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.Signaling mechanisms controlling multicellular behavior of bacteria have attracted much attention in current research. In gram-negative bacteria, homoserine-lactone-based communication systems are prominent. Research in this area led to the term "quorum sensing" for phenomena that are controlled by cell density (12). In gram-positive bacteria, quorum sensing is accomplished by signaling systems that depend on the secretion and sensing of small peptides (11,19). At least two different mechanisms for sensing the presence of pheromone-like peptides are known (21). The first involves import of the peptide and interaction with an intracellular factor (22); the second involves binding to the extracellular portion of a membranebound histidine kinase. This leads to the autophosphorylation of the kinase and subsequent activation, e.g., phosphorylation of a cognate response regulator that mediates changes in gene expression. Quorum-sensing systems regulate a plethora of cellular functions. In Staphylococcus aureus, the AgrC-AgrAsystem is involved in the density-dependent regulation of virulence (18). In Lactobacillus strains, the production of bacteriocins is dependent on peptide-regulated two-component systems (TCS) (4, 10). In Streptococcus pneumoniae, the development of genetic competence (the natural ability to take up DNA) has been shown to be regulated by the comC-DE system (29).The com system of S. pneumoniae was the first quorumsensing system for which a biological function was defined. ...

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Retinoic acid (RA) exerts its pleiotropic effects on cell growth and differentiation through the activation of a family of transcription factors-the RA receptors (RARs). Three subtypes of these receptors exist, RARa, RAR(3, and RARly. The receptors are differentially expressed in different cell types and stages of development, suggesting that they may regulate different sets of genes. We have identified a synthetic retinoid with the characteristics of a selective RARa antagonist. This antagonist counteracts RA effects on HL-60 cell differentiation and on B-lymphocyte polyconal activation.Beyond its potential practical relevance, this and other specific antagonists will be useful to dissect the RAR system and to assign to one given receptor each of the many RA-regulated functions.The natural retinol (vitamin A) derivative retinoic acid (RA) is known to have profound effects on cell growth and differentiation (1) and to be essential for normal embryonic development (2). While RA and some synthetic analogs (retinoids) are useful in the control of some tumors (3) as well as of nonmalignant hyperproliferative conditions of the skin (4), they are, at high concentrations, teratogenic (5).The pleiotropic effects of retinoids are mediated by two known families of nuclear receptors, both belonging to the steroid-thyroid hormone receptor superfamily of ligandinducible transcriptional regulators (6, 7). The RA receptor (RAR) gene family comprises three subtypes-RARa (8, 9), RAR,[8][9][10][11][12], and RAR'y (13, 14)-with each gene encoding a variable number of isoforms arising by differential splicing of two primary . All receptors of the RAR family bind RA with comparable affinity (18). The retinoid receptors of the second family (RXR) do not bind the major form of RA (all-trans-RA) (19). They bind instead the 9-cis stereoisomer of RA (20, 21).Transcription of some RAR genes themselves is RA sensitive (22-25). Also, the expression of some of the cellular retinol-or RA-binding proteins (CRBP and CRABP), putatively involved in the storage, transport, and/or metabolism of retinol and RA, is differentially regulated by RA in a receptor-specific manner (26-28). The RA-related molecules represent, therefore, an autoregulated system. RAR types and isoforms, as well as RXRa and RXRB, are differentially expressed both spatially and temporally (15-18, 29-32). They might therefore regulate different target genes during embryonic and adult life, as well as in specific cell types at different stages of differentiation. RARa is the most ubiquitously expressed, while RAR8 and RARy display a more restricted pattern of distribution, with RARy being predominantly expressed in the skin (31).It seems reasonable to assume that the multiple effects of RA could be dissociated by specific ligands for each of the known receptors, and/or by receptor-specific antagonists, so as to obtain the desired beneficial effects while limiting the unwanted side effects. Retinoids with a good degree of selectivity have been described (33), and we have o...

Microarray technology allows the simultaneous analysis of mRNA expression levels of thousands of genes. In the field of toxicogenomics, this technology could help to identify potentially unsafe compounds based on the changes in mRNA expression patterns they induce. Rodent in vivo and in vitro systems are currently the experimental models of choice for predictive toxicology, especially in early phases of development. This study characterizes several hepatic in vitro systems based on mRNA expression profiles, comparing them to gene expression in liver tissue. The in vitro systems investigated comprise two rat liver cell lines (BRL3A and NRL clone 9), primary hepatocytes in conventional monolayer or in sandwich culture, and liver slices. The results demonstrate that liver slices exhibit the strongest similarity to liver tissue regarding mRNA expression, whereas the two cell lines are quite different from the whole liver. We were able to identify genes with strong changes in expression levels in all or at least one of the in vitro systems relative to whole liver. In particular, for some cytochrome P450s the differences observed on the mRNA expression level were paralleled by protein expression and enzymatic activity. In addition, the effect of time in culture was assessed. We were able to show a profound effect of the duration of culture. Expression patterns change most rapidly soon after cell isolation and culture initiation and stabilize with time in culture. The findings are discussed with respect to the usefulness of the various hepatic in vitro systems for microarray-based toxicological testing of compounds.

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 autophosphory- Prokaryotic organisms commonly sense and respond to changes in their environment using two-component regulatory systems (TCRS). Such systems typically comprise a membraneassociated sensory kinase and a cytoplasmic response regulator. A stimulus is perceived by the sensory domain of the histidine kinase, resulting in its autophosphorylation. Further transmission of the signal is achieved by the phosphorylation of its cognate response regulator. The phosphorylated form of the response regulator binds to promoter regions and thus regulates transcription of genes under its control (22, 31). The TRCS of pathogens have been implicated in detecting conditions favorable for host invasion and activating virulence regulons (29). Virulence of Salmonella enterica serovar Typhimurium is regulated by the phoPQ TCRS. The regulated genes are crucial for survival in macrophages and confer resistance to cationic antimicrobial peptides (19). Several divalent cations have been shown to activate this TCRS, with Mg 2ϩ being the most efficient ion (15). Other TCRS are essential for bacterial growth under laboratory conditions (14,21,26). One example is the divJK system, first identified in the gram-negative aquatic eubacterium Caulobacter crescentus (21). This TCRS controls CtrA, a response regulator essential for transcription of cell cycle-regulated genes and interacting with the principal vegetative sigma factor ( 73 ) of C. crescentus (42). Two-component systems have been recently identified in Streptococcus pneumoniae, a major cause of community-acquired pneumonia (3). One of the best-studied two-component systems in S. pneumoniae is comDE, which is a key regulator of natural competence (9, 33). This system belongs to the quorum-sensing family of two-component systems. A small heptadecapeptide coexpressed with comD-comE was proposed to be the signaling molecule (20,33). A second TCRS, ciaRH, is also involved in regulating competence of S. pneumoniae (18). Recently, the predicted response regulator genes of 13 TCRS found in the nearly complete S. pneumoniae genome sequence were disrupted (26,42). No viable response regulator knockout mutants could be obtained for the vic system. The only other essential TCRS known so far was implicated in cell cycle control (24).In this w...