A nonradioactive high-throughput/high-content assay for measurement of the human serotonin reuptake transporter function in vitro.

Both the tricyclic and specific serotonin reuptake inhibitor classes of antidepressants act primarily by inhibiting the reuptake of released serotonin by the human serotonin reuptake transporter (hSERT). In this article, the authors describe the use of a fluorescent substrate of the transporter (4-(4-(dimethylamino)-styrl)-N-methylpyridinium, ASP) to develop a microplate-based high-throughput screen for hSERT function. The assay is sensitive to known inhibitors of serotonin uptake, including fluoxetine (Prozac), with the correct rank order of potency and IC(50) values close to those reported in the literature for tritiated serotonin uptake.

Gene expression in cortex and hippocampus during acute pneumococcal meningitis.

Background: Pneumococcal meningitis is associated with high mortality (approximately 30%) and morbidity. Up to 50% of survivors are affected by neurological sequelae due to a wide spectrum of brain injury mainly affecting the cortex and hippocampus. Despite this significant disease burden, the genetic program that regulates the host response leading to brain damage as a consequence of bacterial meningitis is largely unknown.

Vasomodulating potential of mediterranean wild plant extracts.

The incidence of cardiovascular disease and endothelial dysfunction is low in the Mediterranean area, where the major proportion of daily calories comes from plant food, high in antioxidant polyphenols. It has been shown that a reduced production or enhanced inactivation of endothelium-derived nitric oxide (NO) is involved in the onset of endothelial dysfunction. We investigated the effects of Mediterranean wild plant, that is, wild artichoke and thyme, phenolic-rich extracts on NO release by porcine aortic endothelial cells (PAECs; by using indirect methods) and by cerebral cell membrane homogenates (by using direct NO detection).

The Streptococcus pneumoniae cia regulon: CiaR target sites and transcription profile analysis.

The ciaR-ciaH system is one of 13 two-component signal-transducing systems of the human pathogen Streptococcus pneumoniae. Mutations in the histidine protein kinase CiaH confer increased resistance to beta-lactam antibiotics and interfere with the development of genetic competence. In order to identify the genes controlled by the cia system, the cia regulon, DNA fragments targeted by the response regulator CiaR were isolated from restricted chromosomal DNA using the solid-phase DNA binding assay and analyzed by hybridization to an oligonucleotide microarray representing the S.

Influence of isolation procedure, extracellular matrix and dexamethasone on the regulation of membrane transporters gene expression in rat hepatocytes.

The influence of the isolation procedure of hepatocytes, extracellular matrix (ECM) configuration and incubation medium supplementation by dexamethasone (DEX) on the cell morphology and on the gene expression of membrane transporters was examined in rat hepatocytes. The mRNA levels were determined using oligonucleotide microarrays, in liver, in suspension and in primary culture in monolayer (CPC), and in collagen gels sandwich (SPC) in absence and presence of DEX (100 and 1000 nM). The results indicated pronounced morphological differences between CPC and SPC in response to DEX demonstrating that the hepatocytes re-formed, as in vivo, multicellular arrays with extensive bile canalicular network only in SPC in presence of DEX.

Visualising gene expression in its metabolic context.

Relative changes in mRNA as well as protein levels induced by sublethal doses of antibiotics on bacteria are measured and results visualised in the context of metabolic pathway diagrams. The mRNA levels present at a given time point after the addition of the antibiotic are measured using microarrays from Affymetrix. Additionally, the relative amount of each protein synthesised during 3 minute intervals sampled at the given times is measured using radio-labelling followed by two-dimensional polyacrylamide gel electrophoresis and the subsequent analysis of the images produced by exposure to a phosphorimager.

Mosaic genes and mosaic chromosomes: intra- and interspecies genomic variation of Streptococcus pneumoniae.

Streptococcus pneumoniae remains a major causative agent of serious human diseases. The worldwide increase of antibiotic resistant strains revealed the importance of horizontal gene transfer in this pathogen, a scenario that results in the modulation of the species-specific gene pool. We investigated genomic variation in 20 S.

Microarray-based identification of a novel Streptococcus pneumoniae regulon controlled by an autoinduced peptide.

We have identified in the Streptococcus pneumoniae genome sequence a two-component system (TCS13, Blp [bacteriocin-like peptide]) which is closely related to quorum-sensing systems regulating cell density-dependent phenotypes such as the development of genetic competence or the production of antimicrobial peptides in lactic acid bacteria. In this study we present evidence that TCS13 is a peptide-sensing system that controls a regulon including genes encoding Blps. Downstream of the Blp TCS (BlpH R) we identified open reading frames (blpAB) that have the potential to encode an ABC transporter that is homologous to the ComA/B export system for the competence-stimulating peptide ComC.

Domain organization and molecular characterization of 13 two-component systems identified by genome sequencing of Streptococcus pneumoniae.

In bacteria, adaptive responses to environmental stimuli are often initiated by two-component signal transduction systems (TCS). The prototypical TCS comprises two proteins: a histidine kinase (HK, hk) and a response regulator (RR rr). Recent research has suggested that compounds that inhibit two-component systems might have good antibacterial activity.

Competence-specific induction of recA is required for full recombination proficiency during transformation in Streptococcus pneumoniae.

Transcriptional activation of the recA gene of Streptococcus pneumoniae was previously shown to occur at competence. A 5.7 kb recA-specific transcript that contained at least two additional genes, cinA and dinF, was identified.

Bacterial transcript imaging by hybridization of total RNA to oligonucleotide arrays.

We have used high-density oligonucleotide probe arrays (chips) for bacterial transcript imaging. We designed a chip containing probes representing 106 Hemophilus influenzae genes and 100 Streptococcus pneumoniae genes. The apparent lack of polyadenylated transcripts excludes enrichment of mRNA by affinity purification and we thus used total, chemically biotinylated RNA as hybridization probe.

The trp RNA-binding attenuation protein (TRAP) regulates the steady-state levels of transcripts of the Bacillus subtilis folate operon.

The Bacillus subtilis folate operon contains nine genes. The first six genes are involved in the biosynthesis of folic acid and tryptophan and have been characterized previously. The 3'-region of the folate operon contains three additional ORFs: orf3, potentially encoding a DNA-binding protein of 68 amino acids, orf4, encoding a protein of 338 amino acids with homology to the Orf1 of the E.

Translation of trpG in Bacillus subtilis is regulated by the trp RNA-binding attenuation protein (TRAP).

trpG of Bacillus subtilis encodes a glutamine amidotransferase subunit that is involved in the synthesis of both folic acid and L-tryptophan. Expression of trpG is negatively regulated by tryptophan even though this gene is located within a folic acid biosynthetic operon. Examination of both transcriptional and translational gene fusions to lacZ involving trpG and direct measurements of trpG mRNA levels and TrpG polypeptide accumulation demonstrated that translation of trpG is regulated by tryptophan whereas transcription is not.

Enzymic characterization of Bacillus subtilis GTP cyclohydrolase I. Evidence for a chemical dephosphorylation of dihydroneopterin triphosphate.

GTP cyclohydrolase I catalyses the first committing step in the biosynthesis of the pterin moiety of folic acid: conversion of GTP to dihydroneopterin triphosphate. GTP cyclohydrolase I of Bacillus subtilis was purified to homogeneity and shown to have a homo-octameric structure. The enzyme had an apparent Km for GTP of 4 microM and, in the absence of cations, a Vmax.

Construction and characterisation of a yeast artificial chromosome library containing three haploid maize genome equivalents.

We have constructed a yeast artificial chromosome (YAC) library using high-molecular-weight DNA prepared from agarose-embedded leaf protoplasts of the maize inbred line UE95. This library contains 79,000 clones with an average insert size of 145 kb and should therefore represent approximately three haploid genome equivalents. The library is organised as an ordered array in duplicate microtitre plates.