IL-33 mediates antigen-induced cutaneous and articular hypernociception in mice

IL-33, a new member of the IL-1 family, signals through its receptor ST2 and induces T helper 2 (Th2) cytokine synthesis and mediates inflammatory response. We have investigated the role of IL-33 in antigen-induced hypernociception. Recombinant IL-33 induced cutaneous and articular mechanical hype rn ociception in a time- and dose-dependent manner. The hypernociception was inhibited by soluble (s) ST2 (a decoy receptor of IL-33), IL-1 receptor antagonist (IL-1ra), bosentan [a dual endothelin (ET)(A)/ETB receptor antagonist], clazosentan (an ETA receptor antagonist), or indomethacin (a cyclooxygenase inhibitor). IL-33 induced hypernociception in IL-18(-/-) mice but not in TNFR1(-/-) or IFN gamma(-/-) mice. The IL-33-induced hypernociception was not affected by blocking IL-15 or sympathetic amines (guanethidine). Furthermore, methylated BSA (mBSA)-induced cutaneous and articular mechanical hypernociception depended on TNFR1 and IFN gamma and was blocked by sST2, IL-1ra, bosentan, clazosentan, and indomethacin. mBSA also induced significant IL-33 and ST2 mRNA expression. Importantly, we showed that mBSA induced hypernociception via the IL-33 -> TNF alpha -> IL-1 beta -> IFN gamma -> ET-1 -> PGE(2) signaling cascade. These results therefore demonstrate that IL-33 is a key mediator of immune inflammatory hype rn ociception normally associated with a Th1 type of response, revealing a hitherto unrecognized function of IL-33 in a key immune pharmacological pathway that may be amenable to therapeutic intervention.

Cysteinyl-leukotriene type 1 receptors transduce a critical signal for the up-regulation of eosinophilopoiesis by interleukin-13 and eotaxin in murine bone marrow

IL-13 and eotaxin play important, inter-related roles in asthma models. In the lungs, CysLT, produced by the 5-LO-LTC4S pathway, mediate some local responses to IL-13 and eotaxin; in bone marrow, CysLT enhance IL-5-dependent eosinophil differentiation. We examined the effects of IL-13 and eotaxin on eosinophil differentiation. Semi-solid or liquid cultures were established from murine bone marrow with GM-CSF or IL-5, respectively, and the effects of IL-13, eotaxin, or CysLT on eosinophil colony formation and on eosinophil differentiation in liquid culture were evaluated, in the absence or presence of: a) the 5-LO inhibitor zileuton, the FLAP inhibitor MK886, or the CysLT1R antagonists, montelukast and MK571; b) mutations that inactivate 5-LO, LTC4S, or CysLT1R; and c) neutralizing mAb against eotaxin and its CCR3 receptor. Both cytokines enhanced GM-CSF-dependent eosinophil colony formation and IL-5-stimulated eosinophil differentiation. Although IL-13 did not induce eotaxin production, its effects were abolished by anti-eotaxin and anti-CCR3 antibodies, suggesting up-regulation by IL-13 of responses to endogenous eotaxin. Anti-CCR3 blocked eotaxin completely. The effects of both cytokines were prevented by zileuton, MK886, montelukast, and MK571, as well as by inactivation of the genes coding for 5-LO, LTC4S, and CysLT1R. In the absence of either cytokine, these treatments or mutations had no effect. These findings provide evidence for: a) a novel role of eotaxin and IL-13 in regulating eosinophilopoiesis; and b) a role for CysLTRs in bone marrow cells in transducing cytokine regulatory signals. J. Leukoc. Biol. 87: 885-893; 2010.

Quercetin Reduces Inflammatory Pain: Inhibition of Oxidative Stress and Cytokine Production

Quercetin (1) is known to have both antioxidant and antinociceptive effects. However, the mechanism involved in its antinociceptive effect is not fully elucidated. Cytokines and reactive oxygen species have been implicated in the cascade of events resulting in inflammatory pain. Therefore, we evaluated the antinociceptive mechanism of 1 focusing on the role of cytokines and Oxidative stress. Intraperitoneal and oral treatments with 1 dose-dependently inhibited inflammatory nociception induced by acetic acid and phenyl-p-benzoquinone and also the second phase of formalin- and carrageenin-induced mechanical hypernociception. Compound I also inhibited the hypernociception induced by cytokines (e.g., TNF alpha and CXCL1), but not by inflammatory mediators that directly sensitize the nociceptor such as PGE(2) and dopamine. On the other hand, 1 reduced carrageenin-induced IL-1 beta production as well as carrageenin-induced decrease of reduced glutathione (GSH) levels. These results suggest that I exerts its analgesic effect by inhibiting pro-nociceptive cytokine production and the oxidative imbalance mediation of inflammatory pain.

Transcription of the Hsp30, Hsp70, and Hsp90 heat shock protein genes is modulated by the PalA protein in response to acid pH-sensing in the fungus Aspergillus nidulans

Heat shock proteins are molecular chaperones linked to a myriad of physiological functions in both prokaryotes and eukaryotes. In this study, we show that the Aspergillus nidulans hsp30 (ANID_03555.1), hsp70 (ANID_05129.1), and hsp90 (ANID_08269.1) genes are preferentially expressed in an acidic milieu, whose expression is dependent on the palA (+) background under optimal temperature for fungal growth. Heat shock induction of these three hsp genes showed different patterns in response to extracellular pH changes in the palA(+) background. However, their accumulation upon heating for 2 h was almost unaffected by ambient pH changes in the palA (-) background. The PalA protein is a member of a conserved signaling cascade that is involved in the pH-mediated regulation of gene expression. Moreover, we identified several genes whose expression at pH 5.0 is also dependent on the palA (+) background. These results reveal novel aspects of the heat- and pH-sensing networks of A. nidulans.

Imitation, mirror neurons and autism

Various deficits in the cognitive functioning of people with autism have been documented in recent years but these provide only partial explanations for the condition. We focus instead on an imitative disturbance involving difficulties both in copying actions and in inhibiting more stereotyped mimicking, such as echolalia. A candidate for the neural basis of this disturbance may be found in a recently discovered class of neurons in frontal cortex, 'mirror neurons' (MNs). These neurons show activity in relation both to specific actions performed by self and matching actions performed by others, providing a potential bridge between minds. MN systems exist in primates without imitative and 'theory of mind' abilities and we suggest that in order for them to have become utilized to perform social cognitive functions, sophisticated cortical neuronal systems have evolved in which MNs function as key elements. Early developmental failures of MN systems are likely to result in a consequent cascade of developmental impairments characterised by the clinical syndrome of autism. Crown Copyright (C) 2001 Published by Elsevier Science Ltd. All rights reserved.

Murine ventricular L-type Ca2+ current is enhanced by zinterol via beta(1)-adrenoceptors, and is reduced in TG4 mice overexpressing the human beta(2)-adrenoceptor

1 The functional coupling of B-2-adrenoceptors (beta (2)-ARs) to murine L-type Ca2+ current (I-Ca(L)) was investigated with two different approaches. The beta (2)-AR signalling cascade was activated either with the beta (2)-AR selective agonist zinterol (myocytes from wild-type mice), or by spontaneously active, unoccupied beta (2)-ARs (myocytes from TG4 mice with 435 fold overexpression of human beta (2)-ARs). Ca2+ and Ba2+ currents were recorded in the whole-cell and cell-attached configuration of the patch- clamp technique, respectively. 2 Zinterol (10 muM) significantly increased I-Ca(L) amplitude of wild-type myocytes by 19+/-5%, and this effect was markedly enhanced after inactivation of Gi-proteins with pertussis-toxin (PTX; 76+/-13% increase). However, the effect of zinterol was entirely mediated by the beta (1)-AR subtype, since it was blocked by the beta (1)-AR selective antagonist CGP 20712A (300 nM). The beta (2)-AR selective antagonist ICI 118,551 (50 nM) did not affect the response of I-Ca(L) to zinterol. 3 In myocytes with beta (2)-AR overexpression I-Ca(L) was not stimulated by the activated signalling cascade. On the contrary, I-Ca(L) was lower in TG4 myocytes and a significant reduction of single-channel activity was identified as a reason for the lower whole-cell I-Ca(L). The beta (2)-AR inverse agonist ICI 118,551 did not further decrease I-Ca(L). PTX-treatment increased current amplitude to values found in control myocytes. 4 In conclusion, there is no evidence for beta (2)-AR mediated increases of I-Ca(L) in wild-type mouse ventricular myocytes. Inactivation of Gi-proteins does not unmask beta (2)-AR responses to zinterol, but augments beta (1)-AR mediated increases of I-Ca(L). In the mouse model of beta (2)-AR overexpression I-Ca(L) is reduced due to tonic activation of Gi-proteins.

Lipid peroxidation in hepatic steatosis in humans is associated with hepatic fibrosis and occurs predominately in acinar zone 3

Background and Aims: Hepatic steatosis has been shown to be associated with lipid peroxidation and hepatic fibrosis in a variety of liver diseases including non-alcoholic fatty liver disease. However, the lobular distribution of lipid peroxidation associated with hepatic steatosis, and the influence of hepatic iron stores on this are unknown. The aim of this study was to assess the distribution of lipid peroxidation in association with these factors, and the relationship of this to the fibrogenic cascade. Methods: Liver biopsies from 39 patients with varying degrees of hepatic steatosis were assessed for evidence of lipid peroxidation (malondialdehyde adducts), hepatic iron, inflammation, fibrosis, hepatic ;stellate cell activation (alpha-smooth muscle actin and TGF-beta expression) and collagen type I synthesis (procollagen a 1 (I) mRNA). Results: Lipid peroxidation occurred in and adjacent to fat-laden hepatocytes and was maximal in acinar zone 3. Fibrosis was associated with steatosis (P < 0.04), lipid peroxidation (P < 0.05) and hepatic iron stores (P < 0.02). Multivariate logistic regression analysis confirmed the association between steatosis and lipid peroxidation within zone 3 hepatocytes (P < 0.05), while for hepatic iron, lipid peroxidation was seen within sinusoidal cells (P < 0.05), particularly in zone 1 (P < 0.02). Steatosis was also associated with acinar inflammation (P < 0.005). α-Smooth muscle actin expression was present in association with both lipid peroxidation and fibrosis. Although the effects of steatosis and iron on lipid peroxidation and fibrosis were additive, there was no evidence of a specific synergistic interaction between them. Conclusions: These observations support a model where steatosis exerts an effect on fibrosis through lipid peroxidation, particularly in zone 3 hepatocytes. (C) 2001 Blackwell Science Asia Pty Ltd.

Cloning of a catalytic subunit of cAMP-dependent protein kinase from the honeybee (Apis mellifera) and its localization in the brain

In the honeybee the cAMP-dependent signal transduction cascade has been implicated in processes underlying learning and memory, The cAMP-dependent protein kinase (PKA) is the major mediator of cAMP action. To characterize the PKA system in the honeybee brain we cloned a homologue of a PKA catalytic subunit from the honeybee,The deduced amino acid sequence shows 80-94% identity with catalytic subunits of PKA from Drosophila melanogaster, Aplysia californica and mammals. The corresponding gene is predominantly expressed in the mushroom bodies, a structure that is involved in learning and memory processes. However, expression can also be found in the antennal and optic lobes,The level of expression varies within all three neuropiles.

Nerve growth factor stimulates proliferation and survival of human breast cancer cells through two distinct signaling pathways

We show here that the neurotrophin nerve growth factor (NGF), which has been shown to be a mitogen for breast cancer cells, also stimulates cell survival through a distinct signaling pathway. Breast cancer cell lines (MCF-7, T47-D, BT-20, and MDA-MB-231) were found to express both types of NGF receptors: p140(trkA) and p75(NTR). The two other tyrosine kinase receptors for neurotrophins, TrkB and TrkC, were not expressed. The mitogenic effect of NGF on breast cancer cells required the tyrosine kinase activity of p140(trkA) as well as the mitogen-activated protein kinase (MAPK) cascade, but was independent of p75(NTR). I, contrast, the anti-apoptotic effect of NGF (studied using the ceramide analogue C2) required p75(NTR) as well as the activation of the transcription factor NF-kB, but neither p140(trkA) nor MAPK was necessary. Other neurotrophins (BDNF, NT-3, NT-4/5) also induced cell survival, although not proliferation, emphasizing the importance of p75(NTR) in NGF-mediated survival. Both the pharmacological NF-KB inhibitor SN50, and cell transfection with IkBm, resulted in a diminution of NGF anti-apoptotic effect. These data show that two distinct signaling pathways are required for NGF activity and confirm the roles played by p75(NTR) and NF-kappaB in the activation of the survival pathway in breast cancer cells.

The role of hedgehog signalling in tumorigenesis

It has long been known from work in both Drosophila and vertebrate systems that the hedgehog signalling pathway is pivotal to embryonic development, but the past 5 years has seen an increase in our understanding of how members of this pathway are crucial to the processes of tumorigenesis. This important link was firmly established with the discovery that mutations in the gene encoding the hedgehog receptor molecule patched are responsible for both familial and sporadic forms of basal cell carcinoma (BCC), as well as a number of other tumour types. It is now known that a number of key members of the hedgehog cascade are involved in tumorigenesis, and dysregulation of this pathway appears to be a key element in the aetiology of a range of tumours. (C) 2001 Elsevier Science Ireland Ltd. All rights reserved.

H-Ras signaling and K-Ras signaling are differentially dependent on endocytosis

dEndocytosis is required for efficient mitogen-activated protein kinase (MAPK) activation by activated growth factor receptors. We examined if H-Ras and K-Ras proteins, which are distributed across different plasma membrane microdomains, have equal access to the endocytic compartment and whether this access is necessary for downstream signaling. Inhibition of endocytosis by dominant interfering dynamin-K44A blocked H-Ras but not K-Ras-mediated PC12 cell differentiation and selectively inhibited H-Ras- but not K-Ras-mediated Raf-1 activation in BHK cells. H-Ras- but not K-Ras-mediated Raf-1 activation was also selectively dependent on phosphoinositide 3-kinase activity. Stimulation of endocytosis and endocytic recycling by wildtype Rab5 potentiated H-Ras-mediated Raf-1 activation. In contrast, Rab5-Q79L, which stimulates endocytosis but not endocytic recycling, redistributed activated H-Ras from the plasma membrane into enlarged endosomes and inhibited H-Ras-mediated Raf-1 activation. Rab5-Q79L expression did not cause the accumulation of wild-type H-Ras in enlarged endosomes. Expression of wild-type Rab5 or Rab5-Q79L increased the specific activity of K-Ras-activated Raf-1 but did not result in any redistribution of K-Ras from the plasma membrane to endosomes. These results show that H-Ras but not K-Ras signaling though the Raf/MEK/MAPK cascade requires endocytosis and enclocytic recycling. The data also suggest a mechanism for returning Raf-1 to the cytosol after plasma membrane recruitment.

Air-water flows down stepped chutes: turbulence and flow structure observations

Interactions between turbulent waters and atmosphere may lead to strong air-water mixing. This experimental study is focused on the flow down a staircase channel characterised by very strong flow aeration and turbulence. Interfacial aeration is characterised by strong air-water mixing extending down to the invert. The size of entrained bubbles and droplets extends over several orders of magnitude, and a significant number of bubble/droplet clusters was observed. Velocity and turbulence intensity measurements suggest high levels of turbulence across the entire air-water flow. The increase in turbulence levels, compared to single-phase flow situations, is proportional to the number of entrained particles. (C) 2002 Elsevier Science Ltd. All rights reserved.

The coactivator-associated arginine methyltransferase is necessary for muscle differentiation - CARM1 coactivates myocyte enhancer factor-2

Studies with the myogenic basic helix-loop-helix and MADS box factors suggest that efficient transactivation is dependent on the recruitment of the steroid receptor coactivator (SRC) and the cofactors p300 and p300/CBP-associated factor. SRCs have been demonstrated to recruit CARM1 (coactivator-associated arginine methyltransferase-1), a member of the S-adenOSyl-L-methionine-dependent PRMTI-5 (protein-arginine N-methyltransferase-1-5) family, which catalyzes the methylation of arginine residues. This prompted us to investigate the functional role of CARM1/PRMT4 during skeletal myogenesis. We demonstrate that CARM1 and the SRC cofactor GRIP-1 cooperatively stimulate the activity of myocyte enhancer factor-2C (MEF2C). Moreover, there are direct interactions among MEF2C, GRIP-1, and CARM1. Chromatin immunoprecipitation demonstrated the in vivo recruitment of MEF2 and CARM1 to the endogenous muscle creatine kinase promoter in a differentiation-dependent manner. Furthermore, CARM1 is expressed in somites during embryogenesis and in the nuclei of muscle cells. Treatment of myogenic cells with the methylation inhibitor adenosine dialdehyde or tet-regulated CARM1 antisense expression did not affect expression of MyoD. However, inhibition of CARM1. inhibited differentiation and abrogated the expression of the key transcription factors (myogenin and MEF2) that initiate the differentiation cascade. This work clearly demonstrates that the arginine methyltransferase CARM1 potentiates myogenesis and supports the positive role of arginine methylation in mammalian differentiation.

Novel genes regulated by Sonic Hedgehog in pluripotent mesenchymal cells

Sonic Hedgehog is a secreted morphogen involved in patterning a wide range of structures in the developing embryo. Disruption of the Hedgehog signalling cascade leads to a number of developmental disorders and plays a key role in the formation of a range of human cancers. The identification of genes regulated by Hedgehog is crucial to understanding how disruption of this pathway leads to neoplastic transformation. We have used a Sonic Hedgehog (Shh) responsive mouse cell line, C3H/10T1/2, to provide a model system for hedgehog target gene discovery. Following activation of cell cultures with Shh, RNA was used to interrogate microarrays to investigate downstream transcriptional consequences of hedgehog stimulation. As a result 11 target genes have been identified, seven of which are induced (Thrombomodulin, GILZ, BF-2, Nr4a1, IGF2, PMP22, LASP1) and four of which are repressed (SFRP-1, SFRP-2, Mip1-gamma, Amh) by Shh. These targets have a diverse range of putative functions and include transcriptional regulators and molecules known to be involved in regulating cell growth or apoptosis. The corroboration of genes previously implicated in hedgehog signalling, along with the finding of novel targets, demonstrates both the validity and power of the C3H/10T1/2 system for Shh target gene discovery.

GATA proteins identify a novel ventral interneuron subclass in the developing chick spinal cord

Members of the GATA transcription factor gene family have been implicated in a variety of developmental processes, including that of the vertebrate central nervous system. However, the role of GATA proteins in spinal cord development remains unresolved. In this study, we investigated the expression and function of two GATA proteins, GATA2 and GATA3, in the developing chick spinal cord. We show that both proteins are expressed by a distinct subpopulation of ventral interneurons that share the same dorsoventral position as CHX10-positive V2 interneurons. However, no coexpression is observed between the two GATA proteins and CHX10. By in vivo notochord grafting and cyclopamine treatment, we demonstrate that the spatially restricted pattern of GATA3 expression is regulated, at least in part, by the signaling molecule Sonic hedgehog. In addition, we further show that Sonic hedgehog induces GATA3 expression in a dose-dependent manner. Using in ovo electroporations, we also demonstrate that GATA2 is upstream of GATA3 in the same epigenetic cascade and that GATA3 is capable of inducing GATA2 expression in vivo. Furthermore, the ectopically expressed GATA proteins can repress differentiation of other ventral cell fates, but not the development of progenitor populations identified by PAX protein expression. Taken together, our findings strongly suggest an important role for GATA2 and GATA3 proteins in the establishment of a distinct ventral interneuron subpopulation in the developing chick spinal cord. (C) 2002 Elsevier Science (USA).