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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. The authors also describe the validation of the assay for natural product screening using a test set of 2400 pure phyto-chemicals and 80 plant extracts. The mean Z of the screened plates was 0.53. Hit rates, confirmation rates, and validation of the hits in a "classical" assay for serotonin uptake are all reported. The assay can also be read in "high-content" mode using a subcellular imaging device, which allows direct detection of possible assay interference by acutely cytotoxic compounds. Among the compounds identified were several previously reported inhibitors of the hSERT, as well as compounds having structural similarity to the tricyclic antidepressant drugs.

Epidemiological data indicate a beneficial effect of Mediterranean diets on human health, especially on the prevalence of cardiovascular disease. These observations are supported by recent intervention studies. However, very little is known about the current role of local Mediterranean food products, which are consumed on a less regular basis and their contribution to a healthy diet. The European consortium “Local Food-Nutraceuticals” collected 127 locally consumed wild or semi-wild plants in three Mediterranean countries, i.e. Greece, Italy, and Spain, in order to assess their ethnobotanical features as well as their biological activities. The project also includes a second line of research, the study of local conceptions about these food resources. All pharmacological assays were conducted with ethanolic extracts prepared from the dried plant material. The biological activities of the extracts were assessed with the following 12 different assays covering a broad range of mechanisms considered crucial in the pathology of chronic, aging-related diseases. Four antioxidant tests: DPPH scavenging, prevention of oxyhaemoglobin bleaching, prevention of lipid peroxidation (malondialdehyde formation), and protection from DNA damage (Comet assay); three enzyme inhibition tests: inhibition of xanthine oxidase, inhibition of myeloperoxidase-catalysed guaiacol oxidation as well as the inhibition of acetylcholine esterase; one test investigating the inhibition of cytokine-induced cell activation (including the extrac ts’ potential cytotoxicity); one assay measuring the anti-proliferation potential; one test assessing the anti-diabetic activity (PPAR ) as well as one assay investigating the extracts’ effect on mood disorder-related biochemical parameters (hSERT). Furthermore, the polyphenol content of all extracts was determined using the Folin–Ciocalteaus method. The assays revealed diverse biological effects for the tested extracts ranging from no activity to almost complete inhibition/activation. Moreover, the experimental matrix led to the identification of a sub-set of extracts, i.e. Berberis vulgaris, Reichardia picroides, Scandix australis, Satureja montana, Thymus piperella, Lythrum salicaria and Vitis vinifera, showing high activity in a broad range of assays. In summary, the in vitro observed modulations and effects exerted by extracts derived from local food plants suggest that these plants may contribute to the observed better aging of rural Mediterranean populations. © 2005 Elsevier Ltd. All rights reserved.

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). NO release by PAECs was significantly potentiated by 234% and 135% by wild artichoke and thyme extracts (10(-6) mol/L), respectively. Direct detection of NO release by brain membranes also showed significantly increased NO production after wild artichoke addition (+35.4%). Further, the release of another vasorelaxant factor by PAECs, that is, prostacyclin, was significantly increased by wild artichoke and thyme (10(-6) mol/L) (+269% and +190%, respectively). Investigation of the mechanism(s) of action of wild artichoke and thyme suggests maintenance of an intracellular reduced environment, as previously shown for ascorbate. Even though these data require in vivo confirmation, they suggest that regular intake of bioactive compounds from Mediterranean wild plants contributes to maintenance of proper vasomotion and to the low incidence of atherosclerosis and endothelial dysfunction recorded in the Mediterranean area.

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. pneumoniae isolates representing major antibiotic-resistant clones and 10 different capsular serotypes. Variation was scored as decreased hybridization signals visualized on a high-density oligonucleotide array representing 1,968 genes of the type 4 reference strain KNR.7/87. Up to 10% of the genes appeared altered between individual isolates and the reference strain; variability within clones was below 2.1%. Ten gene clusters covering 160 kb account for half of the variable genes. Most of them are associated with transposases and are assumed to be part of a flexible gene pool within the bacterial population; other variable loci include mosaic genes encoding antibiotic resistance determinants and gene clusters related to bacteriocin production. Genomic comparison between S. pneumoniae and commensal Streptococcus mitis and Streptococcus oralis strains indicates distinct antigenic profiles and suggests a smooth transition between these species, supporting the validity of the microarray system as an epidemiological and diagnostic tool.

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. In order to identify TCS that are crucial for growth or virulence of Streptococcus pneumoniae, we have examined the genomic sequence of a virulent S. pneumoniae strain for genes that are related to known histidine kinases or response regulators. Altogether 13 histidine kinases and 13 response regulators have been identified. The protein sequences encoded by these genes were compared with sequences deposited in public databases. This analysis revealed that two of the 13 pneumococcal TCSs have been described before (ciaRH and comDE) and two are homologous to the yycFG and the phoRP genes of Bacillus subtilis. All the pneumococcal response regulators contain putative DNA binding motifs within the C-terminal output domain, implying that they are involved in transcriptional control. Two of these response regulators are obviously the first representatives of a new subfamily containing an AraC-type DNA-binding effector domain. To assess the regulatory role of these transcription factors, we disrupted each of the 13 response regulator genes by insertional mutagenesis. All the viable mutant strains with disrupted response regulator genes were further characterized with regard to growth in vitro, competence, and experimental virulence. Two response regulator genes could not be inactivated, indicating that they may regulate essential cellular functions. The possibility of using these systems as targets for the development of novel antibacterials will be discussed.

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. coli fis operon, and a putative lysyl-tRNA synthetase gene (lysS). Four transcripts were identified which encode the first two, eight or all nine proteins or only the last protein LysS. The folate operon contains two promoters, one upstream of the first gene and the second preceding lysS. Transcription of the entire folate operon starts 33 bp upstream of the ATG codon of pab, the first gene of the operon. The mtrB-encoded trp RNA-binding attenuation protein (TRAP) dramatically reduces the steady-state levels of the folate operon transcripts encoding the first eight and all nine proteins, but only has a relatively small effect on the steady-state level of the 2.1 kb transcript encoding the first two genes of the operon, pab and trpG. In addition, transcription of the folate operon is regulated in a growth-phase-dependent manner. Transcripts were present in very low levels after mid-exponential phase, but were dramatically increased directly after transfer of the cells to fresh medium. These results indicate that transcription of the folate operon is regulated by TRAP and also depends on the growth phase of the culture.

The article contains sections titled: 1. Introduction 2. Historical Notes 3. Properties 4. Content in Food and Bioavailability 5. Biosynthesis 6. Chemical Synthesis 7. Metabolism and Biochemical Functions 8. Nutritional Requirements and Medical Use 9. Analysis 10. Economic Aspects

BackgroundPneumococcal meningitis is associated with high mortality (~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.We used an infant rat model of pneumococcal meningitis to assess gene expression profiles in cortex and hippocampus at 22 and 44 hours after infection and in controls at 22 h after mock-infection with saline. To analyze the biological significance of the data generated by Affymetrix DNA microarrays, a bioinformatics pipeline was used combining (i) a literature-profiling algorithm to cluster genes based on the vocabulary of abstracts indexed in MEDLINE (NCBI) and (ii) the self-organizing map (SOM), a clustering technique based on covariance in gene expression kinetics.ResultsAmong 598 genes differentially regulated (change factor ≥ 1.5; p ≤ 0.05), 77% were automatically assigned to one of 11 functional groups with 94% accuracy. SOM disclosed six patterns of expression kinetics. Genes associated with growth control/neuroplasticity, signal transduction, cell death/survival, cytoskeleton, and immunity were generally upregulated. In contrast, genes related to neurotransmission and lipid metabolism were transiently downregulated on the whole. The majority of the genes associated with ionic homeostasis, neurotransmission, signal transduction and lipid metabolism were differentially regulated specifically in the hippocampus. Of the cell death/survival genes found to be continuously upregulated only in hippocampus, the majority are pro-apoptotic, while those continuously upregulated only in cortex are anti-apoptotic.ConclusionTemporal and spatial analysis of gene expression in experimental pneumococcal meningitis identified potential targets for therapy.

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. The mRNA levels of membrane transporters were not affected significantly during isolation procedure. However, plating hepatocytes in CPC resulted in a decrease of major basolateral transporters mRNA level whereas mRNA levels of mdr1b and mrp3 were increased (>100-fold). Similar observations were made in SPC in the absence of DEX demonstrating that the ECM configuration alone did not play a critical role in the regulation of membrane transporters. However, adding DEX to the incubation medium in SPC resulted in an up-regulation of mdr2, oatp2 and mrp2 in a concentration-dependent way for the two latter genes, whereas mdr1b and mrp3 expression were maintained to their baseline liver levels. These data suggested therefore that the combination of ECM and DEX supplementation is essential for the formation of the bile canalicular network and is a determinant factor in the regulation of membrane transporters in cultured rat hepatocytes.

The vic two-component signal transduction system of Streptococcus pneumoniae is essential for growth. The vic operon comprises three genes encoding the following: VicR, a response regulator of the OmpR family; VicK, its cognate histidine kinase; and VicX, a putative protein sharing 55% identity to the predicted product (YycJ) of an open reading frame in the Bacillus subtilis genome. We show that not only is vic essential for viability but it also influences virulence and competence. A putative transcriptional start site for the vic operon was mapped 16 bp upstream of the ATG codon of vicR . Only one transcript of 2.9 kb, encoding all three genes, was detected by Northern blot analysis. VicK, an atypical PAS domain-containing histidine kinase, can be autophosphorylated in vitro, and VicR functions in vitro as a phospho-acceptor protein. (PAS is an acronym formed from the names of the proteins in which the domains were first recognized: the Drosophila period clock protein [PER], vertebrate aryl hydrocarbon receptor nuclear translocator [ARNT], and Drosophila single-minded protein [SIM].) PAS domains are commonly involved in sensing intracellular signals such as redox potential, which suggests that the signal for vic might also originate in the cytoplasm. Growth rate, competence, and virulence were monitored in strains with mutations in the vic operon. Overexpression of the histidine kinase, VicK, resulted in decreased virulence, whereas the transformability of a null mutant decreased by 3 orders of magnitude.

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. The putative translation product of blpC , a small gene located downstream of blpAB , has a leader peptide with a Gly-Gly motif. This leader peptide is typical of precursors processed by this family of transporters. Microarray-based expression profiling showed that a synthetic oligopeptide corresponding to the processed form of BlpC (BlpC*) induces a distinct set of 16 genes. The changes in the expression profile elicited by synthetic BlpC* depend on BlpH since insertional inactivation of its corresponding gene abolishes differential gene induction. Comparison of the promoter regions of the blp genes disclosed a conserved sequence element formed by two imperfect direct repeats upstream of extended −10 promoter elements. We propose that BlpH is the sensor for BlpC* and the conserved sequence element is a recognition sequence for the BlpR response regulator.

An oligonucleotide array of more than 250,000 specific probes representing two complete bacterial genomes including the 1,743 open reading frames of Haemophilus influenzae, and the 1,969 open reading frames of Streptococcus pneumoniae has been designed. When hybridised against labelled genomic DNA, all genes were detected and more than 96% of the signal intensity values were within a factor of three of the mean. For transcript imaging, microarrays were hybridised against total RNA populations quantitatively represented by labelled cDNAs. The measurements of transcript abundance for all genes were shown to be sensitive, specific, quantitative and reproducible. Nearly 85% of all S. pneumoniae mRNAs were found expressed during in vitro exponential growth. Identification of regulons important for S. pneumoniae entry into the stationary phase has been used to validate the approach. We will show, for the first time, how a genome-wide transcriptional analysis is applied to bacterial genomes and is being used to identify signal transduction regulatory networks, and to profile antibiotic compounds.