The bile acid receptor TGR5 does not interact with β-arrestins or traffic to endosomes but transmits sustained signals from plasma membrane rafts.

TGR5 is a G protein-coupled receptor that mediates bile acid (BA) effects on energy balance, inflammation, digestion and sensation. The mechanisms and spatiotemporal control of TGR5 signaling are poorly understood. We investigated TGR5 signaling and trafficking in transfected HEK293 cells and colonocytes (NCM460) that endogenously express TGR5. BAs (deoxycholic acid, DCA, taurolithocholic acid, TLCA) and the selective agonists oleanolic acid (OA) and 3-(2-chlorophenyl)-N-(4-chlorophenyl)-N, 5-dimethylisoxazole-4-carboxamide (CCDC) stimulated cAMP formation but did not induce TGR5 endocytosis or recruitment of β-arrestins, assessed by confocal microscopy. DCA, TLCA and OA did not stimulate TGR5 association with β-arrestin 1/2 or G protein-coupled receptor kinase (GRK) 2/5/6, determined by bioluminescence resonance energy transfer. CCDC stimulated a low level of TGR5 interaction with β-arrestin2 and GRK2. DCA induced cAMP formation at the plasma membrane and cytosol, determined using exchange factor directly regulated by cAMP (Epac2)-based reporters, but cAMP signals did not desensitize. AG1478, an inhibitor of epidermal growth factor receptor (EGFR) tyrosine kinase, the metalloprotease inhibitor batimastat, and methyl-β-cyclodextrin and filipin, which block lipid raft formation, prevented DCA stimulation of extracellular signal regulated kinase (ERK1/2). BRET analysis revealed TGR5 and EGFR interactions that were blocked by disruption of lipid rafts. DCA stimulated TGR5 redistribution to plasma membrane microdomains, localized by immunogold electron microscopy. Thus, TGR5 does not interact with β-arrestins, desensitize or traffic to endosomes. TGR5 signals from plasma membrane rafts that facilitate EGFR interaction and transactivation. An understanding of the spatiotemporal control of TGR5 signaling provides insights into the actions of BAs and therapeutic TGR5 agonists/antagonists.

Computing journey start times with recurrent traffic conditions

n this study, the authors discuss the effective usage of technology to solve the problem of deciding on journey start times for recurrent traffic conditions. The developed algorithm guides the vehicles to travel on more reliable routes that are not easily prone to congestion or travel delays, ensures that the start time is as late as possible to avoid the traveller waiting too long at their destination and attempts to minimise the travel time. Experiments show that in order to be more certain of reaching their destination on time, a traveller has to leave early and correspondingly arrive early, resulting in a large waiting time. The application developed here asks the user to set this certainty factor as per the task in hand, and computes the best start time and route.

A palaeoenvironmental context for terminal upper Palaeolithic and Mesolithic activity in the Colne Valley: offsite records contemporary with occupation at three ways Wharf, Uxbridge

Multi-proxy analyses from floodplain deposits in the Colne Valley, southern England, have provided a palaeoenvironmental context for the immediately adjacent Terminal Upper Palaeolithic and Early Mesolithic site of Three Ways Wharf. These deposits show the transition from an open cool environment to fully developed heterogeneous floodplain vegetation during the Early Mesolithic. Several distinct phases of burning are shown to have occurred that are chronologically contemporary with the local archaeological record. The floodplain itself is shown to have supported a number of rare Urwaldrelikt insect species implying human manipulation of the floodplain at this time must have been limited or episodic. By the Late Mesolithic a reed-sedge swamp had developed across much of the floodplain, within which repeated burning of the in situ vegetation took place. This indicates deliberate land management practices utilising fire, comparable with findings from other floodplain sequences in southern Britain. With similar sedimentary sequences known to exist across the Colne Valley, often closely associated with contemporary archaeology, the potential for placing the archaeological record within a spatially explicit palaeoenvironmental context is great.

The application of social marketing in reducing road traffic accidents amoung young male drivers: an investigation using physical fear threat appeals

There were 338 road fatalities on Irish roads in 2007. Research in 2007 by the Road Safety Authority in Ireland states that young male drivers (17 – 25 years) are seven times more likely to be killed on Irish roads than other road users. The car driver fatality rate was found to be approximately 10 times higher for young male drivers than for female drivers in 2000. Young male drivers in particular demonstrate a high proclivity for risky driving behaviours. These risky behaviours include drink driving, speeding, rug-driving and engaging in aggressive driving. Speed is the single largest contributing factor to road deaths in Ireland. Approximately 40% of fatal accidents are caused by excessive or inappropriate speed. This study focuses on how dangerous driving behaviours may be addressed through social marketing. This study analyses the appropriate level of fear that needs to be induced in order to change young male driving behaviour.

The N-terminal SH2 domain of Syk is required for (hem)ITAM, but not integrin, signaling in mouse platelets

We have used a novel knockin mouse to investigate the effect of disruption of phosphotyrosine binding of the N-terminal SH2 domain of Syk on platelet activation by GPVI, CLEC-2, and integrin αIIbβ3. The Syk(R41Afl/fl) mouse was crossed to a PF4-Cre(+) mouse to induce expression of the Syk mutant in the megakaryocyte/platelet lineage. Syk(R41Afl/fl;PF4-Cre) mice are born at approximately 50% of the expected frequency and have a similar phenotype to Syk(fl/fl;PF4-Cre) mice, including blood-lymphatic mixing and chyloascites. Anastomosis of the venous and lymphatic vasculatures can be seen in the mesenteric circulation accounting for rapid and continuous mixing of the 2 vasculatures. Platelet activation by CLEC-2 and GPVI is abolished in Syk(R41Afl/fl;PF4-Cre) platelets. Syk phosphorylation on Tyr519/20 is blocked in CLEC-2-stimulated platelets, suggesting a model in which binding of Syk via its N-terminal SH2 domain regulates autophosphorylation. In contrast, outside-in signaling by integrin αIIbβ3 is not altered, but it is inhibited in the presence of inhibitors of Src and Syk tyrosine kinases. These results demonstrate that αIIbβ3 regulates Syk through an ITAM-independent pathway in mice and provide novel insight into the course of events underlying Syk activation and hemITAM phosphorylation by CLEC-2.

Stimulation of "stress-regulated" mitogen-activated protein kinases (stress-activated protein kinases/c-Jun N-terminal kinases and p38-mitogen-activated protein kinases) in perfused rat hearts by oxidative and other stresses.

"Stress-regulated" mitogen-activated protein kinases (SR-MAPKs) comprise the stress-activated protein kinases (SAPKs)/c-Jun N-terminal kinases (JNKs) and the p38-MAPKs. In the perfused heart, ischemia/reperfusion activates SR-MAPKs. Although the agent(s) directly responsible is unclear, reactive oxygen species are generated during ischemia/reperfusion. We have assessed the ability of oxidative stress (as exemplified by H2O2) to activate SR-MAPKs in the perfused heart and compared it with the effect of ischemia/reperfusion. H2O2 activated both SAPKs/JNKs and p38-MAPK. Maximal activation by H2O2 in both cases was observed at 0.5 mM. Whereas activation of p38-MAPK by H2O2 was comparable to that of ischemia and ischemia/reperfusion, activation of the SAPKs/JNKs was less than that of ischemia/reperfusion. As with ischemia/reperfusion, there was minimal activation of the ERK MAPK subfamily by H2O2. MAPK-activated protein kinase 2 (MAPKAPK2), a downstream substrate of p38-MAPKs, was activated by H2O2 to a similar extent as with ischemia or ischemia/reperfusion. In all instances, activation of MAPKAPK2 in perfused hearts was inhibited by SB203580, an inhibitor of p38-MAPKs. Perfusion of hearts at high aortic pressure (20 kilopascals) also activated the SR-MAPKs and MAPKAPK2. Free radical trapping agents (dimethyl sulfoxide and N-t-butyl-alpha-phenyl nitrone) inhibited the activation of SR-MAPKs and MAPKAPK2 by ischemia/reperfusion. These data are consistent with a role for reactive oxygen species in the activation of SR-MAPKs during ischemia/reperfusion.

The p38-MAPK inhibitor, SB203580, inhibits cardiac stress-activated protein kinases/c-Jun N-terminal kinases (SAPKs/JNKs).

SB203580 is a recognised inhibitor of p38-MAPKs. Here, we investigated the effects of SB203580 on cardiac SAPKs/JNKs. The IC50 for inhibition of p38-MAPK stimulation of MAPKAPK2 was approximately 0.07 microM, whereas that for total SAPK/JNK activity was 3-10 microM. SB203580 did not inhibit immunoprecipitated JNK1 isoforms. Three peaks of SAPK/JNK activity were separated by anion exchange chromatography, eluting in the isocratic wash (44 kDa), and at 0.08 M (46 and 52 kDa) and 0.15 M NaCl (54 kDa). SB203580 (10 microM) completely inhibited the 0.15 M NaCl activity and partially inhibited the 0.08 M NaCl activity. Since JNK1 antibodies immunoprecipitate the 46 kDa activity, this indicates that SB203580 selectively inhibits 52 and 54 kDa SAPKs/JNKs.

Activation of c-Jun N-terminal kinases and p38-mitogen-activated protein kinases in human heart failure secondary to ischaemic heart disease.

Three well-characterized mitogen-activated protein kinase (MAPK) subfamilies are expressed in rodent and rabbit hearts, and are activated by pathophysiological stimuli. We have determined and compared the expression and activation of these MAPKs in donor and failing human hearts. The amount and activation of MAPKs was assessed in samples from the left ventricles of 4 unused donor hearts and 12 explanted hearts from patients with heart failure secondary to ischaemic heart disease. Total MAPKs or dually phosphorylated (activated) MAPKs were detected by Western blotting and MAPK activities were measured by in gel kinase assays. As in rat heart, c-Jun N-terminal kinases (JNKs) were detected in human hearts as bands corresponding to 46 and 54 kDa; p38-MAPK(s) was detected as a band corresponding to approximately 40 kDa, and extracellularly regulated kinases, ERK1 and ERK2, were detected as 44- and 42-kDa bands respectively. The total amounts of 54 kDa JNK, p38-MAPK and ERK2 were similar in all samples, although 46-kDa JNK was reduced in the failing hearts. However, the mean activities of JNKs and p38-MAPK(s) were significantly higher in failing heart samples than in those from donor hearts (P<0.05). There was no significant difference in phosphorylated (activated) ERKs between the two groups. In conclusion, JNKs, p38-MAPK(s) and ERKs are expressed in the human heart and the activities of JNKs and p38-MAPK(s) were increased in heart failure secondary to ischaemic heart disease. These data indicate that JNKs and p38-MAPKs may be important in human cardiac pathology.

Up-regulation of c-jun mRNA in cardiac myocytes requires the extracellular signal-regulated kinase cascade, but c-Jun N-terminal kinases are required for efficient up-regulation of c-Jun protein.

Cardiac hypertrophy, an important adaptational response, is associated with up-regulation of the immediate early gene, c- jun, which encodes the c-Jun transcription factor. c-Jun may feed back to up-regulate its own transcription and, since the c-Jun N-terminal kinase (JNK) family of mitogen-activated protein kinases (MAPKs) phosphorylate c-Jun(Ser-63/73) to increase its transactivating activity, JNKs are thought to be the principal factors involved in c- jun up-regulation. Hypertrophy in primary cultures of cardiac myocytes is induced by endothelin-1, phenylephrine or PMA, probably through activation of one or more of the MAPK family. These three agonists increased c- jun mRNA with the rank order of potency of PMA approximately endothelin-1>phenylephrine. Up-regulation of c- jun mRNA by endothelin-1 was attenuated by inhibitors of protein kinase C (GF109203X) and the extracellular signal-regulated kinase (ERK) cascade (PD98059 or U0126), but not by inhibitors of the JNK (SP600125) or p38-MAPK (SB203580) cascades. Hyperosmotic shock (0.5 M sorbitol) powerfully activates JNKs, but did not increase c- jun mRNA. These data suggest that ERKs, rather than JNKs, are required for c- jun up-regulation. However, endothelin-1 and phenylephrine induced greater up-regulation of c-Jun protein than PMA and phosphorylation of c-Jun(Ser-63/73) correlated with the level of c-Jun protein. Up-regulation of c-Jun protein by endothelin-1 was attenuated by inhibitors of protein kinase C and the ERK cascade, probably correlating with a primary input of ERKs into transcription. In addition, SP600125 inhibited the phosphorylation of c-Jun(Ser-63/73), attenuated the increase in c-Jun protein induced by endothelin-1 and increased the rate of c-Jun degradation. Thus whereas ERKs are the principal MAPKs required for c- jun transcription, JNKs are necessary to stabilize c-Jun for efficient up-regulation of the protein.