TXTGate: profiling gene groups with text-based information

We implemented a framework called TXTGate that combines literature indices of selected public biological resources in a flexible text-mining system designed towards the analysis of groups of genes. By means of tailored vocabularies, term- as well as gene-centric views are offered on selected textual fields and MEDLINE abstracts used in LocusLink and the Saccharomyces Genome Database. Subclustering and links to external resources allow for in-depth analysis of the resulting term profiles.     

Selective inhibitory DNA aptamers of the human RNase H1

Human RNase H1 binds double-stranded RNA via its N-terminal domain and RNA–DNA hybrid via its C-terminal RNase H domain, the latter being closely related to Escherichia coli RNase HI. Using SELEX, we have generated a set of DNA sequences that can bind efficiently (K_d values ranging from 10 to 80 nM) to the human RNase H1. None of them could fold into a simple perfect double-stranded DNA hairpin confirming that double-stranded DNA does not constitute a trivial ligand for the enzyme. Only two of the 37 DNA aptamers selected were inhibitors of human RNase H1 activity. The two inhibitory oligomers, V-2 and VI-2, were quite different in structure with V-2 folding into a large, imperfect but stable hairpin loop. The VI-2 structure consists of a central region unimolecular quadruplex formed by stacking of two guanine quartets flanked by the 5′ and 3′ tails that form a stem of six base pairs. Base pairing between the 5′ and 3′ tails appears crucial for conferring the inhibitory properties to the aptamer. Finally, the inhibitory aptamers were capable of completely abolishing the action of an antisense oligonucleotide in a rabbit reticulocyte lysate supplemented with human RNase H1, with IC₅₀ ranging from 50 to 100 nM.     

Ultraviolet A radiation transiently disrupts gap junctional communication in human keratinocytes

Ultraviolet A (UVA) (320-400 nm)radiation is known to cause cutaneous aging and skin cancer. We studiedthe effect of UVA (365 nm) radiation on the human epidermis by focusingon keratinocyte gap junction-mediated intercellular communication(GJIC). We observed a dose-dependent 10-fold decrease in GJIC inducedby UVA in normal human keratinocytes. This decrease in GJIC wasassociated with time-dependent internalization of connexin43 (Cx43).UVA radiation also damaged the actin cytoskeleton, as shown bymicrofilament disappearance. Importantly, the decrease in GJIC wastransient when keratinocytes were irradiated with 10 J/cm²UVA, with a return to baseline values after 8 h. Concomitantly, Cx43 was relocalized and the actin cytoskeleton was restored. UVAirradiation and 12-O-tetradecanoylphorbol 13-acetate (TPA) treatment activated protein kinase C and reduced GJIC. However, Cx43localization and phosphorylation were differently regulated by the twotreatments. This suggests that at least two different pathways maymediate the observed fall in GJIC. These findings identify keratinocyteGJIC as a new UVA target that might sensitize human skin to photoagingand cancer formation.

     

Solid-phase micro-extraction-gas chromatography-mass spectrometry and headspace-gas chromatography of tetrahydrocannabinol,amphetamine, methamphetamine,cocaine and ethanol in saliva samples

In the present work, a method was developed aiming at the serial detection of tetrahydrocannabinol (THC), amphetamine, methamphetamine, cocaine and ethanol in saliva. Saliva samples were submitted to an initial headspace procedure for ethanol determination by gas chromatography/flame ionization detector (GC-FID). After this step, two consecutive solid-phase micro-extractions (SPME) were carried out: THC was extracted by submersing a polydimethylsiloxane fiber (100 micro m) in the vial for 20 min; amphetamine, methamphetamine and cocaine were subsequently extracted after alkalinization. Derivatization of the amphetamines was carried out directly in the solution by adding 2 micro l of butylchloroformate. Gas chromatography-mass spectrometry (GC-MS) was used to identify the analytes in selected ion monitoring (SIM) mode. Confidence parameters of validation of the method were: recovery, linearity, intra- and inter-assay precision as well as limits of detection and quantification of the analytes. The limits of quantification (LOQ) obtained were: ethanol (0.010 g/l); amphetamine (5.0 ng/ml); methamphetamine (0.5 ng/ml); cocaine (5 ng/ml) and THC (5 ng/ml). The method proved to be highly precise (coefficient of variation<8%) for all detected substances.     

Expression and role of calcium-ATPase pump and sodium-calcium exchanger in differentiated trophoblasts from human term placenta

Although placental transfer of maternal calcium (Ca(2+)) is a crucial process for fetal development, the biochemical mechanisms are not completely elucidated. Especially, mechanisms of syncytiotrophoblast Ca(2+) extrusion into fetal circulation remain to be established. In the current study we have investigated the characteristics of Ca(2+) efflux in syncytiotrophoblast-like structure originating from the differentiation of cultured trophoblasts isolated from human term placenta. Time-courses of Ca(2+) uptake by differentiated human trophoblasts displayed rapid initial entry (initial velocity (V(i)) of 8.82 +/- 0.86 nmol/mg protein/min) and subsequent establishment of a plateau. Ca(2+) efflux studies with (45)Ca(2+)-loaded cells also showed rapid decline of cell-associated (45)Ca(2+) with a V(i) of efflux (V(ie)) of 8.90 +/- 0.96 nmol/mg protein/min. Expression of membrane systems responsible for intracellular Ca(2+) extrusion from differentiated human trophoblast were investigated by RT-PCR. Messenger RNAs of four known isoforms of PMCA (PMCA 1-4) were detected. Messenger RNAs of two cloned human NCX isoforms (NCX1 and NCX3) were also revealed. More specifically, both splice variants NCX1.3 and NCX1.4 were amplified by PCR with total RNA of differentiated human trophoblast cells. Ca(2+) flux studies in Na-free incubation medium indicated that NCX played a minimal role in the cell Ca(2+) fluxes. However, erythrosine B (inhibitor of PMCA) time- and dose-dependently increased cell associated (45)Ca(2+) suggesting a principal role of plasma membrane Ca(2+)-ATPase (PMCA) in the intracellular Ca(2+) extrusion of syncytiotrophoblast-like structure originating from the differentiation of cultured trophoblast cells isolated from human term placenta.     

PROTICdb: a web-based application to store, track, query, and compare plant proteome data

PROTICdb is a web-based application, mainly designed to store and analyze plant proteome data obtained by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and mass spectrometry (MS). The purposes of PROTICdb are (i) to store, track, and query information related to proteomic experiments, i.e., from tissue sampling to protein identification and quantitative measurements, and (ii) to integrate information from the user's own expertise and other sources into a knowledge base, used to support data interpretation (e.g., for the determination of allelic variants or products of post-translational modifications). Data insertion into the relational database of PROTICdb is achieved either by uploading outputs of image analysis and MS identification software, or by filling web forms. 2-D PAGE annotated maps can be displayed, queried, and compared through a graphical interface. Links to external databases are also available. Quantitative data can be easily exported in a tabulated format for statistical analyses. PROTICdb is based on the Oracle or the PostgreSQL Database Management System and is freely available upon request at the following URL: http://moulon.inra.fr/ bioinfo/PROTICdb.     

Dietary supplementation with (R)-alpha-lipoic acid reverses the age-related accumulation of iron and depletion of antioxidants in the rat cerebral cortex

Accumulation of divalent metal ions (e.g. iron and copper) has been proposed to contribute to heightened oxidative stress evident in aging and neurodegenerative disorders. To understand the extent of iron accumulation and its effect on antioxidant status, we monitored iron content in the cerebral cortex of F344 rats by inductively coupled plasma atomic emission spectrometry (ICP-AES) and found that the cerebral iron levels in 24-28-month-old rats were increased by 80% (p<0.01) relative to 3-month-old rats. Iron accumulation correlated with a decline in glutathione (GSH) and the GSH/GSSG ratio, indicating that iron accumulation altered antioxidant capacity and thiol redox state in aged animals. Because (R)-alpha-Lipoic acid (LA) is a potent chelator of divalent metal ions in vitro and also regenerates other antioxidants, we monitored whether feeding LA (0.2% [w/w]; 2 weeks) could lower cortical iron and improve antioxidant status. Results show that cerebral iron levels in old LA-fed animals were lower when compared to controls and were similar to levels seen in young rats. Antioxidant status and thiol redox state also improved markedly in old LA-fed rats versus controls. These results thus show that LA supplementation may be a means to modulate the age-related accumulation of cortical iron content, thereby lowering oxidative stress associated with aging.     

Do orthologous gene phylogenies really support tree-thinking?

Background  

Since Darwin's Origin of Species, reconstructing the Tree of Life has been a goal of evolutionists, and tree-thinking has become a major concept of evolutionary biology. Practically, building the Tree of Life has proven to be tedious. Too few morphological characters are useful for conducting conclusive phylogenetic analyses at the highest taxonomic level. Consequently, molecular sequences (genes, proteins, and genomes) likely constitute the only useful characters for constructing a phylogeny of all life. For this reason, tree-makers expect a lot from gene comparisons. The simultaneous study of the largest number of molecular markers possible is sometimes considered to be one of the best solutions in reconstructing the genealogy of organisms. This conclusion is a direct consequence of tree-thinking: if gene inheritance conforms to a tree-like model of evolution, sampling more of these molecules will provide enough phylogenetic signal to build the Tree of Life. The selection of congruent markers is thus a fundamental step in simultaneous analysis of many genes.     

Cyclooxygenase-2-issued prostaglandin e(2) enhances the production of endogenous IL-10, which down-regulates dendritic cell functions

PGE(2) is a well-known immunomodulator produced in the immune response by APCs, such as dendritic cells (DCs), the most potent APC of the immune system. We investigated the PGE(2) biosynthetic capacity of bone marrow-derived DC (BM-DC) and the effects of PG on the APC. We observed that BM-DC produce PGE(2) and other proinflammatory mediators, such as leukotriene B(4) and NO, after LPS exposure. Constitutively present in BM-DC, cyclooxygenase (COX)-1 did not contribute significantly to the total pool of PGE(2) compared with the LPS-induced COX-2-produced PGE(2). Treatment of BM-DC with exogenous PGE(2) induced the production of large amounts of IL-10 and less IL-12p70. In addition, selective inhibition of COX-2, but not COX-1, was followed by significant decrements in PGE(2) and IL-10, a concomitant restoration of IL-12 production, and an enhancement of DC stimulatory potential. In contrast, we found no demonstrable role for leukotriene B(4) or NO. In view of the potential of PGE(2) to stimulate IL-10, we examined the possibility that the suppressive effect of PGE(2) is mediated via IL-10. We found that exogenous IL-10 inhibits IL-12p70 production in the presence of NS-398, a COX-2 selective inhibitor, while the inhibitory effects of PGE(2) were totally reversed by anti-IL-10. We conclude that COX-2-mediated PGE(2) up-regulates IL-10, which down-regulates IL-12 production and the APC function of BM-DC.     

Effects of parasitism by Asobara tabida (Hymenoptera: Braconidae) on the development,survival and activity of Drosophila melanogaster larvae

The impact of parasitism by Asobara tabida on Drosophila melanogaster larval development, survival features and larval activity has been investigated using two strains of the parasitoid. The successful parasitism rate of the A1 strain was four times greater than that of the WOPV strain. Both strains induced equivalent mortality rates but hosts parasitized by A1 predominantly died as pupae. The time necessary for the host pupariation and emergence, and the larval weight at 72, 96 and 120 h post-parasitization were measured. Parasitized larvae exhibited longer periods of development and lower weights than controls, especially when parasitized by A1. These results suggest that hosts underwent physiological costs varying with respect to the outcome of the parasitic relationship. Of the parasitoid factors possibly responsible for these costs, we examined venoms for their impact on host mortality. Artificial injections of WOPV venoms induced higher mortality rates than did A1 venoms. Venoms were also found responsible for the induction of a transient paralysis, naturally occuring after parasitization. Again, the strongest effect was observed after parasitization by WOPV or injections of its venoms. This study gives new insights into the intriguing features of A. tabida and constitutes the first report of the paralysing properties of the venoms.