Low Molecular Weight Signal Molecules Research Articles

Chemical-neuroanatomical organization of peripheral sensory-efferent systems in the pond snail (Lymnaea stagnalis)

Perception and processing of chemical cues are crucial for aquatic gastropods, for proper elaboration of adaptive behavior. The pond snail, Lymnaea stagnalis, is a model species of invertebrate neurobiology, in which peripheral sensory neurons with different morphology and transmitter content have partly been described, but we have little knowledge regarding their functional morphological organization, including their possible peripheral intercellular connections and networks. Therefore the aim of our study was to characterize the sensory system of the tentacles and the lip, as primary sensory regions, and the anterior foot of Lymnaea with special attention to the transmitter content of the sensory neurons, and their relationship to extrinsic elements of the central nervous system. Numerous bipolar sensory cells were demonstrated in the epithelial layer of the peripheral organs, displaying immunoreactivity to antibodies raised against tyrosine hydroxylase, histamine, glutamate and two molluscan type oligopeptides, FMRFamide and Mytilus inhibitory peptide. A subepithelial plexus was formed by extrinsic serotonin and FMRFamide immunoreactive fibers, whereas in deeper regions axon processess of different origin with various immunoreactivities formed networks, too. HPLC–MS assay confirmed the presence of the low molecular weight signal molecules in the three examined areas. Following double-labeling immunohistochemistry, close arrangements were observed, formed by sensory neurons and extrinsic serotonergic (and FMRFamidergic) fibers at axo-dendritic, axo-somatic and axo-axonic levels. Our results suggest the involvement of a much wider repertoire of signal molecules in peripheral sensory processes of Lymnaea, which can locally be modified by central input, hence influencing directly the responses to environmental cues.

Abiotic Hydrolysis Kinetics of N‐Acyl‐L‐homoserine Lactones: Natural Silencing of Bacterial Quorum Sensing Signals

Virulence attenuation in pathogenic bacteria without the use of antibiotics reduces the threat of infection while limiting the evolution of multidrug resistant strains. In many bacteria, virulence is regulated by population density dependent genetic switches, a phenomenon known as quorum sensing (QS). QS depends on the synthesis, transmission, and perception of a variety of low molecular weight signal molecules generically classified as autoinducers. Gram‐negative bacteria, such as Pseudomonas aeruginosa, predominantly “communicate” population density using N‐acyl‐L‐homoserine lactones (AHLs). Crucial to developing Gram‐negative QS modulating strategies, as well as modeling the presence/absence of QS under “real‐world” conditions, is the ability to accurately estimate the relative levels of the ring‐closed (bioactive) and ring‐open (bio‐inactive) forms. While some AHLs have been studied for their hydrolysis (ring‐opening) rates, no quantitative and comprehensive model for degradation exists. Here, we employ 1H‐NMR to monitor the hydrolysis of various AHLs under standard laboratory conditions at fixed acidities to develop such a model. Our model provides valuable insight into the availability of bioactive AHLs at relevant pH conditions and is a valuable tool in the development of QS‐control strategies and evaluation of AHL focused chemical assays and studies.

Genes for biosynthesis of butenolide-like signalling molecules in Streptomyces ghanaensis, their role in moenomycin production.

Moenomycins (Mm) - phosphoglycolipid compounds produced by Streptomyces ghanaensis ATCC14672 - are considered a promising model for development of novel class of antibiotics. In this regard it is important to generate Mm overproducing strains which would be a basis for economically justified production of this antibiotic. In this work a set of genes for synthesis and reception of low-molecular weight signaling molecules (LSM) in ATCC14672 were described and their significance for Mm production was studied. The ATCC14672 genome carries structural and regulatory genes for production of LSMs of avenolide and γ-butyrolactone families. Additional copies of LSM biosynthetic genes ssfg_07848 and ssfg_07725 did not alter the Mm production level. ATCC14672 LSMs are not capable of restoring the sporulation of butyrolactone-nonproducing mutant of S. griseus. Likewise, while the heterologous host S. lividans 1326 produced Mm, its mutant M707 (deficient in the butyrolactone synthase gene scbA) did not. Thus, while the natural level of LSMs production by ATCC14672 does not limit Mm synthesis, the former is essential for the synthesis of moenomycins.

α-Hydroxyketone Synthesis and Sensing by Legionella and Vibrio

Bacteria synthesize and sense low molecular weight signaling molecules, termed autoinducers, to measure their population density and community complexity. One class of autoinducers, the α-hydroxyketones (AHKs), is produced and detected by the water-borne opportunistic pathogens Legionella pneumophila and Vibrio cholerae, which cause Legionnaires’ disease and cholera, respectively. The “Legionella quorum sensing” (lqs) or “cholera quorum sensing” (cqs) genes encode enzymes that produce and sense the AHK molecules “Legionella autoinducer-1” (LAI-1; 3-hydroxypentadecane-4-one) or cholera autoinducer-1 (CAI-1; 3-hydroxytridecane-4-one). AHK signaling regulates the virulence of L. pneumophila and V. cholerae, pathogen-host cell interactions, formation of biofilms or extracellular filaments, expression of a genomic “fitness island” and competence. Here, we outline the processes, wherein AHK signaling plays a role, and review recent insights into the function of proteins encoded by the lqs and cqs gene clusters. To this end, we will focus on the autoinducer synthases catalysing the biosynthesis of AHKs, on the cognate trans-membrane sensor kinases detecting the signals, and on components of the down-stream phosphorelay cascade that promote the transmission and integration of signaling events regulating gene expression.

A LuxP-FRET-Based Reporter for the Detection and Quantification of AI-2 Bacterial Quorum-Sensing Signal Compounds

Various bacterial species produce and monitor low-molecular weight signaling molecules that regulate specific sets of genes in a population density-dependent manner. This process is known as quorum sensing (QS). To date, the detection of QS signaling molecules from Gram-negative bacteria has relied primarily on bacterial reporter strains. These bioassays are subject to substantial interference by compounds that affect the growth and metabolism of the reporter strains. In addition, the sensitivity of reporter strains to QS signaling molecules is population density-dependent. Here, we describe the development of an in vitro assay system for the rapid detection and quantification of the furanosyl borate diester (BAI-2) subclass of autoinducer 2 (AI-2), QS molecules. The sensor is based on ligand binding-induced changes in fluorescence resonance energy transfer (FRET) between a cyan and yellow variant of GFP fused to the termini of the BAI-2 receptor, LuxP. Unexpectedly, the addition of synthetic BAI-2 to the purified biosensor induces a decrease in the level of FRET between the terminal fluorophores. Several lines of evidence, including mutation of the ligand binding sites, indicate that the observed FRET changes are BAI-2-dependent. The FRET-based BAI-2 biosensor responded to the addition of culture filtrates from wild-type Vibrio harveyi but exhibited no response to culture filtrates from V. harveyi mutants defective in BAI-2 synthesis. The sensitivity of the biosensor to BAI-2 (apparent Kd = 270 nM) was similar to that of BAI-2 bioassay systems. The limitations of microbial bioassay systems and the advantages and potential applications for the FRET-based BAI-2 biosensor are discussed.

Basic fibroblast growth factor stimulates cytosolic phospholipase A2, phospholipase C-gamma1 and phospholipase D through distinguishable signaling mechanisms.

Fibroblast growth factors (FGFs) stimulate proliferation, differentiation and motility of different cell types. The cellular effects of FGF are transduced by its interaction with any one of four members of a family of high affinity, cell surface FGF receptors (FGFRs) that have autophosphorylating tyrosine kinase activity. Activation of FGFR causes release of various low molecular weight signaling molecules which are required for the pleotropic effects of FGFs. We report here that basic FGF plays critical role in membrane phospholipid hydrolysis in NIH 3T3 cells that are stably transfected with FGFR1. Upon binding to FGFR1, basic FGF stimulates cytosolic form of phospholipase A2 (cPLA2), phospholipase C-gamma1 (PLC-gamma1) and phospholipase D (PLD), the key enzymes for the production of various lipid second messengers, in a tyrosine kinase-dependent manner. In addition to tyrosine phosphorylation, cPLA2 catalytic activation requires serine phosphorylation by p42 mitogen-activated protein (MAP) kinase and possibly pertussis toxin-sensitive G-protein coupling. On the other hand, phosphatidyl inositol 4,5 bisphosphate (PIP2) hydrolysis requires direct phosphorylation at tyrosine residue of the PLC-gamma1 isozyme. The activation of PLD needs direct or indirect receptor tyrosine kinase and protein kinase C (PKC) activities. Additionally, it also requires botulinum toxin C-sensitive Rho-like G-protein activation. All these results suggest that the pleotropic effects of FGF are exerted through its tyrosine kinase receptors and individual effectors are activated via distinguishable signaling mechanisms according to the cell's need.

Activated astrocytes induce nitric oxide synthase-2 in cerebral endothelium via tumor necrosis factor ?

Astrocytes under pathological conditions become activated and produce a variety of cytokines and low molecular weight signal molecules. Previously we demonstrated that activated astrocytes release nitric oxide which can downregulate the expression of nitric oxide synthase (NOS)-2 in co-cultured cerebral endothelium, and also release a transcriptionally regulated factor that can induce NOS-2 expression in endothelium (Borgerding and Murphy: J Neurochem 65:1342, 1995). The activity of this NOS-2-inducing factor was impeded by inhibitors of tyrosine kinases and NF-kappaB activation. Tumor necrosis factor (TNF alpha) alone, or in combination with IL-6, induced NOS-2 expression in endothelial cells. A neutralizing antibody against TNF alpha attenuated the NOS-2 expression in endothelial cells exposed to activated astrocytes. These results imply that cytokine-activated astrocytes release TNF alpha which can induce NOS-2 expression in endothelium and suggest that activated astrocytes within the CNS may induce expression of NOS-2 in cells of the adjacent microvasculature. The ensuing alterations in blood-brain barrier properties may be either beneficial or detrimental.

Activation of Cytosolic Phospholipase A by Basic Fibroblast Growth Factor via a p42 Mitogen-activated Protein Kinase-dependent Phosphorylation Pathway in Endothelial Cells

Basic fibroblast growth factor (FGF) stimulates the proliferation, differentiation, and motility of multiple cell types. Signal transduction by FGF is mediated by high affinity FGF receptors that have autophosphorylating tyrosine kinase activity and also elicit the release of low molecular weight signaling molecules, including inositol 1,4,5-trisphosphate, diacylglycerol, and arachidonate. We have shown previously that basic FGF-stimulated, phospholipase A2 (PLA2)-mediated arachidonate release regulates endothelial cell (EC) motility (Sa, G., and Fox, P.L. (1994) J. Biol. Chem. 269, 3219-3225). Here we identify the phospholipase responsible for basic FGF-mediated arachidonate release as cytosolic PLA2 (cPLA2) by demonstrating in EC lysates a requirement for micromolar Ca2+, dithiothreitol insensitivity, and inactivation by anti-cPLA2 antiserum. The role of cPLA2 is also indicated by the observed mechanisms of activation which show a requirement for p42 mitogen-activated protein kinase activity, cPLA2 phosphorylation, and cPLA2 translocation from cytosol to membranes. Phosphorylation of cPLA2, arachidonate release from prelabeled intact cells, and cell motility all have similar concentration dependencies on basic FGF. Since arachidonate release is required for basic FGF-stimulated motility of EC, our results show that p42 mitogen-activated protein kinase activation of cPLA2 may be a regulatory event in stimulation of cellular release of this important eicosanoid precursor during cellular responses to basic FGF.

Signaling and host range variation in nodulation.

Rhizobium, Bradyrhizobium, and Azorhizobium strains, collectively referred to as rhizobia, elicit on their leguminous hosts, in a specific manner, the formation of nodules in which they fix nitrogen. Rhizobial nod genes, which determine host specificity, infection, and nodulation, are involved in the exchange of low molecular weight signal molecules between the plant and the bacteria as follows. Transcription of the nod operons is under the control of NodD regulatory proteins, which are specifically activated by plant flavonoid signals. The common and species-specific structural nod genes are involved in turn in the synthesis of specific lipo-oligosaccharides that signal back to the plant to elicit root-hair deformations, cortical-cell divisions, and nodule-meristem formation.