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Histidine kinases are key regulators in the bacterial two-component systems that mediate the cellular response to environmental changes. The vast majority of the sensor histidine kinases belong to the bifunctional HisKA family, displaying both kinase and phosphatase activities toward their substrates. The molecular mechanisms regulating the opposing activities of these enzymes are not well understood. Through a combined NMR and crystallographic study on the histidine kinase HK853 and its response regulator RR468 from Thermotoga maritima, here we report a pH-mediated conformational switch of HK853 that shuts off its phosphatase activity under acidic conditions. Such a pH-sensing mechanism is further demonstrated in the EnvZ-OmpR two-component system from Salmonella enterica in vitro and in vivo, which directly contributes to the bacterial infectivity. Our finding reveals a broadly conserved mechanism that regulates the phosphatase activity of the largest family of bifunctional histidine kinases in response to the change of environmental pH.
Signal transduction by the two-component system (TCS) proteins is the primary mechanism for bacteria to detect environmental changes and orchestrate cellular responses1,2,3. Such a process also plays a critical role in bacterial pathogenesis, for example, by mediating pathogenicity genes during the host infection of Salmonella in response to the acidification of the phagocytic vacuole4.
A typical TCS contains a histidine kinase (HK) and a response regulator (RR). HKs are homodimers consisting of an extracellular sensor domain, a transmembrane connector, and a cytoplasmic portion (HKcp) that contains the catalytic activity (Fig. 1a). The cytoplasmic portion of histidine kinases can be further divided into the dimerization and histidine-containing phosphotransfer (DHp) domain and the catalytic and ATP binding (CA) domain1,5. About 80% of all sensor kinases belong to the HisKA family6, including HK853 from Thermotoga maritima, EnvZ from Salmonella, and others.
After sensing the environmental stimuli, the histidine kinase autophosphorylates a conserved histidine residue in the DHp domain (e.g., H260 of HK853; Fig. 1a) by its catalytic CA domain, which then transfers the phosphoryl group to an aspartate residue of the receiver domain of its cognate response regulator (RR) (e.g., D53 of RR468; Fig. 1a). Such an event triggers the interaction of the phosphorylated RR (phospho-RR) with downstream genes or protein targets for regulation of a variety of cellular functions1,7.
Due to the essential role of the phosphorylated response regulator in orchestrating the cellular response, its phosphorylation level is tightly controlled. Such a regulation is predominantly achieved through the opposing kinase and phosphatase activities embedded within the module of bifunctional histidine kinase. The conserved histidine residue involved in the phosphoryl transfer process of the DHp domain has also been implicated in the dephosphorylation reaction; the phosphatase activity is additionally affected by conserved Thr and Asn residues of the E/DxxT/N motif (e.g., T264 of HK853; Fig. 1a) immediately adjacent to the conserved histidine residue8,9. How a bifunctional histidine kinase switches its kinase and phosphatase activities on and off to maintain a balanced phosphorylation level of the response regulator has remained poorly understood.
Here we report a molecular mechanism that gates the phosphatase activity of the bifunctional histidine kinase HK853, a member of the HisKA family, from Thermotoga maritima. Our study reveals a pH-mediated conformational change, involving a sidechain rotameric switch of the catalytic histidine residue that inactivates the phosphatase activity of HK853. Such a mechanism similarly mediates the phosphatase activity of the classical EnvZ bifunctional histidine kinase, also of the HisKA family, from Salmonella enterica in vitro; accordingly, the transcriptional response genes of the EnvZ/OmpR TCS are upregulated at low pH in cells, consistent with the pH-gated inactivation of the EnvZ phosphatase activity and the subsequent accumulation of the phospho-OmpR. We suggest that the regulatory mechanism revealed by this study may represent a universal pH sensor utilized by the largest family of bifunctional histidine kinases in bacteria and other organisms to detect and respond to the change of the host environment to establish pathogenic infection.
19F NMR reveals two conformational states of the complex
BeF3 −, a well-established phosphoryl analog for studying phosphorylated proteins, has been previously used to generate the phosphorylation mimic of the response regulator10,11,12. Using a similar approach, we generated the BeF3 −-conjugated form of RR468, the cognate response regulator of HK853 from Thermotoga maritima, with the beryllium atom forming a covalent bond with the phosphate-accepting residue D53 and with three fluorine atoms (marked as F1, F2, and F3 in Fig. 1b) interacting with the Mg2+ ion, sidechains of K105 and T83, and amide groups of M55 and A84 of RR4685. When RR468 was mixed with an excess amount of BeF3 −, we were able to detect three broad fluorine signals from the protein-bound BeF3 − in the active site of RR468 between −180.0 p.p.m. and −150.0 p.p.m. (Fig. 1c, spectrum A), which were distinct from the strong and narrow signals of the unbound beryllium fluoride molecules13,14.
The identities of the protein-bound fluorine signals were assigned based on the solvent-induced isotopic shift (SIIS) experiment15,16,17 (Supplementary Fig. 1): the upper field 19F signal at −177.18 p.p.m. was the least sensitive to D2O exchange and was identified as the fluorine atom F1 that coordinates Mg2+; the downfield 19F signal at −152.13 p.p.m. displayed the largest shift when the water (H2O) solvent was replaced with 100% D2O (Supplementary Table 1 and Supplementary Fig. 1) and, based on the large SIIS effect, was assigned to fluorine F3 that forms hydrogen bonds with the side chain of T83 and the amide group of M55; the central signal at −156.01 p.p.m. experienced an intermediate SIIS effect and was assigned to fluorine F2 that forms a salt bridge with the sidechain of K105 and a hydrogen bond with the amide group of A84. The identity of this fluorine atom was further validated by the lysine sidechain specific H3(N)F experiment that correlated the F2 fluorine signal with the terminal NH3 proton of K105 (Supplementary Fig. 2).
After identifying individual fluorine signals in BeF3 −-RR468, we examined the fluorine signals of BeF3 −-RR468 in complex with the DHp domain of HK853 (HK853DHp) that possesses both the kinase and phosphatase activities. The DHp domain of HK853 forms a homodimer and interacts with RR468 at an equal molar ratio5. During the titration of HK853DHp at neutral pH, all of the signals in the 19F spectra became broadened, and a set of new peaks emerged downfield of the original ones, indicating the formation of the HK853DHp-BeF3 −-RR468 complex (Fig. 1c, spectrum B). Among the three fluorine signals, F2 was the most perturbed signal with a chemical shift difference of 1.2 p.p.m., indicating that the interaction of the HK853DHp domain significantly alters the salt bridge between F2 and the sidechain of K105.
Interestingly, the 19F NMR spectrum of the HK853DHp-BeF3 −-RR468 complex was highly sensitive to pH changes, a phenomenon that was not observed in BeF3 −-RR468 alone. When pH decreased from 7.0 to 5.0, an additional set of three fluorine signals emerged in the 19F NMR spectra (Fig. 1c, spectrum C and Fig. 1d). These new signals were located next to the original ones and showed higher intensities when the pH dropped below 6.0, indicating a slow exchange between two distinct conformations of the HK853DHp-BeF3 −-RR468 complex. For clarity, we designate the signals of free BeF3 −-RR468 as F x apo (x = 1, 2, 3), the signals of the HK853DHp-BeF3 −-RR468 complex at pH 7.0 as F x ca (x = 1, 2, 3), and the signals under acidic conditions (pH 5.0) as F x ci (x = 1, 2, 3) (Fig. 1b–d). It is important to note that the circular dichroism spectra of HK853cp and its complex with RR468 remained unchanged between pH 5.0 and 8.0, suggesting that the observed changes of the fluorine signals were not caused by denaturation of the proteins under acidic conditions (Supplementary Fig. 3).
H260 is involved in the conformational exchange
A pH-dependent protein conformational change typically occurs in response to the change of the protonation state of a pH-sensitive residue, such as a histidine residue. As the conserved auto-phosphorylated histidine residue of the bifunctional histidine kinases acts as both the phosphate donor in the kinase reaction and the catalytic residue in the phosphatase reaction18,19, we generated the corresponding H260A mutation of the DHp domain of HK853 (HK853DHp) and verified that this mutation indeed displayed diminished phosphatase activity5 (Supplementary Fig. 4; uncropped images shown in Supplementary Fig. 5). Surprisingly, the 19F NMR spectrum of the H260A mutant of the HK853DHp-BeF3 −-RR468 complex displayed only three protein-bound fluorine signals that matched the chemical shifts of the F x ci signals observed in the wild-type protein complex under acidic conditions (Fig. 1c, compare spectra C and D). Furthermore, these fluorine signals no longer displayed pH-dependent changes (Supplementary Fig. 6). Additionally, the signals of the H260A HK853DHp-BeF3 −-RR468 complex were much sharper than the signals from the wild-type complex: the half-width of the F1 signal in the wild-type complex was 306 Hz at pH 7.0, whereas the half-width of the same signal in the H260A mutant complex was only 220 Hz; such an observation supports the notion that the pH-dependent conformational exchange mediated by H260 is suppressed or eliminated in this mutant.
An inactive phosphatase conformation of HK853 at low pH
As our 19F NMR studies revealed the presence of a second conformation that is marginally populated at neutral pH, but becomes the predominant state under acidic conditions, we determined the crystal structure of HK853cp and its cognate response regulator RR468 in the presence of the phosphoryl analog BeF3 − and ATP analog, AMPPNP, under acidic conditions (pH 5.0) in order to visualize this minor conformation at neutral pH. The crystal structure, determined at 2.68 Å resolution (Table 1, a portion of the electron density map is shown in Supplementary Fig. 7), reveals a heterotetrameric complex consisting of two HK853cp and two BeF3 −-RR468. The two antiparallel helices of the N-terminal DHp domains pack into a central four-helix bundle, with the C-terminal CA domains and the RR468 monomers docking onto and surrounding the central four-helix bundle (Fig. 2a). The AMPPNP is captured in the hydrolyzed form of ADP, as previously observed5. The structure of the HK853cp-BeF3 −-RR468 complex is close to symmetric, with the backbone rmsds of 0.4 and 0.2 Å between individual monomers of the kinase and response regulator, respectively. The overall architecture of the HK853cp-BeF3 −-RR468 complex crystallized at pH 5.0 is similar to the previously reported structure of the HK853cp-RR468 complex determined at higher pH (pH 5.6; PDB 3DGE)5.
However, a close examination of the conserved and catalytically important histidine residue involved in the pH-dependent change of NMR signals reveals distinct conformations. In the previously reported structure of the HK853cp-RR468 complex determined at higher pH, the sidechain of HK853cp H260 adopts a trans χ 1 rotameric conformation and points toward D53, the receiving residue of the phosphate group of the response regulator RR468; the imidazole ring of H260 of HK853cp is located within 3.5 Å of a sulfate molecule that mimics the phosphate moiety of the phosphorylated D53; and the overall configuration represents a catalytically active conformation. In contrast, in the structure of the HK853cp-BeF3 −-RR468 complex crystallized at lower pH (pH 5.0), where the conformational state in solution is predominantly the Fci state, the sidechain of H260 adopts a gauche- χ 1 rotameric conformation (Supplementary Fig. 7), with the imidazole ring of H260 located at 11.2 Å away from the beryllium atom of the BeF3 −-modified D53 of the response regulator RR468; such a configuration would prevent H260 from engaging in the phosphatase activity of HK853 and hence represents a catalytically inactive state (Fig. 2b). Accompanying the sidechain movement of H260, the inter-domain arrangement of the inactive complex also changed. The N-terminal half of the first helix, which contains H260, was less bent compared to that of the catalytically active structure solved at a higher pH, with the location of the N-terminus of the helix shifting about 2.1 Å. Such a difference in the packing of the DHp domain caused a clockwise rotation of the C-terminal CA domain and the response regulator RR468 around the central helices, resulting in distance shifts between the catalytically inactive and active conformations of as large as ~6.9 and ~2.7 Å for the CA domain and RR468, respectively (Fig. 2c, d). These structural observations provide a molecular explanation for the lack of the phosphatase activity of HK853 under acidic conditions shown in the Phos-tag assay (Fig. 2e, f; uncropped images shown in Supplementary Fig. 8a, b, respectively) and suggest that the phosphatase activity of HK853 is regulated by a pH-gated conformational switch.
T264 is required for the H260-mediated phosphatase activity
Beside H260, T264 is another conserved residue that is critical for the transmitter phosphatase activity of HK853. When we titrated the T264A mutant of HK853DHp into the BeF3 −-RR468 solution, the fluorine signal (F2) interacting with K105 of RR468 was unchanged; however, the other two fluorine signals interacting with T83 and Mg2+ of RR468 were perturbed and were in good agreement with the fluorine signals of the inactive Fci state of the HK853-RR468 complex (Fig. 1c, spectrum E). These observations suggest that the T264A HK853 mutant still formed a complex with the response regulator RR468, but the critical chemical entity participating in the RR468 K105-fluorine (F2) interaction was absent in the T264A mutant of the HK853-RR468 complex.
As T264 of HK853cp is at least 5 Å away from either K105 or the K105-interacting fluorine atom (F2) of BeF3 −-modified D53, we suggest that the chemical entity causing the chemical shift perturbation of the K105-interacting fluorine atom (F2) is a T264-mediated water molecule, which in turn forms a hydrogen bond with the F2 fluorine atom; furthermore, this water molecule is likely the catalytic water molecule that additionally interacts with H260 of HK853 in the trans χ 1 rotameric configuration of the catalytically active state at high pH (Fig. 3, upper right panel). Supporting our model of dual interactions of T264 and H260 with the catalytic water molecule, a sidechain-flipped configuration of H260 captured under the acidic condition in our crystal structure of the HK853cp-BeF3 −-RR468 complex would result in a loss of the critical hydrogen bond between H260 and the catalytic water molecule and the perturbation of the chemical shift of the K105-interacting fluorine F2 to the catalytically inactive state (Figs. 1c, 3, lower right panel). Further elimination of the hydroxyl group in the T264A HK853 mutant would result in a complete loss of the catalytic water molecule (Fig. 3, lower left panel), returning the chemical environment of the F2 fluorine atom in the T264A HK853-BeF3 −-RR468 complex to that of the substrate state of BeF3 −-RR468 (Fig. 3, upper left panel). A similar water molecule has been previously hypothesized to interact with a conserved glutamine residue of the DxxQ motif in the nitrogen sensor NarX of the HisKA_3 family based on mutagenesis studies, though the phospho-accepting histidine residue was not thought to be involved in the water interaction8.
In order to probe the chemical environment of the HK853cp T264A mutant and gain further structural support for our proposed model of dual interactions of HK853cp T264 and H260 with the catalytic water molecule, we crystallized the T264A HK853cp-BeF3 −-RR468 complex under the same condition as the wild-type complex at pH 5.0, and determined the structure at 3.6 Å resolution (Table 1, a portion of the electron density map is shown in Supplementary Fig. 9). Consistent with our proposed model (Fig. 3), we found that the structure of the T264A mutant complex indeed captured the same catalytically inactive conformation as the wild-type complex at pH 5.0, with H260 adopting a gauche- χ 1 rotameric conformation (Fig. 4a, Supplementary Fig. 9). The overall backbone rmsd between the two structures is 0.32 Å, confirming that the T264A mutation did not affect the binding interface or the stability of the protein complex. However, with the loss of the hydroxyl sidechain in the T264A mutation and with H260 swung away from the active site in a catalytically inactive, gauche- χ 1 rotameric conformation (Fig. 4b), none of the water bridging functional groups is retained in the mutated HK853cp-BeF3 −-RR468 complex, leaving the F2 fluorine in a chemical environment identical to that in apo BeF3 –-RR468. Taken together, these observations provide strong support for the dual engagement model of the catalytic water molecule by both H260 and T264 of HK853 (Fig. 3).
pH-regulated phosphatase activity in the EnvZ-OmpR TCS
In Salmonella, expression of the type III secretion system genes encoded by the Salmonella Pathogenicity Island (SPI) 2 is required for systemic infection in mice and is regulated by the ssrA-ssrB genes20 (Fig. 5a). Active transcription of the ssrA-ssrB genes occurs after Salmonella enters host cells in an acidified vacuole, accompanied by the accumulation of phospho-OmpR, the response regulator of the classical EnvZ/OmpR two-component system21. EnvZ belongs to the same HisKA subfamily as HK853 and shares a similar bifunctional motif (Fig. 5b). It has recently been reported that intravacuolar Salmonella is acidified in response to external acid stress4, suggesting that the cytoplasmic pH could be an important regulator of the bifunctional histidine kinase EnvZ that leads to the accumulation of phospho-OmpR.
In order to test this scenario, we first evaluated the pH-dependent phosphatase activity of EnvZ by the Phos-tag assay using the purified cytoplasmic portion of EnvZ (EnvZcp) and the receiver domain of the response regulator OmpR (OmpRr). Similar to HK853, we found that EnvZcp is a less efficient phosphatase under acidic conditions than under neutral pH conditions (Fig.5c; uncropped image shown in Supplementary Fig. 10). Based on these in vitro observations, we went on to examine whether the pH-regulated phosphatase activity of the bifunctional histidine kinase EnvZ is responsible for the accumulation of the phospho-OmpR and transcriptional activation of ssrA and ssrB in vivo using a quantitative real-time PCR assay. As OmpR is the specific response regulator of EnvZ, the catalytically inactive H243A EnvZ mutant does not generate phospho-OmpR; therefore the background transcriptional levels of ssrA and ssrB were normalized to one in the envZ-H243A mutant strain at pH 5.0 and 7.0, respectively, for comparison with the wild-type strain and the envZ-T247A mutant strain under different pH conditions. In a neutral pH environment (pH 7.0), we observed a modest enhancement of the expression of both ssrA and ssrB due to the presence of phospho-OmpR generated by the kinase activity in the wild-type bifunctional EnvZ enzyme (Fig. 5d); however, the enhancements of mRNA levels of ssrA and ssrB under neutral pH were significantly lower than those under the acidic environment (pH 5.0). Such an in vivo observation is consistent with our in vitro discovery that the phosphatase activity of bifunctional histidine kinases is diminished by a pH-gated conformational switch, resulting in the accumulation of a higher level of phospho-OmpR in the acidic environment. In contrast, although the mRNA levels of ssrA and ssrB in the envZ-T247A mutant strain deficient in the phosphatase activity are enhanced in comparison to those in the wild-type EnvZ strain, the variation in the mRNA levels of ssrA and ssrB under different pH environments is statistically insignificant, confirming that the observed changes in the mRNA levels of ssrA and ssrB under the control of wild-type EnvZ is directly caused by the differential phosphatase activity mediated by a pH-gated conformational switch, but not other factors.
Taken together, our data suggest that a pH-gated inactivation of the phosphatase activity of EnvZ directly regulates the accumulation of the phosphorylated response regulator OmpR and expression of SPI2-encoded genes of the two-component system (ssrA and ssrB) and the type III secretion system for enhanced bacterial infectivity. Such a model is further supported by the Salmonella host invasion experiment: ablation of the kinase activity in the H243A EnvZ mutant eliminated the production of phospho-OmpR and profoundly diminished the macrophage infectivity of the mutant bacterium compared to the wild-type strain, whereas eliminating the phosphatase activity in the T247A EnvZ mutant resulted in an enhanced macrophage infectivity due to the accumulation of phospho-OmpR (Fig. 5e, Supplementary Fig. 11).
The bifunctional histidine kinases of bacterial two-component systems possess both phosphoryl transfer activity (kinase) and phosphoryl removal activity (phosphatase) toward their cognate response regulators. Despite the essential roles of these enzymes in regulating bacterial pathogenesis in response to the change of the host environment during infection, the molecular mechanisms that regulate the opposing kinase and phosphatase activities are only starting to be elucidated. In the ChpT-CtrA two-component system of Brucella abortus 22, it was suggested that the quaternary structure of the ChpT2–CtrA2 complex enforces an asymmetric mechanism of phosphoryl transfer between ChpT and CtrA, and only one of the two asymmetric binding modes is competent in the phosphotransfer activity of ChpT. However, the molecular mechanism regulating the phosphatase activity in this system remains entirely unclear. Recently, the regulatory mechanism of the bifunctional histidine kinase CckA from Caulobacter crescentus was revealed23. In this case, binding of the second messenger cyclic di-guanosine monophosphate (c-di-GMP) suppresses the default kinase activity of CckA through noncovalent cross-linking of the catalytic domain with the dimerization histidine phosphotransfer domain. Our study represents a complementary regulatory mechanism of bifunctional histidine kinases that inactivates the phosphatase activity through a pH-gated conformational switch (Fig. 3). It is important to note that the conformation-dependent inactivation process occurs on the slow exchange time scale for NMR measurements (μs-to-ms) and yields two sets of signals corresponding to the conformations of the active and inactivate states between pH 5.2 and 6.5 (Fig. 1d). Such a mechanism is distinct from the general reduction of the catalytic activity due to protonation of the catalytic base, where the fast protonation-deprotonation process yields a single set of population-weighted signals as observed in many enzymes24. Interestingly, although the catalytically inactive conformation reported here has not been previously captured in wild-type enzymes, it has been observed in a "rewired" HK853cp*-RR468* complex, in which the interface residues of HK853 and RR468 from Thermotoga maritima were mutated to match those of the two-component system in Escherichia coli, PhoR and PhoB, respectively25. As these mutations also perturbed the arrangement and relative orientation of the histidine kinase with respect to the response regulator, the critical factor that trapped the chimeric complex into the inactive state has not been isolated. In comparison, by combining solution NMR and crystallographic studies of the wild-type HK853-RR468 complex, we have been able to establish the critical role of pH in regulating the conformation-dependent phosphatase activity of the HisKA family of bifunctional histidine kinases. Whether the inactive conformation of the wild-type HK853-RR468 complex observed at low pH could resemble snapshots of transient intermediates of the phosphotransfer reaction as previously suggested for the rewired complex25 remains to be investigated.
In summary, our combined solution NMR and crystallographic studies have revealed a pH-gated conformational switch that modulates the phosphatase activity of the largest family of bifunctional histidine kinases. Given the broad conservation of residues involved in this conformation switch, such a regulatory mechanism likely reflects a general pH-response mechanism for the vast majority of the two-component signal transduction systems that function in bacteria, plants and fungi.
Protein preparation and purification
Plasmids encoding HK853cp (residues 232–489), RR468, EnvZcp (residues 215–450), OmpR receiver domain (OmpRr; residues 1–125) and His6-MBP-HK853DHp (residues 250–315) were transformed into E. coli BL21(DE3) Gold cells (Novagen). For unlabeled protein, the transformed bacterial cells were cultivated in the LB medium containing either ampicillin (100 μg mL−1) or kanamycin (50 μg mL−1) at 37 °C, and then induced with IPTG (isopropyl β-d-1-thiogalactopyranoside; 1 mM) at 20 °C for 20 h before collecting. 15N-labeling of RR468 was achieved by growing cells in the M9 minimal medium containing 15N NH4Cl as the sole nitrogen source.
HK853cp was purified by ammonium sulfate precipitation (40% w/v) followed by anion-exchange and size-exclusion chromatography. RR468 was purified by incubation at 60 °C followed by anion-exchange and size-exclusion chromatography5,26. Similar protocols were used to purify EnvZcp and OmpRr. The H260A and T264A point mutants of HK853DHp were generated by point mutagenesis (primers summarized in Supplementary Table 2). Wild-type and mutant HK853DHp were expressed as His6-MBP fusion proteins. Each fusion protein was purified by using the amylose affinity chromatography and eluted with the buffer containing 20 mM Tris (pH 7.6), 20 mM maltose and 300 mM NaCl. The His6-MBP tag was cleaved by the tobacco etch virus (TEV) protease digestion and removed by passing the cleavage sample through an amylose column in a buffer containing 20 mM Tris (pH 7.6) and 300 mM NaCl. The target HK853DHp bound weakly to the Ni2+-NTA column and was separated from the His6-tagged TEV by elution in a buffer containing 50 mM phosphate (pH 8.0), 300 mM NaCl, and 30 mM imidazole. All protein samples were further purified to homogeneity using size-exclusion chromatography.
NMR experiments
Protein samples were dissolved in the NMR buffer containing 20 mM HEPES (pH 5.0–7.0), 50 mM KCl, and 10 mM MgCl2, with various levels of D2O. For NMR samples without D2O, a hermetical tube containing D2O was inserted into the NMR tube for the field lock.
BeF3 –-RR468 was generated by adding 5 mM BeCl2 and 50 mM NaF to the RR468 solution, which was incubated at room temperature for 30 min. The protein complexes were generated by adding HK853DHp or mutants at a concentration 10-fold higher than the substrate BeF3 –-RR468.
The 19F NMR experiments13,14 were performed on a 600 MHz Bruker spectrometer (equipped with a 5 mm CPQCI 1H/19F-−13C/15N/D Z-GRD cryoprobe) at 298 K, and the chemical shifts were referenced to CFCl3 (the free fluorine signal F− at −119.0 p.p.m.).
The pulse sequence for the H3(N)F experiment was adapted from the H3(N)P experiment originally designed to analyze the 3h J NP coupling27. For the 19F dimension, the carrier position was set to −156.0 p.p.m. The evolution decay in the 15N dimension was set to 35.0 ms. The chemical shift of lysine sidechain NH3 was assigned by 2D 1H−15N HISQC, 3D (H)CCENH3, and 3D (H)CCNH experiments28,29.
NMR data were processed with NMRpipe30 and analyzed by Topspin 3.2.
Protein crystallization and structural analysis
The protein complex (10 mg mL−1 HK853cp and 7.5 mg mL−1 RR468) was dissolved in a buffer containing 10 mM Tris (pH 8.0), 7 mM MgCl2, 5 mM BeCl2, 30 mM NaF, and 4 mM AMPPNP5. Crystallization of the wild-type complex was carried out by using the sitting drop vapor diffusion method at 20 °C in drops containing equal volumes of the protein complex and mother liquor consisting of 0.1 M citric acid (pH 4.0), 0.8 M ammonium sulfate, with the final pH adjusted to 5.0. For the T264A mutant complex, the mother liquor contained 0.1 M citric acid (pH 5.0), 0.208 M ammonium sulfate, and crystallization was carried out at 15 °C by using the hanging drop vapor diffusion method. The crystals were cryoprotected by a solution containing 50% mother liquor (v/v) and 50% glycerol (v/v) for the wild-type complex and 50% mother liquor (v/v) and 50% sucrose (v/v) for the T264A mutant complex, respectively. The data were collected at the SER-CAT 22-ID and 22-BM lines at Argonne National Laboratory for the wild-type and T264A mutant complexes, respectively; diffraction data were processed with XDS31 or HKL200032 and truncated and scaled with the University of California Los Angeles anisotropy diffraction server33,34. The previously reported structure of the HK853cp-RR468 complex crystallized at pH 5.6 (PDB: 3DGE)5 was used as the search model for molecular replacement by using the PHENIX suite35. The structural model was iteratively built by COOT36 and refined by PHENIX. In the crystal structure of the wild-type HK853cp-RR468 complex, additional electron density was observed near one of the two H260 residues, which was interpreted as a glycerol molecule from the cryo-protectant.
Dephosphorylation assay in vitro
Samples of RR468 and OmpRr were phosphorylated at a protein concentration of 4 mg mL−1 in the presence of 12 mM acetylphosphate at 25 °C for 1 h. The free phosphate was removed by using a GE desalting column. Samples of phosphorylated RR468 and phosphorylated OmpRr were exchanged into desired reaction buffers (20 mM Tris, 10 mM MgCl2, 100 mM NaCl at pH 8.0; and 20 mM NaAc, 10 mM MgCl2, 100 mM NaCl at pH 5.0), flash-frozen, and stored at −80 °C.
A reaction mixture of phospho-RR468 and HK853DHp or mutant enzymes at a molar ratio of 6:1 was incubated at 4 °C for the detection of the phosphatase activity by the native polyacrylamide gel electrophoresis (PAGE) assay, whereas a molar ratio of 13.8:1 was used for the Phos-tag gel assay. The same protocol was used to assay the phosphatase activity of the EnvZcp and mutants towards phospho-OmpRr. Samples at different time points were taken by flash-freezing at −80 °C. For the native-PAGE detection, gels were run on ice at 90 V for 5 h. The Phos-tag gel was made by adding 75 uM Phos-tag acrylamide and 150 μM MnCl2 into the SDS-PAGE37. All the gels used for assays were stained by Coomassie blue.
Construction of Salmonella EnvZ mutant strains
S. typhimurium strains containing the envZ H243A or T247A mutations were constructed from the LT2 strain38,39. Briefly, the envZ gene in LT2 strain was first replaced by a kanamycin resistance gene (kan) to obtain the envZ::kan strain using the pKD46-mediated gene disruption system40. The kanamycin resistance gene in the envZ::kan strain was subsequently replaced with the H243A or T247A mutant of the envZ gene using a homologous recombination method as previously described41. Detailed procedures are described in Supplementary Methods.
RNA extraction and qRT-PCR
Cells of S. typhimurium strains were grown in a modified N-minimal medium (MgM) buffered with 20 mM Tris (pH 7.0) or 20 mM MES (pH 5.0) containing 7.5 mM (NH4)2SO4, 5 mM KCl, 0.5 mM K2SO4, 1 mM KH2PO4, 10 mM MgCl2, 2 mM glucose, and 0.1% Casamino acids4. When OD600 reached 0.5, cells were collected by centrifugation. Total RNA of each collection was extracted with the TRIzol reagent (Invitrogen) according to the manufacturer's protocol. After treatment with RNase-free DNase I (Promega), 2 μg of each RNA sample was used as the template to obtain the cDNA by RNA PCR kit (AMV) ver. 3.0 (TaKaRa). Real-time PCR was performed using Universal SYBR Green Supermix Reagent (Bio-Rad). Results were normalized to the 16S rRNA level. For each strain, wild-type or mutants, all tests were repeated four times independently. Mean values and standard deviations (SD) of these experiments were calculated. Detailed information of PCR primers is listed in Supplementary Information.
Immunofluorescence detection of macrophage infection
S. typhimurium LT2 cells were grown in the LB medium overnight at 37 °C until OD600 reached 1.0. The RAW 264.7 host cells (FineTest, Wuhan) were cultured at 37 °C and then infected by LT2 at a multiplicity of infection of 100 for 30 min followed by PBS washes. The host cells were further incubated for 4 h, then fixed with 4% formaldehyde, permeabilized in 0.2% Triton X-100, and treated with 5% FBS before antibody incubation. LT2 cells were incubated with anti-Salmonella Csa-1 primary antibody (KPL) and DyLight 549-conjugated AffiniPure rabbit anti-sheep IgG (EarthOx) second antibody at 4 °C overnight. The host cells were visualized by FITC-conjugated phalloidin (Sigma-Aldrich) that stains actin filaments. The excitation wavelengths of the two different fluorescent dyes were 495 and 552 nm, respectively. The final images were visualized and processed by confocal microscopy.
Data availability
The coordinates and structure factors for the X-ray structures of the wild-type HK853cp-BeF3 −-RR468 complex and the T264A mutant complex at pH 5.0 have been deposited to the Protein Data Bank (PDB) with accession codes 5UHT and 6AZR, respectively. Other data are available from the corresponding author upon request.
Additional information
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We would like to thank Prof. Honggao Yan from the Michigan State University for providing the HK853DHp and RR468 plasmids, Mr. Zhongwei Wang from the Wuhan Institute of Virology for assistance with the qRT-PCR experiment, Prof. Lin Guo and Dr. Lizhi Hu from the Wuhan University for help with the infectivity experiment, and Dr. Chenyu Cao from the Northwest A&F University for optimizing the protocols of HK853DHp and RR468 purification. Use of the Advanced Photon Source was supported by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences, under Contract No. W-31–109-Eng-38. This work was supported by grants from National Key R&D Program of China (#2017YFA0505400 awarded to Dr. Xu Zhang from Wuhan Institute of Physics and Mathematics), Ministry of Science and Technology of China (#2013CB910200 awarded to Dr. Chun Tang from Wuhan Institute of Physics and Mathematics) and the Natural Science Foundation of China (#21573280 awarded to L.J. and #21603268 awarded to Y.L.) and the National Institute of General Medical Sciences (GM115355 awarded to P.Z.).
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text:
Landmark Native American Lawsuit Settled
Navajo Times, Posted: Jan 05, 2010
After 13 litigious years, the landmark case Cobell v. Salazar was settled Dec. 7, reports the Navajo Times.
The $3.4 billion settlement is the largest ever by the U.S. government, yet it's a mere fraction of the $40 billion some accountants estimate the feds owe individual Indian plaintiffs for mismanaging their trust assets.
Lead plaintiff Elouise Cobell said she had mixed feelings about the settlement, but wanted to see the case resolved before any more plaintiffs pass away.
For most of the 500,000 plaintiffs in the class action lawsuit, it will mean a payment of $1,500 with the remaining funds divvied up when the lawyers are paid and all the claims are in.
The government has also set up a $2 billion fund that tribes can use to buy back allotted land they would like the Interior Department to take back into trust, and $60 million for scholarships for Native American students.
But for Ervin Chavez, president of Shii Shi Keyah, the organization that represents the Navajo Nation's 42,000 allottees, the best part of the settlement is that the feds have agreed to some major changes in their accounting practices that will hopefully prevent future mismanagement.
"The settlement doesn't make up for our losses," Chavez said in a phone interview. "Indian people have taken a lot more loss than most people will ever know. Hopefully what we've done is to spend $4 or $5 billion to redo the trust issue."
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subset: commoncrawl


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1. Mr. Rajah Aiyar's argument is that the application alone under Order XXI, Rule 89, of the Code of Civil Procedure need be made within 30 days as required by Article 166 of the Limitation Act but the deposit might be made either within the 30 days mentioned in Order XXI, Rule 92, Clause (2), of the Code of Civil Procedure, or within such further time as the Court might choose to give in virtue of its alleged inherent powers under Section 151 of the Code of Civil Procedure.
2. In the first place, it is clear from the decision in Karunakara Menon v. Krishna Menon 27 Ind. Cas. 952: 2 L.W.196 that the 'provision in Order XXI, Rule 89, is in the nature of an indulgence to the judgment-debtors' and that 'Courts are bound to see that the provisions of law in this respect are very strictly conformed to.' That the deposit within 30 days is much more important than the application to set aside has been held in some cases see Mariappa Annam v. Hari Hara Iyer 22 Ind. Cas. 291 and that even an oral application to sot aside is sufficient and can be presumed from an affidavit accompanying the deposit, provided the deposit itself is made within 30 days. Mr. Rajah Aiyar's argument, if accepted, would make the deposit within 30 days a secondary matter.
3. Further we are of opinion that the words 'on his depositing' in Order XXI, Rule 89, of the Code of Civil Procedure qualify 'apply' and that such deposit is a condition precedent to the making of the application.
4. Lastly, we hold that the Court has no power to extend the time given by the Code (and not by an order of the Court) for making the deposit and that the requirements of Order XXI, Rule 92, Clause (2), of the Code of Civil Procedure that the deposit should be made within 30 days is not merely directory but mandatory.
5. Order XXI, Rule 92, Clause (2), of the Code of Civil Procedure is not happily worded. An order allowing an application to set aside the sale is the same thing as an order setting aside the sale and it is, therefore, superfluous to say that on the allowing of such an application, the Court shall make an order setting aside the sale.
6. If, again, the deposit should precede or be contemporaneous with the application, it was unnecessary to prescribe 30 days for the deposit when 30 days are prescribed by the Limitation Act for the application, unless the Legislature intended not to allow to the making of the deposit the extension of time permitted to applications by the Limitation Act in certain contingencies, such as the Court being closed on the last day of limitation. However, we are clear that the learned Subordinate Judge was right in his view that the Court had no power to extend the 30 days fixed in the Code for making the required deposit, as Section 151 of Civil Procedure Code cannot be legitimately invoked for extending periods of limitation and for overruling Legislative provisions.
7. We dismiss the appeal with costs.


text:
Det här är en gammal arkiverad spelning
Goran Kajfes + Antenna + Josef & Erika + Midaircondo + Michala Østergaard (DK)
Källa: ticnet.se
Goran Kajfes
This is an incorrect tag for Goran Kajfeš. If this non-artist appears in your charts, do last.fm and yourself a favor. Fix your artist tags. Read more on Last.fm . User-contributed text is available under the Creative Commons By-SA License; additional terms may apply.
Josef & Erika
The Swedish duo Josef och Erika, with the instruments doubble bass/electric bass, vocals and harmonium, is known for their intimate and fascinating appearences. They introduced their second album "Small small small small sounds"in september 2007 and recived brilliant reviews and a nomination for a Swedish grammy. Their first album "Wallpaper stories" from 2005 was appointed one of the top ten albums of 2005 by the Swedish newspaper Svenska Dagbladet. Josef och Erika has, during a couple of years, toured mostly in Sweden but also in other Scandinavian contries, Germany and New York. Their music is influenced by both singer/songwriter and modern improvised music. Somewhere between these genres they've created music where stories are in foc...
midaircondo is a constellation which explores beat, sound, voice, improvisation in an audio-visual environment. The group was founded in Gothenburg, Sweden in December 2002. The starting point was the yearning to explore new and unexpected ways to create music.
midaircondo is a new making within the swedish electronic music scene, based on the idea of playful music, working in between the acoustic and electronic area.
midaircondo combines the music with their own visuals, where the projections interact with the music performed on stage.
midaircondo is:
Lisa Nordström - flutes, voice, computer etc.
Lisen Rylander - saxophones, voice, computer etc.
Malin Dahlstrom left the group in 2006...


text: tag:blogger.com,1999:blog-6325522722526721789.post8496462576170784780..comments2017-03-13T20:01:49.907-04:00Comments on The Dead Baby Club: Ways to Memorialize Your BabyHeatherhttp://email@example.com:blogger.com,1999:blog-6325522722526721789.post-44821016949588932782012-09-04T11:42:24.563-04:002012-09-04T11:42:24.563-04:00A different approach to remembering a baby or love...A different approach to remembering a baby or loved one that has passed away inside a beautiful keepsake.Cremation Rings for Womenhttp://email@example.com:blogger.com,1999:blog-6325522722526721789.post-40394567288158204582012-07-16T13:01:37.734-04:002012-07-16T13:01:37.734-04:00Beautiful keepsakes to honor family and any member...Beautiful keepsakes to honor family and any member of the family.Family Cremation Jewelryhttp://firstname.lastname@example.com:blogger.com,1999:blog-6325522722526721789.post-13782441614139275052010-11-17T21:24:32.739-05:002010-11-17T21:24:32.739-05:00The idea of having gardens and creating some water...The idea of having gardens and creating some water ponds is great too. It can make the house better..Cremation Pendantshttp://email@example.com:blogger.com,1999:blog-6325522722526721789.post-67145301303020927802010-08-10T22:39:58.014-04:002010-08-10T22:39:58.014-04:00Emily, over at http://mumblingsfromtroyohio.blogsp...Emily, over at http://mumblingsfromtroyohio.blogspot.com/ (you'll need an invite) told me that to commenorate her daughter's one year she sent up balloons with "Forget-Me-Not" flower seeds in them.Heatherhttps://email@example.com:blogger.com,1999:blog-6325522722526721789.post-48263541947854760592009-12-10T11:31:28.955-05:002009-12-10T11:31:28.955-05:00We had a tree planted in our local park and we pla...We had a tree planted in our local park and we placed Will's ashes there (3 1/2 yrs ago). We put origami cranes in it on his birthday and for others who need honoring. It has become a place to be with who he has become to us. We also put our son Tiger's ashes there this fall- sadly to be with his brother. I have an intense vertical scar up my front that will forever remind me of Tiger. My husband wanted something permanent to remind him of our boys as well so we both got an amazing tattoo of a tree and a tiger in the bark of the tree. The ritual and memorializing of our sons has been very integrative and comforting to firstname.lastname@example.com:blogger.com,1999:blog-6325522722526721789.post-66366237218212366992009-10-08T07:03:59.121-04:002009-10-08T07:03:59.121-04:00Thank you for sharing these sites. I am looking in...Thank you for sharing these sites. I am looking into them as I can never do enough for Lukas.Maryhttps://firstname.lastname@example.com


text:
Commune: Busko-Zdrój (pow. buski, woj. świętokrzyskie)
Framework element or context represented:
Sequences of marine sediments of Upper Jurassic, Upper Cetaceous and Neogene (Badenian) of Nida basin separated with stratigraphic gaps and discordances.
Primary geological/geomorphological interest:
Stratigraphic sequence with Alpine tectonic gaps and unconformities.
Comparative assessment justification:
The most illustrative outcrop documenting stratigraphic-tectonic evolution of the Nida basin during Alpine cycle (Jurassic-Neogene).
Protection status and accessibility:
Protected as a documentary site (since 1993) – accessible.
Character of site: Abandoned quarry.
Area: 11.19 ha
Altitude: 270-285 m a.s.l.
Lithology: sandstones, conglomerates, breccias, other siliceous rocks, limestones, marls, shales
Discipline: Palaeoenvironment, Stratigraphy of Phanerozoic, Tectonics
Process Age: Quaternary, *all periods
Bedrock Age: Neogene, Cretaceous, Jurassic, *all periods
Description of primary interest:
The sequence exposed in the abandoned quarry comprises the Upper Jurassic limestones and Upper Cretaceous rocks overlain by transgressive facies of Miocene. Two phases of Alpine tectonic movements are perceptible here. The first discontinuity (gap with traces of erosion and low angle discordance) is observed between the Oxfordian limestones and Cenomanian sandstones. The Jurassic is represented with bedded limestones dipping 35º to the north. Thin Cenomanian glauconitic sandstone layer (less than 1 m) contains phosphorite nodules and inoceramian shells. It is overlain by the Turonian limestones grading to thick complex of Turonian and Senonian marls and opokas bearing numerous flynt nodules and horizons. Occurrence of relics of submarine hot water springs are supposed in the Cretaceous rocks. Paleo-surface of the Cretaceous and Jurassic rocks, shaped by marine abrasion, is discordantly covered by Badenian breccia-conglomerate, which is irregularly intercalated with sandy limestone. The breccia is composed of weakly cemented fragments of its substratum (limestones, flints and marls) - boulders, rubble, pebbles, often with animal borings (sponges, lamellibranchs, polychaetes, gastropods) and incrustations (bryozoans, polychaetes, lithothamnia). Biogenic material (foraminifera, algae) occurs also in matrix of the breccia. The main body of breccia represents allochtonous sediment of littoral fore-cliff depression. They are overlain by biodetrital and lithothamnian (algal) limestones containing also other fossils: foraminifers, gastropods, lamellibranchs (pelecypods), echinoids, and teeth of fishes. Miocene rocks slightly dip to the north (the discordance angle – ca. 20º).
JURKIEWICZ W., MORAWIECKI A. 1956–On phosphorite-bearing Cenomanian conglomerate from Mt Zajęcza Góra near Busko (English sum.). Arch. Miner. 19 (1955), 2: 181-200. MAŁECKI J. 1966–Miocene Bryozoa from the borings of lithopags of Skotniki near Busko, Central Poland (English sum.). Roczn. Pol. Tow. Geol. 36, 4. 481-495. MIGASZEWSKI Z.M., HAŁAS S., DURAKIEWICZ T. 1996–The age and origin of the calcite mineralization in the Holy Cross Mts based on lithologic-petrographic and isotopic evidence (English sum.). Przegl. Geol. 44: 275-281. MIGASZEWSKI Z.M., SIUDA T., WALASZCZYK I. 1987–Upper Cretaceous pyroclastic and hydrothermal formations of the southwestern borderland of the Świętokrzyskie Mts (Busko-Pińczów area). (English sum.). Przegl. Geol. 35, 8-9: 425-431. RADWAŃSKI A. 1965–Additional notes on Miocene littoral structures of Southern Poland. Bull. Acad. Pol. Sc., ser. Sc. Geol.-Geogr. 13, 2: 167-173. RADWAŃSKI A. 1967–Pitting processes in clastic and oolitic sediments (English sum.) Roczn. Pol. Tow. Geol. 35, 2: 179-210. RADWAŃSKI A. 1969–Lower Tortonian transgression onto the southern slopes of the Holy Cross Mts (English sum.). Acta Geol. Pol., 19: 1-164. URBAN J., WRÓBLEWSKI T. 1999–Representative geosites of the Góry Świętokrzyskie (Holy Cross Mts.) and the Nida Basin, Central Poland. Pol. Geol. Inst., Special Papers, 2: 61-70. WALASZCZYK I. 1992–Turonian through Santonian deposits of the Central Polish Uplands, their facies development, inoceramid paleontology and stratigraphy. Acta Geol. Pol., 42: 1-122.


text:
Twice this year I've been told by people I admire and respect that all I have to do is get out of my own way. The first time I thought: Why of course! That makes perfect sense. The second time I realized I have no idea what that actually means.
Get out of my own way.
How does one do that exactly? And is that meant as a constructive criticism? Because I'll admit, on the face of it, it doesn't exactly feel like it.
The truth is, for the last year I would argue that is exactly what I've been doing by acknowledging my triggers and learning to set and keep personal barriers. But now I'm not so sure. Because after all of it, the change that I've made my life and the calming of emotions, I continue to struggle every single day with one important fact: I still don't trust myself.
So last night, when I heard this again, in a more intimate and significant way, I felt shaken and confused. I realized that the hard work I've done to pull myself out of the trenches of depression and anxiety has been important and valuable, but hasn't yet fully addressed the root of my struggles—my sense of self-worth and my ability to love and honour myself.
I'm not very good at seeing the forest for the trees and tend to get wrapped up in the little things and then let them eat away at me.
Like my own thoughts, and the words and emotions that sit lodged in my head and that seek to negate all the progress that I've made.
And so I realized that that is what is meant by this well-meaning advice: To get out of my own way is to acknowledge those thoughts and then trust myself enough to be able to move beyond them. What's more, I know that it has less to do with my day-to-day happiness, but rather with day to day life management.
Seems simple enough stated here, crisp and bold in black and white. But in reality I think this will be the biggest hurdle of my life.


text:
I have been busy making things this week! And its only Tuesday! Busy Busy.
I've been designing an apron for kids crayons. I hope to have a photo of it on the blog today or tomorrow at the latest. Its so cute! I made a girl apron, now I'm working on a boy one. It should be done today. (I hope) But until then here are some photos of some key chains. "Mommy" and one of the polk a dot key chains were custom orders. The bag I just did because I thought the little gymnastics girl was too cute. And the scrapbooking pages was a custom order for a sweet lady in FL. Its a 1st year album I did for her baby girl Reagan.


text:
03 March 2003, 06:34 PM
I just got the WD 200GB HD, and I hooked it up to my ECS K7AMA 1.2 Athalon. When I boot up, the bios reads the HD as 138 GB instead of 200. I checked the IDE cable to make sure it was ATA100, and that was fine, my only guess is the bios. Any other ideas? Thanks,


text:
Question about Computers & Internet
Posted by bania on
If you are having an issue with a virus go to: http://onecare.live.com/site/en-us/default.htm and run the scans. Try to remove any viruses found.
If it is a virus call Microsoft and they will help you remove it. It shouldn't cost to remove a virus.
Posted on Dec 01, 2007
SOURCE: I have freestanding Series 8 dishwasher. Lately during the filling cycle water hammer is occurring. How can this be resolved
A 6ya expert can help you resolve that issue over the phone in a minute or two.
Best thing about this new service is that you are never placed on hold and get to talk to real repairmen in the US.
The service is completely free and covers almost anything you can think of (from cars to computers, handyman, and even drones).
click here to download the app (for users in the US for now) and get all the help you need.
Good luck!
Posted on Jan 02, 2017
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text:
Our whole team would like to gladly welcome you to the Bauernhof Weingut-Gästezimmer zum Schwalbennest . Winter Sports Hotels abound, but the Bauernhof Weingut-Gästezimmer zum Schwalbennest is a great choice to fulfill all of your needs and expectations. Book with us and find out! It doesn't matter whether you're staying for one night or more, you will find lots of parking spaces available at our comfy hotel. You won't have to leave your four-legged friends at home when staying in this hotel - bring them along, the manager of the hotel is fine with it. Our acclaimed restaurant and leisurely hotel bar provide wholesome meals and a variety of liquid refreshments. Refresh in our hotel's sauna. In case you happen to have some free time, try out some of the leisure activities, for example climbing upon one of our horses that provide a fun-filled horseback-riding experience.
- Parking
- free
- Pets allowed
- Restaurant/cafe


text:
Apparently I think I'm some sort of artistic genius. During Cam's afternoon nap I decided to play around with my camera and take snap a few shots of Christmas details around the house.
I also took some of our favorite pictures from Hawaii, mostly of Cameron, and turned them into a similar collage. I guess I have nothing better to do at the moment...?
By the way, here's a set of interesting pictures from today. I was in the middle of folding some laundry and found our son with women's underwear on his head. Not by accident either. It was completely intentional and he thought it was the funniest thing EVER. I'm not sure how we are supposed to interpret this...? But it was pretty cute!


text:
Greetings Kankas and Kankies.
Just read in the Weekly Telegraph today that Robbie Fowler (Liverpool's finest) is in Oz speaking terms with Australia's most famous sons............ FC Sydney.
Do I detect a smile forming on Oz Kanka's handsome features ???
Watch this space for more on this momentous sporting occurrence.
All the best from Eski Kanka Jim


text:
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Just a thread for fun...
WHAT was your first handgun that you ever aquired for use in Alaska?
Where you happy with it? Why/Why not?
.22 Ruger Bearcat (circa 1973ish)
12th Birthday present, first handgun.
Couldn't have BEEN happier.
Just a thread for fun...
WHAT was your first handgun that you ever aquired for use in Alaska?
Where you happy with it? Why/Why not?
.22 Ruger Bearcat (circa 1973ish)
12th Birthday present, first handgun.
Couldn't have BEEN happier.
Funny as I sit here trying to go through all my guns in my head. I can remember the first gun I bought over the counter at 18. I'm not sure on the hand gun I think my first hand gun my grandfather gave to me. It is a .357 S&W that he bought new in the early 50's he also gave me a Randell knife to go with it. I love that gun shoots where you point it and is still in 95% condition. I still take it out of the safe on occasion. Let me tell you as my seven year old son says "when my dad dies I get all his guns". Kind of funny to hear from a seven year old but you now what, he is right.
Dad gave me a S&W model 19 in .357 mag when I turned 14. I shot a moose with it up on the Koyukuk back in the early 80's. When I graduated high school he gave me a Ruger SBH in .44mag which I've never shot anything with to this day, but I sure love it! I still have 'em both though I tend to carry others more often nowadays. I've never been able to pay him back for what he's done for me in giving me this love for guns and hunting. I did get to fly him and my uncle out on a cool caribou hunt out west one year, and I did have one of my sheep horns carved out and mounted on an oak base engraved "Father of the Year 2000" "For taking a kid hunting" which he seemd to like ok. Hopefully he lives plenty long for me to come up with some other ways to show him my appreciation... or to borrow some more money from if nothign else!
My first Ak hand gun was a Ruger BH 41Mag. Got it in 1983 when we moved from Washington to Nome. It's still the handgun I carry most when I'm in the woods.
I arrived in 1975 with about a dozen, mostly 357's, but a SBH and a BH in 41, plus a K-22, a Woodsman, a Ruger Single Six and a semi. Got the most use out of an M-19 6", the K-22 and the SBH.
In 1970 A Ruger Blackhawk .44 Mag. from the "Silver Dollar Loan Co." it came with a "Lawrence" Quick Draw Rig. Lesson learned: Quick Draw Rigs ain't how one packs big heavy iron all day through Alder thickets. It was a short love affair. The whole package cost: $125.00 New.
Since I don't live in nor have never been to Alaska I can't elate to the the topic. However, when I read stuff like Fozz's 7 year old son saying "When my dad dies I get all his guns." And Chriso mentions the model 19 that his dad gave him and how much he appreciates and how he has expressed that appreciation in return....well it gives me more warm fuzzies than a whole sack full of bird dog pups and I like them a lot.
I'll never, ever get tired of reading stories in regards to our hunting and shooting traditions getting passed from one generation to the next.
Thanks for that guys!
I bought a S&W model 27, 357 when I move to Ak in 1973. Still have it and it occupies a permanent place in my vault.
Thank you, keep it UP! ps: my first rifle was ALSO from Dad, a Brand New Steven's Favorite commemorative single shot for my 7th Birthday! Gold trigger/medallian, etc, purchased with me standing with him at the counter of what was "Caribou" Wards at that time (now REI) on the corner of Spenard and Northern Lights, across from the "original" Gary Kings with the flashing neon sign of the skiier going downhill. 10th birthday was a new Ruger M77 in .243.
Chriso, you hit the 10 ring, Bro!!!
First Alaska pistol was a Ruger Blackhawk 44 mag, I bought it from a friend I worked with (1969). I promised to sell it back to him when I returned from my summer job on Kodiak Island. I carried the pistol and about 300 rounds of ammo in my gym bag on the airplane between my feet on the floor. No one even cared about what was in the bag. I carried the pistol and my 30-06 rifle when ever we left the fish camp on hikes. Saw lots of bears, shot birds, Sold the gun back to my friend when I returned. Now a 4" 500 S.& W. is my carry gun. Gerberman
As with many others here my first pistol was a Ruger Super Blackhawk 44, and it has a kinda interesting story behind it, at least I think so. I found out I was moving up here, and of course I needed a 44 mag before they would let me cross the border,right? So I started to look for one, and they ain't a common item in SW Oklahoma. I found two, a 7.5in Redhawk a buddy had but it was definately not for sale being a Christmas present from his dad. The other one was a S&W 29 Classic 8 3/8 my dad's buddy had and it was for sale, trouble being I couldn't come up with a whole 300 bucks to buy it, with it's holster, dies, ammo, and 2 sets of grips.
So as I sat around sulking that I was gonna have to go without and would surely be eaten by a bear, a fellow I went to gunsmith school with offered to help. Seems his father owned a big gunshop on the east coast and got guns from police departments, trade ins and confiscations. A quick phone call confirmed he had a SS SBH that had been used for some kind of crime that he would let me have for 100 dollars. I was in business, or so I thought. When it arrived in my possesion I found that 4 of the 5 grip frame screws were broken off in the frame, o'boy. Well a couple of hours in the machine shop and I had managed to fish them out without any damage to the frame. The only replacement screws I could find were blued and it still has 4 blued screws along with one stainless one but it works and it is the pistol I grab before I head out to hunt or fish or waalk the dog to this day.
And low and behold I've never had to even draw it on a bear, I guess they know better than to mess with a hardened criminal gun.
I grew-up here as a rifle kid, Dad only had one handgun, a Ruger Single-Six in .22lr that he still owns to this day.
My First Handgun purchase was after I was WELL into my twenties, a Walther 22lr pistol that was so picky about ammo that I immediately traded it for a well-kept 4" S&W model 19 that I've had for over ten years now. To this day it has the best trigger I have ever felt, and is as accurate as I can shoot.
My wife worked as an armed security-guard when we met in college, and she carried a Model 19 then. I pained her to return that fine revolver when she left the job and moved back to Alaska with me, now she has one again and she will never let me part with it. Not that I would anyway!
You can probably guess what my last handgun purchase was, a stainless Single-Six, just like Dad's
Yup, to me...this is what MAKES a gun.
Not so much the factory spec's or the ballistics, etc...but the STORY/memories attached to it. (Alaska, or otherwise).
This may sound funny, but I BELIEVE that there's a karma that gets attached that actually effects performance...it has a "soul" if you will. (crazy, I know...). For example...the story about old Joe Zentner's Colt Woodsman .22 in another thread.
Great posts, folks! Keep 'em coming.
Ruger Single Six, 22 LR.
Smitty of the North
Walk Slow, and Drink a Lotta Water.
Has it ever occurred to you, that Nothing ever occurs to God? Adrien Rodgers.
You can't out-give God.
Ruger Single Six .22 LR,
Not many stories yet, just bought it, my first handgun in AK so I'm a handgun rookie (well I do have a Beretta 92 but not for the woods) so handgun "Hunting" rookie is more like it
lovin' this gun tho, what a great little rig to carry around the woods
Never could get too jacked on the semiauto deal but this revolver is one excellent gun (glad I asked around on this forum while deciding what to buy)
Dad gave me his 5.5" 44 Redhawk that I always remember hanging on his hip when fishing or hiking. I guess he figured he wouldn't need it down in "Bama" where he retired so he gave it to me when I decided to move back to AK. Oddly enough I rarely carry it, I picked up a 10mm last year and it just packs better so the RH mostly gets range time and regular cleanings.
Taurus tracker 44 mag that I bought in 2006. Carry it all summer, every summer I have had it.
I like the gun and will continue to carry it even though I have several other options.
Mine was a S&W Model 629 .44 mag. I bought with a shoulder holster within my first week of being stationed at Ft. Greely in August 1994. Took it every where I went. About 1 month before I PCS'd in Dec 86 I traded it for a 3006 Model 70 Winchester XTR Sporter which I still have today. Wish I never would have sold the 629 but I needed a better Deer Rifle at the time more than I needed a pistol.
my first hand gun was a S&W model 10 38.special 4.inch barrel and bad sights that i learned to shoot with my uncle carried as a cop in Dallas he fell and the revolver got banged up pretty good from going down the steps in the apt building and after the fall down the steps dureing the fight he told the sights got a littel banged up and he did not want to carry it any more and went got a new duty revolver a model 27 ..he fixed the sights for me and would shoot basic point of aim and i carryed it in the back county areas of the farm when out on horse back and in the truck ..wore it in a old cross draw holster along with two police dump pouch fill of ammo for them on the pants belt ..
OK my first pistols to bring were #1 a 1976 roll marked BH 4 5/8" 41mag with a SS SBH grip frame #2 was my engraved S&W mod 63 first year with pinned barrel. My daughter now keeps the Smith under her pillow and a very good friend packs the Ruger


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As you may already know Future of Web Design registration is now open 🙂
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Click here to get your pass but hurry as there are only a limited number of Early Bird tickets available.
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We are doing things a little differently this year in that we are looking at the whole design process from concept to completion.
Check out the site for all the info and the location of the Official FOWD after party!! 🙂


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Aurora kinase A is a possible target of OSU‑03012 to destabilize MYC family proteins
- Authors:
- Andres Silva
- Jennie Wang
- Sarah Lomahan
- Tuan-Anh Tran
- Laura Grenlin
- Akiko Suganami
- Yutaka Tamura
- Naohiko Ikegaki
- Published online on: July 11, 2014 https://doi.org/10.3892/or.2014.3325
- Pages:901-905
Copyright: © Silva et al. This is an open access article distributed under the terms of Creative Commons Attribution License [CC BY_NC 3.0].
Neuroblastoma is the most common pediatric extracranial solid tumor of neural crest origin. Characteristically, it can exhibit either a favorable or an unfavorable phenotype. Favorable neuroblastomas are treatable with minimal interventions, whereas unfavorable neuroblastomas are aggressive and require extensive treatments, including autologous stem cell rescue (1). Long-term survival of children with unfavorable neuroblastoma is hence the lowest among childhood cancers. Approximately half of unfavorable neuroblastoma cases have MYCN amplification, which is associated with high MYCN expression, older age of onset, advanced stage disease, rapid tumor progression, and the worst prognosis (2,3). A recent study also showed that non-MYCN amplified neuroblastomas of the unfavorable subset express high levels of MYC instead of MYCN, which appears to be the determining factor of their aggressiveness (4).
OSU-0312 is a celecoxib-derived PDK1 inhibitor that has a growth suppressive effect on various cancer cell lines (5,6). When RAS is activated through external stimuli or mutation, two distinct cellular pathways are activated: the RAF/MEK/ERK and PI3/PDK1/AKT/GSK3 pathways. These pathways in turn affect the stability of MYC family proteins (7–9) (Fig. 1). In various cancer cell lines, when MEK is inhibited by U0126, there is a marked reduction in MYC protein expression levels with concomitant growth suppression (10). Based on these observations, it is plausible that inhibition of any point in the RAF/MEK/ERK and/or PI3K/PDK1/AKT pathways can destabilize MYC and MYCN. Hence, OSU-0312 was predicted to destabilize MYC and MYCN protein in neuroblastoma cells.
Figure 1
A model of the regulation of MYC family protein stability by the RAS, RAF/MEK/ERK and PI3K/PDK1/AKT/GSK3 pathways. Inhibition of RAF/MEK/ERK and/or PI3K/PDK1/AKT pathways by small-molecule inhibitors [e.g., U0126 (10) and PI-103 (21)] results in the destabilization of MYC and MYCN. Inhibition of the RAF/MEK/ERK pathway prevents phosphorylation of S62, which otherwise stabilizes MYC family proteins. S62 phosphorylation is a prerequisite for T58 phosphorylation. On the other hand, inhibition of the PI3K/PDK1/AKT pathway augments the phosphorylation of T58 by maintaining GSK3 activity, which then facilitates the degradation of MYC family proteins through proteasome proteolysis action (7–9). OSU-03012 inhibits Aurora kinase A, but not PDK1, leading to the activation of GSK3. This in turn enhances MYC family protein degradation. Alternatively, OSU-03012 binding to Aurora A causes a structural change to Aurora kinase A. This then results in the dissociation of Aurora kinase A from the MYC or MYCN protein, making them more susceptible to proteasome-mediated degradation (22).
However, since PDK1 phosphorylates AKT to activate it, and OSU-03012 is an inhibitor of PDK1, it was puzzling that under normal cell culture conditions, AKT was barely phosphorylated and OSU-03012 did not affect the p-AKT status in neuroblastoma cells (as confirmed below). This observation suggests that PDK1 is not the main target of OSU-03012 in neuroblastoma cells. In addition, it has been reported that GSK3β is phosphorylated by Aurora kinase A, and that Aurora A knockdown results in reduced MYC levels (11). These observations collectively suggest that OSU-03012 inhibits Aurora kinase A, which in turn destabilizes MYC and MYCN in neuroblastoma cells (Fig. 1). In the present study, we investigated this possibility.
Materials and methods
Neuroblastoma cell lines
The neuroblastoma cell lines were grown in RPMI-1640 supplemented with 5% (v/v) fetal bovine serum and OPI (1 mM oxaloacetate, 0.45 mM pyruvate, 0.2 unit/ml insulin, at final concentrations). These cell lines tested negative for mycoplasma, and their identity was validated by the original source or by microsatellite analysis (P.S. White, Children's Hospital of Philadelphia, Philadelphia, PA, USA; unpublished data). The IMR5 (a clone of IMR32) and CHP134 cell lines were obtained from Dr Roger H. Kennett (Wheaton College, Wheaton, IL, USA). The SKNBE(2)C cell line was from Robert Ross (Fordham University, New York, NY, USA). The SKNAS cell line was from Dr C. Patrick Reynolds (The Texas Tech University Health Sciences Center, Lubbock, TX, USA). CHP134, IMR5 and SKBBE(2)C are MYCN-amplified cell lines, whereas SKNAS is a non-MYCN-amplified cell line.
MTS assay
An MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt] assay (a water-soluble form of the MTT assay) was performed as described in our previous study (12). OSU-03012 was purchased from Cayman Chemical Company (Ann Arbor, MI, USA). VX-680 was purchased from LC Laboratories (Woburn, MA, USA). The stock solutions of the inhibitors were prepared at 10 mM in DMSO, and stored at −20°C.
Western blot analysis
Western blotting was performed as previously described (13,14). Light emission signals were captured by an LAS-3000 digital image analyzer (Fujifilm). Cell extracts were made in 2D gel sample buffer (9 M urea, 2% Nonidet-P40, 2% 2-mercaptoethanol and 0.32% pH 3.0 to 10.0 2D Pharmalyte), and the protein content of the samples was determined by the Bio-Rad protein assay kit using bovine serum albumin as a standard and the sample buffer as the blank. Antibodies used to detect proteins of interest are described in the figure legends.
Molecular docking analysis in silico
The 3D structure of AURKA (PDB ID: 3DAJ) was obtained from the Brookhaven Protein Databank. The structures of FXG and OSU-03012 were constructed using MOE (version 2007; Chemical Computing Group, Montreal, Canada). The docking simulations and interaction energy calculations were performed using MOE Dock of MOE. The most stable docking structures of FXG and OSU-03012 with AURKA were displayed by MOE.
Cell-free Aurora kinase A assay
The HTScan® Aurora A kinase assay kit (Cell Signaling Technology Inc., Danvers, MA, USA) was used to examine the inhibitory activity of OSU-03012 and VX-680 against recombinant Aurora kinase A, according to the manufacturer's instructions.
OSU-03012 and VX-680 suppress the growth of neuroblastoma cells
First, we examined the dose response of OSU-03012 and VX-680, a potent Aurora kinase inhibitor (16), on the growth of neuroblastoma cell lines (with and without MYCN amplification). As shown in Fig. 2A, both compounds were significantly growth inhibitory against the neuroblastoma cells in dose-dependent manners.
Figure 2
(A) OSU-03012 and VX-680 suppress the growth of neuroblastoma cells. CHP134, IMR5, SKNBE(2)C and SKNAS neuroblastoma cells were treated with OSU-03012 and VX-680 for 2 days and then subjected to MTS assay. (B) OSU-03012 and VX-680 destabilized MYC and MYCN, while AKT phosphorylation was not affected. CHP134, IMR5, SKNBE(2)C and SKNAS neuroblastoma cells were treated with OSU-03012 and VX-680 for 1 day and subjected to western blot assay. Anti-pan MYC monoclonal antibody, NCM II 143, was used to detect MYC and MYCN proteins. The rabbit anti-AKT (polyclonal) and anti-pAKTT308 (monoclonal, clone D25E6) antibodies were from Cell Signaling Technology Inc. (Danvers, MA, USA). Calyculin-treated (for 30 min) CHP134, IMR5, SKNBE(2)C and SKNAS cells were used to show that the anti-pAKTT308 could detect endogenous levels of pAKTT308.
OSU-03012 and VX-680 destabilize MYC and MYCN
We next assessed the effect of OSU-03012 and VX-680 on the stability of MYC and MYCN in the neuroblastoma cell lines. As shown in Fig 2B, both compounds destabilized MYC and MYCN at low μM concentrations following one day of drug treatment.
OSU-03012 and VX-680 do not affect the phosphorylation status of AKT
The status of AKT phosphorylation and the effect of OSU-03012 and VX-680 on it were examined next. As shown in Fig. 2B, under the normal culture condition, AKT was not phosphorylated at detectable levels nor was its phosphorylation status affected following drug treatment. The anti-pAKTT308 antibody used was functional, as it detected pAKTT308 upon treatment of the neuroblastoma cell lines with a protein phosphatase inhibitor, calyculin. This observation suggests that PDK1 is not the main target of OSU-03012 in neuroblastoma cells.
In silico analysis reveals that OSU-03012 inhibits Aurora kinase A
FXG is a derivative of compound 6, an Aurora kinase inhibitor, discovered through site-specific dynamic combinatorial chemistry by Cancilla et al (15). Furthermore, co-crystallization of FXG and Aurora A has been performed and the coordinates have been deposited in the Brookhaven Protein Database (PDB ID: 3DAJ). Therefore, we used this complex (FXG docking into Aurora A) as the positive control to examine whether OSU-03012 has any likelihood of binding to Aurora kinase A. We performed in silico docking analysis of OSU-03012 and Aurora kinase A. As shown in Fig. 3A, OSU-03012 exhibited a lower calculated binding energy in comparison to a positive control compound (FXG) against Aurora kinase A. This result suggests that OSU-03012 has enough potential to bind to and exhibit an inhibitory effect on Aurora kinase A.
Figure 3
(A) Docking simulation of OSU-03012 with Aurora kinase A. The docking simulations and interaction energy calculations were performed by MOE Dock of MOE. The resulting most stable docking structures between Aurora kinase A and FXG (a positive control) or OSU-03012 were displayed by MOE. (B) OSU-03012 inhibited Aurora A kinase activity in vitro. Aurora A kinase inhibitory activity of OSU-03012 was assessed by the HTScan® Aurora A kinase assay kit (Cell Signaling Technology) according to the manufacturer's instructions. VX-680, a potent and specific Aurora kinase inhibitor was used as a positive control.
OSU-03012 inhibits Aurora kinase A in an in vitro assay
To further assess if, in fact, OSU-03012 can inhibit Aurora kinase A, we performed an in vitro Aurora kinase A inhibition assay. As shown in Fig. 3B, OSU-03012 inhibited Aurora kinase A, although its efficacy was less than that of the potent Aurora kinase A inhibitor, VX-680 (16).
It has been well documented that the stability of MYC family proteins are in part regulated through their phosphorylation status. ERK-mediated serine 62 (S62) phosphorylation appears to be the first signal regulating the MYC protein stability, which stabilizes MYC proteins. However, S62 phosphorylation triggers phosphorylation of MYC family proteins at threonine 58 (T58) via GKS3. This leads to dephosphorylation of S62. MYC proteins phosphorylated at T58 are then degraded through the proteasome (Fig. 1) (7,9).
As shown in Fig. 1, inhibition at any point in the RAF/MEK/ERK and PI3K/PDK1/AKT/GSK3 pathways by small-molecule inhibitors would therefore destabilize MYC family proteins. We initially became interested in OSU-03012 as this small-molecule inhibitor was reported to inhibit PDK1. OSU-03012 is a derivative of celecoxib, which has a weak PDK1 inhibitory activity (6). Based on this observation, Zhu et al developed OSU-03012 as a more potent PDK1 inhibitor through structural optimization of celecoxib.
In fact, treatment of neuroblastoma cells with OSU-03012 resulted in destabilization of MYC family proteins (Fig. 2). However, AKT, the downstream effector of PDK1, was barely phosphorylated under normal cell culture conditions, and OSU-03012 did not affect the status of AKT phosphorylation. Thus, PDK1 is not a likely target of OSU-03012, and this compound may affect another point of the signaling cascades that regulate the stability of MYC family proteins. Yacoub et al (17) reported a similar observation that OSU-03012 activity was not closely correlated with inhibition of PDK-1 and the phosphorylation status of AKT, and argued that OSU-03012 must have additional targets apart from PDK-1 in its cytotoxic actions in lung cancer cells. Thus, the potential target of OSU-03012, apart from PDK1, that could affect the stability of MYC family proteins remained unknown.
Notably, it was reported that knockdown of Aurora kinase A by RNAi considerably reduced the expression of MYC (11). This observation and the findings mentioned above collectively indicated that OSU-03012 could inhibit Aurora kinase A, hence reducing the stability of MYC family proteins. In the present study, we investigated this possibility. In fact, in silico docking analysis and an in vitro Aurora kinase A inhibition assay demonstrated that OSU-03012 can bind and inhibit Aurora kinase A.
To date, many small-molecule inhibitors of protein kinases have been reported (18). These inhibitors often have potencies of nM IC50 values against cognate protein kinases in cell-free assay (19), and they are considered highly selective. For example, VX-608 has an IC50 value of 36 nM against Aurora kinase A in cell-free assay (20). However, Bain et al (19) demonstrated that all of the small-molecule protein kinase inhibitors that they tested had substantial off-target effects on protein kinases other than their cognate kinases. Collectively, our results suggest that OSU-03012 affects multiple cellular targets, including Aurora kinase A, to exhibit its growth suppressive and MYC and MYCN-destabilizing effects in neuroblastoma and other cancer cells.
The present study was supported by the NIH grant CA127571 and a grant from the St. Baldrick's Foundation.
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Question about Apple iPod touch
Posted by haverdegrace on
1. Plug the iPod into the primary computer linked to that iPod, the first computer you synchronized it with unless you have designated another computer as the primary computer.
2. Open iTunes and sync the iPod as normal.
3. Remove the iPod, following the normal removal procedure. The iPod unlocks automatically as you remove it.
Posted on Oct 08, 2009
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Posted on Jan 02, 2017
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