrophotometric method, based on the same reference standard, the Trolox molecule, has a linearity interval between 0.2 and 2.5 mmol l(-1) of Trolox (y=-0.01x+4.54; r=0.95; n=5); the thiol groups assay has a linearity interval between 0.1 and 1 mmol l(-1) of l-cysteine (y=1.68x-47.09; r=0.998; n=5). Different clinical samples of plasma from healthy individuals, obese subjects and patients with liver diseases were tested. Interesting correlations were obtained among the three methods, but no significant correlations emerged between antioxidant capacity and clinical parameters. Significant differences were there only between men and women among obese subjects and between drinkers and non-drinkers among liver disease patients.