Liquid chromatography-tandem mass spectrometry characterization of ergocristam degradation products

The UPLC method with diode array UV detection was developed for qualitative determination of ergocristine and ergocristam including degradation products. The mechanism of the ergocristam disruptive reaction was described based on MS/MS characterization of ammonolytic product, N-(d-lysergyl)-l-valinamide (A1) and two methanolytic products, methyl ester of N-(d-lysergyl)-l-valine (M2), and N-[N-(d-lysergyl)-l-valyl]-l-phenylalanyl-d-prolyl methyl ester (M1). The influence of extraction conditions on epimerization and degradation of ergocristine and ergocristam was tested and conditions for reproducible decomposition of ergocristam were found. The presented method could potentially be applied for ergot alkaloids determination in sclerotia, fermentation broth, mycelium, and possibly contaminated food products, i.e. corn, flour, bread, etc., and feeding stuffs containing ungrounded cereals.     

Evolutionary origins and functions of the carotenoid biosynthetic pathway in marine diatoms

Carotenoids are produced by all photosynthetic organisms, where they play essential roles in light harvesting and photoprotection. The carotenoid biosynthetic pathway of diatoms is largely unstudied, but is of particular interest because these organisms have a very different evolutionary history with respect to the Plantae and are thought to be derived from an ancient secondary endosymbiosis between heterotrophic and autotrophic eukaryotes. Furthermore, diatoms have an additional xanthophyll-based cycle for dissipating excess light energy with respect to green algae and higher plants. To explore the origins and functions of the carotenoid pathway in diatoms we searched for genes encoding pathway components in the recently completed genome sequences of two marine diatoms. Consistent with the supplemental xanthophyll cycle in diatoms, we found more copies of the genes encoding violaxanthin de-epoxidase (VDE) and zeaxanthin epoxidase (ZEP) enzymes compared with other photosynthetic eukaryotes. However, the similarity of these enzymes with those of higher plants indicates that they had very probably diversified before the secondary endosymbiosis had occurred, implying that VDE and ZEP represent early eukaryotic innovations in the Plantae. Consequently, the diatom chromist lineage likely obtained all paralogues of ZEP and VDE genes during the process of secondary endosymbiosis by gene transfer from the nucleus of the algal endosymbiont to the host nucleus. Furthermore, the presence of a ZEP gene in Tetrahymena thermophila provides the first evidence for a secondary plastid gene encoded in a heterotrophic ciliate, providing support for the chromalveolate hypothesis. Protein domain structures and expression analyses in the pennate diatom Phaeodactylum tricornutum indicate diverse roles for the different ZEP and VDE isoforms and demonstrate that they are differentially regulated by light. These studies therefore reveal the ancient origins of several components of the carotenoid biosynthesis pathway in photosynthetic eukaryotes and provide information about how they have diversified and acquired new functions in the diatoms.     

Apoptosis and inhibition of gap-junctional intercellular communication induced by LA-12, a novel hydrophobic platinum(IV) complex

A new hydrophobic platinum(IV) complex, LA-12, a very efficient anticancer drug lacking cross-resistance with cisplatin (CDDP), is now being tested in clinical trials. Here we investigated the apoptogenic activity of LA-12 and its effect on gap-junctional intercellular communication (GJIC) in the rat liver epithelial cell line WB-F344. LA-12 induced apoptosis much more efficiently than did CDDP due to a combination of rapid penetration into the cell and attack on DNA, leading to fast activation of p53 and caspase-3. Exposure of WB-F344 cells to LA-12 led to rapid induction of the time- and dose-dependent decrease in GJIC. On the molecular level, loss of GJIC induced by LA-12 was mediated by activation of extracellular signal-regulated kinase (ERK)-1 and ERK-2, as demonstrated by the use of inhibitors of ERK activation. Inhibition of GJIC was linked to rapid hyperphosphorylation of connexin-43 and disappearance of connexon clusters from membranes, which was not observed in the case of CDDP.     

Purification and characterization of a nitrilase from Fusarium solani O1

An intracellular nitrilase was purified from a Fusarium solani O1 culture, in which the enzyme (up to 3000 U L⁻¹) was induced by 2-cyanopyridine. SDS-PAGE revealed one major band corresponding to a molecular weight of approximately 40 kDa. Peptide mass fingerprinting suggested a high similarity of the protein with the putative nitrilase from Gibberella moniliformis. Electron microscopy revealed that the enzyme molecules associated into extended rods. The enzyme showed high specific activities towards benzonitrile (156 U mg⁻¹) and 4-cyanopyridine (203 U mg⁻¹). Other aromatic nitriles (3-chlorobenzonitrile, 3-hydroxybenzonitrile) also served as good substrates for the enzyme. The rates of hydrolysis of aliphatic nitriles (methacrylonitrile, propionitrile, butyronitrile, valeronitrile) were 14–26% of that of benzonitrile. The nitrilase was active within pH 5–10 and at up to 50 °C with optima at pH 8.0 and 40–45 °C. Its activity was strongly inhibited by Hg²⁺ and Ag⁺ ions. More than half of the enzyme activity was preserved at up to 50% of n-hexane or n-heptane or at up to 15% of xylene or ethanol. Operational stability of the enzyme was examined by the conversion of 45 mM 4-cyanopyridine in a continuous and stirred ultrafiltration-membrane reactor. The nitrilase half-life was 277 and 10.5 h at 35 and 45 °C, respectively.     

The influence of polymorphism of -493G/T MTP gene promoter and metabolic syndrome on lipids, fatty acids and oxidative stress

The aim of this study was to investigate the effect of the microsomal triglyceride transfer protein (MTP) -493G/T polymorphism on clinical and biochemical parameters in relation to the presence of metabolic syndrome (MS). A group of 270 participants, 143 men and 127 women [50 men/36 women fulfilled the International Diabetes Federation (IDF) criteria of MS], was categorized on the basis of the MTP -493G/T polymorphism: GG homozygotes (Group GG) and carriers of the T allele (Group TT+TG). In men with MS, the presence of the T allele was associated with elevated concentrations of plasma insulin (by 48%, P<.01) and nonesterified fatty acids (by 49%, P<.05); homeostasis model assessment for insulin resistance index was higher by 64% (P<.05). Carriers of the T allele were further characterized by elevated plasma concentrations of total cholesterol (by 14%, P<.05) and by increased triglycerides in plasma (by 95%, P<.01) and in very low-density lipoprotein (by 106%, P<.01). They also had lower concentrations of n-6 polyunsaturated fatty acids in plasma phospholipids (by 3.5%, P<.05), lower Delta5-desaturase activities (by 18%, P<.05) and elevated concentrations of conjugated dienes in low-density lipoprotein (by 29%, P<.01). No significant differences between Groups GG and TT+TG were found in men without MS and in women with and without MS. Our results imply evidence for interactive effects of genetic, metabolic and gender-specific factors on several components of metabolic syndrome, which can increase the risk for cardiovascular disease.     

Two types of aspartic proteinases from buckwheat seed--gene structure and expression analysis

Two types of aspartic proteinase (AP) genes have been isolated from the cDNA library of developing buckwheat seeds. Analysis of their sequences showed that one of these, FeAP9, resembled the structure and shared high homology with the so-called typical plant APs characterized by the presence of a plant-specific insert (PSI), an element unique among APs. The other cDNA, FeAPL1, encoded an AP-like protein lacking that domain. Different expression profiles were observed for FeAP9 and FeAPL1. FeAPL1 mRNAs were restricted to the seeds only, whereas FeAP9 mRNAs were also present in the other plant tissues - leaves, roots, and flowers. Higher levels of FeAP9 were observed in senescent leaves compared with green leaves. The differential expression pattern of these two unique APs raises the interesting possibility that these proteinases have unique substrate specificity and may have different roles in plant development and other physiological processes.     

Gender-specific growth patterns for stature,sitting height and limbs length in Croatian children and youth (3 to 18 years of age)

In a cross-sectional study of growth, 5,155 children (2,591 females, 2,564 males) from the town of Zagreb (Croatia) were measured. Four traits of linear dimensionality (stature, sitting height, arm and leg lengths) were studied in the age span of 3 to 18 years. A significant average annual increase of all four anthropometric parameters were observed up to 14 and 15 years of age in girls and 16 years of age in boys, showing that girls had a shorter growing period. In the prepubertal period until 9 years of age, gender differences were negligible. At the age of 10, boys were overgrown by girls in all parameters due to the earlier onset of puberty in girls. The growth gains for girls, when compared with those for boys, show a different pattern across variables. The female growth advantage remained in a two years period for the limbs length, but in a three year period for stature and the longest, for 4 years, for sitting height. The male predominance in size had an onset at the age of 13 for the limbs and in the age of 14 for stature and sitting height. The patterns of sexual dimorphism in stature and sitting height during growing years are similar to those observed in other populations of Europe. Growth of Croatian children and youth is very similar to that of the tallest European populations.