Staphylococcus aureus lipoteichoic acid inhibits platelet activation and thrombus formation via the Paf receptor

Impaired healing is common in wounds infected with the major human pathogen Staphylococcus aureus, although the underlying mechanisms are poorly understood. Here, we show that S.aureus lipoteichoic acid (LTA) inhibits platelet aggregation caused by physiological agonists and S. aureus and reduced platelet thrombus formation in vitro. The presence of D-alanine on LTA is necessary for the full inhibitory effect. Inhibition of aggregation was blocked using a monoclonal anti-platelet activating factor receptor (PafR) antibody and Ginkgolide B, a well-defined PafR antagonist, demonstrating that the LTA inhibitory signal occurs via PafR. Using a cyclic AMP (cAMP) assay and a western blot for phosphorylated VASP, we determined that cAMP levels increase upon platelet incubation with LTA, an effect which inhibits platelet activation. This was blocked when platelets were preincubated with Ginkgolide B. Furthermore, LTA reduced haemostasis in a mouse tail-bleed assay.

Cox-dependent fatty acid metabolites cause pain through activation of the irritant receptor TRPA1.

Prostaglandins (PG) are known to induce pain perception indirectly by sensitizing nociceptors. Accordingly, the analgesic action of nonsteroidal anti-inflammatory drugs (NSAIDs) results from inhibition of cyclooxygenases and blockade of PG biosynthesis. Cyclopentenone PGs, 15-d-PGJ(2), PGA(2), and PGA(1), formed by dehydration of their respective parent PGs, PGD(2), PGE(2), and PGE(1), possess a highly reactive alpha,beta-unsaturated carbonyl group that has been proposed to gate the irritant transient receptor potential A1 (TRPA1) channel. Here, by using TRPA1 wild-type (TRPA1(+/+)) or deficient (TRPA1(-/-)) mice, we show that cyclopentenone PGs produce pain by direct stimulation of nociceptors via TRPA1 activation. Cyclopentenone PGs caused a robust calcium response in dorsal root ganglion (DRG) neurons of TRPA1(+/+), but not of TRPA1(-/-) mice, and a calcium-dependent release of sensory neuropeptides from the rat dorsal spinal cord. Intraplantar injection of cyclopentenone PGs stimulated c-fos expression in spinal neurons of the dorsal horn and evoked an instantaneous, robust, and transient nociceptive response in TRPA1(+/+) but not in TRPA1(-/-) mice. The classical proalgesic PG, PGE(2), caused a slight calcium response in DRG neurons, increased c-fos expression in spinal neurons, and induced a delayed and sustained nociceptive response in both TRPA1(+/+) and TRPA1(-/-) mice. These results expand the mechanism of NSAID analgesia from blockade of indirect nociceptor sensitization by classical PGs to inhibition of direct TRPA1-dependent nociceptor activation by cyclopentenone PGs. Thus, TRPA1 antagonism may contribute to suppress pain evoked by PG metabolites without the adverse effects of inhibiting cyclooxygenases.

Critical role for an acidic amino acid region in platelet signaling by the HemITAM (hemi-immunoreceptor tyrosine-based activation motif) containing receptor CLEC-2 (C-type lectin receptor-2)

CLEC-2 is a member of new family of C-type lectin receptors characterized by a cytosolic YXXL downstream of three acidic amino acids in a sequence known as a hemITAM (hemi-immunoreceptor tyrosine-based activation motif). Dimerization of two phosphorylated CLEC-2 molecules leads to recruitment of the tyrosine kinase Syk via its tandem SH2 domains and initiation of a downstream signaling cascade. Using Syk-deficient and Zap-70-deficient cell lines we show that hemITAM signaling is restricted to Syk and that the upstream triacidic amino acid sequence is required for signaling. Using surface plasmon resonance and phosphorylation studies, we demonstrate that the triacidic amino acids are required for phosphorylation of the YXXL. These results further emphasize the distinct nature of the proximal events in signaling by hemITAM relative to ITAM receptors.

The role of proliferator-activated receptor gamma coactivator-1 alpha in the fatty-acid -dependent transcriptional control of interleukin-10 in hepatic cells of rodents

Interleukin-10 (IL-10) is an endogenous factor that restrains hepatic insulin resistance in diet-induced steatosis Reducing IL-10 expression increases proinflammatory activity in the steatotic liver and worsens insulin resistance As the transcriptional coactivator proliferator-activated receptor gamma coactivator-1 alpha (PGC-1 alpha) plays a central role in dysfunctional hepatocytic activity in diet-induced steatosis, we hypothesized that at least part of the action of PGC-1 alpha could be mediated by reducing the transcription of the IL-10 gene Here, we used immunoblotting, real-time polymerase chain reaction, immunocytochemistry, and chromatin immunoprecipitation assay to investigate the role of PGC-1 alpha in the control of IL-10 expression in hepatic cells First, we show that, in the intact steatotic liver, the expressions of IL-10 and PGC-1 alpha are increased Inhibiting PGC-1 alpha expression by antisense oligonucleotide increases IL-10 expression and reduces the steatotic phenotype. In cultured hepatocytes, the treatment with saturated and unsaturated fatty acids increased IL-10 expression. This was accompanied by increased association of PGC-1 alpha with c-Maf and p50-nuclear factor (NF) kappa B, 2 transcription factors known to modulate IL-10 expression In addition, after fatty acid treatment. PGC-1 alpha, c-Maf, and p50-NF kappa B migrate from the cytosol to the nuclei of hepatocytes and bind to the IL-10 promoter region Inhibiting NF kappa B activation with salicylate reduces IL-10 expression and the association of PGC-1 alpha with p50-NF kappa B Thus, PGC-1 alpha emerges as a potential transcriptional regulator of the inflammatory phenomenon taking place in the steatotic liver (C) 2010 Elsevier Inc All rights reserved

Folic acid derivatives for PET imaging and therapy addressing folate receptor positive tumors

Folic acid, also known as vitamin B9, is the oxidized form of 5,6,7,8-tetrahydrofolate, which serves as methyl- or methylene donor (C1-building blocks) during DNA synthesis. Under physiological conditions the required amount of 5,6,7,8-tetrahydrofolate for survival of the cell is accomplished through the reduced folate carrier (RFC). In contrast, the supply of 5,6,7,8-tetrahydrofolate is insufficient under pathophysiological conditions of tumors due to an increased proliferation rate. Consequently, many tumor cells exhibit an (over)expression of the folate receptor. This phenomenon has been applied to diagnostics (PET, SPECT, MR) to image FR-positive tumors and on the other hand to treat malignancies related to a FR (over)expression. Based on this concept, a new 18F-labeled folate for PET imaging has been developed and was evaluated in vivo using tumor-bearing mice. The incorporation of oligoethylene spacers into the molecular structure led to a significant enhancement of the pharmacokinetics in comparison to previously developed 18F-folates. The liver uptake could be reduced by one sixth by remaining a tumor uptake of 3%ID/g leading to better contrast ratios. Encouraged by these results, a clickable 18F-labeled serine-based prosthetic group has been synthesized, again with the idea to improve the metabolic and pharmacokinetic profile of hydrophilic radiotracers. Therefore, an alkyne-carrying azido-functionalized serine derivative for coupling to biomolecules was synthesized and a chlorine leaving group for 18F-labeling, which could be accomplished using a microwave-assisted synthesis, a [K⊂2.2.2]+/carbonate system in DMSO. Radiochemical yields of 77±6% could be achieved.rnThe promising results obtained from the FR-targeting concept in the diagnostic field have been transferred to the boron neutron capture therapy. Therefore, a folate derivative was coupled to different boron clusters and cell uptake studies were conducted. The synthesis of the folate-boron clusters was straightforward. At first, a linker molecule based on maleic acid was synthesized, which was coupled to the boron cluster via Michael Addition of a thiol and alkene and subsequently coupled to the targeting moiety using CuAAC. The new conjugates of folate and boron clusters led to a significant increase of boron concentration in the cell of about 5-times compared to currently used and approved boron pharmaceuticals. rnMoreover, azido-folate derivatives were coupled to macromolecular carrier systems (pHPMA), which showed an enhanced and specific accumulation at target sites (up to 2.5-times) during in vivo experiments. A specific blockade could be observed up to 30% indicating an efficient targeting effect. A new kind of nanoparticles consisting of a PDLLA core and p((HPMA)-b-LMA)) as surfactants were developed and successfully radiolabeled via 18F-click chemistry in good RCYs of 8±3%rnThe nanoparticles were obtained via the miniemulsion technique in combination with solvent evaporation. The 18F-labeled nanoparticles were applied to in vivo testing using a mouse model. PET imaging showed a “mixed” biodistribution of low molecular weight as well as high molecular weight systems, indicating a partial loss of the 18F-labeled surfactant.rnIn conclusion, the presented work successfully utilized the FR-targeting concept in both, the diagnostic field (PET imaging) and for therapeutic approaches (BNCT, drug delivery systems). As a result, the high potential of FR-targeting in oncological applications has been shown and was confirmed by small animal PET imaging.rn

Control of Steroid 21-oic Acid Synthesis by Peroxisome Proliferator-activated Receptor and Role of the Hypothalamic-Pituitary-Adrenal Axis

A previous study identified the peroxisome proliferator-activated receptor alpha (PPARalpha) activation biomarkers 21-steroid carboxylic acids 11beta-hydroxy-3,20-dioxopregn-4-en-21-oic acid (HDOPA) and 11beta,20-dihydroxy-3-oxo-pregn-4-en-21-oic acid (DHOPA). In the present study, the molecular mechanism and the metabolic pathway of their production were determined. The PPARalpha-specific time-dependent increases in HDOPA and 20alpha-DHOPA paralleled the development of adrenal cortex hyperplasia, hypercortisolism, and spleen atrophy, which was attenuated in adrenalectomized mice. Wy-14,643 activation of PPARalpha induced hepatic FGF21, which caused increased neuropeptide Y and agouti-related protein mRNAs in the hypothalamus, stimulation of the agouti-related protein/neuropeptide Y neurons, and activation of the hypothalamic-pituitary-adrenal (HPA) axis, resulting in increased adrenal cortex hyperplasia and corticosterone production, revealing a link between PPARalpha and the HPA axis in controlling energy homeostasis and immune regulation. Corticosterone was demonstrated as the precursor of 21-carboxylic acids both in vivo and in vitro. Under PPARalpha activation, the classic reductive metabolic pathway of corticosterone was suppressed, whereas an alternative oxidative pathway was uncovered that leads to the sequential oxidation on carbon 21 resulting in HDOPA. The latter was then reduced to the end product 20alpha-DHOPA. Hepatic cytochromes P450, aldehyde dehydrogenase (ALDH3A2), and 21-hydroxysteroid dehydrogenase (AKR1C18) were found to be involved in this pathway. Activation of PPARalpha resulted in the induction of Aldh3a2 and Akr1c18, both of which were confirmed as target genes through introduction of promoter luciferase reporter constructs into mouse livers in vivo. This study underscores the power of mass spectrometry-based metabolomics combined with genomic and physiologic analyses in identifying downstream metabolic biomarkers and the corresponding upstream molecular mechanisms.

5-Hydroxytryptamine-4 receptor messenger ribonucleic acid levels and densities in gastrointestinal muscle layers from healthy dairy cows

Serotonin (5-hydroxytryptamine, 5-HT) is involved in gastrointestinal tract (GIT) motor functions through binding to specific receptors located in the GIT walls. The objectives of the current study were to compare mRNA levels and binding sites of 5-HT(4) receptors (5-HTR(4)) in smooth muscle layers from the fundus abomasi, pylorus, ileum, cecum, proximal loop of the ascending colon (PLAC), and external loop of the spiral colon (ELSC) of healthy dairy cows, and to verify whether mRNA and protein expression were correlated. Smooth muscle samples were prepared by scraping the mucosa and submucosa from full-thickness intestinal wall samples. The mRNA levels of 5-HTR(4) were measured by real-time PCR and expressed relative to those of the housekeeping gene glyceraldehyde phosphate dehydrogenase. Binding studies were performed using the 5-HTR(4) antagonist [(3)H]GR113808. The mRNA levels of 5-HTR(4) were affected (P < 0.05) by location along the GIT. The mRNA levels of 5-HTR(4) in the ELSC and the ileum were greater than in the PLAC (P = 0.05 and P = 0.07, respectively) but similar to those of all other locations. The competitive binding of [(3)H]GR113808 to suspended membranes from the fundus abomasi, pylorus, cecum, and ELSC was best fit by a 2-site receptor model, whereas it was best fit by a 1-site receptor model in the ileum and PLAC. The mRNA levels and numbers of 5-HTR(4) were not correlated (r = 0.14; P = 0.71). In conclusion, mRNA and binding sites for 5-HTR(4) are present in the smooth muscle layer of the entire GIT of dairy cows and may play a role with respect to motility. The effects of activation of this receptor subtype may be different among GIT locations due to differences in the amount of high- relative to low-affinity binding sites.

Nuclear receptor and nuclear receptor target gene messenger ribonucleic acid levels at different sites of the gastrointestinal tract and in liver of healthy dogs

Nuclear receptors (NR) are ligand-activated transcription factors that regulate different metabolic pathways by influencing the expression of target genes. The current study examined mRNA abundance of NR and NR target genes at different sites of the gastrointestinal tract (GIT) and the liver of healthy dogs (Beagles; n = 11). Samples of GIT and liver were collected postmortem and homogenized, total RNA was extracted and reverse transcribed, and gene expression was quantified by real-time reverse-transcription PCR relative to the mean of 3 housekeeping genes (beta-actin, glyceraldehyde-3-phosphate dehydrogenase, and ubi-quitin). Differences were observed (P < or = 0.05) in the mRNA abundance among stomach (St), duodenum (Du), jejunum (Je), ileum (Il), and colon (Col) for NR [pregnane X receptor (Du, Je > Il, Col > St), peroxisome proliferator-associated receptor gamma (St, Du, Col > Je, Il), constitutive androstane receptor (Je, Du > Il, Col), and retinoid x receptor alpha (Du > Il)] and NR target genes [glutathione-S-transferase A3-3 (Du > Je > St, Il; St > Col), phenol-sulfating phenol sulfotransferase 1A1 (Du, Je > Il, St; Col > St), cytochrome P450 3A12 (Du, Je > St, Il, Col), multiple drug resistance gene 1 (Du, Je, Il, Col > St), multiple drug resistance-associated protein 2 (Je, Du > Il > St, Col), multiple drug resistance-associated protein 3 (Col > St > Il; Du > Je, Il; St > Il), NR corepressor 2 (St > Il, Col), and cytochrome P450 reductase (St, Du, Je > Il, Col)], but not for peroxisome proliferator-associated receptor alpha. Differences (P > 0.05) in mRNA abundance in the liver relative to the GIT were also observed. In conclusion, the presence of numerous differences in expression of NR and NR target genes in different parts of the GIT and in liver of healthy dogs may be associated with location-specific functions and regulation of GIT regions.

Syntheses, receptor bindings, in vitro and in vivo stabilities and biodistributions of DOTA-neurotensin(8-13) derivatives containing β-amino acid residues - a lesson about the importance of animal experiments

Neurotensin(8-13) (NTS(8-13)) analogs with C- and/or N-terminal β-amino acid residues and three DOTA derivatives thereof have been synthesized (i.e., 1-6). A virtual docking experiment showed almost perfect fit of one of the 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) derivatives, 6a, into a crystallographically identified receptor NTSR1 (Fig.1). The affinities for the receptors of the NTS analogs and derivatives are low, when determined with cell-membrane homogenates, while, with NTSR1-exhibiting cancer tissues, affinities in the single-digit nanomolar range can be observed (Table 2). Most of the β-amino acid-containing NTS(8-13) analogs (Table 1 and Fig.2), including the (68) Ga complexes of the DOTA-substituted ones (6; Figs.2 and 5), are stable for ca. 1 h in human serum and plasma, and in murine plasma. The biodistributions of two (68) Ga complexes (of 6a and 6b) in HT29 tumor-bearing nude mice, in the absence and in the presence of a blocking compound, after 10, 30, and 60 min (Figs. 3 and 4) lead to the conclusion that the amount of specifically bound radioligand is rather low. This was confirmed by PET-imaging experiments with the tumor-bearing mice (Fig.6). Comparison of the in vitro plasma stability (after 1 h) with the ex vivo blood content (after 10-15 min) of the two (68) Ga complexes shows that they are rapidly cleaved in the animals (Fig.5).

Stereochemistry of Amino Acid Spacers Determines the Pharmacokinetics of (111)In-DOTA-Minigastrin Analogues for Targeting the CCK2/Gastrin Receptor.

The metabolic instability and high kidney retention of minigastrin (MG) analogues hamper their suitability for use in peptide-receptor radionuclide therapy of CCK2/gastrin receptor-expressing tumors. High kidney retention has been related to N-terminal glutamic acids and can be substantially reduced by coinjection of polyglutamic acids or gelofusine. The aim of the present study was to investigate the influence of the stereochemistry of the N-terminal amino acid spacer on the enzymatic stability and pharmacokinetics of (111)In-DOTA-(d-Glu)6-Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH2 ((111)In-PP11-D) and (111)In-DOTA-(l-Glu)6-Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH2 ((111)In-PP11-L). Using circular dichroism measurements, we demonstrate the important role of secondary structure on the pharmacokinetics of the two MG analogues. The higher in vitro serum stability together with the improved tumor-to-kidney ratio of the (d-Glu)6 congener indicates that this MG analogue might be a good candidate for further clinical study.

Directing gene expression to cerebellar granule cells using γ-aminobutyric acid type A receptor α6 subunit transgenes

Expression of the γ-aminobutyric acid type A receptor α6 subunit gene is restricted to differentiated granule cells of the cerebellum and cochlear nucleus. The mechanisms underlying this limited expression are unknown. Here we have characterized the expression of a series of α6-based transgenes in adult mouse brain. A DNA fragment containing a 1-kb portion upstream of the start site(s), together with exons 1–8, can direct high-level cerebellar granule cell-specific reporter gene expression. Thus powerful granule cell-specific determinants reside within the 5′ half of the α6 subunit gene body. This intron-containing transgene appears to lack the cochlear nucleus regulatory elements. It therefore provides a cassette to deliver gene products solely to adult cerebellar granule cells.

The interaction of the general anesthetic etomidate with the γ-aminobutyric acid type A receptor is influenced by a single amino acid

The γ-aminobutyric acid type A (GABAA) receptor is a transmitter-gated ion channel mediating the majority of fast inhibitory synaptic transmission within the brain. The receptor is a pentameric assembly of subunits drawn from multiple classes (α1–6, β1–3, γ1–3, δ1, and ɛ1). Positive allosteric modulation of GABAA receptor activity by general anesthetics represents one logical mechanism for central nervous system depression. The ability of the intravenous general anesthetic etomidate to modulate and activate GABAA receptors is uniquely dependent upon the β subunit subtype present within the receptor. Receptors containing β2- or β3-, but not β1 subunits, are highly sensitive to the agent. Here, chimeric β1/β2 subunits coexpressed in Xenopus laevis oocytes with human α6 and γ2 subunits identified a region distal to the extracellular N-terminal domain as a determinant of the selectivity of etomidate. The mutation of an amino acid (Asn-289) present within the channel domain of the β3 subunit to Ser (the homologous residue in β1), strongly suppressed the GABA-modulatory and GABA-mimetic effects of etomidate. The replacement of the β1 subunit Ser-290 by Asn produced the converse effect. When applied intracellularly to mouse L(tk−) cells stably expressing the α6β3γ2 subunit combination, etomidate was inert. Hence, the effects of a clinically utilized general anesthetic upon a physiologically relevant target protein are dramatically influenced by a single amino acid. Together with the lack of effect of intracellular etomidate, the data argue against a unitary, lipid-based theory of anesthesia.

Defective γ-aminobutyric acid type B receptor-activated inwardly rectifying K+ currents in cerebellar granule cells isolated from weaver and Girk2 null mutant mice

Stimulation of inhibitory neurotransmitter receptors, such as γ-aminobutyric acid type B (GABAB) receptors, activates G protein-gated inwardly rectifying K+ channels (GIRK) which, in turn, influence membrane excitability. Seizure activity has been reported in a Girk2 null mutant mouse lacking GIRK2 channels but showing normal cerebellar development as well as in the weaver mouse, which has mutated GIRK2 channels and shows abnormal development. To understand how the function of GIRK2 channels differs in these two mutant mice, we compared the G protein-activated inwardly rectifying K+ currents in cerebellar granule cells isolated from Girk2 null mutant and weaver mutant mice with those from wild-type mice. Activation of GABAB receptors in wild-type granule cells induced an inwardly rectifying K+ current, which was sensitive to pertussis toxin and inhibited by external Ba2+ ions. The amplitude of the GABAB receptor-activated current was severely attenuated in granule cells isolated from both weaver and Girk2 null mutant mice. By contrast, the G protein-gated inwardly rectifying current and possibly the agonist-independent basal current appeared to be less selective for K+ ions in weaver but not Girk2 null mutant granule cells. Our results support the hypothesis that a nonselective current leads to the weaver phenotype. The loss of GABAB receptor-activated GIRK current appears coincident with the absence of GIRK2 channel protein and the reduction of GIRK1 channel protein in the Girk2 null mutant mouse, suggesting that GABAB receptors couple to heteromultimers composed of GIRK1 and GIRK2 channel subunits.

Role of brain allopregnanolone in the plasticity of γ-aminobutyric acid type A receptor in rat brain during pregnancy and after delivery

The relation between changes in brain and plasma concentrations of neurosteroids and the function and structure of γ-aminobutyric acid type A (GABAA) receptors in the brain during pregnancy and after delivery was investigated in rats. In contrast with plasma, where all steroids increased in parallel, the kinetics of changes in the cerebrocortical concentrations of progesterone, allopregnanolone (AP), and allotetrahydrodeoxycorticosterone (THDOC) diverged during pregnancy. Progesterone was already maximally increased between days 10 and 15, whereas AP and allotetrahydrodeoxycorticosterone peaked around day 19. The stimulatory effect of muscimol on 36Cl− uptake by cerebrocortical membrane vesicles was decreased on days 15 and 19 of pregnancy and increased 2 days after delivery. Moreover, the expression in cerebral cortex and hippocampus of the mRNA encoding for γ2L GABAA receptor subunit decreased during pregnancy and had returned to control values 2 days after delivery. Also α1,α2, α3, α4, β1, β2, β3, and γ2S mRNAs were measured and failed to change during pregnancy. Subchronic administration of finasteride, a 5α-reductase inhibitor, to pregnant rats reduced the concentrations of AP more in brain than in plasma as well as prevented the decreases in both the stimulatory effect of muscimol on 36Cl− uptake and the decrease of γ2L mRNA observed during pregnancy. These results indicate that the plasticity of GABAA receptors during pregnancy and after delivery is functionally related to fluctuations in endogenous brain concentrations of AP whose rate of synthesis/metabolism appears to differ in the brain, compared with plasma, in pregnant rats.