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S-Nitrosothiols modulate G protein-coupled receptor signaling in a reversible and highly receptor-specific manner
Recent studies indicate that the G protein-coupled receptor (GPCR) signaling machinery can serve as a direct target of reactive oxygen species, including nitric oxide (NO) and S-nitrosothiols (RSNOs). To gain a broader view into the way that receptor-dependent G protein activation – an early step in signal transduction – might be affected by RSNOs, we have studied several receptors coupling to the Gi family of G proteins in their native cellular environment using the powerful functional approach of [35S]GTPγS autoradiography with brain cryostat sections in combination with classical G protein activation assays.
We demonstrate that RSNOs, like S-nitrosoglutathione (GSNO) and S-nitrosocysteine (CysNO), can modulate GPCR signaling via reversible, thiol-sensitive mechanisms probably involving S-nitrosylation. RSNOs are capable of very targeted regulation, as they potentiate the signaling of some receptors (exemplified by the M2/M4 muscarinic cholinergic receptors), inhibit others (P2Y12 purinergic, LPA1lysophosphatidic acid, and cannabinoid CB1 receptors), but may only marginally affect signaling of others, such as adenosine A1, μ-opioid, and opiate related receptors. Amplification of M2/M4 muscarinic responses is explained by an accelerated rate of guanine nucleotide exchange, as well as an increased number of high-affinity [35S]GTPγS binding sites available for the agonist-activated receptor. GSNO amplified human M4 receptor signaling also under heterologous expression in CHO cells, but the effect diminished with increasing constitutive receptor activity. RSNOs markedly inhibited P2Y12 receptor signaling in native tissues (rat brain and human platelets), but failed to affect human P2Y12 receptor signaling under heterologous expression in CHO cells, indicating that the native cellular signaling partners, rather than the P2Y12 receptor protein, act as a molecular target for this action.
These in vitro studies show for the first time in a broader general context that RSNOs are capable of modulating GPCR signaling in a reversible and highly receptor-specific manner. Given that the enzymatic machinery responsible for endogenous NO production is located in close proximity with the GPCR signaling complex, especially with that for several receptors whose signaling is shown here to be modulated by exogenous RSNOs, our data suggest that GPCR signaling in vivo is likely to be subject to substantial, and highly receptor-specific modulation by NO-derived RSNOs.
G protein-coupled receptors (GPCRs) represent the largest group of integral membrane proteins involved in signal transduction and are the most important targets of clinically marketed drugs
Nitric oxide (NO) is a unique gaseous messenger generated in vivo by three isoforms of NO synthases (NOS). The established mode of NO signaling is through the activation of the hemoprotein, soluble guanylyl cyclase, resulting in increased production of the second messenger cGMP. However, accumulating evidence points towards cGMP-independent mechanisms by which NO can react with proteins, forming covalent post-translational modifications
To begin to address these issues, we have studied how exogenous RSNOs affect receptor-mediated G protein activity – a very proximal step of GPCR signal transduction – by studying the signaling of several receptors that couple to the Gi family of heterotrimeric G proteins. This family consists of both pertussis toxin sensitive (Gαi1-3, Gαo, transducin, gustducin) and insensitive (Gαz) members. We applied the powerful functional approach of [35S]GTPγS autoradiography in brain cryostat sections, as this technique allows selective detection of receptor-dependent G protein activity simultaneously in multiple brain regions with minimal disturbance of the GPCR microenvironment
Exogenous RSNOs modulate GPCR signaling via mechanisms likely involving S-nitrosylation
We used the functional approach of [35S]GTPγS autoradiography, as this technique allows selective detection of receptor-stimulated Gi protein activity simultaneously in multiple brain structures with minimal disturbance of the GPCR microenvironment
Figure 1
S-nitrosoglutathione (GSNO) reversibly modulates basal and receptor-dependent G protein activity in rat brain cryostat sections
S-nitrosoglutathione (GSNO) reversibly modulates basal and receptor-dependent G protein activity in rat brain cryostat sections. [35S]GTPγS autoradiography of sagittal brain sections was conducted using a 3-step protocol with DPCPX (10-6 M) present throughout steps 2 and 3, as detailed in the
Figure 2
GSNO modulates GPCR signaling in a dose-dependent manner
GSNO modulates GPCR signaling in a dose-dependent manner. [35S]GTPγS autoradiography was conducted using a 3-step protocol with DPCPX (10-6 M) present throughout steps 2 and 3, as detailed in the
Additional File 1
Supplementary material (Supplementary Figures 1, 2, 3, 4, 5, 6, 7 and Supplementary Tables 1, 2) is provided as a single file. This pdf-file (size 0.75 MB) is readable using Adobe Acrobat.
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GSNO is present in significant amounts (~15 pmol/mg protein) in the brain tissue and it is thought to act as a physiological carrier of NO for S-nitrosylation reactions
Various RSNOs, including S-nitrosocysteine (CysNO) (Figure
Figure 3
S-nitrosocysteine (CysNO) mimics the effects of GSNO in modulating GPCR signaling, whereas sodium nitroprusside (SNP) and 8-bromo cyclic GMP (8Br-cGMP) do not
S-nitrosocysteine (CysNO) mimics the effects of GSNO in modulating GPCR signaling, whereas sodium nitroprusside (SNP) and 8-bromo cyclic GMP (8Br-cGMP) do not. [35S]GTPγS autoradiography was conducted using a 3-step protocol with DPCPX (10-6 M) present throughout steps 2 and 3, as detailed in the
Table 1
Effects of GSNO treatment on agonist dose-response parameters in [35S]GTPγS binding assays of various Gi-coupled receptors in their native cellular environment. Membranes or lysates were preincubated in control conditions or in the presence of 0.5 mM GSNO for 30 min. Values are mean ± SE from three to four independent experiments performed in duplicate. Emax is expressed in percentage over basal with nonspecific binding subtracted.
Receptor (agonist)
Emax (%)
Emax (%)
M2/M4 mAChRs (CCh)
-4.94 ± 0.05
176 ± 2
-5.55 ± 0.11**
207 ± 4**
-6.84 ± 0.26
140 ± 7
-6.53 ± 0.40
116 ± 3*
Cannabinoid CB1 (CP55940)
-7.84 ± 0.07
223 ± 3
-7.69 ± 0.08
183 ± 2***
Adenosine A1 (2ClAdo)
-6.90 ± 0.04
256 ± 3
-6.87 ± 0.12
234 ± 7*
μ-opiate (DAMGO)
-6.85 ± 0.09
167 ± 3
-7.31 ± 0.19
164 ± 5
ORL1 (Nociceptin)
-8.66 ± 0.12
173 ± 3
-8.78 ± 0.10
161 ± 2*
-7.37 ± 0.11
202 ± 4
-7.05 ± 0.12
176 ± 4**
P2Y12 (MeSADP)
-8.19 ± 0.03
279 ± 2
-8.30 ± 0.08
195 ± 3***
α2A-adrenoceptor (NA)
-5.33 ± 0.08
161 ± 2
-5.49 ± 0.09
182 ± 3**
* Statistically different from control (P < 0.05)
** Statistically different from control (P < 0.01)
*** Statistically different from control (P < 0.001)
Figure 4
GSNO only marginally affect opiate related receptor (ORL1), μ opioid receptor (MOR) and adenosine A1 (Ado A1) receptors signaling in brain sections
GSNO only marginally affect opiate related receptor (ORL1), μ opioid receptor (MOR) and adenosine A1 (Ado A1) receptors signaling in brain sections. [35S]GTPγS autoradiography was conducted using a 3-step protocol with adenosine deaminase (ADA, 1 U/ml) present throughout steps 2 and 3, as detailed in the
Figure 5
The P2Y12 receptor is not a direct target of RSNO action
The P2Y12 receptor is not a direct target of RSNO action. Human P2Y12 receptor was stably expressed in CHO cells and agonist-stimulated G protein activity was determined in control conditions and in membranes pretreated for 30 min with 0.5 mM GSNO or CysNO, as detailed in the
Previous studies have suggested that RSNOs can act as NO+, NO., and NO- donors under physiological conditions
The above results demonstrate that the effects of NO-related species was shared by -and restricted to – different classes of RSNO compounds, suggesting that S-nitrosylation rather than other types of NO reactions, or cGMP-dependent mechanisms, were involved. According to the S-nitrosylation scheme, treatment with exogenous RSNOs should result in transnitrosylation of potential protein thiols (R-SNO + Protein-SH ↔ R-SH + Protein-SNO). To demonstrate the presence of SNO moieties in GSNO-treated brain section, we used the indirect approach where heterolytic cleavage of S-NO bond with HgCl2 generates nitrite which can be measured by a colorimetric method. To this end, brain sections were treated with GSNO (0.5 mM), and after thorough washes, the sections were incubated further in the absence or presence of HgCl2 (10-4 M). These experiments (shown in Supplementary Figure 6 [see
Figure 6
GSNO accelerates the rate of M2/M4 AChR-stimulated [35S]GTPγS binding to rat forebrain membranes but has no effect on basal guanine nucleotide exchange rate
GSNO accelerates the rate of M2/M4 AChR-stimulated [35S]GTPγS binding to rat forebrain membranes but has no effect on basal guanine nucleotide exchange rate. Membranes were preincubated for 30 min in control conditions or in the presence of 0.5 mM GSNO, and time-response for basal and CCh-stimulated (10-4 M] [35S]GTPγS binding was determined, as detailed in the
RSNOs modulate GPCR signaling in native tissues in a highly receptor-specific manner
Although [35S]GTPγS autoradiography offers the advantage of monitoring G protein activity simultaneously in multiple brain regions with minimal disturbance of the GPCR microenvironment, generating quantitative data from the autoradiography images is relatively tedious. As a complementary approach, we tested the effect of RSNOs on agonist potency and efficacy for several additional Gi-coupled receptors using classical membrane and lysate [35S]GTPγS binding assays. The results of these experiments are summarized in Table
Figure 7
GSNO increases the number of M2/M4 AChR interacting high-affinity [35S]GTPγS binding sites in rat forebrain membranes
GSNO increases the number of M2/M4 AChR interacting high-affinity [35S]GTPγS binding sites in rat forebrain membranes. Membranes were preincubated for 30 min in control conditions or in the presence of 0.5 mM GSNO, and incubated thereafter for 90 min with 0.15 nM [35S]GTPγS, 10-5 M GDP and indicated concentrations of unlabeled GTPγS in the presence and absence of CCh (10-4 M), as detailed in the
Table 2
Effects of GSNO treatment on agonist (CCh) dose-response parameters in [35S]GTPγS binding assays of hM4 cell line membranes. Membranes were preincubated in control conditions or in the presence of 0.5 mM GSNO for 30 min. Values are mean ± SE from three to four independent experiments performed in duplicate. Emax is expressed in percentage over basal with nonspecific binding subtracted.
CHO cell line
Emax (%)
Emax (%)
-4.87 ± 0.14
586 ± 31
-4.94 ± 0.09
674 ± 23
-4.87 ± 0.05
213 ± 3
-4.98 ± 0.13
276 ± 10**
-5.21 ± 0.20
134 ± 3
-5.27 ± 0.20
154 ± 8*
-5.13 ± 0.10
198 ± 4
-5.12 ± 0.11
235 ± 6**
* Statistically different from control (P < 0.05)
** Statistically different from control (P < 0.01)
CHO cells express endogenous Gi-coupled LPA receptors
The inhibitory effect of GSNO on basal and P2Y12 receptor-dependent G protein activity was not due to a nonspecific action on platelet membranes, since the signaling of another Gi-linked platelet receptor, the α2A-adrenoceptor, was significantly amplified in GSNO-treated membranes (Table
RSNOs amplify muscarinic responses by increasing the rate of GDP/GTP exchange and the number of high-affinity GTP binding sites
The amplification of M2/M4 responses by RSNOs was clearly evident in native brain tissue. Further experiments were designed to address the mechanism of this action. Results of these studies are presented in Figures
RSNO amplification of M4 receptor responses are preserved under heterologous expression but the amplification is lost with constitutive receptor activity
To investigate the signaling of the human M4 receptor (hM4) under heterologous expression system, the receptor was stably transfected into CHO cells. The effects of GSNO on agonist-stimulated G protein activity were compared in rat forebrain membranes and three cell lines expressing the wild-type (WT) hM4 receptor with increasing capability to activate G proteins. The results of these experiments are shown in Figure
Figure 8
GSNO-evoked potentiation of muscarinic signaling is preserved under heterologus expression but the effect diminishes with increasing constitutive activity
GSNO-evoked potentiation of muscarinic signaling is preserved under heterologus expression but the effect diminishes with increasing constitutive activity. The human M4 (hM4) receptor was stably transfected into CHO cells and wild type (WT) or mutant (C133S) cell lines with differential G protein activation capacity were compared with that in rat forebrain membranes. CCh-stimulated G protein activity was determined in control conditions and in membranes pretreated for 30 min with 0.5 mM GSNO, as detailed in the
The final experiments were intended to clarify whether cysteine 133 (C133), located adjacent to the G protein-interacting DRY-motif in the intracellular end of transmembrane helix 3 of the Gi-coupled muscarinic receptors (M2/M4), could serve as the molecular target of RSNO action. To this end, C133 was mutated into serine to reveal if the C133S mutation would abolish the effect of GSNO on the efficacy of CCh. However, GSNO treatment significantly increased Emax also in the mutant C133S-hM4 cell line (Figure
[35S]GTPγS autoradiography of brain cryostat sections revealed a highly receptor-specific modulation of GPCR signaling by RSNOs in several receptor-enriched anatomical structures. This modulation was fully reversible upon addition of excess thiols. We have provided evidence indicating that S-nitrosylation, rather than other types of NO reactions or the NO – guanylyl cyclase – cGMP signaling pathway, was responsible for the observed effects. The RSNOs effects were receptor-specific, as signaling of some receptors was markedly potentiated (M2/M4 AChRs and α2-adrenoceptors), whereas that of others was clearly inhibited (P2Y12, LPA and cannabinoid CB1 receptors), while signaling of other receptors was only marginally affected (adenosine A1, MOR, and ORL1 receptors) by comparable treatments. We further demonstrated that RSNOs can amplify M2/M4 receptor responses by increasing the rate of GDP/GTP exchange as well as the number of high-affinity G protein α subunits capable of interacting with the agonist-activated receptors. The potentiating effect of RSNOs on hM4 receptor responses was preserved when this was studied in a heterologous expression system but was diminished in constitutively active hM4 receptors. We also demonstrated that the GPCR itself or its native signaling partners serve as potential targets of this modulation, as it was attenuated, or even lost, when receptor signaling was studied under heterologous expression. Our study suggests that GPCR signaling is subject to a highly receptor-specific modulation by NO-derived RSNOs.
The GPCRs and their proximal signaling partners as likely targets of RSNO action
Since [35S]GTPγS binding assays monitor G protein activation, one of the earliest measurable steps in GPCR signal transduction, it is obvious that the molecular targets of RSNO action are the receptors, their cognate G proteins and/or additional signaling partners, whose thiol modification can directly regulate guanine nucleotide binding and hence G protein activation.
It is interesting that RSNO treatment of brain sections consistently resulted in thiol-sensitive increases in basal Gi protein activity throughout the gray matter regions. However, no such effect was present in brain membrane [35S]GTPγS binding assays, nor was it detected in CHO cell membranes but in platelet membranes, RSNOs inhibited basal G protein activity by ~20%. As receptor input should be minimal in basal conditions, the differential behavior of RSNOs in cryostat sections and various membrane preparations likely reflects direct action on the Gi proteins and/or their proximal regulatory partners. It has been known for some time that G proteins can serve as direct targets of ROS, including NO
In platelet membranes, RSNOs inhibited both basal and P2Y12 receptor-dependent G protein activity, but clearly potentiated α2A-adrenoceptor responses. Platelet P2Y12 receptors couple to Gαi2
Although the basic module of GPCR signaling is traditionally considered to be the receptor, its cognate G protein, and the effector, recent studies have identified a wide range of proteins that can directly interact with the receptor and/or G proteins. These can modulate signaling efficiency, cellular localization, or the regulation of the GPCRs or G proteins
Most Gα proteins are palmitoylated at a cysteine near the amino terminus and this modification is required for G protein targeting to lipid rafts
Functional implications
Specialized plasma membrane microdomains act as unique platforms with specific enrichment of GPCRs, their signaling partners, and the enzymatic machinery for NO biosynthesis
In the heart, endothelial NOS (eNOS) is localized n caveolin-enriched myocyte membrane fractions and it has been shown that lipid draft-disrupting agents severely compromise NO-dependent inhibition of adenylyl cyclase types 5 and 6
The P2Y12 receptor plays a central role in platelet activation and aggregation
One of the novel findings in this study was that RSNOs strongly inhibited Gi-mediated LPA receptor signaling in the brain and in CHO cells. The relevance of this finding with respect to brain LPA1 receptor signaling remains to be established. In vivo, peripheral LPA receptor signaling is closely associated with NO. In bovine aortic endothelial cells, LPA stimulates endothelial NOS via Gi-coupled LPA receptors
In conclusion, this study revealed that G protein activation, an early step of GPCR signal transduction, is subject to a reversible and highly receptor-specific modulation by exogenous RSNOs at physiologically relevant concentrations. Since NOS synthases (and thus NO production) have been shown to reside in close proximity with the GPCR signaling machinery, especially for many of the receptors whose signaling is subject to modulation by exogenous RSNOs, these findings suggest that GPCR signaling in vivo is likely to be finely tuned by NO-derived RSNO species. Future studies should aim at pinpointing the precise molecular targets of these actions, and at understanding the specific modifications (S-nitrosylation and/or S-thiolation) involved, as well as revealing the physiological and/or pathophysiological relevance in vivo.
All drugs and chemicals were from Sigma (St. Louis, MO) or Merck (Darmstadt, Germany), unless otherwise stated. Cell culture media, sera, and antibiotics were from Euroclone (Pero, Italy). Protein concentrations were determined with Bio-Rad protein assay (Bio-Rad, Hercules, CA, USA). Adenosine deaminase (ADA) was purchased from Roche (Mannheim, Germany) and guanosine-5'-O-(3-[35S]-thio)-triphosphate ([35S]GTPγS; initial specific activity 1250 Ci/mmol) from NEN (Boston, MA). CP55940, DAMGO, nociceptin, and SNAP were purchased from Tocris Cookson Ltd. (Bristol, UK).
DNA constructs
Human M4 muscarinic receptor (hM4, gift from Dr. Johnny Näsman, University of Kuopio) was subcloned from pBluescript into pcDNA3 mammalian expression vector and a triple hemagglutinin (HA) epitope tag was subcloned after the initiating Met codon of the hM4 gene. This construct was used to create a C133S mutant hM4 receptor with QuickChange Site Directed Mutagenesis Kit (Stratagene, La Jolla, CA). Human P2Y12 purinergic receptor (hP2Y12) was amplified from QuickClone human brain cDNA (Stratagene) using RT-PCR with gene-specific primers. The PCR product was ligated into pcDNA3 and a N-terminal hemagglutinin (HA) epitope tag was inserted in a PCR reaction with 5' primer containing the HA tag DNA sequence. All receptor constructs were confirmed by restriction analyses and DNA sequencing prior to transfections.
Cell culture and transfection
Recombinant plasmids were introduced into Chinese hamster ovary (CHO) cells with Lipofectamine 2000 transfection reagent (Gibco, Paisley, UK). Transfected cells were placed under G-418 selection (600 μg/ml) and several cell lines originating from single G-418 resistant cells were isolated. The G-418 resistant cell lines were cultured as monolayers with 100 μg/ml G-418 in Ham's F-12 nutrient mixture, containing 10% fetal calf serum, 100 U/ml penicillin and 100 μg/ml streptomycin at 37°C in a humidified atmosphere of 5% CO2 / 95% air. Stable cell lines were analyzed for HA tag (and thus receptor) expression using receptor ELISA with mouse anti-HA primary antibody
Preparation of cryostat sections, membranes, and cell lysates
Naïve, four-week-old male Wistar rats were used for the preparation of brain cryostat sections essentially as described earlier
Preparation of S-nitrosothiols
S-nitroso-N-acetyl-D,L-penicillamine (SNAP) was purchased from Tocris Cookson Ltd. (Bristol, UK). All other RSNOs were synthesized from the respective thiols using acidified NaNO2. For example, S-nitrosoglutathione (GSNO) was prepared by mixing 100 μl sodium nitrite (100 mM) with 100 μl HCl (150 mM) and adding 100 μl reduced glutathione (100 mM). Reactions were allowed to proceed for 10 min at room temperature, protected from light. Reaction mixtures were neutralized with 150 μl NaOH (100 mM) and used immediately in the experiments. Millipore-quality water was used throughout and the assay buffer routinely contained 1 mM EDTA. The concentrations of RSNOs were determined by UV spectroscopy using previously published
[35S]GTPγS autoradiography
The assay was conducted under optimized conditions, where basal noise due to tonic adenosine A1 receptor activity has been eliminated
[35S]GTPγS membrane binding assays
The incubations were carried with slight modifications to previously published protocols
Data analysis
[35S]GTPγS-membrane binding data were analyzed with GraphPad Prism software (GraphPad, San Diego, CA) using non-linear fitting for sigmoid dose-response curves. Statistical analyses were made with one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test. When comparison was made between only two groups, unpaired T-test was used.
List of abbreviations
2MeSADP, 2-methylthio-ADP; [35S]GTPγS, guanosine-5'-O-(3-[35S]-thio)-triphosphate; 5-HT, 5-hydroxytryptamine; ADA, adenosine deaminase; AGS, activator of G protein signaling; CCh, carbacholine; CHO, Chinese hamster ovary; CP-55940, (-)-3-[2-hydroxy-4-(1,1-dimethylheptyl)-phenyl]-4-[3-hydroxypropyl]cyclohexan-1-ol; CysNO, S-nitrosocysteine; CysNO-Gly, S-nitroso-cysteinyl-glycine; DAMGO, [D-Ala2, N-Me-Phe4, Gly5-ol]-enkephalin; DPCPX, 8-cyclopentyl-1,3-dipropylxanthine; DTT, dithiotreitol; Glu-CysNO, L-γ-glutamyl-S-nitrosocysteine; GSH, glutathione; GPCR(s), G protein-coupled receptor(s); GSNO, S-nitrosoglutathione; HA, hemagglutinin; hM4, human muscarinic receptor subtype 4; hP2Y12, human P2Y12 purinergic receptor; LPA, lysophosphatidic acid; NA, noradrenaline; NO, nitric oxide; NOBF4, nitrosodium tetrafluoroborate; NOS, NO synthase; RGS, regulator of G protein signaling; RT, reverse transcriptase; SNAP, S-nitroso-N-acetyl-D,L-penicillamine; RSNO, S-nitrosothiol; SNP, sodium nitroprusside
Authors' contributions
TK carried out the cell culture, molecular biology and mutagenesis studies, participated in the membrane and lysate [35S]GTPγS binding assays, participated in the design of the study and drafted the manuscript. JRS and MDR carried out most of the membrane [35S]GTPγS binding assays and performed the statistical analyses for these. KSM carried out G protein activation assays with human platelets. JTL conceived of the study, its design and coordination and conducted [35S]GTPγS autoradiography experiments. All authors read and approved the final manuscript.
MDR is a medical student from Madrid, Spain and participated in this study as an IFMSA (International Federation of Medical Students' Association) exchange student. We wish to thank Dr. Johny Näsman (University of Kuopio) for the hM4 receptor cDNA, and Prof. Mika Scheinin (University of Turku) for the CHO cell lines expressing α2-adrenoceptor subtypes. Reija Heikkinen (M.Sc.) is acknowledged for her contribution in the generation and functional testing of hM4 cell lines. Dr. Ewen MacDonald is acknowledged for the revision of the language of this paper. We are thankful to Mrs. Taina Vihavainen, Mrs. Tiina Räsänen and Mrs. Taija Vaarala for skillful technical assistance and to Mrs. Merja Saastamoinen for secretarial help.
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The G protein-coupled receptor repertoires of human and mouse
Target validation of G-protein coupled receptors
Seven-transmembrane receptors
Regulators of G-protein signalling as new central nervous system drug targets
Accessory proteins for G protein-signaling systems: activators of G protein signaling and other nonreceptor proteins influencing the activation state of G proteins
Novel effects of nitric oxide
S-Transnitrosation reactions are involved in the metabolic fate and biological actions of nitric oxide
NO+, NO, and NO- donation by S-nitrosothiols: implications for regulation of physiological functions by S-nitrosylation and acceleration of disulfide formation
S-nitrosylation and S-glutathiolation of protein sulfhydryls by S-nitroso glutathione
Modification of creatine kinase by S-nitrosothiols: S-nitrosation vs. S-thiolation
Inhibition of papain by S-nitrosothiols. Formation of mixed disulfides
S-nitrosylation: spectrum and specificity
Nitrosylation. The prototypic redox-based signaling mechanism
cGMP and S-nitrosylation: two routes for modulation of neuronal excitability by NO
The biochemistry and physiology of S-nitrosothiols
Protein S-glutathiolation triggered by decomposed S-nitrosoglutathione
Cysteine regulation of protein function – as exemplified by NMDA-receptor modulation
S-nitrosylation in health and disease
Protein S-nitrosylation: purview and parameters
Mono ADP-ribosylation of transducin catalyzed by rod outer segment extract
Nitric oxide signaling: a possible role for G proteins
Nitric oxide-regulated endogenous ADP-ribosylation of rod outer segment proteins
Modification of cysteine residues within G(o) and other neuronal proteins by exposure to nitric oxide
Endogenous ADP-ribosylation of a G(alpha i) protein in bovine ciliary body is stimulated by nitric oxide
Modulation of bradykinin receptor ligand binding affinity and its coupled G-proteins by nitric oxide
Adenylyl cyclase, a coincidence detector for nitric oxide
Effects of S-nitroso-cysteine on proteins that regulate exocytosis in PC12 cells: inhibitory effects on translocation of synaptophysin and ADP-ribosylation of GTP-binding proteins
G alpha(i) and G alpha(o) are target proteins of reactive oxygen species
Nitric oxide selectively inhibits adenylyl cyclase isoforms 5 and 6
Nitric oxide regulates adenylyl cyclase activity in rat striatal membranes
Reactive oxygen species potentiate the negative inotropic effect of cardiac M2-muscarinic receptor stimulation
Activation mechanism of Gi and Go by reactive oxygen species
Pulmonary vasoconstriction by serotonin is inhibited by S-nitroso-glutathione
Nitric oxide inhibition of adenylyl cyclase type 6 activity is dependent upon lipid rafts and caveolin signaling complexes
[35S]GTPγS Autoradiography: A powerful functional approach with expanding potential for neuropharmacological studies on receptors coupled to Gi family of G proteins
Localization of adenylyl cyclase isoforms and G protein-coupled receptors in vascular smooth muscle cells: expression in caveolin-rich and noncaveolin domains
delta-Opioid receptors exhibit high efficiency when activating trimeric G proteins in membrane domains
Unbiased quantitative proteomics of lipid rafts reveals high specificity for signaling factors
Location, location, location
[35S]GTPγS autoradiography reveals a wide distribution of Gi/o-linked ADP receptors in the nervous system: close similarities with the platelet P2YADP receptor
2-Alkylthio-substituted platelet P2Y12 receptor antagonists reveal pharmacological identity between the rat brain Gi-linked ADP receptors and P2Y12
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Growth factor-like effects of lysophosphatidic acid, a novel lipid mediator
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Dexras1 inhibits adenylyl cyclase
Dexras1/AGS-1 inhibits signal transduction from the Gi-coupled formyl peptide receptor to Erk-1/2 MAP kinases
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Palmitoylation is required for signaling functions and membrane attachment of Gq alpha and Gs alpha
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Nitric oxide modulates beta(2)-adrenergic receptor palmitoylation and signaling
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Sodium nitroprusside induces internalization of muscarinic receptors stably expressed in Chinese hamster ovary cell lines
NO donors potentiate the beta-adrenergic stimulation of I(Ca,L) and the muscarinic activation of I(K,ACh) in rat cardiac myocytes
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Lack of correlation between the observed stability and pharmacological properties of S-nitroso derivatives of glutathione and cysteine-related peptides
S-nitroso-glutathione inhibits platelet activation in vitro and in vivo
Evidence for a cyclic GMP-independent mechanism in the anti-platelet action of S-nitrosoglutathione
Deficient platelet-derived nitric oxide and enhanced hemostasis in mice lacking the NOSIII gene
Inhibition of platelet P2Y12 and alpha2A receptor signaling by cGMP-dependent protein kinase
Lysophosphatidic acid and receptor-mediated activation of endothelial nitric-oxide synthase
Lysophospholipids and the cardiovascular system
Athero- and thrombogenic actions of lysophosphatidic acid and sphingosine-1-phosphate
Important amino acids for the function of the human MT1 melatonin receptor
Selective detection of adenosine A1 receptor-dependent G-protein activity in basal and stimulated conditions of rat brain [35S]guanosine 5'-(gamma-thio)triphosphate autoradiography
Gamma-35S]GTP autoradiography allows region-specific detection of muscarinic receptor-dependent G-protein activation in the chick optic tectum
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subset: commoncrawl


text:
May 8, 2004
Baltimore, Md. - Loyola rallied from a seven-goal second-half deficit and put a scare in No. 1 Johns Hopkins, but it wasn't nearly enough as the Blue Jays ended the regular season with an 11-7 win over the Greyhounds at Diane Geppi-Aikens Field.
Senior Stephen Brundage scored in his last game at Evergreen, ending the season with a team-high 20 goals. The story, however, was Loyola's freshmen, who tallied five of the 'Hounds seven goals. All five coming in a 6-2 Loyola run that cut Johns Hopkins lead from seven goals to three.
The Blue Jays went up early with two goals from Conor Ford and two goals from Kyle Barrie. The Jays held a 5-0 lead before Brundage knocked in Loyola's first goal, a turn-around shot that caught Hopkins' goalie Scott Smith off-guard. The Blue Jays scored the next three and early in the third quarter the lead stood at 8-1.
Freshman Tony Ferreira gave the 'Hounds some life with an unassisted goal at 9:55 of the third quarter. Hopkins answered and as the fourth quarter began, the score was 9-2.
"Our guys never gave up," says Loyola Head Coach Bill Dirrigl. "They battled the whole game and I'm proud of them for that."
Sticking to their game plan, Loyola played a possession game and when the opportunity arose, freshman Patrick Kennedy converted, scoring an extra-man goal off a dish from Brundage. Freshmen Joe Landry and Greg Leonard scored the next two goals, Leonard's goal being Loyola's second man-up goal of the period, and the 'Hounds trailed 9-5.
Kyle Harrison ended Loyola's 3-0 run, scoring seven seconds after Leonard's goal. The 'Hounds still battled and when freshman Jordan Rabidou fired home a goal at 6:27, the score was 10-6. Junior Pat Shek made it 10-7 with a goal at 5:36, but Loyola would not get any closer. Matt Rewkowski scored the game's final goal at 2:06 for the final four-goal margin.
Sophomore Michael Fretwell registered 13 saves in goal for Loyola while picking up a season-high five groundballs. Brundage, Kennedy and Rabidou all added assists to their goals and finished the game with two points, freshman Matt Cassalia had five groundballs while winning 8-of-17 faceoffs.
"Since I've been coach at Loyola, we've had problems with Syracuse, Georgetown and Johns Hopkins," says Dirrigl. "Those games have gotten out of hand and this season, although the wins don't show it, we've hung in there. I think we have a lot to look forward to with Loyola Lacrosse."
Johns Hopkins, 11-1 overall, ends the season with the nation's top ranking and will have the No. 1 seed in the NCAA Tournament. The Greyhounds end the year at 4-8 with seven losses to ranked opponents. Brundage, who led the 'Hounds in scoring with 31 points this season, ends his collegiate career with 88 goals and 143 total points.


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JERUSALEM — What does the word "mud" connote? To most people it simply means a soft, wet deposit left on the ground after rain. To Auschwitz survivors, however, "mud" has a far deeper significance.
The camp inmate's day involved moving rapidly from living quarters to the toilets to the soup line; it involved long treks from the camp to the work areas and entailed hours of standing motionless. The people were weak, and the mud on the ground made walking and standing difficult. Corpses lay uncovered in the mud. The mud was an enemy.
Thus, when an Auschwitz survivor refers to "mud" in a testimony, it is far more than a climatic or topographical description.
Historian and linguist Itzhak Attia is researching, in the framework of the International Institute for Holocaust Research, the semantics involving the deeper meaning of the nouns used in testimonies.
When a survivor describes his or her Auschwitz experience, Attia explains, there is a lack of suitable language to sensitively and accurately describe the situation without making it sound banal. Elie Wiesel commented that Auschwitz is incomprehensible to someone who was not there.
In an effort to give deeper meaning to the terms used in testimonies, and consequently to draw a more comprehensive picture of camp life, Attia has compiled a list of 50 words used repetitively in testimonies, which he explains based on their context. This list amounts to a dictionary of Auschwitz survivors' terminology.
"Words are signals," explains Attia. "Simple nouns such as 'soup,' 'walk,' 'food' and 'door' encompass a whole world of connotations. Words, he says, have both structural and cognitive semantics. His interest lies with the former. "In order to talk about a noun in a particular time period, one has to understand the semantic [or the explicit] traits attached to that noun, at that time."
One word that has especially interested Attia is "door." More accurately, the door to the train that transported Jews into the camp. When a survivor refers to the opening of the [transport] "door," he or she is recalling a bombardment of sensations: the immense noise of the simultaneous opening of all the train doors; the sudden transferal from pitch black to glaring light; the contrast between the intense heat suffered for anything between three and 10 days in the stuffy carriage to the ice-cold chill of the raw elements; and the change from an intensely claustrophobic environment to a vast open expanse.
Attia calls the "door" the "missing air chamber," because it should be the space where those being transported can prepare or readjust to the stark difference between life on either side of it. Instead of alighting through this door, the inmates are dragged from the carriage and marched into a horrific new reality. And the more their reality is contrasted to ours, the harder it is for us to understand the depth of language they use in recalling events.
Attia says that those who read the testimonies need to be initiated into the language used by the survivors because, largely for psychological reasons, the survivors do not explain them.
"Soup" is another word that Attia has attempted to describe explicitly. Soup was more than nutrition and very temporary warmth. It was an instrument of reward and punishment. The Nazis decided who received soup with stock — which could be bartered for essential items — and who received just tasteless hot water. Fighting for a place in the soup line was also part of the feeding process. Soup was a source of life.
The term "arrival" (at the camp) also has specific connotations to the Auschwitz inmate. With arrival, came assault by the SS, terrifying dogs, selection and registration.
In order to fully comprehend the memory being recalled by survivors and in order not to trivialize their testimonies, these hidden meanings must be acknowledged and understood. Attia's lexicon will go a long way toward providing this understanding.


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What we desire is to find writers of songs that are already writing and capturing the heart of God for His people, and help them grow in their craft and passion. So we would love to get an MP3 of just one worship song that you have written either sung by the people at your church or yourself, just a recording from the house desk, or the tune (with words) recorded on your phone. Email it to email@example.com
We would like also to receive a short note, letter, email, or phone call from your senior pastor acknowledging your ministry in your local home church. See the contact page for details. We will also need a full financial commitment up front to secure your place.


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What Website Design Makes Your Visitors Chicken Out
What scares them?
Yes, you might have an artistic website design, but do you know that some of your ideas simply freak your visitors out? Read on and see what design errors must be avoided.
Pop-up surveys: You know the importance of a customer feedback in your website. That's why you cannot leave it out of your website design. But when it pops up and asks your visitor to complete a survey, it simply scares them out.
Trendy web 2.0 interface: Simplicity and consistency still holds the basic principles of website design. Informational and entertainment-based websites must provide contents that can be easily accessed by visitors. Keeping up with a consistent interface then, is a must.
Contents that are impossible to read: You may have created a fancy website design, but your contents are just not readable. Check your text size, your font face, your font color and your background color.
Too much animation: Visitors hate a website design that has too many animations in it. There's also the presence of those videos that start loading without the need to click a play button. This consumes too much bandwidth and makes the other tabs of your visitors to load slower.
Too loud: Adding a loud audio advertisement, announcement, or any music in your website design can be really irritating. If you want to be remembered and to be catchy using an intense audio, then you are on the wrong track.
Remember, when it comes to your business and your companies exposure on internet, take you time locating a quality web design Philippines company. Your future may depend on it. A technically sound website design company takes care of all these factors. But as a client, you must be entirely satisfied with the services of a particular web design Philippines company before you make your pick.
Do you have fears facing what your visitors hate to see in your web design? Did I scare you? Fear not, because there is a solution. WebFocus Solutions, Inc. When you see a website with stunning graphics and easy navigation, it must definitely be one of our masterpieces. It must definitely be one of our many genius website designs.
Need I say more?


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We help you search for a Longniddry double bass tutor. Double Bass lessons are an excellent method to learn a fun new hobby, acquire a new skill and gain confidence with double bass. Use our service to search for Longniddry double bass teachers and determine which best suits your requirements so you can start your double bass lessons in Longniddry straight away!
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Stockton Heath
Nestled in a leafy South Warrington village, surrounded by boutiques and restaurants, our Stockton Heath bistrot finds itself in a vibrant, bustling location.
Our 'raison d'être' is great value French cooking and seasonal menus. We like to combine French bistrot classics such as steak-frites, boeuf bourguignon and crème brûlée with our own take on regional favourites like tarte flambée, confit de canard and pâté de campagne. Our relaxed and friendly bistrots are the place to go for any occasion.
It's warm, welcoming and a great place to relax and enjoy authentic French cooking anytime of day or night and is a local favourite amongst residents and businesses alike.
We have something for everyone, from our two course Prix fixe lunch for £11.95 and our two course Pre théâtre menu for £14.95 to our evening À la carte menu. We also offer gluten-free dishes and a children's menu, which comes with our bespoke 14 page activity pack.
Visit Us
85 London Road
Victoria Square
Stockton Heath
View on google maps
Opening hours
Saturday 9–11.30am
Sunday 9.30–11.30am
Full breakfast menu available.
Monday to Friday 12–3pm
Saturday & Sunday 12–5pm
Monday to Wednesday 5–9.30pm
Thursday to Saturday 5–10pm
Sunday 5–9pm
Bank Holiday Monday opening hours are the same as Sunday opening hours.
Also nearby
Walton Gardens
With its spacious lawns, children's play area, children's zoo and pottery in the park it's the perfect place for a family day out in Warrington. Click Here
Pyramid & Parr Hall
Situated in the heart of Warrington's Cultural Quarter, Pyramid & Parr Hall are two of the North West's leading arts & entertainment venues for music, comedy, dance, drama and much more. Click Here
Warrington Museum and Art Gallery
Warrington Museum and Art Gallery has plenty to see and do, including a dinosaur, Time Tunnel for hands-on history, changing exhibitions and kids Crafternoons. Click Here
Head Chef - Corneliu
Corneliu completed his Chef's training in Romania before joining our bistrot family in 2007 as a Chef de Partie in Nottingham. Since then he has worked in our Stratford-upon-Avon and Leamington Spa bistrots and is now head chef at our Stockton Heath bistrot.
The food was very lovely and I'm looking forward to visiting again. The service was outstanding and was of a higher standard than many restaurants I've visited.Anna Micklewright via Online Feedback


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How to Teach Thankfulness to Your Children
November 24, 2016 | Home and Family
Thankfulness is more than just teaching your child to be polite when someone gives him or her a gift. It is a trait that, when learned, can have a much larger impact on a child. Research shows that there are both physical and emotional benefits for people who are thankful. They experience things like stronger immune systems, lower blood pressure, better sleep, higher levels of positive emotions and more joy and optimism.
Being thankful can also enhance a person's ability to have healthy relationships. It can make you more helpful, generous, compassionate, forgiving and outgoing. It can also reduce things like envy, aggression and loneliness.
Children Need Your Help to Learn Thankfulness
Thankfulness is something that requires intentionality. It's taught. Amy McCready, founder of PositiveParentingSolutions.com and author of The "Me, Me, Me" Epidemic – A Step-by-Step Guide to Raising Capable, Grateful Kids in an Over-Entitled World, says, "Children are naturally very self-centered, but we can teach them to think beyond their own needs and wants."
She adds that the hectic pace of life can sometimes push the topic of thankfulness aside, but it's never too late to start. McCready says bringing thankfulness front and center for your children may be easier than you think. Here are her best tips to teach your children to be thankful:
Pre-School Children: McCready says, "This is the age at which children are beginning to be a little less egocentric, so it's a great time to start teaching them to be thankful." She adds that very young children will learn about thankfulness best by doing. Here are some ways to get them going.
- When the child is given a gift, have him or her draw a thank-you picture. Young children may not be able to write a note yet, but they can certainly draw a nice colorful picture instead.
- Read books about being thankful.
- Role-play thankfulness. Use dolls or stuffed animals to practice "thankful conversations" with your kids. Create cues in those conversations that can transition to real life so that after a playdate, or when your child is given a gift, she is reminded to say "thank you" without being prompted.
Elementary School Children: At this age, McCready says children can have a little more ownership in how they show thankfulness. Consider these tips:
- Personalize thank-you notes. A thank-you note from children in this age group is appropriate, but it doesn't need to stop there. If your child likes cooking, let him bake cookies as a thank-you gift. If she is crafty, let her dream up a project. If he's chatty, let him make a thank-you phone call. The concept of the thank you stays the same, but the method changes.
- Begin serving in the home. Children in this age group can help fold laundry, dust, vacuum, rake leaves, water plants and more. McCready says when children help, they will feel like they can make a difference in their family. She says, "This will also help them appreciate not only what they have, but what others do for them."
- Begin neighborhood service projects. Children can pick up trash, help an elderly neighbor with yard work or shovel for the single mom down the street. Let them see that they can tangibly impact their immediate surroundings. McCready says, "These children are ready to have conversations about the idea of serving others. When we help other people, we learn to be more thankful for what we have."
Middle and High School Children: By the time children reach this age group, they should reach another level of ownership in deciding how to show thankfulness. McCready says, "Their skill set is different now, so they can do some really fun and different things to express thankfulness." Thank-you notes could turn into thank-you videos they film and edit themselves.
- Send a daily thank-you text. Have your child pick anyone they know and take one minute to text that person a note about why they're thankful for, or thinking about, him or her.
- Serve at home in bigger ways. Children should now be able to take on higher-level tasks like making a meal or doing all of their own laundry. This builds on the idea that by contributing to the home, children learn to be thankful for what others do for them, not just what they have.
- Step up neighborhood service. Encourage your teenager to offer to help by babysitting, tutoring, doing yard work, etc.—and be sure to do it for free. When they are done, talk about how others reacted to their efforts. Seeing others be thankful for something they had a hand in can go a long way in teaching them to express their own thankfulness when the time comes.
- Serve an organization. McCready says actions like these allow for a change of conversation at home. You can talk to your children about how your family has more and is thankful for it, so you feel called to give back and to serve others in need. Allow older teenagers to choose a community organization they are passionate about and volunteer there once a month or create a fundraiser that can benefit e organization.
Making Thankfulness a Routine
If you'd like to build thankfulness in your children all year round, McCready says, "The key is to make thankfulness a routine and part of your family culture. That means parents role-modeling it and being willing to create family rituals that support thankfulness as a value." At dinner, go around the table and have everyone list three things they're thankful for. Once you get started, you may never look back. Here are a few more ideas.
- Use a white board in your home where everyone can post things he or she is thankful for. Or use a fish bowl: Put thankful notes inside, and read them once a week as a group.
- Complete a service project once a month. Take turns deciding whom and how to serve. On the drive home or in the days after you have served, reinforce the idea that when we serve others in need, it makes us more thankful for what we have. Ask them what they're most thankful for.
- Make do with less. Pick a period of time to go without something basic: air conditioning, cable or Netflix, or any other luxury so your family can feel truly thankful for it when it returns.
Raising thankful children doesn't have to be tough, but it does require intentionality from parents and a commitment to establishing the concept as a value within your family unit. Hopefully you've found a few simple tips here to give you a great start on a thankful holiday season and beyond.
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Featuring an alto saxophone or trumpet player in a tasty, rock ballad setting, this creative original features a gorgeous solo theme that is completely written. The ensemble provides a lush foundation
In Your Dreams... is an exceptional ballad feature for solo trombone or tenor saxophone. The rhythmic and technical demands are minimal allowing rehearsal time to focus on musicality and
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Tomorrow Is Always a Good Day is a sensational ballad feature for solo trombone or tenor saxophone. This lovely composition begins in a minor key, keeps building to a modulation to major, then
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text: Smart Home revenues to reach $71bn by 2018 predicts Juniper Research 11 Feb 2014 05:50 PM Network modernisation essential to service delivery if mobile networks are to be able to meet demand for OTT content services, such as NetflixRevenues generated from Smart Home services are set to reach a global market value of $71 billion by 2018, rising from $33 billion last year, a new report from Juniper Research forecasts.The new report 'Smart Home Ecosystems & the Internet of Things' found that nearly 80% of total Smart Home service revenues will come from entertainment services by the end of the forecast period. As connection speeds have increased, service providers are able to offer greater volumes of content, delivered with increasing convenience.According to the report, growth has been spurred by the emergence of high-profile OTT (Over-The-Top) content providers such as Netflix, LOVEFiLM and Amazon Instant Video, while demand is being further fuelled by mass adoption of connected TVs.While the smart TV market is just beginning to take hold, the Set-Top Box and the console remain popular options for users to connect their TVs to the Internet and access both subscription services as well as pay-per-view, download-to-own and rentals.Maintaining the Smart Home connection The report found that 4K video services and an array of 'Internet of Things' smart home devices will place the network under additional strain. Hence, stakeholders will need to rapidly ensure that their networks are modernised and transitioned from legacy technologies, such as IPv4 and CGNAT, if they are to remain a viable player in meeting consumers' expectations.Other key findings include:Industry collaboration between stakeholders is crucial to realising the potential of the 'Internet of Things'; no single stakeholder is likely to be able to dominate thanks to the number of verticals within the home.Security and control elements of the Smart Home will reveal a service provider opportunity approaching $12 billion by 2018 as automation elements are added to service propositions. Nevertheless, providers must examine the real 'value add' in order to capitalise on this market.The whitepaper, 'Smart Homes - It's an Internet of Things Thing,' is available to download from the Juniper Research website together with full details of the report. James Atkinson EditorLinkedIn |Google + |Email Leave a CommentPlease write code to prove you're humanMore on... Smart homesSmart home industry must tackle security issue if it is to growConsumers confused over what constitutes smart home technology SoftAtHome joins the Z-Wave Alliance for smart home IoT offeringsMore on... Smart CitiesNokia unveils wireless router for power distribution networks ETSI forms new industry group for smart city solutionsLatest in IoTAltair Semiconductor introduces narrowband CAT-M1 and NB1 chipsetTelefónica, Huawei and Kamstrup launch in NB-IoT project in Chile oneM2M standard holds third IoT interoperability event


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|Nathan Scott <nscott@xxxxxxxxxx>
|Re: review: allocate bmapi args
|David Chinner <dgc@xxxxxxx>
|Fri, 20 Apr 2007 15:34:11 +1000
|David Chinner <dgc@xxxxxxx>, xfs-dev <xfs-dev@xxxxxxx>, xfs-oss <xfs@xxxxxxxxxxx>
|<20070419072505.GS48531920@xxxxxxxxxxxxxxxxx> <1176969062.6273.169.camel@edge> <20070419082331.GW48531920@xxxxxxxxxxxxxxxxx> <1177044117.6273.203.camel@edge>
On Fri, Apr 20, 2007 at 02:41:57PM +1000, Nathan Scott wrote: > On Thu, 2007-04-19 at 18:23 +1000, David Chinner wrote: > > ... > > > Are you sure this is legit though? > > > > It *must* be. We already rely on being able to do substantial > > amounts of allocation in this path.... > > Not necessarily, we only sometimes (for some values of BMAPI flags > I mean) do memory allocations. > > > ... > > We modify the incore extent list as it grows and shrinks in this > > path. It is critical that we are able to allocate at least small > > Well, not always. For cases where we modify the extents we must > call in here with Steve's funky I'm-in-a-transaction process-flag > set, which is the secret handshake for the memory allocator to not > use GFP_FS. *nod* > For cases where we are only reading the extent list, > we would not be doing allocations before and we'd not be protected > by that extra magic. So now those paths can start getting ENOMEM > in places where that wouldn't have happened before.. I guess that > filesystem shutdowns could result, possibly. Well, with a sleeping allocation we'll get hangs, not shutdowns. Quite frankly, hangs are far easier to debug than shutdowns. If it is really does become an issue, we could use mempools here - we can guarantee that we will return the object to the pool if we don't hang on some other allocation and hence we'd always be able to make progress. However, I'm not sure I want to go that far without having actually seen a normal allocation cause a problem here and right now I think that saving ~250 bytes of stack (~10-15% of XFS's stack usage on ia32!) through the paths we know blow is substantial. > > FWIW, I have done low memory testing and I wasn't about to trigger > > any problems..... > > It would be a once-in-a-blue-moon type problem though, unfortunately; > one of the really-really-hard to trigger types of problem. Yes, that it is. But a sysrq-t will point out the problem immediately as we'll see processes hung trying to do memory allocation. > > > (Oh, and why the _zalloc? Could just do an _alloc, since previous > > > code was using non-zeroed memory - so, should have been filling in > > > all fields). > > > > Habit. And it doesn't hurt performance at all - we've got to take > > Hrmmm... is there any point in having a non-zeroing interface at > all then? Sorry - i should have said that "for small allocations like this it doesn't hurt performance" - the cycles consumed by the allocation and lost in the initial cacheline fetch are far, far greater than those spent in zeroing part of a cacheline once it's accessable. > I thought the non-zeroing version was about all using > the fact that you know you're going to overwrite all the fields > anyway shortly, so theres no point zeroing initially... Zeroing causes sequential access to cachelines and hence hardware prefetchers can operate quickly and reduce the number of CPU stalls compared to filling out the structure in random order. And some CPUs can zero-fill cachelines without having fetched them from memory (PPC can do this IIRC) so you don't even stall the CPU.... But there's still plenty of cases where you don't want to touch some or all of the allocated region (e.g. you're about to memcpy() something into it) so we still need the non-zeroing version of the allocator... Cheers, Dave. -- Dave Chinner Principal Engineer SGI Australian Software Group
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First-Article Inspections
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Let us know when your receive great advice so we can thank our lawyers and point others with similar questions in the right direction.Got it!
My ex-husband was abusive, including threats against our son, and I moved out of state. He ran me out of money so I was unrepresented at trial and he was given unsupervised visits in Spokane. We are now one month past that ruling and he had his first visit. He refused to tell me where he lives, but I know he is not at the address he has on file with the courts. He also refuses to tell me who he is living with or introduce me to her (his new girlfriend). Our son is two and cannot tell me what happens when he is there. My ex is also not responding when I ask questions about how he is doing, etc. None of this is specifically required in our parenting plan - do I have grounds to modify?
Absent extraordinary circumstances, an introduction to the girlfriend is definitely not required, and who your ex lives with is also unlikely to be an issue that concerns the court. If the father has day visits only, then his home address probably won't be something you are entitled to either; however, if the visits include overnights, you probably have a right to know your child's whereabouts. As such, I recommend you consult with a local attorney to determine whether this issue is something for which you should initiate a court proceeding. Good luck!
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You should schedule a brief paid consultation with a family attorney to review your orders, interview you about the details, and then advise you on your options.
This AVVO Answer is provided for general educational purposes only. By using or participating in this site you agree and understand that there is no attorney client relationship between you and the attorney responding, and no attorney-client confidentiality. The law changes frequently, and varies from jurisdiction to jurisdiction. The information provided in this Answer is general in nature and may not apply to the factual circumstances described in your question. The applicable law and the appropriate answer may be different in the State or States where the relevant facts occurred. For a definitive answer you should seek legal advice from an attorney who (1) is licensed to practice in the state which has jurisdiction; (2) has experience in the area of law you are asking about, and (3) has been retained as your attorney for representation or consultation. Your question and the attorney's answer may be used for promotional or educational purposesAsk a similar question
On the pattern order of child support (and several other forms usually filed in a dissolution of marriage) are these provisions:
The Obligor Parent Must Immediately File With the Court and the Washington State Child Support Registry, and Update as Necessary, the Confidential Information Form Required by RCW 26.23.050.
The Obligor Parent Shall Update the Information Required by Paragraph 3.2 Promptly After any Change in the Information. The Duty to Update the Information Continues as long as any Support Debt Remains due Under This Order.
There are similar provisions for the other parent. Part of the required information is the service address and the residential address of each parent.
In some special circumstances (such as domestic violence), providing the information can be waived.
The courts in WA run background checks on all adults living in a parent's household before signing a final parenting plan. So, you are entitled to know information about the adults living in the other parent's household. However, there is no law that requires that you be introduced to those persons.
"My ex is also not responding when I ask questions about how he is doing, etc." If the "he" in the sentence refers to the ex, he has no legal obligation to tell you how he is doing. If "he" refers to the child, presumably, you know how your child is doing since you appear to be the custodial parent, someone who is frequently with the child. Unless something out of the ordinary happens to the child (such as suddenly being ill or injured), a parent usually does not have a legal duty to provide a daily report of the child's every day activities to the other parent. (Cooperative parents do discuss the daily activities of the children with each other.)
You should review the specific facts with your attorney to find out your legal options.Ask a similar question
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text:
- 12 total posts
- 12 comments
Recent comments from Errn76
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You look great! Thank you for updating. READ COMMENT
Thank you for the update! Can't wait to see final result. I can see your butt developing nicely. Yay READ COMMENT
How are you doing? Are you pleased with results? READ COMMENT


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Good For SomethingFavorites [+]
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text:
Steve sprays one squirt of Aqua Di Gio on the left side of his neck before heading out of the door. Cell phone, check. Wallet, check. Breath mints, double check. He's headed downtown to meet Lisa, an adventurous 4th grade teacher who spends her summers traveling, tasting wine, and reading. They've communicated several times via email, text, and phone, but this will be the first time they've met in person. They've met online and finally felt comfortable enough to make it a date. It beats waiting for a lover to fall out of the sky. Just like all matters of life, the experience will be what you make it.
I know several people (myself included) that met people online. The experiences shared among us range from disaster to happily married. Either way, credit is due for stepping out the comfort zone and for try a new way to make a connection. According to a survey commissioned by Match.com, 17 percent of marriages between 2007 and 2010 met online, making it the third-most-frequent method of introduction, behind meeting through a mutual acquaintance or at work or school.
Here are 2 personal stories that were shared with me while researching this post.
Anonymous Female – plentyoffish.com
"I found it very entertaining, just searching the website. My 1st experience was really good, super, super nice guy. He was Ex military and a God-fearing man. He made 6 figures, had his own home, 2 dogs, engineer, couple of motorcycles, etc. BUT he was going way to fast! After a month he was talking about marriage/kids (both things I want), guessing the size of my ring finger, etc. It was just a little much for me. He scared me a little, so I ended it. My 2nd experience, the guy was too whiny. So, nothing after the 1st date. I talked 3 of my girlfriends into joining it, and everyone loves it. They've all been on dates, and finds it to be a great pass time. I think the 1st couple of dates should be very lighthearted. Nothing to serious. So, people won't get scared off like me. 2 Words: Great Topic!"
Craig and Sarah – Match.com
"Craig and I met on Match. I had also done eHarmony in the past (with very little success) and he had also done Plenty of Fish. I did not like eHarmony because I never felt like I had much control… I just got what they gave me, in terms of options. I think Craig met people on Plenty of Fish, but nothing was ever very serious. I'm not sure it's a very serious site anyway for dating.
Craig and Sarah had been in so many of the same places at the same times, but never met each other before. They were both in Greenville, NC from '98-'99 and both lived in Chicago around the same time as well. At the time Sarah was married. Things did not work out in her first marriage and after bouncing around a bit they both ended up in Charlotte, NC. Craig went back to school to earn his engineering degree at UNC-Charlotte. After receiving his degree, he landed a job at The Center for the Environment at Catawba College. Sarah finally got the job of her dreams in Charlotte with Mecklenburg County's Children's Developmental Services Agency as an evaluating speech-language pathologist. After a little over a year in Charlotte, she decided to do match.com. Craig sent her an email through the site and they are now happily engaged. Craig knew from the first telephone call that he was going to marry her. He also said, "God had a hand in our meeting when we did. Although we crossed paths so many times, I truly believe God was making us wait until we were both ready to meet each other…"
They were exactly what they had been looking for in terms of age, education level, interests, etc. However, they both agreed that there's got to be more there in order for something to last. Sarah said, "As we got to know each other, we found out more that was written in the profile. Warmth, family connections, personality…none of that translates in an online dating profile. We definitely got lucky. We are very honest with people about how we met. At first I was a tad embarrassed, but online dating really is a part of our culture now, particularly with people over a certain age who are not bar people."
Become Better lessons:
- Nothing is 100% so research it before putting yourself out there and before paying money.
- When meeting new people in general you should always proceed with caution.
- Meeting people online can be a wonderful experience if you're willing to take a chance.
Whether you meet in church, the grocery story, or online, relationships are what you make them to be. Know better. Do better. Be better.
Here are my reviews of some of the most popular online dating sites. (SN: It's funny how they all say their #1 or the leading matchmaking site in the world).
Cost: $40 per month and sometimes they run specials. There's plenty of people to choose from which makes it a little more competitive, but it's one of the more popular dating sites. Very professional. You don't have to be matched up to browse profiles.
Cost: 1 month = $59.95/month. 3 months = $39.95/month. 6 months = $29.95/month. 12 months = $19.95/month. After completing a questionaire, they send you the matches. You can only view profiles of matches they send you. It kinda stinks if they don't have any matches, but the good thing is you're not wasting your time. If there is no match you don't have to pay (Money back guarantee). Does not offer any connections for LGBT community.
Cost: 1 month = $29.95/month. 3 months = $19.99/month. 12 months = $9.99/month. Popular with the 30 and 40 something crowd. Integrates online dating and social media. Zoosk can gather information about you from your social networking sites (i.e. facebook)
Cost: Free but you can upgrade your account for $9.99/month, which in my opinion isn't necessary. It's similar to Match.com being that there's a lot of people so it's competitive to meet people. I personally know people who have great things to say about this site.
Cost: 1 month = $29.99/month. 3 months = $16.99/month. 6 months = $13.99/month. A great website to meet other Christians. You can meet Christians on the other sites, but this site is specifically for Christians.
Cost: 1 month = $39.99/month. 3 months = $33.33/month. 6 months = $24.99/month. A great website to meet people of the Jewish faith. You can meet Jewish people on the other sites, but this site is specifically for Jewish dating.
Cost: 1 month = $24.95/month. 3 months = $16.65/month. 6 months = $12.49/month. A great website to meet other Catholics. You can meet Catholics on the other sites, but this site is specifically for people of Catholic faith.
Cost: 1 month = $19.99/month. 6 months = 11.99/month. Profile is free, but you can't communicate unless you have a paid membership. The site is geared towards (but not limited to) singles over 50. Very easy site to use.
Cost: EXPENSIVE AS HELL! I'm talking $1,500+ expensive! This site connects you with a match and helps plan your first date. They help you find a date and then schedule a time and place to meet for lunch or drinks after work. I've read that customer service isn't great and they do not off refunds. I've also heard their selection pool is limited. It sounds like such a great concept, but it looks like you'd be better off going on Yelp and seeing who complained about IJL and then asking THEM out on a date.
Cost: Free! It's literally PLENTY. Similar to Match in the # of people. This dating site has great reviews and it sounds like people love it because there are so many people to connect with and it's free.
Cost: Free. Not my first choice of sites to find someone, but it's free. There's a big stigma associated with Craig's List because of the "Craig's List" killer, but when using ANY of these sites you should proceed with caution.
Here are some other sites to search. Chemistry.com, Loveandseek.com (Christian), Blackpeoplemeet.com, Bbpeoplemeet.com (Big and Beautiful), Singleparentmeet.com.


text:
by Sperry Top Sider
Order now to get it by: Saturday August 12 - Monday August 14
This product ships with limited tracking information, and may take longer than usual to be delivered.
Condition: New
Product ID: 357512
Delivery Information |Returns & Exchanges |Payment Methods
Set your little sailor off in style with the Sperry Top-Sider Charter. This adorable oxford looks just like the
full-sized version, but it's made just for your first mate. They don't need to know how to hoist the main to look great.
Just outfit your little one with some khaki shorts and a plaid button down and you're ready to set sail.


text:
3. The Earth and The moon
Salvador Carrión // Colegio Madrigal
28 days
How big is the moon?
28 days
Phases of the moon
New moon
First quarter
Full moon
Last quarter
The tides
The sun
Big mass
Big distance
The moon
Small mass
Short distance
The eclipses
Solar eclipse
Moon eclipse
Main ideas
What are the craters of the moon?
What is the distance Earth-moon?
Name and describe the movements of the Moon.
Draw a diagram with the phases of the moon and name it.
What are the tides?
When we have a high tide? and a low tide?
When an eclipse occurs?
Vocabulary (page 30 and 31 )
Copy the title!
You know what you have to do...
No atmosphere
Extreme temperatures
28 days
Low gravity
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Home > Home & Garden > Garden Supplies > material fabric wuxi artificial turf for landscaping manufacturing
|Product Name:
material fabric wuxi artificial turf for landscaping manufacturing
|Packaging Details:
|Black PP Cloth Width
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|7-20 days
material fabric wuxi artificial turf for landscaping manufacturing
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text:
About Us
Branch Network
WEA Cymru is democratic by nature. Learners are active partners in the construction and organisation of their learning wherever appropriate and the Association as a whole runs under the guidance of elected committee officers and committee members who are themselves students. The governance is thus firmly in the hands of the elected representatives of learners.
Wherever you are in Wales, you are never far from a WEA branch or group or class. Branch members are citizens who have joined together to speak up for their community's educational needs.
In addition to WEA classes and informal groups reach out into communities and to students everywhere, offering a fresh start through learning.
To find out more contact the Learning Manager in your area
Anglesey & Bangor 01766 800443 firstname.lastname@example.com
Blaenau Gwent & Monmouthshire 01495 369869 firstname.lastname@example.com
Cardiff, Vale of Glamorgan & Bridgend 02920 382431 firstname.lastname@example.com
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Swansea, Neath and Port Talbot 01792 467791 firstname.lastname@example.com
Wrexham & Flintshire 01766 800486 firstname.lastname@example.com


text:
class and marbled patterns
Expand Messages
- Hi All,
Just wanted to let you know that I'll be teaching traditional marbling at
Artiscape in Columbus, Ohio on Friday, April 16th. There are several spaces
available if you or a friend are interested. Also, although The Ultimate
Marbling Handbook is out of print and the remaining copies are very expensive, I
have published the patterning section in color and am selling it for $20.
Details about my classes and the book are on my website www.dianemaurer.com
Best regards,
[Non-text portions of this message have been removed]
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text:
classes & ensembles
contact us
support us
classes & ensembles
contact us
support us
Portsmouth Music and Arts Center, 973 Islington Street, Portsmouth, NH 03801, 603-431-4278
To build community through the arts by providing all people with the opportunity to achieve their full creative potential, regardless of their age, ability, or economic status. We accomplish this through high quality music and visual arts education programs and by creating ensemble, performance, and exhibition opportunities for students, professionals, and audiences.
Contact Us
Portsmouth Music and Arts Center is a 501(c)(3) nonprofit community music and arts school. There is a place for everyone at PMAC - we welcome your involvement as a student, parent, volunteer or member. Through music and art, PMAC helps our students meet and accomplish important life goals. We are committed to excellence in arts education and work daily to build community through the arts.
Since our founding in 2002, the Portsmouth Music and Arts Center has successfully motivated and inspired thousands of music and art students of all ages. Our faculty's commitment to the highest educational standards, combined with the passion and dedication of our students, is at the core of this success.
In 2014 we opened our new facility at 973 Islington Street, which features two new visual arts classrooms, two music ensemble classrooms, a digital classroom for both art and music, and the Haas Family Gallery and Recital Hall, in addition to many studios for private music instruction. The building was renovated specifically for our special use as a music and arts school, with architectural design by Lisa DeStefano and Robert Harbeson of DeStefano Architects and acoustical design by acoustical engineer Eric Reuter.
At PMAC we offer a wide variety of educational programs that meet the needs and aspirations of students of all ages and ability levels. Our teaching, regardless of musical genre or artistic medium, encourages the development of strong foundations. Our music curriculum has grown in recent years to include classical, jazz, blues, bluegrass, folk and rock, and many of our students explore other styles beyond our core curriculum. We frequently welcome guest artists who introduce students to a wide array of culturally diverse traditions. Our visual arts program focuses on strong foundations through a wide variety of media and cultural traditions. Our goal is always to provide the student with the opportunity to achieve their full creative potential.
PMAC's warm and supportive faculty provides mentoring support and guidance for all students. Whether you aspire to study music or art at the college level and enter a career in the arts, or just want to follow through on your New Year's resolution to learn piano, we're here to help you with each step.
Portsmouth Music and Arts Center is the proud home of more than twenty student music ensembles ranging from young children to adults. These groups perform regularly throughout the community. If you are interested in having a group play at your event, please download the PMAC Community Outreach Performance Guidelines and Application form. Please return the completed application form to PMAC , c/o Russ Grazier, CEO, 973 Islington Street, Portsmouth, NH 03801 for approval. All applicants will be notified within 14 days of application. If you have any questions, please contact Russ Grazier at 603-431-4278 or via e-mail at firstname.lastname@example.com.
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Sketch Boss Mega Bundle
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Average Customer Rating
(based on 2 reviews)
Reviewed by 2 customers
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Beautiful wave curling with sunset peeking through
By MotoGPDude
from Santa Cruz
About Me Budget Shopper
Purchase Reason
I measured the overall frame and glass size and found the closet sizes to fit it. Then I saw this wave and thought it will look so amazing and it really turned out nice.The frame was about an inch too short on either edge. I used the old artwork to draw cut lines and cut the wave to fit! It looks so good out in the hall people love it.Thank you so much
Bottom Line Yes, I would recommend this to a friend
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(4 of 4 customers found this review helpful)
Nice picture but drab printed colours
By Stuart
from northamptonshire
This picture looks really nice but the colours on screen have not translated well to paper. I'm happy with the shot (after I cut the writing off the bottom!) and the paper quality is good but I wouldn't buy pictures online again, I think you really need to see a better representation of the final quality!
Bottom Line No, I would not recommend this to a friend
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Includes jacket, belt with attached gun holster, trousers, hat and boot covers.
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text:
Minish Cap 4 Koma-1 by Sage-of-WindsWatch
©2005-2017 Sage-of-Winds
Hee heee, 4 KOMAS RAWK !
Now that kinstone will fit in between his teeth!
lol the pie one is good but i dont understand the other
Minish are tiny and therefore impossible to see for most people of our size. One misstep and you'll easily crush a village.
i knew it was a bad idea to put rocks in the pie
kawaii!! poor link and poor minish poeples!
How cute~
Toon Link is crazy. XD
The first time I heard the idea of putting kinstones in food, I thought it was silly XD
Ah man! how nostolgic! I love this game, I only beat it like 100 times!!! Once I beat it the first time, I wanted to beat it again.
hahaha great ideas XDD
I always thought that when you walk around wouldnt the minish want to run away so much? xD poor minish.
Just one tooth!c ^_^ That's funny though, to see Link with no teeth.XD
Awww..man! You beat me to the idea of the kinstone in bread comic! TT__TT (actually,one of my friends gave me the idea,but still!) Really, how else would you find out that there are kinstone pieces in the bread? By eating the bread, of course! My friend went on to say that Link would lose a tooth from the bread, place it under his bed at night, and when the toothfairy came, Link would catch it in a bottle,so......there!
Ezlo is like navi....He won't shutup.
lol funny poor Minish!!!! love the style
Seriously funny!
HAHAHA XD!! Greatness..
X3 Nice! I never thought about that Hee hee, I love The Minish Cap. ^^
Hee hee!
These are so funny!! XD
Merrr...I know...I always have trouble with that version of Link's hair e-e....
Well, anything can be imporoved with time and effort. Just keep at it and you'll get it for sure.
Hehe. That was really funny
Now this was a treasured moment. I'm glad I didn't miss it. It was well worth seeing.
Lol, I love the expressions you give Link! They are perfect!
Nice coloring!!! So GOOD!!!
Phew, I'm glad I'm not the only one to get Ezlo's name wrong ^_^ Is the shop sign random menu? Isn't it more fun to use real modern Hylian lettering? Nonetheless, great job. If I had time and art skills, I'd definitely draw these all the time!
Lol! Oh that's a good one ^^ I always get annoyed if there isn't one in my baguette!
OOOOOOOOOOOOOOHHHHHHHHHHHHHHHHHHHHH SCREAM AND RUN AWAY!!!!!!!!!!!!!!!!!!!IT"S LINK!!!!!!!!lol, nice job.
Lmao,Funny. The The Hat seems to be high. What a bad influence for Link. x3
I like it!!!! Its realy funny!!!!!!!!!!
These comics are awesome!
O_o That hat scares me.. a talking hat what next? A talking pie? Wait.. that would be kewl..
Hmm. Very good.. hehe! Theres so many good Zelda artists <3!!
Hmm. Very good.. hehe! Theres so many good Zelda artists <3!!
Very cute. Poor Minish and Link look before you bite into it, It me reminds when someone put coins in bownies once XD.
LOL! Nice. Always wondered what would have happened if he snacked down on that pie with the kinstone in it.
0.0 -dies laughing- gahaha, i thought about Link crushing the Minish too
lol this is so funny! XD
XD That's hilarious...I love your comics. ^^
Hahaha! That's just funny... Poor little near-microscopic Minsih....
Heh heh heh...funny! <3
ahahah this is so funny XD love it!