RGS2, a GTPase-activating protein (GAP) for Gqα, regulates vascular relaxation and blood pressure. RGS2 can be phosphorylated by type Iα cGMP-dependent protein kinase (cGKIα), increasing its GAP ...activity. To understand how RGS2 and cGKIα regulate vascular smooth muscle signaling and function, we identified signaling pathways that are controlled by cGMP in an RGS2-dependent manner and discovered new mechanisms whereby cGK activity regulates RGS2. We show that RGS2 regulates vasoconstrictor-stimulated Ca2+ store release, capacitative Ca2+ entry, and noncapacitative Ca2+ entry and that RGS2 is required for cGMP-mediated inhibition of vasoconstrictor-elicited phospholipase Cβ activation, Ca2+ store release, and capacitative Ca2+ entry. RGS2 is degraded in vascular smooth muscle cells via the proteasome. Inhibition of cGK activity blunts RGS2 degradation. However, inactivation of the cGKIα phosphorylation sites in RGS2 does not stabilize the protein, suggesting that cGK activity regulates RGS2 degradation by other mechanisms. cGK activation promotes association of RGS2 with the plasma membrane by a mechanism requiring its cGKIα phosphorylation sites. By regulating GAP activity, plasma membrane association, and degradation, cGKIα therefore may control a cycle of RGS2 activation and inactivation. By diminishing cGK activity, endothelial dysfunction may impair RGS2 activation, thereby blunting vascular relaxation and contributing to hypertension.
The safety and pharmacokinetics ofcolistin were determined after first dose (n = 30) and again under steady‐state conditions (n = 27) in 31 patients with cystic fibrosis receiving the drug as a ...component of their treatment for an acute pulmonary exacerbation of their disease. Patients ranged in age from 14 to 53 years and received colistin for 6 to 35 days. Each patient was started on colistin 5 to 7 mg/kg/day administered intravenously in three equally divided doses. Elimination half‐life (t1/2), mean residence time (MRT), steady‐state volume of distribution (Vdss), total body clearance (Cl), and renal clearance (Clr) after first‐dose administration averaged 3.4 hours, 4.4 hours, 0.09 l/kg, and 0.35 and 0.24 ml/min/kg, respectively. No differences in colistin disposition characteristics between first‐dose and steady‐state evaluations were observed. Sputum sampling was incomplete and confounded by previous aerosol administration but revealed colistin concentrations that markedly exceeded observed plasma concentrations. Twenty‐one patients experienced one or more side effects attributed to colistin administration. The most common reactions involved reversible neurologic manifestations, including oral and perioral paresthesias (n = 16), headache (n = 5), and lower limb weakness (n = 5). All of these apparent colistin‐induced neurologic adverse effects, though bothersome, were benign and reversible. Intermittent proteinuria was observed on urinalysis in 14 patients, and 1 patient developed reversible, colistin‐induced nephrotoxicity. No relationship between the occurrence of any colistin‐associated adverse effect and plasma colistin concentration or colistin pharmacokinetic parameter estimate was observed. These data provide no basis for routine monitoring of colistin plasma concentrations to guide dosing for patient safety and suggest slow upward dose titration to minimize the incidence and severity of associated side effects.
A new design of a cryogenic germanium detector for dark matter search is presented, taking advantage of the coplanar grid technique of event localisation for improved background discrimination. ...Experiments performed with prototype devices in the EDELWEISS II setup at the Modane underground facility demonstrate the remarkably high efficiency of these devices for the rejection of low-energy β, approaching 105. This opens the road to investigate the range beyond 10−8 pb in the WIMP–nucleon collision cross-sections, as proposed in the EURECA project of a one-ton cryogenic detector mass.
During the cell cycle of the yeast Saccharomyces cerevisiae, the actin cytoskeleton and cell surface growth are polarized, mediating bud emergence, bud growth, and cytokinesis. We have determined ...whether p21-activated kinase (PAK)-family kinases regulate cell and actin polarization at one or several points during the yeast cell cycle. Inactivation of the PAK homologues Ste20 and Cla4 at various points in the cell cycle resulted in loss of cell and actin cytoskeletal polarity, but not in depolymerization of F-actin. Loss of PAK function in G1 depolarized the cortical actin cytoskeleton and blocked bud emergence, but allowed isotropic growth and led to defects in septin assembly, indicating that PAKs are effectors of the Rho-guanosine triphosphatase Cdc42. PAK inactivation in S/G2 resulted in depolarized growth of the mother and bud and a loss of actin polarity. Loss of PAK function in mitosis caused a defect in cytokinesis and a failure to polarize the cortical actin cytoskeleton to the mother-bud neck. Cla4-green fluorescent protein localized to sites where the cortical actin cytoskeleton and cell surface growth are polarized, independently of an intact actin cytoskeleton. Thus, PAK family kinases are primary regulators of cell and actin cytoskeletal polarity throughout most or all of the yeast cell cycle. PAK-family kinases in higher organisms may have similar functions.
A general property of signal transduction pathways is that prolonged stimulation decreases responsiveness, a phenomenon termed desensitization. Yeast cells stimulated with mating pheromone activate a ...heterotrimeric G-protein-linked, MAP-kinase-dependent signalling pathway that induces G1-phase cell-cycle arrest and morphological differentiation (reviewed in refs 1, 2). Eventually the cells desensitize to pheromone and resume growth. Genetic studies have demonstrated the relative importance of a desensitization mechanism that uses the SST2 gene product, Sst2p. Here we identify a mammalian gene family termed RGS (for regulator of G-protein signalling) that encodes structural and functional homologues of Sst2p. Introduction of RGS family members into yeast blunts signal transduction through the pheromone-response pathway. Like SST2 (refs 8-10), they negatively regulate this pathway at a point upstream or at the level of the G protein. The RGS family members also markedly impair MAP kinase activation by mammalian G-protein-linked receptors, indicating the existence and importance of an SST2-like desensitization mechanism in mammalian cells.
BACKGROUNDWe herein investigate critical ischemia times and the effect of novel preservation solutions such as new histidine-tryptophan-ketoglutarate (HTK-N) and TiProtec on the individual tissues of ...a rat limb isograft.
METHODSOrthotopic hind-limb transplantations were performed in male Lewis rats after 2 hours, 6 hours, or 10 hours of cold ischemia (CI). Limbs were flushed and stored in HTK-N, TiProtec, HTK, or saline solution. Muscle, nerve, vessel, skin, and bone samples were procured on day 10 for histology, immunohistochemistry, confocal and electron microscopy, and quantitative real-time polymerase chain reaction analysis.
RESULTSHistomorphology of the muscle showed a mainly perivascular inflammatory infiltrate, fibrotic degeneration, and neovascularization after 6 hours and 10 hours of CI. However, centrally aligned nuclei observed in muscle fibers suggest for muscle regeneration in these samples. In addition to Wallerian degeneration, nerve injury was significantly aggravated (P = 0.032) after prolonged CI. Proinflammatory and regulatory cytokines were most significantly upregulated after 2-hour CI. Our data suggest no superiority of novel perfusates HTK-N and TiProtec in terms of tissue preservation, compared with HTK and saline.
CONCLUSIONSLimiting CI time for less than 6 hours is the most significant factor to reduce tissue damage in vascularized tissue transplantation. Signs of muscle regeneration give rise that ischemic muscle damage in limb transplantation might be reversible to a certain extent.
RGS family members are GTPase-activating proteins (GAPs) for heterotrimeric G proteins. There is evidence that altered RGS gene expression may contribute to the pathogenesis of cardiac hypertrophy ...and failure. We investigated the ability of RGS4 to modulate cardiac physiology using a transgenic mouse model. Overexpression of RGS4 in postnatal ventricular tissue did not affect cardiac morphology or basal cardiac function, but markedly compromised the ability of the heart to adapt to transverse aortic constriction (TAC). In contrast to wild-type mice, the transgenic animals developed significantly reduced ventricular hypertrophy in response to pressure overload and also did not exhibit induction of the cardiac "fetal" gene program. TAC of the transgenic mice caused a rapid decompensation in most animals characterized by left ventricular dilatation, depressed systolic function, and increased postoperative mortality when compared with nontransgenic littermates. These results implicate RGS proteins as a crucial component of the signaling pathway involved in both the cardiac response to acute ventricular pressure overload and the cardiac hypertrophic program.
Regulators of G protein signaling (RGS) proteins of the B/R4 family are widely expressed in the cardiovascular system where their role in fine-tuning G protein signaling is critical to maintaining ...homeostasis. Among members of this family, RGS2 and RGS5 have been shown to play key roles in cardiac and smooth muscle function by tightly regulating signaling pathways that are activated through Gq/11 and Gi/o classes of heterotrimeric G proteins. This chapter reviews accumulating evidence supporting a key role for RGS2 in vascular function and the implication of changes in RGS2 function and/or expression in the pathogenesis of blood pressure disorders, particularly hypertension. With such understanding, RGS2 and the signaling pathways it controls may emerge as novel targets for developing next-generation antihypertensive drugs/agents.
Uveal melanoma (UM) is the most common intraocular tumor in adults. Nearly half of UM patients develop metastatic disease and often succumb within months because effective therapy is lacking. A novel ...therapeutic approach has been suggested by the discovery that UM cell lines driven by mutant constitutively active Gq or G11 can be targeted by FR900359 (FR) or YM-254890, which are bioavailable, selective inhibitors of the Gq/11/14 subfamily of heterotrimeric G proteins. Here, we have addressed the therapeutic potential of FR for UM. We found that FR inhibited all oncogenic Gq/11 mutants reported in UM. FR arrested growth of all Gq/11-driven UM cell lines tested, but induced apoptosis only in a few. Similarly, FR inhibited growth of, but did not efficiently kill, UM tumor cells from biopsies of primary or metastatic tumors. FR evoked melanocytic redifferentiation of UM tumor cells with low (class 1), but not high (class 2), metastatic potential. FR administered systemically below its LD50 strongly inhibited growth of PDX-derived class 1 and class 2 UM tumors in mouse xenograft models and reduced blood pressure transiently. FR did not regress xenografted UM tumors or significantly affect heart rate, liver function, hematopoiesis, or behavior. These results indicated the existence of a therapeutic window in which FR can be explored for treating UM and potentially other diseases caused by constitutively active Gq/11.
RGS proteins negatively regulate heterotrimeric G proteins at the plasma membrane. RGS2-GFP localizes to the nucleus, plasma
membrane, and cytoplasm of HEK293 cells. Expression of activated G q ...increased RGS2 association with the plasma membrane and decreased accumulation in the nucleus, suggesting that signal-induced
redistribution may regulate RGS2 function. Thus, we identified and characterized a conserved N-terminal domain in RGS2 that
is necessary and sufficient for plasma membrane localization. Mutational and biophysical analyses indicated that this domain
is an amphipathic α-helix that binds vesicles containing acidic phospholipids. However, the plasma membrane targeting function
of the amphipathic helical domain did not appear to be essential for RGS2 to attenuate signaling by activated G q . Nevertheless, truncation mutants indicated that the N terminus is essential, potentially serving as a scaffold that binds
receptors, signaling proteins, or nuclear components. Indeed, the RGS2 N terminus directs nuclear accumulation of GFP. Although
RGS2 possesses a nuclear targeting motif, it lacks a nuclear import signal and enters the nucleus by passive diffusion. Nuclear
accumulation of RGS2 does not limit its ability to attenuate G q signaling, because excluding RGS2 from the nucleus was without effect. RGS2 may nonetheless regulate signaling or other processes
in the nucleus.
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