Diverse mechanisms underlying the regulation of ion channels by carbon monoxide

Carbon monoxide (CO) is firmly established as an important, physiological signalling molecule as well as a potent toxin. Through its ability to bind metal-containing proteins, it is known to interfere with a number of intracellular signalling pathways, and such actions can account for its physiological and pathological effects. In particular, CO can modulate the intracellular production of reactive oxygen species, NO and cGMP levels, as well as regulate MAPK signalling. In this review, we consider ion channels as more recently discovered effectors of CO signalling. CO is now known to regulate a growing number of different ion channel types, and detailed studies of the underlying mechanisms of action are revealing unexpected findings. For example, there are clear areas of contention surrounding its ability to increase the activity of high conductance, Ca2+ -sensitive K+ channels. More recent studies have revealed the ability of CO to inhibit T-type Ca2+ channels and have unveiled a novel signalling pathway underlying tonic regulation of this channel. It is clear that the investigation of ion channels as effectors of CO signalling is in its infancy, and much more work is required to fully understand both the physiological and the toxic actions of this gas. Only then can its emerging use as a therapeutic tool be fully and safely exploited.

Novel probiotics and prebiotics : road to the market

Novel probiotics and prebiotics designed to manipulate the gut microbiota for improving health outcomes are in demand as the importance of the gut microbiota in human health is revealed. The regulations governing introduction of novel probiotics and prebiotics vary by geographical region. Novel foods and foods with health claims fall under specific regulations in several countries. The paper reviews the main requirements of the regulations in the EU, USA, Canada and Japan. We propose a number of areas that need to be addressed in any safety assessment of novel probiotics and prebiotics. These include publication of the genomic sequence, antibiotic resistance profiling, selection of appropriate in vivo model, toxicological studies (including toxin production) and definition of target population.

The impact of secondary pests on Bacillus thuringiensis (Bt) crops

The intensification of agriculture and the development of synthetic insecticides enabled worldwide grain production to more than double in the last third of the 20th century. However, the heavy dependence and, in some cases, overuse of insecticides has been responsible for negative environmental and ecological impacts across the globe, such as a reduction in biodiversity, insect resistance to pesticides, negative effects on nontarget species (e.g. natural enemies) and the development of secondary pests. The use of recombinant DNA technology to develop genetically engineered (GE) insect resistant crops could mitigate many of the negative side effects of pesticides. One such genetic alteration enables crops to express toxic crystalline (Cry) proteins from the soil bacteria Bacillus thuringiensis (Bt). Despite the widespread adoption of Bt crops, there are still a range of unanswered questions concerning longer term agro-ecosystem interactions. For instance, insect species that are not susceptible to the expressed toxin can develop into secondary pests and cause significant damage to the crop. Here we review the main causes surrounding secondary pest dynamics in Bt crops and the impact of such outbreaks. Regardless of the causes, if non-susceptible secondary pest populations exceed economic thresholds, insecticide spraying could become the immediate solution at farmers’ disposal, and the sustainable use of this genetic modification technology may be in jeopardy. Based on the literature, recommendations for future research are outlined that will help to improve the knowledge of the possible longterm ecological trophic interactions of employing this technology.

CLEC-2 is not required for platelet aggregation at arteriolar shear

The C-type lectin receptor CLEC-2 is expressed primarily on the surface of platelets, where it is present as a dimer, and is found at low level on a subpopulation of other hematopoietic cells, including mouse neutrophils [1–4] Clustering of CLEC-2 by the snake venom toxin rhodocytin, specific antibodies or its endogenous ligand, podoplanin, elicits powerful activation of platelets through a pathway that is similar to that used by the collagen receptor glycoprotein VI (GPVI) [4–6]. The cytosolic tail of CLEC-2 contains a conserved YxxL sequence preceded by three upstream acidic amino acid residues, which together form a novel motif known as a hemITAM. Ligand engagement induces tyrosine phosphorylation of the hemITAM sequence providing docking sites for the tandem-SH2 domains of the tyrosine kinase Syk across a CLEC-2 receptor dimer [3]. Tyrosine phosphorylation of Syk by Src family kinases and through autophosphorylation leads to stimulation of a downstream signaling cascade that culminates in activation of phospholipase C γ2 (PLCγ2) [4,6]. Recently, CLEC-2 has been proposed to play a major role in supporting activation of platelets at arteriolar rates of flow [1]. Injection of a CLEC-2 antibody into mice causes a sustained depletion of the C-type lectin receptor from the platelet surface [1]. The CLEC-2-depleted platelets were unresponsive to rhodocytin but underwent normal aggregation and secretion responses after stimulation of other platelet receptors, including GPVI [1]. In contrast, there was a marked decrease in aggregate formation relative to controls when CLEC-2-depleted blood was flowed at arteriolar rates of shear over collagen (1000 s−1 and 1700 s−1) [1]. Furthermore, antibody treatment significantly increased tail bleeding times and mice were unable to occlude their vessels after ferric chloride injury [1]. These data provide evidence for a critical role for CLEC-2 in supporting platelet aggregation at arteriolar rates of flow. The underlying mechanism is unclear as platelets do not express podoplanin, the only known endogenous ligand of CLEC-2. In the present study, we have investigated the role of CLEC-2 in platelet aggregation and thrombus formation using platelets from a novel mutant mouse model that lacks functional CLEC-2.

Syk-dependent phosphorylation of CLEC-2: a novel mechanism of hem-immunoreceptor tyrosine-based activation motif signalling

The C-type lectin-like receptor CLEC-2 signals via phosphorylation of a single cytoplasmic YXXL sequence known as a hem-immunoreceptor tyrosine-based activation motif (hemITAM). In this study, we show that phosphorylation of CLEC-2 by the snake toxin rhodocytin is abolished in the absence of the tyrosine kinase Syk but is not altered in the absence of the major platelet Src family kinases, Fyn, Lyn, and Src, or the tyrosine phosphatase CD148, which regulates the basal activity of Src family kinases. Further, phosphorylation of CLEC-2 by rhodocytin is not altered in the presence of the Src family kinase inhibitor PP2, even though PLCγ2 phosphorylation and platelet activation are abolished. A similar dependence of phosphorylation of CLEC-2 on Syk is also seen in response to stimulation by an IgG mAb to CLEC-2, although interestingly CLEC-2 phosphorylation is also reduced in the absence of Lyn. These results provide the first definitive evidence that Syk mediates phosphorylation of the CLEC-2 hemITAM receptor with Src family kinases playing a critical role further downstream through the regulation of Syk and other effector proteins, providing a new paradigm in signaling by YXXL-containing receptors.

Renal cells activate the platelet receptor CLEC-2 through podoplanin.

We have recently shown that the C-type lectin-like receptor, CLEC-2, is expressed on platelets and that it mediates powerful platelet aggregation by the snake venom toxin rhodocytin. In addition, we have provided indirect evidence for an endogenous ligand for CLEC-2 in renal cells expressing HIV-1. This putative ligand facilitates transmission of HIV through its incorporation into the viral envelope and binding to CLEC-2 on platelets. The aim of the present study was to identify the ligand on these cells which binds to CLEC-2 on platelets. Recombinant CLEC-2 exhibits specific binding to HEK-293T (human embryonic kidney) cells in which the HIV can be grown. Furthermore, HEK-293T cells activate both platelets and CLEC-2-transfected DT-40 B-cells. The transmembrane protein podoplanin was identified on HEK-293T cells and was demonstrated to mediate both binding of HEK-293T cells to CLEC-2 and HEK-293T cell activation of CLEC-2-transfected DT-40 B-cells. Podoplanin is expressed on renal cells (podocytes). Furthermore, a direct interaction between CLEC-2 and podoplanin was confirmed using surface plasmon resonance and was shown to be independent of glycosylation of CLEC-2. The interaction has an affinity of 24.5+/-3.7 microM. The present study identifies podoplanin as a ligand for CLEC-2 on renal cells.

Regulation of Ras.GTP loading and Ras-Raf association in neonatal rat ventricular myocytes by G protein-coupled receptor agonists and phorbol ester. Activation of the extracellular signal-regulated kinase cascade by phorbol ester is mediated by Ras.

The small G protein Ras has been implicated in hypertrophy of cardiac myocytes. We therefore examined the activation (GTP loading) of Ras by the following hypertrophic agonists: phorbol 12-myristate 13-acetate (PMA), endothelin-1 (ET-1), and phenylephrine (PE). All three increased Ras.GTP loading by 10-15-fold (maximal in 1-2 min), as did bradykinin. Other G protein-coupled receptor agonists (e.g. angiotensin II, carbachol, isoproterenol) were less effective. Activation of Ras by PMA, ET-1, or PE was reduced by inhibition of protein kinase C (PKC), and that induced by ET-1 or PE was partly sensitive to pertussis toxin. 8-(4-Chlorophenylthio)-cAMP (CPT-cAMP) did not inhibit Ras.GTP loading by PMA, ET-1, or PE. The association of Ras with c-Raf protein was increased by PMA, ET-1, or PE, and this was inhibited by CPT-cAMP. However, only PMA and ET-1 increased Ras-associated mitogen-activated protein kinase kinase 1-activating activity, and this was decreased by PKC inhibition, pertussis toxin, and CPT-cAMP. PMA caused the rapid appearance of phosphorylated (activated) extracellular signal-regulated kinase in the nucleus, which was inhibited by a microinjected neutralizing anti-Ras antibody. We conclude that PKC- and Gi-dependent mechanisms mediate the activation of Ras in myocytes and that Ras activation is required for stimulation of extracellular signal-regulated kinase by PMA.

Regulation of mitogen-activated protein kinases in cardiac myocytes through the small G protein Rac1.

Small guanine nucleotide-binding proteins of the Ras and Rho (Rac, Cdc42, and Rho) families have been implicated in cardiac myocyte hypertrophy, and this may involve the extracellular signal-related kinase (ERK), c-Jun N-terminal kinase (JNK), and/or p38 mitogen-activated protein kinase (MAPK) cascades. In other systems, Rac and Cdc42 have been particularly implicated in the activation of JNKs and p38-MAPKs. We examined the activation of Rho family small G proteins and the regulation of MAPKs through Rac1 in cardiac myocytes. Endothelin 1 and phenylephrine (both hypertrophic agonists) induced rapid activation of endogenous Rac1, and endothelin 1 also promoted significant activation of RhoA. Toxin B (which inactivates Rho family proteins) attenuated the activation of JNKs by hyperosmotic shock or endothelin 1 but had no effect on p38-MAPK activation. Toxin B also inhibited the activation of the ERK cascade by these stimuli. In transfection experiments, dominant-negative N17Rac1 inhibited activation of ERK by endothelin 1, whereas activated V12Rac1 cooperated with c-Raf to activate ERK. Rac1 may stimulate the ERK cascade either by promoting the phosphorylation of c-Raf or by increasing MEK1 and/or -2 association with c-Raf to facilitate MEK1 and/or -2 activation. In cardiac myocytes, toxin B attenuated c-Raf(Ser-338) phosphorylation (50 to 70% inhibition), but this had no effect on c-Raf activity. However, toxin B decreased both the association of MEK1 and/or -2 with c-Raf and c-Raf-associated ERK-activating activity. V12Rac1 cooperated with c-Raf to increase expression of atrial natriuretic factor (ANF), whereas N17Rac1 inhibited endothelin 1-stimulated ANF expression, indicating that the synergy between Rac1 and c-Raf is potentially physiologically important. We conclude that activation of Rac1 by hypertrophic stimuli contributes to the hypertrophic response by modulating the ERK and/or possibly the JNK (but not the p38-MAPK) cascades.