A validated enantiospecific method for determination and purity assay of clopridogrel

A new and accurate HPLC method using β‐cyclodextrin chemically bonded to spherical silica particles as chiral stationary phase (CSP) was developed and validated for determination of S‐clopidogrel and its impurities R‐enantiomer and S‐acid as a hydrolytic product. The effects of acetonitrile and methanol content in the mobile phase and temperature on the resolution and retention of enantiomers were investigated. A satisfactory resolution of S‐clopidogrel active form and its impurities was achieved on ChiraDex® column (5 μm, 4 × 250 mm) at a flow rate of 1.0 ml/min and 17°C using acetonitrile, methanol and 0.01 M potassium dihydrogen phosphate solution (15:5:80 v/v/v) as mobile phase. The detection wavelength was set at 220 nm. The method was validated in terms of accuracy, precision, linearity, and robustness. The limit of detection for R‐enantiomer and S‐acid were 0.75 and 0.09 μg/ml, respectively, injection volume being 20 μl. Finally, the molecular modeling of the inclusion complexes between the analytes and β‐cyclodextrin was performed to investigate the mechanism of the enantiorecognition and to study the quantitative structure–retention relationships. Chirality, 2009. © 2008 Wiley‐Liss, Inc.     

Gender differences in nutritional behavior and weight status during early and late adolescence

The current study aimed to determine gender differences in nutritional habits, eating behaviour, weight status, body image and weight control practices during early and late adolescence. 677 Viennese pupils (253 boys and 424 girls) between the ages 10 and 18 years (x = 14.1 yrs; +/- 2.2) were enrolled in the study. Weight status was determined by means of body mass index percentiles. To assess eating behavior, food preferences, body image and weight control practices, a 48 item questionnaire was developed. Significant gender differences in weight status were observable during late adolescence only. Girls are significantly less satisfied with their body weight. Furthermore, girls practice dieting and weight control to avoid any weight gain more frequently than boys. Gender differences in eating behavior intensified from early to late adolescence. From early to late adolescence, meal size decreased among girls, while it remains stabile or increased among boys. Boys eat generally more than girls. Furthermore, boys preferred meat and fast food while girls consumed fruits, vegetables and healthy food significantly more frequently. These gender differences are explained by gender specific energetic demands and culture typical beauty ideals.     

Differential expression of heat-shock proteins Hsp70 and Hsp90 in vegetative and reproductive tissues of Iris pumila

Tissue-specific variation in Hsp70 and Hsp90 expression was studied in vegetative (leaf) and reproductive organs (floral tube, ovary and stamen) of Iris pumila plants originating from a sun-exposed and a shaded natural population, which experienced similar growth conditions in an experimental garden. Western blot analysis revealed the presence of both the Hsps in all examined tissues, but at different amounts. In addition to Hsp90a and Hsp90b that were previously detected in vegetative tissues, three new immunospecific bands, designated herein as Hsp90c, Hsp90d and Hsp90e, were recognized with the same anti-Hsp90 antibody in the reproductive tissues. Apart from showing tissue-specific differences in the relative amount of Hsp70 and Hsp90, our study provides evidence that the degree of Hsps expression within the same tissue also depended on the habitat type that the I. pumila plants were derived from.     

Boron atom as an electron density relay

The electronic structure of tetrapyrrolidinodiborane [(C₄H₈N)₂B]₂ has been investigated by HeI and HeII UV photoelectron spectroscopy (UPS) and DFT/OVGF calculations. Our results indicate that the title molecule has very low first ionization energy and can be readily oxidized in solution. We estimated the magnitudes of alkyl substituent effects on nitrogen and boron atoms and the efficiency of boron atom to act as an electron density relay facilitating through-bond interactions between the nitrogen lone pairs.     

Somatic embryogenesis and shoot organogenesis from the hypocotyl slices and free radical scavenging activity of regenerants of collard greens (Brassica oleracea L. var. acephala)

Brassica oleracea var. acephala is an important leafy vegetable that has been widely consumed as a high-nutrient, low-calorie food. Because of the plant’s biennial and self-incompatibility nature, biotechnological approaches are alternative way for propagation and breeding improvements. Since tissue culture studies have been focused in other B. oleracea representatives, the aim of the present study was to achieve effective regeneration protocol distinctive for collard greens, and evaluate the total phenolic content and antioxidant activity of regenerants. The effect of 3 cytokinins [thidiazuron (TDZ), 6-benzyladenine (BA) and 6-furfuryladenine (kinetin, KIN)] at increasing concentrations (0, 5, 7.5, 10, 20 or 30 µM) in combination with tenfold lower concentration of 1-naphtaleneacetic acid (NAA) (0, 0.5, 0.75, 1, 2 or 3 µM, respectively) on the regeneration from hypocotyl slices was studied. Histological analysis revealed the two regeneration pathways, somatic embryogenesis and shoot organogenesis, simultaneously occurred in the same explant, regardless of the cytokinin/NAA combinations used. The regeneration frequency of 95.9%, with 7.5 morphogenic structures regenerated per explant, and the healthy appearance of regenerated plants indicated the optimal combination 20 µM TDZ?+?2 µM NAA. TDZ at 5 µM provided the high somatic embryo proliferation rate by generation of secondary embryos (7.79) along with the lowest rate of their abnormalities. Embryo-developed plants were successfully acclimatised (above 90%). The plants regenerated and proliferated on TDZ-containing media had higher total phenolic content that correlated with the highest free radical scavenging activity (IC₅₀?=?19.09 µg ml^??1).

     

Light-controlled root elongation in in vitro cultures of Dracaena fragrans Ker-Gawl

Haploid plants have been obtained from unpollinated ovules of onion cultured in vitro as here reported for the first time. Callus was not formed at any step of the process and the young developing embryos sprouted by splitting the ovules. From a total of about 26000 cultured ovules, 13 embryos were obtained and 7 of them developed into plants. Cytological observation of root apexes showed that 3 embryos out of 7 regenerated haploid plants (8 chromosomes). The yield of embryo induction was quite low (0.28% the best treatment) so further experiments are in progress to increase it.Abbreviations NAA naphthaleneacetic acid - IAA indole-3yl-acetic acid - 2iP N⁶-²-isopentenyladenine - TIBA 2-3-5 trijodobenzoic acid - adenine SO₄ adenine sulphate · 2H₂O - ABA abscisic acid - GA₃ gibberellic acid     

The rat leukocyte-type 12-lipoxygenase exhibits an intrinsic hepoxilin A3 synthase activity

Hepoxilins are biologically relevant eicosanoids formed via the 12-lipoxygenase pathway of the arachidonic acid cascade. Although these eicosanoids exhibit a myriad of biological activities, their biosynthetic mechanism has not been investigated in detail. We examined the arachidonic acid metabolism of RINm5F rat insulinoma cells and found that they constitutively express a leukocyte-type 12S-lipoxygenase. Moreover, we observed that RINm5F cells exhibit an active hepoxilin A(3) synthase that converts exogenous 12S-HpETE (12S-5Z,8-Z,10E,14Z-12-hydro(pero)xy-eicosa-5,8,10,14-tetraenoic acid) or arachidonic acid predominantly to hepoxilin A(3). 12S-lipoxygenase and hepoxilin A(3) synthase activities were co-localized in the cytosol; immunoprecipitation with an anti-12S-lipoxygenase antibody co-precipitated the two catalytic activities. These data suggested that hepoxilin A(3) synthase activity may be considered an intrinsic catalytic property of the leukocyte-type 12S-lipoxygenase. To test this hypothesis we cloned the leukocyte-type 12S-LOX from RINm5F cells, expressed it in Pichia pastoris, and found that the recombinant enzyme exhibited both 12S-lipoxygenase and hepoxilin A(3) synthase activities. The recombinant human platelet-type 12S-lipoxygenase and the porcine leukocyte-type 12S-lipoxygenase also exhibited hepoxilin A(3) synthase activity. In contrast, the native rabbit reticulocyte-type 15S-lipoxygenase did not convert 12S-HpETE to hepoxilin isomers. These data suggest that the positional specificity of lipoxygenases may be crucial for this catalytic function. This hypothesis was confirmed by site-directed mutagenesis studies that altered the positional specificity of the rat leukocyte-type 12S- and the rabbit reticulocyte-type 15-lipoxygenase. In summary, it may be concluded that naturally occurring 12S-lipoxygenases exhibit an intrinsic hepoxilin A(3) synthase activity that is minimal in lipoxygenase isoforms with different positional specificity.     

Cholinesterase unit establishment and issuing of >Warning Cards< for carriers of suxamethonium sensitive serum butyrylcholinesterase variants

Recognition of butyrylcholinesterase (EC 3.1.1.8) variants in human serum is essential to identify patients who may be susceptible to a prolonged reaction of suxamethonium and mivacurium, short-acting muscle relaxants. Thus they can be given appropriate advice along with their relatives who may be similarly affected. Therefore, Cholinesterase Unit for detection of individuals, carriers of inherited suxamethonium sensitive butyrylcholinesterase variants was established at the Institute for Clinical Chemistry of the Clinical Hospital >Merkur<, Zagreb, Croatia. A study was conducted on sera from patients referred to the Unit. Butyrylcholinesterase variants were determined by measuring the enzyme activity and inhibition by specific inhibitors in the sera of 384 patients and of the members of seven families. Cholinesterase Unit issued >Warning Cards< to the carriers of inherited serum butyrylcholinesterase variants in order to avoid prolonged apnea that suxamethonium might cause.     

Fitness level of adult economically active population in the Republic of Croatia estimated by EUROFIT system

The study presents the results of the EUROFIT CROATIA project that was conducted according to the instructions of Council of Europe. The aim of the study was evaluation of fitness level of the employed Croatian population. The measurements comprised EUROFIT battery of tests as well as Baecke's questionnaire for evaluation of habitual physical activity. The sample was comprised of 1628 active inhabitants, 18-60 years of age, all of who were employed and living in Croatia. The obtained results of functional, motor and morphological characteristics were presented according to the sex and age. The results shower poor aerobic capacity, high obesity indicators and low motor performance in both sexes, which pointed to the increased risk for developing many cardiovascular or musculo-skeletal diseases. The overall fitness level of Croatian population showed to be unsatisfactory and it should be improved by engagement in sports-recreational activities.