Recent evidence suggests a role for phosphatidylinositol (PI) 3-kinase in various inflammatory responses. In this study, the consequences of LPS-induced PI 3-kinase activation on cytokine and chemokine expression and the intracellular mechanisms of inflammatory activation were examined in mouse macrophages. LPS stimulation induced a complex formation between PI 3-kinase and myeloid differentiation factor 88 (MyD88), which was followed by an induction of IL-1 g , tumor necrosis factor- § (TNF- § ) and macrophage inflammatory protein (MIP)-2. The induction of IL-1 g , but not of MIP-2 or TNF- § , was blocked by the PI 3-kinase inhibitors LY294002 and wortmannin. The nuclear factor-‹ B (NF-‹ B) inhibitor pyrrolidinedithiocarbamate (PDTC) blocked the induction of IL-1 g and TNF- § , but had no effect on MIP-2 expression. Inhibition of PI 3-kinase decreased the LPS-induced transcriptional activity of NF-‹ B, but it had no effect on the nuclear DNA binding activity of NF-‹ B. These findings suggest that, while NF-‹ B nuclear localization and DNA binding are necessary, they are not sufficient for transcriptional activation of the IL-1 g gene in the absence of PI 3-kinase activity. Taken together, our results demonstrate that activation of Toll-like receptor (TLR)-4 results in PI 3-kinase-MyD88 complex formation, and that PI 3-kinase activity selectively leads to cytokine induction downstream of TLR4.
Toll-like receptors (Tlr) have recently been linked to the immunostimulatory function of microbial toxins in human and mice. Tlr signals activation of nuclear factor B that leads to the production of a number of proinflammatory mediators. Tlr4 mediates the endotoxin-induced inflammatory response, whereas Tlr2 may be involved in the response to yeast and Gram-positive bacterial products. To better understand age-related changes in acute inflammatory response, we studied the ontogeny of Tlr2 and Tlr4 mRNA in murine fetal lung, liver, and placenta by quantitative reverse transcriptase-PCR. Different expression patterns were seen between the tissues and between the Tlr. This is in accordance with the evidence that there are differences in the receptors for different microbial toxins and that the response is organ specific. We additionally show that the expression of Tlr was dependent on the stage of differentiation. In the liver, the levels of Tlr2 and Tlr4 were high regardless of the age. In the lung, Tlr2 and Tlr4 expression levels were barely detectable in immature fetus (d 14 -15). Tlr2 and Tlr4 were increased severalfold during prenatal development and further increased after birth. The present results support the finding of a deficient inflammatory response of the immature lung to microbial toxins. Bacterial LPS (also known as endotoxin) as a constituent of the cell wall of Gram-negative bacteria is a major causative agent of septic shock. LPS starts a complex cascade of events in responsive cells, particularly in monocytes and macrophages, that leads to the production of endogenous mediators such as proinflammatory cytokines IL-1, tumor necrosis factor-␣, IL-6, IL-8, and a number of other mediators. In Grampositive bacteria, the major immunostimulatory components of the cell wall include peptidoglycan and lipoteichoic acid (1, 2).A cell membrane component required for LPS-induced immunostimulation was recently identified to be a Tlr (3-5). According to the latest studies, Tlr2 and Tlr4 recognize different bacterial cell wall components. Tlr4 has been shown to mediate LPS-induced signal transduction (6, 7), whereas Tlr2 may mediate the response to yeast and Gram-positive bacteria (2, 8). Macrophages contain a surface protein called CD14, which binds ligands such as LPS (5, 7). However, CD14 does not participate directly in signaling. Rather, Tlr are essential for the innate immune response. Whereas the extracellular domain of Tlr, compatible with CD14, discriminates between pathogens, the cytoplasmic tail of Tlr triggers the cascade of intracellular mediators, leading to the activation of the nuclear factor B and of the inflammatory response. Tlr are the mammalian homologues of the Drosophila Toll family that controls the dorsoventral patterning in the developing embryo and the antimicrobial response in the adult fly (9). So far, at least six of Tlr Drosophila have been identified in humans and mice (10). It has been proposed that Tlr control the switch from the innate to adaptive immune response (11). Tlr2 an...
The luteinizing hormone receptor (LHR) is a G protein-coupled receptor involved in regulation of ovarian and testicular functions. Here we show that the receptor is present also in specific areas of the peripheral and central nervous system and may thus have a broader functional role than has been anticipated. Full-length LHR mRNA and two receptor protein species of M r 90,000 and 73,000, representing mature and precursor forms, respectively, were expressed in adult and developing rat nervous tissue, starting at fetal day 14.5. The receptor was capable of ligand binding because it was purified by ligand affinity chromatography, and human chorionic gonadotropin and LH were able to displace 125 I-labeled human chorionic gonadotropin binding to fetal head membranes in a dose-dependent manner. Finally, two 5-flanking sequences (ϳ 2 and 4 kb) of the rat LHR gene were shown to direct expression of the lacZ reporter to specific areas of the peripheral and central nervous system in fetal and adult transgenic mice, especially to structures associated with sensory, memory, reproductive behavior, and autonomic functions. Importantly, the transgene activity was confined to neurons and colocalized with the cytochrome P450 side chain cleavage enzyme. Taken together, these results indicate that the neuronal LHR is a functional protein, implicating a role in neuronal development and function, possibly by means of regulating synthesis of neurosteroids.
A method for the analysis of intact glucuronides and sulfates of common neurotransmitters serotonin (5-HT) and dopamine (DA) as well as of 5-hydroxy-3-indoleacetic acid (5-HIAA), 3,4-dihydroxyphenylacetic acid (DOPAC), and homovanillic acid (HVA) in rat brain microdialysates by liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed. Enzyme-assisted synthesis using rat liver microsomes as a biocatalyst was employed for the production of 5-HT-, 5-HIAA-, DOPAC-, and HVA-glucuronides for reference compounds. The sulfate conjugates were synthesized either chemically or enzymatically using a rat liver S9 fraction. The LC-MS/MS method was validated by determining the limits of detection and quantitation, linearity, and repeatability for the quantitative analysis of 5-HT and DA and their glucuronides, as well as of 5-HIAA, DOPAC, and HVA and their sulfate-conjugates. In this study, 5-HT-glucuronide was for the first time detected in rat brain. The concentration of 5-HT-glucuronide (1.0-1.7 nM) was up to 2.5 times higher than that of free 5-HT (0.4-2.1 nM) in rat brain microdialysates, whereas the concentration of DA-glucuronide (1.0-1.4 nM) was at the same level or lower than the free DA (1.2-2.4 nM). The acidic metabolites of neurotransmitters, 5-HIAA, HVA, and DOPAC, were found in free and sulfated form, whereas their glucuronidation was not observed.
The solid-phase synthesis of 1,2,3-triazoles via 1,3-dipolar cycloaddition of polymer-bound azides to various alkynes is reported. Polymer-bound azides were synthesized from polymer-bound halides and sodium azide and reacted with alkynes to produce polymer-bound 1,2,3-triazoles. Cleavage of the triazoles was performed with trifluoroacetic acid. A traceless synthesis of 1,2,3-triazoles was developed using 2-methoxy-substituted resin (polymer-bound 4-hydroxy-2-methoxybenzyl alcohol). In addition, a synthesis of 4-hydroxybenzyl-substituted 1,2,3-triazoles from the bromo-Wang resin (4-(bromomethyl)phenoxymethyl polystyrene) was achieved.
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