Determination of ximelagatran,an oral direct thrombin inhibitor,its active metabolite melagatran,and the intermediate metabolites,in biological samples by liquid chromatography-mass spectrometry

Analytical methods for the determination of ximelagatran, an oral direct thrombin inhibitor, its active metabolite melagatran, and intermediate metabolites, melagatran hydroxyamidine and melagatran ethyl ester, in biological samples by liquid chromatography (LC) positive electrospray ionization mass spectrometry (MS) using selected reaction monitoring are described. Isolation from human plasma was achieved by solid-phase extraction on octylsilica. Analytes and isotope-labelled internal standards were separated by LC utilising a C(18) analytical column and a mobile phase comprising acetonitrile-4 mmol/l ammonium acetate (35:65, v/v) containing 0.1% formic acid, at a flow-rate of 0.75 ml/min. Absolute recovery was approximately 80% for ximelagatran, approximately 60% for melagatran ethyl ester and >90% for melagatran and melagatran hydroxyamidine. Limit of quantification was 10 nmol/l, with a relative standard deviation <20% for each analyte and <5% above 100 nmol/l. Procedures for determination of these analytes in human urine and breast milk, plus whole blood from rat and mouse are also described.     

Identification of the hydroxyl radical and other reactive oxygen species in human neutrophil granulocytes exposed to a fragment of the amyloid beta peptide

A fragment of the amyloid beta protein, βA(25-35), was investigated for its effect on production of reactive oxygen species (ROS) in human neutrophil granulocytes. The formation and identification of ROS were examined by using a 2',7'-dichlorofluorescin (DCF) fluorescence assay, a luminol chemiluminescence assay, electron paramagnetic resonance (EPR) spectroscopy with DEPMPO as a spin trap, and hydroxylation of 4-hydroxybenzoate (4-HBA). The DCF assay showed that βA(25-35) stimulated formation of ROS in a concentration and time dependent manner. The inverted peptide, βA(35-25), gave no response. Also, luminol-amplified chemiluminescence was stimulated by βA(25-35). Incubation with diethyldithiocarbamate (a superoxide dimustase inhibitor) and salicylhydroxamate (SHA; a myeloperoxidase inhibitor) reduced the chemiluminescence. This indicates that hypochlorous acid (HOCl) is formed after exposure to βA(25-35). The EPR spectra indicated a concentration dependent formation of superoxide ( O 2 • - ) - and hydroxyl ( •OH)- radicals. Hydroxylation of 4-HBA to 3,4,-dihydroxybenzoate confirmed production of •OH. This response was attenuated by SHA, indicating involvement of HOCl in formation of •OH. The DCF fluorescence was inhibited with U0126 (an extracellular signal regulated protein kinase (ERK) inhibitor). Further analysis with western blot confirmed phosphorylation of ERK1/2 after exposure to βA(25-35). The phospholipase A 2 (PLA 2 ) inhibitor 7,7-dimethyl-(5Z,8Z)-eicosadienoic acid, and diphenyleneiodonium, which inhibits the NADPH oxidase, also led to a reduction of the DCF fluorescence. The present findings indicate that βA(25-35) stimulates the NADPH oxidase by activating the ERK pathway and PLA 2 . Production of O 2 • - can lead to HOCl and further formation of •OH, which both have a cytotoxic potential.     

Phytoseiid predators of whiteflies feed and reproduce on non-prey food sources

Two phytoseiid species, Euseius scutalis (Athias-Henriot) and Typhlodromips swirskii (Athias-Henriot), are able to suppress whitefly populations on single plants and are candidate biological control agents for whiteflies such as Bemisia tabaci (Gennadius). These species can feed on pollen and insect-produced honeydew and these food sources are likely to be available in crops. If the utilization of these food types results in increased reproduction or survival, populations of predators can persist when whitefly prey is scarce or absent. We studied the impact of pollen and whitefly-produced honeydew on the life history of the two phytoseiids. Cattail pollen allowed for survival, development and reproduction of both predators. Whitefly-produced honeydew greatly increased survival of E. scutalis, allowed for development into adulthood and for a sustained low rate of oviposition. The survival of adult T. swirskii was high on cucumber leaf tissue, either with or without pollen or honeydew. Oviposition by adults and juvenile survival of T. swirskii was very low in presence of honeydew. Biological control of whiteflies may benefit from both pollen and honeydew because these non-prey food sources have a positive effect on the life history of the two predator species, especially E. scutalis.     

A simple system for monitoring biodiversity in protected areas of a developing country

The achievements of initiatives to strengthen biodiversity conservation in developing countries may be difficult to assess, since most countries have no system for monitoring biodiversity. This paper describes a simple and cost-effective, field-based biodiversity monitoring system developed specifically for areas where specialist staff is lacking. We discuss the preliminary lessons learned from protected areas in the Philippines. Whilst the monitoring system aims to identify trends in biodiversity and its uses so as to guide management action, it also promotes the participation of local people in the management, stimulates discussions about conservation amongst stakeholders and builds the capacity of park staff and communities in management skills. In addition, it seeks to provide people with direction regarding the aims of protected areas, and reinforces the consolidation of existing livelihoods through strengthening community-based resource management systems. The field methods are: (1) standardised recording of routine observations, (2) fixed point photographing, (3) line transect survey, and (4) focus group discussion. Both bio-physical and socio-economic data are used and given equal importance. The system can be sustained using locally available resources. The approach is useful in countries embarking on shared management of park resources with local communities, where rural people depend on use of natural ecosystems, and where the economic resources for park management are limited. We hope this paper will encourage other countries to develop their own biodiversity monitoring system, letting its development become a means for capacity building whilst at the same time supporting the creation of ownership.     

An approximation to the phylogeny of Sclerotinia and related genera

Phylogenies are constructed based on nuclear ribosomal internal transcribed spacer (ITS) DNA sequences from an ingroup consisting of 50 isolates representing 24 species of the discomycete family Sclerotiniaceae and an outgroup consisting of five related taxa of the same family. The ingroup taxa are: three Botrytis spp., two Botryotinia spp., one Ciborinia sp., one Dumontinia sp., one Grovesinia sp., six Myriosclerotinia spp., nine Sclerotinia spp. and one Sclerotium sp. The outgroup taxa are: one Ciboria sp., one Encoelia sp. and three Monilinia spp. The type species is included for all taxa except for Ciborinia and Encoelia. Several of the included taxa are important plant pathogens. The resulting phytogenies are discussed with regard to morphology, life history and taxonomy. A suspected relationship between Sclerotinia borealis and S. tetraspora , and Myriosclerotinia is rejected, while a suspected relationship between Ciborinia ciborium and Myriosclerotinia is strongly supported. Sclerotinia ulmariae , previously synonymized with Dumontinia tuberosa , is reinstated as an independent species of Dumontinia. Two new combinations, Dumontinia ulmariae and Myriosclerotinia ciborium , are proposed. The imperfectly known taxon Sclerotium cepivorum seems most closely related to Dumontinia. We conclude that Dumontinia , and Myriosclerotinia , as currently conceived, are monophyletic, and that Botryotinia along with Botrytis anamorphs probably also constitute a monophyletic lineage. The genus Sclerotinia is probably polyphyletic and characterized by symplesiomorphies rather than synapomorphies. Two putatively new taxa, Sclerotinia sp. 1, and Sclerotinia sp.2 are most closely related to S. minor, S. sclerotiorum and S. trifoliorum , and to S. borealis , respectively.     

High- and low-affinity GABA-receptors in cultured cerebellar granule cells regulate transmitter release by different mechanisms

The ability of high- and low-affinity GABA_A-receptors, respectively to inhibit depolarization coupled transmitter release was studied in cultured glutamatergic cerebellar granule cells which, depending on the culture conditions, express either high-affinity GABA_A-receptors alone or high-affinity receptors together with low-affinity receptors. In order to gain information about the coupling of these receptors to chloride channels the effect of picrotoxin and binding of [³⁵S]t-butylbicyclophosphorothionate, both of which interact specifically with such channels were studied. Moreover, the influence of Flunitrazepam on the GABA-mediated inhibition of transmitter release was investigated to see if the GABA-receptors are coupled to benzodiazepine binding sites. Under conditions where the granule cells express only high-affinity GABA_A-receptors it was found that GABA was able to inhibit transmitter release elicited by mild depolarization induced either by 30 mM KCl or 25 μM glutamate. This effect of GABA could be enhanced by Flunitrazepam and blocked by picrotoxin. However, transmitter release from these neurones induced by a more pronounced depolarization (55 mM KCl) could not be inhibited by GABA. Under conditions where the neurons express both high- and low-affinity GABA_A-receptors transmitter release elicited by 55 mM KCl could be inhibited by GABA but this inhibitory effect of GABA could not be blocked by picrotoxin, nor could it be enhanced by Flunitrazepam. These results strongly suggest that while the action of the high-affinity GABA_A-receptors is coupled to chloride channels and benzodiazepine binding sites, the physiological action of the low-affinity GABA_A-receptors is not. This lack of coupling between the low-affinity GABA_A-receptors and chloride channels is further supported by the finding that the K_D and B_max values for [³⁵S]TBPS binding to the granule cells were independent of whether or not the cells expressed low-affinity GABA_A-receptors. While the results clearly show that the inhibitory action of GABA mediated by low-affinity GABA_A-receptors is not coupled to chloride channels, the exact mechanism of action of these receptors still remains to be elucidated.     

A Study on the Fundamental Mechanism and the Evolutionary Driving Forces behind Aerobic Fermentation in Yeast

Baker’s yeast Saccharomyces cerevisiae rapidly converts sugars to ethanol and carbon dioxide at both anaerobic and aerobic conditions. The later phenomenon is called Crabtree effect and has been described in two forms, long-term and short-term effect. We have previously studied under fully controlled aerobic conditions forty yeast species for their central carbon metabolism and the presence of long-term Crabtree effect. We have also studied ten steady-state yeast cultures, pulsed them with glucose, and followed the central carbon metabolism and the appearance of ethanol at dynamic conditions. In this paper we analyzed those wet laboratory data to elucidate possible mechanisms that determine the fate of glucose in different yeast species that cover approximately 250 million years of evolutionary history. We determine overflow metabolism to be the fundamental mechanism behind both long- and short-term Crabtree effect, which originated approximately 125–150 million years ago in the Saccharomyces lineage. The “invention” of overflow metabolism was the first step in the evolution of aerobic fermentation in yeast. It provides a general strategy to increase energy production rates, which we show is positively correlated to growth. The “invention” of overflow has also simultaneously enabled rapid glucose consumption in yeast, which is a trait that could have been selected for, to “starve” competitors in nature. We also show that glucose repression of respiration is confined mainly among S. cerevisiae and closely related species that diverged after the whole genome duplication event, less than 100 million years ago. Thus, glucose repression of respiration was apparently “invented” as a second step to further increase overflow and ethanol production, to inhibit growth of other microbes. The driving force behind the initial evolutionary steps was most likely competition with other microbes to faster consume and convert sugar into biomass, in niches that were semi-anaerobic.     

Molecular Subtypes in Stage II-III Colon Cancer Defined by Genomic Instability: Early Recurrence-Risk Associated with a High Copy-Number Variation and Loss of RUNX3 and CDKN2A

Objective

We sought to investigate various molecular subtypes defined by genomic instability that may be related to early death and recurrence in colon cancer.

Methods

We sought to investigate various molecular subtypes defined by instability at microsatellites (MSI), changes in methylation patterns (CpG island methylator phenotype, CIMP) or copy number variation (CNV) in 8 genes. Stage II-III colon cancers (n = 64) were investigated by methylation-specific multiplex ligated probe amplification (MS-MLPA). Correlation of CNV, CIMP and MSI, with mutations in KRAS and BRAFV600E were assessed for overlap in molecular subtypes and early recurrence risk by uni- and multivariate regression.

Results

The CIMP phenotype occurred in 34% (22/64) and MSI in 27% (16/60) of the tumors, with noted CIMP/MSI overlap. Among the molecular subtypes, a high CNV phenotype had an associated odds ratio (OR) for recurrence of 3.2 (95% CI 1.1-9.3; P = 0.026). Losses of CACNA1G (OR of 2.9, 95% CI 1.4-6.0; P = 0.001), IGF2 (OR of 4.3, 95% CI 1.1-15.8; P = 0.007), CDKN2A (p16) (OR of 2.0, 95% CI 1.1-3.6; P = 0.024), and RUNX3 (OR of 3.4, 95% CI 1.3-8.7; P = 0.002) were associated with early recurrence, while MSI, CIMP, KRAS or BRAF V600E mutations were not. The CNV was significantly higher in deceased patients (CNV in 6 of 8) compared to survivors (CNV in 3 of 8). Only stage and loss of RUNX3 and CDKN2A were significant in the multivariable risk-model for early recurrence.

Conclusions

A high copy number variation phenotype is a strong predictor of early recurrence and death, and may indicate a dose-dependent relationship between genetic instability and outcome. Loss of tumor suppressors RUNX3 and CDKN2A were related to recurrence-risk and warrants further investigation.     

Activity assays of mammalian thioredoxin and thioredoxin reductase: Fluorescent disulfide substrates,mechanisms, and use with tissue samples

Thioredoxin (Trx) is a protein disulfide reductase that, together with nicotinamide adenine dinucleotide phosphate (NADPH) and thioredoxin reductase (TrxR), controls oxidative stress or redox signaling via thiol redox control. Human cytosolic Trx1 has Cys32 and Cys35 as the active site and three additional cysteine residues (Cys62, Cys69, and Cys73), which by oxidation generates inactive Cys62 to Cys69 two-disulfide Trx. This, combined with TrxR with a broad substrate specificity, complicates assays of mammalian Trx and TrxR. We sought to understand the autoregulation of Trx and TrxR and to generate new methods for quantification of Trx and TrxR. We optimized the synthesis of two fluorescent substrates, di-eosin–glutathione disulfide (Di-E–GSSG) and fluorescein isothiocyanate-labeled insulin (FiTC–insulin), which displayed higher fluorescence on disulfide reduction. Di-E–GSSG showed a very large increase in fluorescence quantum yield but had a relatively low affinity for Trx and was also a weak direct substrate for TrxR, in contrast to GSSG. FiTC–insulin was used to develop highly sensitive assays for TrxR and Trx. Reproducible conditions were developed for reactivation of modified Trx, commonly present in frozen or oxidized samples. Trx in cell extracts and tissue samples, including plasma and serum, were subsequently analyzed, showing highly reproducible results and allowing measurement of trace amounts of Trx.