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A regulator of G Protein signaling, RGS3, inhibits gonadotropin-releasing hormone (GnRH)-stimulated luteinizing hormone (LH) secretion
Jimmy D Neill1Email author,
L Wayne Duck1,
Jeffrey C Sellers1,
Lois C Musgrove1 and
John H Kehrl2
© Neill et al; licensee BioMed Central Ltd. 2001
Luteinizing hormone secreted by the anterior pituitary gland regulates gonadal function. Luteinizing hormone secretion is regulated both by alterations in gonadotrope responsiveness to hypothalamic gonadotropin releasing hormone and by alterations in gonadotropin releasing hormone secretion. The mechanisms that determine gonadotrope responsiveness are unknown but may involve regulators of G protein signaling (RGSs). These proteins act by antagonizing or abbreviating interaction of Gα proteins with effectors such as phospholipase Cβ. Previously, we reported that gonadotropin releasing hormone-stimulated second messenger inositol trisphosphate production was inhibited when RGS3 and gonadotropin releasing hormone receptor cDNAs were co-transfected into the COS cell line. Here, we present evidence for RGS3 inhibition of gonadotropin releasing hormone-induced luteinizing hormone secretion from cultured rat pituitary cells.
A truncated version of RGS3 (RGS3T = RGS3 314–519) inhibited gonadotropin releasing hormone-stimulated inositol trisphosphate production more potently than did RSG3 in gonadotropin releasing hormone receptor-bearing COS cells. An RSG3/glutathione-S-transferase fusion protein bound more 35S-Gqα than any other member of the G protein family tested. Adenoviral-mediated RGS3 gene transfer in pituitary gonadotropes inhibited gonadotropin releasing hormone-stimulated luteinizing hormone secretion in a dose-related fashion. Adeno-RGS3 also inhibited gonadotropin releasing hormone stimulated 3H-inositol phosphate accumulation, consistent with a molecular site of action at the Gqα protein.
RGS3 inhibits gonadotropin releasing hormone-stimulated second messenger production (inositol trisphosphate) as well as luteinizing hormone secretion from rat pituitary gonadotropes apparently by binding and suppressing the transduction properties of Gqα protein function. A version of RGS3 that is amino-terminally truncated is even more potent than intact RGS3 at inhibiting gonadotropin releasing hormone-stimulated inositol trisphosphate production.
Pituitary Cell
Luteinizing Hormone Secretion
GnRH Secretion
A near-universal feature of cell signaling via seven transmembrane, G protein-coupled receptors is attenuation of a cellular response upon prolonged exposure to an extracellular stimulus [1]. This desensitization process is well-established for GnRH-stimulated LH secretion from pituitary gonadotropes [2, 3], and occurs after about 6 h of continued exposure to GnRH in rats [3]; removal of GnRH permits recovery from the profound suppression of LH secretion, albeit rather slowly (2–4 days; 4). The molecular mechanisms involved in GnRH-induced desensitization are poorly understood, but they do not appear to be due to alterations in GnRH receptor expression [5, 6] or to changes in LH concentrations in the pituitary gland [7, 8].
Earlier, we presented evidence that a regulator of G protein signaling, RGS3, might participate in the regulation of GnRH receptor signaling [9]. The mechanism of RGS action is antagonism of the Gα protein either by accelerating its hydrolysis of GTP, thereby prematurely terminating signaling due to reassociation of the Gα and Gβγ complexes [10], or by antagonizing interaction of the Gα protein with its effector such as phospholipase Cβ [11, 12]. A large number of RGS genes have been identified in mammals based on the presence of a 120 amino acid core domain [13–16]. RGS proteins have been described that interact with the Giα and Gqα members of the G protein family [17–21].
Previously, we showed that RSG3 was expressed endogenously in rat pituitary cells and, when experimentally co-expressed together with GnRH receptors in a heterologous cell line (COS1), it suppressed GnRH-stimulated inositol trisphosphate production [9]. Here, those studies have been extended to include GnRH-stimulated LH secretion using adenovirus-mediated gene transfer of RGS3 complementary DNA into normal rat pituitary gonadotropes. RGS3 was found to profoundly inhibit GnRH-stimulated LH secretion and 3H-inositol phosphate accumulation. However, a calcium ionophore stimulated LH secretion in RGS3-expressing gonadotropes, confirming a specific site of action occurring at phospholipase Cβ or earlier in the signal transduction pathway.
The initial experiments were performed to confirm our earlier finding that RGS3 suppressed GnRH-stimulated IP3 production in COS-1 cells [9] and to test a truncated form of RGS3, RGS3T (RGS3 314–519) [24] for suppressive activity in the same assay (Fig. 1). cDNAs representing the GnRH receptor and RGS3 or RGS3T were co-transfected into COS-1 cells for GnRH-A treatment 72 h later; intracellular IP3 concentrations were measured at 0, 10, 20, and 30 sec after addition of 10-7M GnRH-A (Fig. 1). As expected, RGS3 exerted a profound suppression of IP3 production at each of the time points during the 30 sec after agonist treatment (p < 0.05) (Fig. 1). RGS3T is comprised primarily by the consensus RGS domain and was originally reported to decrease the growth rate of yeast and to inhibit IL-8 receptor mediated activation of MAP kinase by a factor of 3–10 fold as compared to RGS3 [24] and was subsequently reported to be a normally expressed variant of RGS3 [37]. Therefore, we compared it to RGS3 for attenuation of GnRH-stimulated IP3 production in COS-1 cells (Fig. 1). As reported by others [24, 37], we found that RGS3T was more potent than RGS3 (p < 0.05) (Fig. 1). Identification of a C-terminal 205 amino acid variant of RGS3 that has equal or greater potency than the full-length 519 amino acid RGS3 will facilitate the identification of RGS3 functional protein domains that are important for its activity.
RGS3 and RGS3T suppress GnRH-stimulated IP3 concentrations in COS-1 cells. The cells were co-transfected with cDNAs representing the GnRH receptor and RGS3 or RGS3T; 72 h later the cells were treated with 10-7M GnRH for 0, 10, 20, or 30 sec before being collected for IP3 measurements using receptor radioassay in which IP3 and 3H-IP3 competed for binding to IP3 receptors prepared from calf cerebellum. Data presented are the mean ± SEM of three independent experiments. The SEM is not shown for some data points because it was smaller than the symbol. The RGS3 and RGS3T groups are significantly lower than the control group (p < 0.05), and the RGS3T group is significantly lower than the RGS3 group (p < 0.05).
The family of RGS proteins are established to interact with various Gα-protein subunits, thereby antagonizing their activities [13–18, 24]. Indeed, we previously reported that RGS3 bound Gqα [9], the G protein subunit that mediates GnRH receptor signaling [38]. Here, we have extended those binding studies to include other Gα proteins (Fig. 2). An RGS3-GST fusion protein was incubated with an 35S-Met-Gα-protein, and the amount of labeled Gα-protein was determined using glutathione-coupled beads to "pull down" the RGS3-GST/35S-Gα-protein complex that was then separated and quantified by SDS/PAGE (Fig. 2). More Gqα was bound by RGS3 than any other protein, although its binding was not significantly different from that of Gzα and Giα (p > 0.05). Significant (p < 0.05) amounts of Gi2α, Gi1α, Goα, and Gsα also were bound but were not as high as Gqα, Gzα, and Gi3α. These results confirm our previous findings that RGS3 interacts with Gqα but also show that it has significant interaction with all other Gα-proteins studied.
In vitro binding of RGS3 to Gα proteins. An RGS3-GST fusion protein was incubated with 35S-Met-Gα proteins, and glutathione beads were used to "pull-down" the RGS3-GST/Gα protein complex. The amount of 35S-Gα protein bound was determined by SDS/PAGE separation, and quantification using a Phosphor imaging device. The data point for each G protein is the fraction of its total cpm that were bound to RGS3-GST fusion protein after subtraction of the cpm bound to control GST fusion protein. Mean ± SEM of three independent experiments. Gqα, Gzα, and Gi3α were not significantly different from the other Gα-proteins (p < 0.05) that, in turn, were significantly higher than a group to which 35S-Met alone was added (p < 0.05).
The findings of the current, and an earlier study [9], demonstrate clearly that RGS3 suppresses GnRH receptor signaling in a heterologous cell system. Because of the evidence that the signal transduction pathway for GnRH in gonadotropes involves the GnRH receptor, Gqα, phospholipase Cβ, and cellular IP3 release [39, 40], we determined the effect of RGS3 on GnRH-stimulated LH secretion using adenoviral-mediated gene transfer in rat pituitary cells (Fig. 3). In these experiments, we performed a dose-response test that included three experimental groups: uninfected, adeno-β galactosidase, and adeno-RGS3 infected. Adeno-βgal infection was used for determination of the fraction of cells infected at each dose of virus (Fig. 3, top panel) and as a control for the effects of adeno-RGS3 on GnRH-stimulated LH secretion (Fig. 3, middle panel). Cell viability, measured as trypan blue exclusion, varied from 98.5–100.0% (Fig. 3, top panel). The fraction of cells infected by the adenovirus depended on viral dose (0–5.0 M.O.I.; 1 M.O.I. = 1 viral particle/pituitary cell) and ranged from about 2% (no adenovirus) to 99.5% (5.0 M.O.I.). Adeno-βgal had no effect on LH secretion at any dose compared to the uninfected group (Fig. 3, middle panel). In contrast, an inverse dose-related effect was observed between adeno-RGS3 and GnRH-stimulated LH secretion (Fig. 3, bottom panel). RGS3 0.2 M.O.I. was the lowest dose to inhibit LH secretion (p < 0.05), and 2.0 and 5.0 M.O.I. exerted near complete inhibition of LH secretion; i.e. LH concentrations in these two latter GnRH-treated groups did not differ from their cohort controls (p > 0.05).
RGS3 inhibits GnRH-stimulated LH secretion. Rat anterior pituitary cells were infected or not with adeno-βgal or adeno-RGS3, and 72 h later treated with GnRH to stimulate LH secretion. Upper panel: Adeno-βgal infection was used to determine the fraction of total pituitary cells infected at each dose of adenovirus. Cell viability was measured as trypan blue exclusion. Middle panel: Adeno-βgal infection at various doses was used to control for the effect of RGS3 on GnRH-stimulated LH secretion. No dose of adeno-βgal had an effect on LH release (p < 0.05). Bottom panel: Dose-response relationship between dose of adeno-RGS3 and GnRH-stimulated LH secretion. Adeno-RGS3 doses of 0.2 M.O.I. and higher significantly inhibited LH release (p < 0.05); doses of 2.0 and 5.0 M.O.I, suppressed LH secretion to levels that were not significantly different from their cohorts not treated with GnRH (p < 0.05). Data are mean ± SEM of three independent experiments.
The profound inhibition of GnRH-stimulated LH secretion observed in Fig. 3 required verification that RGS3 expression was induced by adeno-RGS3 infection. To address this issue, we performed RGS3 immunoblots (Fig. 4) which showed an increase in RGS3 protein expression in adeno-RGS3 (5.0 M.O.I.) infected pituitary cells as compared to low to negligible levels of protein in the uninfected and adeno-βgal infected groups.
One immunoblot representative of 2 showing RGS3 expression in rat pituitary cells that remained uninfected (control) or were infected with 5 M.O.I, adeno-βgal or of adeno-RGS3. The ~70 kDa band is RGS3 (24); the bands at ~54 kDa are non-specific because they appear in all 3 lanes.
As evidenced earlier, the molecular locus of RGS protein action is thought to be the G protein α subunit, specifically Gqα in the case of GnRH receptor activation. The results shown in Fig. 5 are consistent with this conclusion: adeno-RGS3 infection of rat pituitary cells profoundly suppressed GnRH-stimulated 3H-inositol phosphate accumulation. These results are compatible with a molecular site of action for RGS3 being at phospholipase C or earlier in the signal transduction pathway.
3H-inositol phosphate accumulation stimulated by GnRH was inhibited by RGS3. Pituitary cells were infected with adeno-βgal or adeno-RGS3 (5 M.O.I.) and 72 h later were treated with GnRH agonist (10-7 M for 1 h). Data presented are the means ± SEM (n = 4). The letter a above the results of the treatment groups indicates that a group is not statistically significantly different (p > 0.05) from its control whereas the letter b indicates difference (p < 0.05) from its control group.
Another experiment conducted to test the specificity and molecular locus of RGS3 action in inhibiting GnRH-stimulated LH secretion was treatment of adeno-RGS3 infected cells with an agent that increases intracellular calcium (Fig. 6). Calcium is an established stimulator of LH secretion that is thought to act at sites downstream of phospholipase C [39, 40]. Therefore, adeno-RGS3 infected pituitary cells were treated with a calcium ionophore (A23187). Adeno-RSG3 inhibited LH secretion in the GnRH-treated control group as expected (Fig. 6). However, the calcium ionphore stimulated LH secretion in RGS3-expressing gonadotropes (Fig. 6), an effect that would be expected if the RGS3 inhibition was exerted at the level of the Gqα protein.
Calcium influx stimulated LH secretion in rat pituitary cells expressing RGS3. Calcium influx was achieved by calcium ionophore (A23187) treatment (100 μM for 3 h) of pituitary cells infected with 5 M.O.I, of adeno-RGS3. Adeno-RGS3 significantly inhibited LH secretion stimulated by GnRH (p < 0.05) but not by calcium ionophore (p > 0.05).
In two final control studies, we investigated the effect of adeno-RGS3 on LH concentrations in pituitary cells and on GnRH receptor binding (Fig. 7). In neither case did RGS3 affect these parameters of gonadotrope function (p > 0.05).
Effect of RGS3 on LH content and GnRH receptor binding. Pituitary cells were infected or not with adeno-RGS3 or adeno-βgal (5 M.O.I.) and 72 h later were used for measurement of LH content or of GnRH receptors. Upper panel: LH content of pituitary cells was measured after repeated freezing and thawing to release LH for measurement by RIA. There were no significant differences among the groups (p > 0.05). Lower panel: Pituitary cell membranes were prepared for measurement of GnRH receptors by binding to 125I-GnRH-A. There were no significant differences among the groups. Our earlier report that adeno-βgal (60 M.O.I.) increased receptor binding by 100% (23) was not observed here because of the lower dose of adeno-βgal (5 M.O.I.) used. Data are mean ± SEM of three independent experiments.
The quantity of LH secreted by the anterior pituitary gland is the outcome of an interaction between the amount of hypothalamic GnRH secretion and the level of gonadotrope responsiveness to GnRH [7]. Quantitatively, gonadotrope responsiveness is more important than the level of GnRH secretion; for example, during the estrogen-stimulated preovulatory surge of LH secretion in the rat there is a 5-fold increase in GnRH secretion but a 50-fold increase in LH secretion [7]. Moreover, a quantity of GnRH that will evoke a normal preovulatory LH surge during the period preceding ovulation will induce only a small increase in LH secretion at other stages of the estrous cycle [7]. Similarly, termination of the preovulatory LH surge appears to be due to desensitization of the gonadotrope to GnRH action; in the sheep, a decrease in LH secretion precedes the decrease in GnRH secretion [41], whereas in the rat, although the decrease in GnRH and LH secretion are co-incident [7], there is evidence that the gonadotrope is already desensitized to GnRH [42, 43].
The mechanisms that alter gonadotrope responsiveness to GnRH are unknown but they do not appear to be due to GnRH receptor number or LH stores in the pituitary gland [5–8]. However, the fraction of LH that is released by a maximal dose of GnRH does vary positively with gonadotrope responsiveness [7], and elucidation of the mechanisms altering the fraction of releasable LH is likely to illuminate mechanisms of gonadotrope responsiveness to GnRH [44].
Two molecular loci recently have been implicated as potential modulators of GnRH receptor signaling: one is the receptor itself [23], and the other is the G protein that transduces the signal generated by binding of the receptor to its agonist [9]. With respect to the former locus, G protein coupled receptor kinases (GRKs) act in an agonist-specific manner to phosphorylate intracellular regions of the receptor thus permitting β arrestin binding that sterically inhibits G protein association with the receptor [1, 45]. We have reported previously that experimental expression of GRKs in GnRH receptor-expressing heterologous cells (COS-1) suppressed GnRH-stimulated IP3 production, and that co-expression of GRK2 and β-arrestin 2 suppressed GnRH receptor signaling more than that of either alone [23]. Stronger evidence that the GRK/β arrestin paradigm may play a role in regulating GnRH receptor signaling is our recent finding that adenovirus-mediated gene transfer of GRK2 into normal pituitary gonadotropes suppressed GnRH-stimulated LH secretion and IP3 production [31]. However, no evidence has been presented for a direct interaction between β-arrestin and the GnRH receptor.
With respect to the G protein as a locus for modulation of GnRH receptor signaling, we reported earlier that experimental expression of RGS3 but not RGS1, 2, or 4 in GnRH receptor expressing COS-1 cells suppressed GnRH-stimulated IP3 production [9]. In the current report, we present evidence strengthening the hypothesis of a regulatory role for RGS3 in GnRH receptor signaling: adenovirus-mediated expression of RGS3 in normal pituitary gonadotropes profoundly inhibited GnRH-stimulated LH secretion and 3H-inositol phosphate accumulation. Arguing for the specificity of this effect is the stimulation of LH secretion by a calcium ionophore and the failure of LH stores and GnRH-receptor binding to be affected by RGS3. Moreover, our finding that GnRH-stimulated IP3 production was inhibited supports the locus of the RGS3 effect as residing at phospholipase C or earlier in the transduction pathway, most likely at the Gα protein because of our studies both earlier [9] and here showing that RGS3 binds Gqα in vitro. Finally, the stronger inhibitory effect of RGS3T than RGS 3 on COS cell function was presumed to be true also for gonadotrope function and therefore was not tested here.
The determination of RGS specificity for receptor-G protein pairs are currently unknown; i.e., we and others have found that several RGSs interact with Gqα but only one, RGS3, has potent inhibitory activity on GnRH receptor signaling. For instance, RGS4 and RGS-GAIP [11], RGS3 [9], RGS2 [46], and RGS7 (our unpublished findings) all bind Gqα, the established G protein that transduces GnRH receptor signaling [38]. Nevertheless, RGS2 and 4 [9], and RGS-GAIP and RGS7 (our unpublished data) are weak or null inhibitors of GnRH receptor signaling. A recent report seems to have clarified this issue: RGS4 must bind not only Gqα but also selected Gq-coupled receptors before it can inhibit receptor signaling [47, 48]. Based on this finding, we propose that RGS3's binding to the GnRH receptor as well as to Gqα accounts for its specific inhibiting of GnRH receptor signaling.
The results of the current investigation as well as those presented earlier [31] nominate two molecular mechanisms as playing roles in setting the responsiveness level of GnRH receptor signaling; i.e., the RGS3 system acts at the G protein transducing step and the GRK/β arrestin system acts at the level of the receptor. Future studies must address whether both, neither, or only one of these mechanisms are physiologically relevant.
cDNA transfection of COS-1 cells and measurement of IP3 production
cDNA representing the rat GnRH receptor was cloned into pcDNA3 as described and validated previously [22, 23]. cDNAs representing RGS3 and RGS3T (RGS3 amino acids 314–519) were cloned into the eukaryotic expression vector, rcCMV, as described [24]. COS-1 cells cultured as detailed before [23] were co-transfected with GnRH receptor cDNA and RGS3 or RGS3T cDNA using Lipofectamine (Life Technologies, Gaithersburg, MD) [23], and 72 h later, treated with 10-7 M GnRH-A for 0, 10, 20, or 30 sec before being collected for IP3 measurements [22, 23].
IP3 measurements were performed on COS-1 cell cytosolic preparations using a radioreceptor assay in which IP3 and 3H-IP3 compete for binding to IP3 receptors prepared from calf cerebellum membranes [22, 23].
RGS3 binding of Gα-proteins in vitro
RGS3 protein fused to glutathione-S-transferase (RSG3-GST fusion protein) was produced in the pGEX-5x-1 system of Pharmacia as detailed previously [9]. 35S-Met labeled Gα-proteins were prepared in vitro using a transcription/translation protocol [9]. cDNAs for Gqα [25] and Gzα [26] were obtained from M. I. Simon; for Gsα (ref. No. 63315) and Gi2α (ref. No. 63311) were obtained from the American Type Culture Collection (ATCC); for Gαo [27] was obtained from Eva G. Neer; for Gi1α, [28] from G. R. Post; and, for Gi3α [29] from M. G. Farquhar. Binding studies of recombinant RGS3-GST fusion protein to 35S-Met-Gα-proteins were performed as detailed [9]. Glutathione-coupled beads were used to select 35S-Gα proteins bound to RGS3-GST, and were then eluted for separation by SDS/PAGE [9]. The amount and molecular size of 35S-Gα protein were determined using a Phosphor imaging device (Molecular Dynamics).
Preparation of Adeno-RGS3
Recombinant adenovirus containing RGS3 cDNA was prepared using procedures previously described in detail by Becker et al.[30] and by us [31]. Human RGS3 cDNA [24] was restricted from the pRC/CMV vector with EcoRI, and then subcloned into the corresponding site of the pACCMVpLpa plasmid. The proper orientation of the RGS3 cDNA was confirmed by nucleotide sequencing. The procedure for preparation of the adenovirus was co-transfection of a human embryonic kidney cell line (HEK 293) with the pACCMVpLpa plasmid and another plasmid (pJM17) containing most of the viral genome [30]. A relatively rare recombination of the two plasmids occurs in the HEK 293 cells that reconstitutes the adenoviral genome. The recombination event reconstituting adeno-RGS3 was confirmed using PCR analysis to detect the presence of RGS3 cDNA. Next, adeno-RGS3 was isolated by clonal selection in HEK 293 cells followed by a second PCR to confirm the presence of RGS3 cDNA. Finally, large-scale amplification and titering of adeno-RGS3 was performed in HEK 293 cells. This virus preparation was used for infection of rat pituitary cells [31].
Culture and infection of rat pituitary cells
Anterior pituitary glands were obtained from female Charles River CD rats that were maintained in accordance with the NIH Guide for the Care and Use of Laboratory Animals using an experimental protocol that had been approved by the Institutional Animal Care and Use Committee. The pituitaries were dispersed with trypsin as described before [32]. The mono-dispersed cells were plated into poly-L-lysine coated 60 mm plastic Petri dishes in DMEM/10% gelding horse serum containing 10-9 M estradiol to optimize the GnRH-responsiveness of the gonadotropes [33], and incubated overnight at 37°C in 8% CO2-92% air.
Pituitary cells were infected using a volume of adeno-RGS3 or adeno-β galactosidase necessary to achieve the chosen multiplicity of infection (M.O.I.) diluted to 0.3 ml; this was further diluted with 1.2 ml of TS buffer (8.0 gm/L NaCI, 380 mg/L KCl, 100 mg/L Na2HPO4, 3.0 gm/L Tris HCI, 200 mg/L CaCl2· 2H2O, 100 mg/L MgCl2 · 6H2O; pH 7.4). The pituitary cell culture medium was aspirated and replaced with the virus solution, and the cells were incubated at 37°C in a CO2 incubator for approximately 72 h.
Next, the cells were collected by trypsinization and counted using a hemocytometer; then, they were checked for viability using trypan blue [31]. The cells were replated for 2 h at 37°C in DMEM -0.1% bovine serum albumin (BSA) and subjected to x-gal staining for determination of the fraction of cells infected by the virus [31], or to stimulation by GnRH-A for measurement of LH release.
GnRH-stimulated LH secretion
Pituitary cells that had been re-trypsinized 72 h after infection and then replated (100,000 cells/well in a 24-well poly-L-lysine coated plate) for a 2 h preincubation were subsequently treated with a GnRH agonist (1 ml of 10-7M D-Ala6 – des Gly10 – GnRH ethylamide; GnRH-A, Sigma, St. Louis, MO) or a calcium ionophore (A23187, Sigma) for 3 h as described by Conn et al.[34]. The medium was removed and centrifuged at 200 × g for 10 min to pellet any detached cells. The supernatant solution was either used immediately or stored at -20°C prior to RIA.
LH RIAs were performed using reagents and instructions provided by Dr. A. F. Parlow on behalf of the National Hormone and Pituitary Program, National Institute of Diabetes and Digestive and Kidney Diseases, NIH. Rat LH (NIDDK-rLH-l-9) was radioiodinated, the LH antiserum (NIDDK-anti-rLH-S-11) was prepared in rabbits, and the reference standard was NIDDK-rLH-RP3. The mean EC50 of the standard curves (n = 10) was 0.59 ng with a coefficient of variation of 4.4%.
GNRH-stimulated 3H-inositol phosphate accumulation in pituitary cells
Total [3H] inositol phosphate accumulation was determined in [3H] myoinositol-labeled pituitary cells as described earlier [22]. At 48 h after infection of the pituitary cells, the incubation medium was removed and replaced with a balanced salt solution (10 mM Hepes, pH7.4, 135 mM NaCI, 4.5 mM KCL, 1.5 mM CaCl2, 0.5 mM MgCL2, 1.0 mM NaH2PO4, 5.6 mM glucose, 0.3% BSA, and 10-9M estradiol) containing 5 μCi of [3H]-myoinositol/ml of medium. After a further 24 h, the pituitary cells were recovered by trypinization, counted, and replated in balanced salt solution (400,000 – 500,000 cells/well in a 24-well plate) for a 2 h preincubation. Then, the cells were stimulated with GnRH-A (10-7M for one hr) in the presence of 5 mM LiCI. [3H]-inositol phosphate accumulation in the cells was then measured using ion exchange chromatography followed by liquid scintillation spectroscopy [22].
Immunoblots for RGS3
An antibody to hexa-histidine-tagged human RGS3T was prepared in chickens. RGS3T [24] is the carboxy terminal 206 amino acids (a.a. 314–519) of RGS3 and is represented by nt 1227–1847 of RGS3 cDNA (Genbank Accession #U27655). To prepare hexa-histidine amino terminally-tagged RGS3T, we modified RGS3T cDNA [24] using PCR with primers containing restriction sites (the 5' primer incorporated a Bam HI site, whereas the 3' primer bore an Nde 1 site). The PCR product was then cloned into the pET15b vector which carries an N-terminal Histag sequence followed by BamH1 and Nde 1 cloning sites (Novagen Inc., Madison, WI). The resulting plasmid DNA was nucleotide-sequenced to confirm the presence of RGS3T cDNA. The cDNA was then used to transform E. coli of the BL21(DE3) strain. Protein induction was performed using isopropyl-β-D-thiogalactopyranoside (IPTG) as described by Novagen Inc. Inclusion bodies containing the protein were prepared and then solubized in 6 M guanidine. The Histag RGS3T protein was purified on a Ni2+ resin using a Pharmacia Hi-Trap 1 ml Chelating Column (Pharmacia Biotech, Piscataway, NJ).
Antibodies to Histag RGS3T protein were prepared in chickens (Aves Labs, Inc., Tigard, OR). A chicken was injected with 145 μg of RGS3T into the breast muscle on four separate occasions; the first injection was in complete Freund's adjuvant, whereas injections 2–4 were made in a 1:1 dilution of complete and incomplete Freund's adjuvant. One week after the last injection, 6 immune eggs were collected, and the IgY fraction purified from the 6 eggs as well as from 3 pre-immune eggs collected from the same hen.
Immunoblots for RGS3 expression in pituitary cells were performed as described previously by us [22, 23]. In brief, rat pituitary cells at 72 h after adeno-RGS3 infection (5 M.O.I.) were collected by trypsinization, homogenized, and a protein determination made. Fifty μg protein samples were heated in SDS-PAGE sample buffer containing β-mercaptoethanol and then electrophoresed on a 10% SDS-polyacrylamide gel. Separated proteins were electroblotted onto nylon membranes (Millipore Corp., Bedford, MA) which were then incubated for 2 h in a blocking solution containing 5% non-fat dry milk in phosphate buffered saline containing 0.02% Tween-20. The primary antibody (chicken IgY, 62.5 μg/ml) was diluted in blocking solution and incubated overnight at 4°C with the nylon membrane. Blots were then incubated for 2 h at room temperature in alkaline phosphatase-conjugated goat anti-chicken IgY (0.1 μg/ml diluted in blocking solution; Aves Labs, Tigard, OR). Color development was achieved by incubating the membrane in substrate [(5-bromo-4-chloro-3-indolyl phosphate-nitrobluetetra-zolium chloride) (BCIP-NBT)].
Assay of LH contents in pituitary cells
For LH RIA measurements of cell contents, 1 × 105 anterior pituitary cells infected, or not, 72 h previously with adeno-RGS3 or adeno-βgal were trypsinized and plated for 2 h. LH was extracted from the cells [36] by removing and discarding the medium and adding 1 ml extraction buffer (150 mM NaCI, 50 mM Tris, 5 mM EDTA, and 1 mM bacitracin, pH 7.4) followed by freeze-thawing twice. After centrifugation, the supernatant was removed and subjected to LH RIA as described above.
Measurements of GnRH receptors by receptor radioassay (RRA)
About 3 × 106 pituitary cells infected, or not, 72 h previously with adeno-βgal or adeno-RGS3 were trypsinized, and cell membranes containing the GnRH receptors were prepared by placing the cells in a Dounce homogenizer (Kontes Co., Vineland, NJ) and disrupting them with 30 strokes of the pestle. The cell membranes were pelleted by centrifugation at 26,000 × g and then resuspended in 10 mM Tris buffer. The GnRH RRA on the membranes was performed as described previously [22, 23]. Cell membranes and 1 × 105 cpm [125I]GnRH-A were incubated at 0°C for 90 min. Nonspecific binding was determined as counts per min bound in the presence of 10-6 M unlabeled GnRH-A. Bound and free [125I]GnRH-A were separated by filtration. The filters were counted by γ-scintillation spectrometry.
The results are presented as the mean ± SEM of at least three independent experiments unless indicated otherwise. The statistical tests were one-way or two-way analyses of variance with significant differences (p < 0.05) identified by the Student – Newman-Keuls method. Statistical analysis was performed using Sigma Stat Statistical Software for Windows (Jandel Scientific, San Rafael, CA).
Supported by NIH Grant 1-R01-HD 34862.
Department of Physiology and Biophysics, University of Alabama School of Medicine, Birmingham, AL 35294, USA
Laboratory of Immunoregulation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA
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What to be a member of a group of high school students that come together to have fun and promote healthy lifestyle choices?
CHILL Youth Task Force is the group for YOU!
Maintain acceptable school attendance and G.P.A.
Applications are available in the guidance office or main office during the application process time, usually March, April of each school year. Check with your guidance counselor or the main office for more information.
The application directions will be included in the packet. Fill out the application, get 2 teacher recommendations, accept the contract requirements (including parent/guardian permission) and attend the interview night for new CHILL prospects!
Because there are usually more applicants than available spots open in CHILL, there is a selection process based on application, teacher recommendations, and the interview. CHILL prospects will be sent a letter within 7-10 days after the interview takes place letting them know whether or not they have been accepted into CHILL.
After being accepted, there is one last step. To become "official" new members MUST attend a three-day training session during the summer. Your letter of acceptance will have a list of several training dates to choose from.


text:
Q: Updating list of dictionaries with another list of dictionaries. Is there a faster way? I have a list of dictionaries that I need to update with information from another list of dictionaries. My current solution (below) works by taking every dictionary from the first list and comparing it to every dictionary in the second list. It works, but is there a faster, more elegant way of achieving the same result?
a = [ { "id": 1, "score":200 }, { "id": 2, "score":300 }, { "id":3, "score":400 } ]
b = [ { "id": 1, "newscore":500 }, { "id": 2, "newscore":600 } ]
# update a with data from b
for item in a:
    for replacement in b:
        if item["id"]==replacement["id"]:
            item.update({"score": replacement["newscore"]})

A: Create a dictionary indexed by id using the first array.
Loop through the second array using the id.
for replacement in b:
   v = lookup.get(replacement['id'], None)
   if v is not None:
      v['score'] = replacement['newscore']

This converts an O(n^2) problem to an O(n) problem.

A: Instead of doing a len(a) * len(b) loop, process b into something easier to work with:
In [48]: replace = {d["id"]: {"score": d["newscore"]} for d in b}

In [49]: new_a = [{**d, **replace.get(d['id'], {})} for d in a]

In [50]: new_a
Out[50]: [{'id': 1, 'score': 500}, {'id': 2, 'score': 600}, {'id': 3, 'score': 400}]

Note that the {**somedict} syntax requires a modern version of Python (>= 3.5.)

A: List Comprehension:
[i.update({"score": x["newscore"]}) for x in b for i in a if i['id']==x['id']]
print(a)

Output:
[{'id': 1, 'score': 500}, {'id': 2, 'score': 600}, {'id': 3, 'score': 400}]

Timing:
%timeit [i.update({"score": x["newscore"]}) for x in b for i in a if i['id']==x['id']]

Output:
100000 loops, best of 3: 3.9 µs per loop

A: If you are open to using pandas and a, b are pandas dataframes then here is a oneliner
a.loc[a.id.isin(b.id), 'score'] = b.loc[b.id.isin(a.id), 'newscore']

Converting a, b to dataframes is simple, just use pd.DataFrame.from_records

Another way of doing this if you can change "newscore" to "score"
a = pd.DataFrame.from_records(a, index="id")
b = pd.DataFrame.from_records(b, index="id")
a.update(b)

Here are the timeit results
In [10]: %timeit c = a.copy(); c.update(b)
702 µs ± 37.3 µs per loop (mean ± std. dev. of 7 runs, 1000 loops each)

A: First create a dict of scores to update:
>>> new_d={d['id']:d for d in b}
>>> new_d
{1: {'id': 1, 'newscore': 500}, 2: {'id': 2, 'newscore': 600}}

Then iterate over a and update by id:
for d in a:
    if d['id'] in new_d:
        d['score']=new_d[d['id']]['newscore']

>>> a
[{'id': 1, 'score': 500}, {'id': 2, 'score': 600}, {'id': 3, 'score': 400}]

Which could be even simpler as:
new_d={d['id']:d['newscore'] for d in b}
for d in a:
    if d['id'] in new_d:
        d['score']=new_d[d['id']]

redpajama_set_name: RedPajamaStackExchange


Durability-loss at clang should depend on what weapons are used.
Discussion in 'General Discussion' started by Zael, Sep 1, 2018.
It's ridiculous for a knife to survive more than one clang with a powerfist or powermaul.
One guy has a state-of-the-art piece of high-tech that can punch tanks around.
The other guy has a pointy piece of metal.
Grammaticus_John and Data8671 like this.
Combat Knife not breaking the instant it makes contact with the Power Fist, then partially severing head from shoulders before planting skull 3 feet into the wall behind it completing the severance is the equivalent to someone expecting to parry Exterminatus with their Fist.
DrDooManiC and Atsidas like this.
Common sense is the collection of prejudices acquired by the age of eighteen , it varies between people.
Sumshine and Atsidas like this.
Aislinn and Krayt like this.


Jenzabar Introduces New Advancement Solution to Propel Fund-Raising Success.
BOSTON -- Jenzabar, Inc., a leading provider of software, services and strategies for higher education, announced today a pilot program for FrontRunner by Jenzabar[R], a new institutional advancement system with progressive features to manage advancement activity, engage constituents, and cultivate stewardship. The FrontRunner by Jenzabar system provides comprehensive advancement functionality in a flexible service-oriented architecture to help organizations achieve fund-raising success in this new era of constituent and donor engagement.
Users of FrontRunner by Jenzabar interact with the system anytime, anywhere through popular Web browsers. Advancement professionals can improve their effectiveness with quick and easy access to their schedules and complete prospect and constituent profile information. The integrated reporting in FrontRunner equips staff and leaders with the data and business intelligence needed to execute successful campaigns. Networking features, such as online communities and events management, help institutions strengthen alumni affinity and increase constituent engagement.
Four institutions are participating in the pilot program including Spring Arbor University, which offers programs at 14 locations throughout Michigan and in Ohio.
"We are excited to be partnering with Jenzabar as one of the pilot schools for FrontRunner by Jenzabar. This new institutional advancement solution will greatly enhance the work of the Office of Advancement at Spring Arbor University," said Rhonda Saurbek, Assistant Vice President for Advancement Operations. "This new Web-based product will allow our casual user to navigate the alumni, donor, and prospect database with more ease, with the information displayed in a way that is easy to disseminate. For the power user, data entry and gift receipting will be more intuitive and will cut down extensively on the amount of time needed to enter and process information."
FrontRunner by Jenzabar runs on multiple hardware platforms and works with multiple database languages. It can be implemented as a stand-alone advancement system or as a fully-integrated component of Jenzabar's Java enterprise resource planning system. In addition to the full set of advancement functionality, the extensible technology platform of FrontRunner by Jenzabar enables integration and partnering; institutions can easily incorporate other components into FrontRunner, including services that run in the cloud.
"Jenzabar has embraced a new product development paradigm that combines our expertise in the advancement needs of higher education with the best open standards tools available to deliver highly functional, highly adaptable technology," said Robert A. Maginn, Jr., Jenzabar Chairman and Chief Executive Officer. "FrontRunner by Jenzabar's service-oriented architecture delivers an extensible platform to harness the value of institutional data with the power and flexibility of technology to achieve advancement success."
Newegg and Zayo Enter into Multi-Year Colo Agreement.
David V. Campbell Joins Avidbank Board of Directors.


text:
package fi.uta.fsd.metka.storage.repository;

import fi.uta.fsd.metka.enums.ConfigurationType;
import fi.uta.fsd.metka.model.configuration.Configuration;
import fi.uta.fsd.metka.model.general.ConfigurationKey;
import fi.uta.fsd.metka.model.guiconfiguration.GUIConfiguration;
import fi.uta.fsd.metka.storage.repository.enums.ReturnResult;
import org.apache.commons.lang3.tuple.Pair;
import org.springframework.transaction.annotation.Transactional;

import java.util.List;

@Transactional(readOnly = true)
public interface ConfigurationRepository {
    @Transactional(readOnly = false) public ReturnResult insert(Configuration configuration);
    @Transactional(readOnly = false) public ReturnResult insert(GUIConfiguration configuration);/*
    @Transactional(readOnly = false) public ReturnResult insertDataConfig(String text);
    @Transactional(readOnly = false) public ReturnResult insertGUIConfig(String text);*/
    public Pair<ReturnResult, Configuration> findConfiguration(String type, Integer version);
    public Pair<ReturnResult, Configuration> findConfiguration(ConfigurationType type, Integer version);
    public Pair<ReturnResult, Configuration> findConfiguration(ConfigurationKey key);
    public Pair<ReturnResult, Configuration> findLatestConfiguration(ConfigurationType type);/*
    public Pair<ReturnResult, Configuration> findLatestByRevisionableId(Long id);
    public Pair<ReturnResult, Configuration> findConfigurationForRevision(Long id, Integer revision);
    public Pair<ReturnResult, GUIConfiguration> findGUIConfiguration(String type, Integer version);*/
    public Pair<ReturnResult, GUIConfiguration> findGUIConfiguration(ConfigurationType type, Integer version);
    public Pair<ReturnResult, GUIConfiguration> findGUIConfiguration(ConfigurationKey key);
    public Pair<ReturnResult, GUIConfiguration> findLatestGUIConfiguration(ConfigurationType type);

    public List<ConfigurationKey> getDataKeys();
    public List<ConfigurationKey> getGUIKeys();
    public Pair<ReturnResult, String> getDataConfiguration(ConfigurationKey key);
    public Pair<ReturnResult, String> getGUIConfiguration(ConfigurationKey key);
}

redpajama_set_name: RedPajamaGithub


a. the supplier(s) is selected.
b. price and terms are determined.
c. potential suppliers are analyzed.
d. needs are recognized and described.
e. the buyer follows-up and expedites the order.
a. request for quotation (RFQ).
b. request for proposal (RFP).
c. request for information (RFI).
d. request for bid (RFB).
e. request for suggestions (RFS).
b. the advantages from economies of scale.
c. visibility up- and downstream in the supply chain.
d. the ability to automate and facilitate transactions.
a. to give users a way to authorize buyers to acquire goods and services.
b. to solicit price quotes for goods or services from suppliers.
c. to give finance a way to have the final say in any decision to spend money.
d. to clarify the description of need before communicating with potential suppliers.
e. to request or requisition supplies from external suppliers.
a. eliminate bar coding and manual counting.
b. eliminate further investment in information technology.
c. supplement bar coding to reduce theft.
d. be more cost effective to implement than bar coding.


BipCoin Windows GUI wallet v.0.0.0.4 is out now. (Linux users can also build Linux version from our source.) Unlike our CLI wallets, this GUI wallet will work on both 32-bit and 64-bit Windows. You cannot mine on our GUI wallet (that requires the CLI version), but they are much easier to transact on than the CLI version.
If you never had a previous version of BipCoin wallet, skip this part. if you DID have a previous version of BipCoin wallet, backup your wallet files from your old wallet folder, these wallet files (bipcoinwallet.wallet, bipcoinwallet.addressbook, bipcoinwallet.cfg). Then delete your old blockchain files from Windows at C:\Users\(ComputerUserName)\AppData\Roaming\bipcoin (deleteeverything in that folder). Because your new install will re-download the blockchain. This will prevent errors associated with .0003. This shouldn't take that long at this point, maybe 60-90 min, depending on network speed.
Download and unzip to a folder.
You will probably get some warnings, depending on what anti-virus software you have, and what your firewall settings are (this is common with a new program). You'll get something like this, below.
For some other anti-virus programs, it may require you to click "allow" or something like that.
Check both boxes, then click Allow Access. The wallet with take about 30 seconds to open.
If you have already installed the CLI wallet v0.0.0.4, you already have the blockchain downloaded, and your wallet should sync almost immediately to the correct block height.
After the first time, it will take a lot less time to sync when you open your wallet, especially if you've opened your wallet recently.
Once you're synced you can begin transacting.
Allow them to overwrite the files already in that folder from installing the new wallet.
If you never had the wallet before, you're fine and don't need to do that.
The wallet has an address book, where you can keep frequently used BipCoin addresses.
If you also have the CLI wallet, please note that on most systems you can open the GUI while the CLI wallet is running. But you cannot open bipcoind to open the CLI wallet while GUI is running.
Note the "anonymity level" slider on the GUI wallet is equivalent to the "mixin" in the CLI wallets.
Happy transacting, enjoy, and tell two friends!
-The BipCoin team of happy hamsters.
The BipCoin will roll right in!
Posted byCatBot October 28, 2016 November 18, 2016 Posted inNewTags: I AM THE CAPTAIN NOW!


text:
My name is Patrick and I am writing about things.
The world's best croisasnt
February 14, 2019 ~ Pat Boxall
A writer at the New York Times declared Lune Croissanterie, in Fitzroy, the home of the world's best croissant and the Melbourne institution has now reached unprecedented levels of pastry prominence. I've heard rumours of these croissants: croissants so buttery you'd wish yourself bulimic just to taste them twice, croissants so flaky you wouldn't bother inviting them anywhere. The French Government vehemently denies their existence, but I'm about to have one in my grubby little hands.
I take my place in line at the intersection of two inner-city streets. A group of girls have gathered across the road, taking photos of the fools lining up for a croissant. It was embarrassing, being stuck in this line, until moments ago a little girl pushed past on a Razor scooter and fell flat on her face. She stood up, kept going and fell over again. The tears came ¬– hers, not mine – and I whispered thank you, convinced that she's an angel sent to make me feel like less of a dickhead. She walked away with her dad, both her nose and dreams broken, and perfect tears streamed down her angelic face. I can't be sure, but at one point she turned back, winked and said: 'Get that pastry, Patrice.'
I've never seen so many Insta-hungry croissant connoisseurs, henceforth known as cronnoisseurs, in my life. In fact, I've never seen so much as one until today. They're patiently waiting for a chance to get a photo next to the Lune logo on the building's brick exterior, while the order of the day inside is croissants, naturally, with a side of Boomerangs. These people are Boomerang-ing everything.
'Is this a fucking joke?' says the guy behind me, 'why are people lining up for this, do they even serve food?'
'It's a croissanterie babe,' says his partner, 'they serve croissants.'
She's done her research.
In the middle of the converted warehouse sits a glass cube in which a chef forms fresh, buttery treats. Tourists press their noses to the glass like children at the zoo, phones held high to capture the excitement of the morning bake. To the left, there's a rack of official merchandise: not only does Lune make branded shirts, tote bags and socks, but people are actually buying them, buying a pair of socks branded with a croissanterie which, while it's a fun word to say, doesn't historically scream fashion icon.
There's a young guy double-parked near the merchandise, with a sugar-dusted almond croissant in his left hand and a traditional butter beauty in his right. He takes a bite of each then swaps with his friend, whose fingers drip with the innards of a greasy ham and cheese number. There are, it seems, no rules in Croissant Club. It's disgustingly beautiful.
After 20 minutes and 15 seconds of waiting, I bite into the world's best croissant and you know what? It's fairly good. The world's best? It's up there, but the 20 minute wait leaves a sour, rather than buttery, taste in the mouth and besides, I'm uncomfortable with people proclaiming something as the world's best. It happens all the time in country towns, and while I don't doubt that the pie shop in Kangaroo Valley, NSW, makes phenomenal pies, I'm sceptical that anyone could sample a pie from every bakery and make an informed decision on the "World's Best Pie" without suffering a coronary. At least they'd die doing what they loved.
Maybe that's why I'm not in sales. "Really Decent Pies, Depending on Your Taste in Pies," isn't the most impressive tagline. Based off no evidence other than my own observations, it seems that the less qualified someone is to proclaim something as the world's best, the more likely they will. To definitively rank something as broad and personal as the taste of croissants, or even pies, it helps to have experienced as few as possible because unshakable conviction often depends on a lack of exposure.
There's a bakery near my house with a sign that reads "The best croissants in the world: as voted by my cousin and two of his mates." Though it's certainly convincing, questions arise like a curious erection.
What authority, for example, do the two mates lend to the cousin's original claim? And furthermore, if only one of his mates agreed, could the cousin still justify his claim? After all, the New York Times writer is just one person, how can his views stack up to a group consensus? Regardless, I've stopped going because I feel uncomfortable. Not so much with the hyperbole, it's more that something smells off and that something, it would seem, is not me.
I went in a few months ago for a flaky fix and the young girl serving asked what perfume I was wearing. I didn't know the name but I promised to let her know next time, and like most promises I make to the important women in my life, of which there aren't many, I soon forgot about it.
The next time I saw her, it became immediately apparent that she had not, like me, forgotten about our conversation. We were two dogs at the park: me trying to go about my business as she sniffed around, subtly at first, but becoming more and more aggressive until finally, unexpectedly, her nose was in my metaphorical arsehole. I stepped back from the counter in a state of confusion and concern.
'Your perfume,' she said, 'what is it? You said you'd find out.'
'It's called Fuel for Life,' I said, waiting for her to hand me my literal fuel for life, the world's best croissant. It's a simple action, the handover, and one might say it's key to running a successful bakery, but the more I saw her the more difficult this transaction became.
I went in one day and she was working with another girl. 'This is the guy,' she said to her colleague when I ordered, 'the one who smells nice. Smell him.'
I stood still, awkward, but no more awkward than usual.
'You'll have to stop coming here,' she continued, 'you smell too good.'
'I will if you don't give me my croissant soon,' I said.
I haven't been back since.
I remember the day my family realised I needed to wear deodorant. I was 13, a good decade before I hit puberty, and we were in a hotel room in Berlin. I was wearing a Wallabies' rugby jersey – probably had been for several days – and they were trying to locate the source of a horrific funk, which turned out to be my armpits. I had never had such a powerful tool at my disposal and they begged me to stop as I ran around the room putting everything I could find under my armpit. Nothing was safe, nothing was sacred and nothing was spared. It was, they remind me, the world's worst smell.
I like to think I've come a long way since Berlin. Geographically, yes, but also when it comes to musk. I now realise that it's nice to smell nice, but it's not nice to be smelt no matter how nice you may smell. There's a sandalwood cross that's mine to bear and the weight of expectation is too heavy, which has left me, predictably, in a pastry predicament.
On the one hand, I have a local croissant that was voted the world's best by three unverified sources, but to enjoy said croissant I must maintain a level of personal hygiene I'm not willing to commit to and feel uncomfortable seeking my food-based comfort.
On the other hand, I have the world's best croissant as judged by a respected publication, but the consumption of a croissant, ultimately, comes down to experience rather than taste. It should be simple, it should be stress-free, and having to wait 20 minutes for a croissant consequently discounts that croissant from being the world's best.
I guess I'm in a bit of a jam.
croissantfoodfrancemelbourne
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Look: I'm throwing all the hopes and dreams of my twenties in the air and they're tumbling down the valley with everyone else's. It's an unstoppable avalanche of regrets and failures! Hoorah! But now that I'm spending the first day of my 30s in bed with fake malaria/exhaustion/aggressive syphilis (I wish hehe 🙈), now's as good as time as any to say thanks for not only your messages, but also your general existence. I've been pretty lucky to spend an inordinate amount of time travelling and working overseas and it sometimes feels like I've got a thousand acquaintances and minimal mates left, but y'all are always there when needed, even if we don't see each other for 1/2/3/5/10 years. If it weren't for you I'd be tumbling down that valley too so thanks for, I dunno, agreeing to get a beer with me every 9 months. It means a lot. Ps. I now live in Melbourne again and need one wife, one daughter and one affordable property by the end of the year. Let me know if you know someone xo
And that's just about a wrap for Vanuatu. Four weeks of planting the seeds of existential dread in hopeful young hearts and teaching them that life comes in swings and roundabouts, but mainly swings, and we should attack said swings with enthusiasm and, more importantly, Birkenstocks. But now it's time to swing into the silly season and let it be known that I swing some ways - see you shortly 'straya.
This is Faith, and this is Destiny. I asked them to paint me like one of their colonialist French girls. One drew a cat, the other drew a bright pink Patrick. Safe to say they nailed the brief. #hotcreativesbeinghotandcreative #vanuatu #lelepa #moso
Welcome to Vanuatu. I'm waiting for you. #hotguysonasolohoneymoon #livelovelife #travelchangedme
Went full Britney, shaved me head and made tracks to Vanuatu. Haven't slept in 36 hours but my new best mate Seheety is taking me out at 3am to catch some 60kg Yellowfin and I'm assuming that he's referring to fishing. Man stuff, y'know? I've also made really good friends with Donovan and Ricky and Freddie and this kid called Rambo, I think? They're not in the photo because they're in their underpants. Probably for the best. If you need me I'll be working on Lelepa Island and around Vanuatu for the next four weeks. Drop by. The sunsets are better than my fat dumb head. Promise. Actually here are photos of my new friends. They are shouting Captain America and jumping into shallow water. I feel like a good influence.
Now is as good a time as any. ⠀⠀⠀⠀⠀⠀⠀⠀⠀ It's an important announcement. @ellielinaker and I have adopted @osmancanaltan as our son. ⠀⠀⠀⠀⠀⠀⠀⠀⠀ We are renaming him Scott. ⠀⠀⠀⠀⠀⠀⠀⠀⠀ He's going to play cricket. ⠀⠀⠀⠀⠀⠀⠀⠀⠀ And study medicine. ⠀⠀⠀⠀⠀⠀⠀⠀⠀ Not really. ⠀⠀⠀⠀⠀⠀⠀⠀⠀ Says he wants to be an artist. ⠀⠀⠀⠀⠀⠀⠀⠀⠀ But we are going to have a talk. ⠀⠀⠀⠀⠀⠀⠀⠀⠀ This is actually an announcement that my favourite shirt has been officially declared lost by Norwegian Airlines. ⠀⠀⠀⠀⠀⠀⠀⠀⠀ I just don't have the emotional strength to talk about it. ⠀⠀⠀⠀⠀⠀⠀⠀⠀ But if I could hide the pain like that shirt hid my love handles. ⠀⠀⠀⠀⠀⠀⠀⠀⠀ I'd handle losing the love of my life better. ⠀⠀⠀⠀⠀⠀⠀⠀⠀ Enjoy your curry-soaked grave, shirt. ⠀⠀⠀⠀⠀⠀⠀⠀⠀ I'll never forget what we had. #hotguystearingup #hotguysmakinghottereuolgies PS. Cheers @spence_luke for the shorts. Your loss goes some way to finding peace.
On the surface, this is me mansplaining thermodynamics and how safe paragliding is despite having absolutely no experience and a well-documented history of hating heights. . Dig deeper, however, and this is me telling you that I'll be touching down in Australia on Friday morning and gee I'm looking forward to seeing some of your beautiful and slightly weathered faces. . Dig even deeper, and this is me telling you that Europe has changed me and other lies, which also happens to be the name of my soon-to-be-considered-for-publication-and-ultimately-rejected collection of technically lacking poems and overly wordy short stories. . Dig deepest, and this is me proving that nothing changes: I still don't know what to do with my hands. And you know what the saddest thing is? That shirt, as well as my sensible hat, has been lost in transit along with my entire hiking pack somewhere in Norway. And you know what's actually even sadder than the saddest thing? The pack in question contains a delicious green curry that I cooked the night before it went missing and now, 15 days later, I'm starting to worry that even if my bag does turn up, the curry may no longer be edible. Fuck Europe. . #hotguysmissinghotcurries #hotguyscominginhottohotcountries #hotguyshanginguptheirboots #whichareactuallylostintransit
Voted Europe's most professional and least knowledgable tour guides one season in a row. Coming soon to a pub near you. #busaboutcrew
Cornered by the media in the shadow of Nidaros Cathedral, Mr Boxall was asked to comment on his transformation from the Turkish @osher_gunsberg to what some sources are calling the favourite to become the first Australian Pope. . 'It took 24 days, 650km and 4.5 tubs of peanut butter (crunchy) but I learnt a lot about both the world and myself,' he said, before launching into a philosophical yet ultimately incoherent speech that confused basic elements of Christianity, Buddhism and Islam. He also managed to somehow butcher Jesse McCartney's timeless pop classic in an attempt to define the singular Beautiful Soul of humanity as a collective. . As for the eternal future, his Holiness plans to launch the 'How I Absolved All Sins And Replaced Them With Monounsaturated Fats And You Can Too!' program in the coming months. Devoted followers may, however, be waiting a while to receive this holy sermon as unconfirmed reports suggest Mr Boxall was seen mere hours ago in Trondheim's infamous university district, dancing in a light drizzle naked save for his boots, which he had set alight under the influence of alcohol-free beer. Though the boots have faithfully carried him for thousands of kilometres across four continents, they will not, it would appear, be taking him to Rome. . Upon hearing of Mr Boxall's candidature for Chief Ruler of Christiandom and Them Other Religions, a Vatican spokesperson suggested that Boxall should 'take a hike,' proving once and for all that the Catholic Church does have a sense of humour, if not a sense of morality. More to come. . #hotguysgoingthedistance #justpilgrimthings #jessemccartney #inrodwetrust
He had a brainless head and a frost-bitten nose, and why he found these things fun not one person knows. He wore matching wool thermals and socks with his shoes, and he as a partner not one person would choose. With a three-week-old beard that was barely pubescent, he walked and he walked while he learnt some new lessons. . He was nature itself, a disturbance in its truest sense. He was the ripples across the lake and the rustle of leaves, the crunch of gravel and squelch of moss underfoot. He wielded both power untamed and a lime-green spork but was hesitant to use either. His coffee was the antithesis to his quest for meaning: instant. And when the rain came and dampened his clothes and his mood, he found solace and warmth in a cabin in the woods. So he took a photo and put it on Instagram and the rest is history. . #hotguysinoversizedchairs #poetsofinstagram
Between the uphills and the downed beers - just $19 for your home-brewed stout? Take my money! - the past 450km have been utterly draining in every possible way. But a break in weather means a break in walking and, with just under 200km to go and a feast of bananas and chocolate digestives in me sweaty little lap, things are looking up. Deadset. How much further up can this thing go? Send help and more biscuits to Hjerkinnhøe. It's a hill, I think, and it has turned out to be both a hjerk and a høe. #hotguyscrushinghøes
Day 12 (300km) ~ Ringebu stavkirke is the only stave church on the pilegrimsleden and dates back to the 12th century. The icy blue waters of the Gudbrandsdalslågen flow through the valley below but despite the chill, I'm told this is a bit of a hotspot for God and such things. The locals also like to play silly buggers and lock all eight of public toilets. Most importantly, it's a cracking spot for a banana and peanut butter wrap. Cheers Jesus 🍻 . #gudbrandsdalen #pilgrimage #norway
This is Bente. Her favourite dance is The Twist and she's been threatening to knit trousers to cover this little pilgrim's penis for years now (the statue, not me). She caught me setting up camp and cooking in a nearby public toilet (a clean one I swear) after walking 30km and finding nowhere to set up a tent, so she took me back to the community centre and gave me full run of the place. Hot showers, sauna, a kitchen and the comfiest couch in the world to sleep on. Then she turned up in the morning with a jug of coffee and a bag of eggs for my walk. . The centre is run by volunteers and was built with funds raised by the local women selling cakes and running auctions for many years. Next week it celebrates its 10th anniversary while also hosting the local election. Bente is voting for the current mayor because 'she's always smiling and we need more of that.' So if you ever find yourself in Hoff, which I'm sure you will, go and say g'day. And make sure you're wearing pants. . #hotguysbeingsaved #hotguysgoingthedistance #gudbrandsdalen #stolavways #pilgrim #norway
Cannot even begin to describe how good it was to come down to this free shelter, what the Norwegians call a gapahuk, after a backbreaking day lugging 17kg over the mountains. What the guidebook described as a casual, 18km stroll with uphill sections ended up being a touch over 26km, eight of which were uphill. But with pine forests stretching into the blue horizon and luxury accommodation on the fjord's edge, life is looking pretty decent even if my posture isn't. . #hotguysalltuckeredout #shelteredguyswhoarealsohot #gudbrandsdalen #pilgrimage #visitnorway #pilgrim #norway #fjord
Day 4: slept in a graveyard last night and it was kinda scary but nowhere near as terrifying as the night before. . See Thor put on a tantrum one could Marvel at and this storm swept across the fjords and crackled into the valley where we were camped. We were in these open fields and it was all flashes and cracks and then lightning licked the farmhouses and barns behind us and man I've never been so scared. . So like any good pilgrim I prayed. I rolled onto my back and I prayed: O Christ my spine hurts. . Then I rolled onto my side and I prayed: O Christ my hips hurt too. . Then I rolled onto my stomach and I prayed: O Christ my neck O good Lord this is agony. . And I'm not one for miracles but those flashes of lightning just turned into flashes of pack-induced pain and they rumbled down the valley as I grumbled in my tent, sore but not scorched, frazzled but not fried, with enough peanut butter to last the week and enough healthy vertebrae to last the month (maybe). . #hotguyssleepingwithdeadpeople #gudbrandsdalen #visitnorway #pilgrimage #pilgrim #buenbloodycamino
The first thing that I noticed about Norway is that everybody is tall and beautiful and tall. The second thing I noticed is that a cold pint - a single refreshing beer - costs roughly the same as a round of espresso martinis with the gals back home because hell, we just want to dance tonight. What I'm trying to say is this: Norway ain't a place conducive to either my romanticisms or my fondness for a refreshing ale. . But I'm in Oslo regardless and they weren't lying when they said it's expensive here. I paid 2 euros for the privilege of peeing at the bus station and 5 euros for the privilege of hostel sheets. And now that I've checked out it's become apparent that I should've peed in the sheets in protest of it all but I didn't, because that's not what this trip is about. It's not about Ivan either, my new Brazilian roommate, who near had a conniption when telling me how much a Burger King Whopper costs, answering a question I never asked. So I left him to eat his oven pizza and when he asked where I was going I told him to get a Whopper because I'm an alpha and he needed to know that as much as I needed to know the price of said Whopper. . Now what I am doing, between taking very expensive toilet breaks, is heading off to walk the Gudbrandsdalen (good brands darrrrlin', like them ones the Kardashians wear). Don't be ashamed if you haven't heard of it, I hadn't either until two weeks ago, but apparently it's a 650ish kilometre pilgrimage from Oslo to Trondheim through the mountains and farmlands and winding valley pathways that them tall Scandis used to walk to honour Saint Olav, Norway's patron saint and Viking king. He's said to be buried in Nidaros Cathedral - where this trek ends - but he's probably not. It's nice to have a reason to walk for a month though, cheers Olav. . I don't really know much about it so to summarise, I probably won't be particularly communicative for the next month or so and for that I apologise. I'll put up some photos and have a yarn but otherwise I'll see y'all once I emerge from my expensive silk cocoon as a tall blonde man holding a Whopper and preaching what I've learnt. . #hotguysgoingthedistance #visitnorway #gudbrandsdalen
Sitting here in rainy Belfast and I haven't seen the sun in three days and I could be reflecting on so many things about Turkey: the sunshine, the people, the effort @taylaroxene made to come visit and develop this film. Even the extraordinarily dangerous Long Island iced teas and the soul-destroying replays of Mambo No. 5 from dusk till dawn. But instead I find myself wondering how many times I wore this god damn shirt and I've realised, oh so cruelly, that I'm wearing it now. And maybe there's something poetic in that or maybe, and perhaps more likely, it's time to peruse the aisles and shelves and the gutters and bins for a new shirt. Or, at the very least, it's time undo one more button and let some brown sugar sweeten up the world. . . #hotguysonfilm #hotgirlsundermywing #howmanymmisthisfilm #35mm #40mm #85mm
Had a real hot and cool caption to describe how nice it was to see this guy but I've been waiting in a pub for 45 minutes as he was buying soap and I'll tell ya what nothing will scrub the bitterness off now y'know? #hotguysdisagreeing
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Our school hockey tours to Spain offer a wide variety of cities to base your tour in Lloret de Mar and Benidorm, both offering the warm Mediterranean climate, good standard of local teams and cosmopolitan lifestyle for everyone to enjoy. A Nation proud of their Spanish sports and vibrant lifestyle, Spain will certainly test your school hockey team competitively both on and off the pitch and will certainly provide you with Sun, Sea and plenty of Sport!
For school hockey tours, Spain offers you the opportunity to explore the coastline, play sports on the spectacular beaches and enjoy the local culture. A tour to Lloret de Mar also offers you the opportunity to have a day trip to Barcelona and visit the Nou Camp Stadium, home of Barcelona FC or if your group is more energetic a day trip to Portaventura Theme Park or the local Water Park is a must. Similarly, Terra Mitica Theme Park and Aqualandia Water Park are great excursions for those touring to Benidorm.
Fixtures, accommodation and coach service were all great and everyone enjoyed themselves. Brilliant tour!


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Behind many smiling faces there hides a hurting heart, and for what may appear to be glamorous, there is usually a high price to pay. Mrs. Walton gives us a glimpse into the life of a disillusioned young actress. In her fabricated world of glamour and glitz, Rosalie finds solace in the words of a gentle old man who gives her a picture of a Shepherd who loves and cares for her. Her travels take on new meaning as she shares with the "down-and-outers" about this wonderful Shepherd who loves them, too. This profound century-old story is still relevant in today's entertainment-enmeshed culture. A Peep Behind the Scenes outsold The Scarlet Letter by over two million copies in the 1800s.
From Jack Snavely of Castle Rock, Colorado on 7/30/2018 .
I can't say enough about how good Lamplighter books are. I enjoy them much more than modern books. Characters like Mother Manikin just make you smile. For those that often think the grass is greener on the other side of the fence or covet the lives of others around them, this is a must read! Things are not often what they appear! A very balanced book that is enjoyable to the last sentence.
From Janice Robinson of Neosho, Missouri on 3/14/2017 .
This book is very good!
From Sarah of Morenci, Michigan on 7/14/2014 .
From Anonymous of St. Clair, Michigan on 3/7/2014 .
I really enjoyed this book. The tension was incredibly well done. It moved slowly enough to make you wonder what's next, but not so pokey as to lose your interest. I loved the characters. I think I finished this in one day! Recommended.