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We have identified a novel pathway of ataxia telangiectasia mutated (ATM) and DNA-dependent protein kinase (DNA-PK) signaling that results in nuclear factor κB (NF-κB) activation and chemoresistance in response to DNA damage. We show that the anthracycline doxorubicin (DOX) and its congener N-benzyladriamycin (AD 288) selectively activate ATM and DNA-PK, respectively. Both ATM and DNA-PK promote sequential activation of the mitogen-activated protein kinase (MAPK)/p90rsk signaling cascade in a p53-independent fashion. In turn, p90rsk interacts with the IκB kinase 2 (IKK-2) catalytic subunit of IKK, thereby inducing NF-κB activity and cell survival. Collectively, our findings suggest that distinct members of the phosphatidylinositol kinase family activate a common prosurvival MAPK/IKK/NF-κB pathway that opposes the apoptotic response following DNA damage.
Resistance to cancer chemotherapy may be achieved in multiple ways. In addition to transmembrane-mediated export mechanisms, the disruption of the apoptotic pathways initiated by antineoplastic drugs may lead to chemoresistance (30, 40). Recent evidence indicates that an additional pathway involved in inducible chemoresistance is triggered by anticancer drug-mediated activation of the NF-κB transcription factor, which potently inhibits the apoptotic response following DNA damage (31). Typically, activation of NF-κB involves its release from an IκB molecule in the cytoplasm and translocation to the nucleus. In response to DNA double-strand breaks (DSBs) generated by topoisomerase (topo) I and II poisons, IκB-α is phosphorylated at Ser32 and Ser36 by the IκB kinase (IKK) complex (6, 10, 15, 37), which is formed by the IKK-1 and IKK-2 catalytic subunits and by a scaffold subunit termed IKK-γ/NEMO (32). As a consequence of phosphorylation, the IκB-α protein is rapidly ubiquitinated on lysine residues 21 and 22 and degraded through the proteasome pathway, thereby allowing for migration of NF-κB to the nucleus, where it regulates the expression of a variety of genes involved in cell survival (6, 52). Consistent with this mechanism, inhibition of NF-κB activity through ectopic expression of a degradation-resistant mutant IκB-α sensitized chemoresistant tumors to the topo I poison camptothecin (CPT) (51).
Although the prosurvival role of NF-κB in response to anticancer drug treatment has been well documented, the mechanism of NF-κB activation upon drug- or irradiation (IR)-induced DNA DSBs remains poorly understood. Previously, two independent studies have reported an essential role of the ataxia telangiectasia (A-T) mutated (ATM) gene during NF-κB activation following IR or CPT-induced DSBs (24, 38). Furthermore, activation of the IKK complex upon IR or neocarzinostatin treatment was found to be severely impaired in A-T lymphoblasts, suggesting that IKKs are required for ATM-mediated induction of NF-κB in response to DNA DSBs (26).
ATM is a nuclear serine-threonine kinase that belongs to the family of phosphatidylinositol kinase (PIK)-related kinases, which, following DNA DSBs, activate multiple pathways involved in stress response, cell cycle progression, and DNA DSB repair (17, 47). For example, in response to DNA damage, ATM regulates p53 transcriptional activity through direct phosphorylation on serine 15 and promotes p53 stabilization through phosphorylation of Chk2 and Mdm2 (47). Importantly, in gamma-irradiated A-T cells, defective activation of MAP kinases (MAPKs), including extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38γ, has been documented (18, 23, 46, 48, 53). Because the MEK/MAPK signaling pathway has been implicated in NF-κB activation in response to DNA damage (41) and oncogenic Ras transformation (1), here we tested whether ATM promoted NF-κB activation through MEK/ERK in response to drug-induced DNA damage.
We show that, in response to doxorubicin (DOX)-induced DNA DSBs, ATM activates a p53-independent MEK/ERK/p90rsk/IKK signaling pathway that leads to NF-κB activation and cell survival. Importantly, we report that the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs), but not ATM, mediates NF-κB activation via the same MEK-to-IKK signaling pathway in response to treatment with the catalytic inhibitor of topo II N-benzyladriamycin (AD 288).
Overall, our findings suggest that PIK-related kinases can activate both proapoptotic (p53 and JNK) and antiapoptotic (IKK) pathways through common upstream mediators and that the balance between these opposing signals modulates the cellular response to DNA damage.
Cell culture and treatment conditions.Murine embryo fibroblasts (10)1 (14) and NIH 3T3 RelA and 3T3 RelA−/− (5) were grown in high-glucose Dulbecco's modified Eagle medium (DMEM; BioWhittaker, Walkersville, Md.) with 10% fetal bovine serum (FBS; Gibco/BRL, Rockville, Md.). (10)1 val cells express a temperature-sensitive mutant p53 and were described previously (55). HEK 293 cells were grown in RPMI medium (Cellgro, Herndon, Va.) with 10% FBS. Simian virus 40-transformed human fibroblast lines from a healthy A-T heterozygote (GM00637) and a patient with A-T (GM09607) were obtained from Coriell Cell Repositories (Camden, N.J.) and grown at low passage in Eagle minimal essential medium plus Earle salt (Sigma, St. Louis, Mo.) with 15% FBS. V-3F18 (gift from D. J. Chen, Lawrence Berkeley National Laboratory, Berkeley, Calif.), AA8, and V-3 cells (8) were grown in high-glucose DMEM with 10% FBS and 94 μM β-mercaptoethanol.
Cells were incubated with either 5 μM DOX (Sigma), 5 μM AD 288 (29) dissolved in dimethyl sulfoxide (DMSO), carrier DMSO as a control, or 20 ng of tumor necrosis factor alpha (TNF-α; Sigma)/ml. For treatment, cells were also incubated for periods of time specified below with 50 to 100 μM PD98059, 10 to 20 μM UO126, 5 mM caffeine, 50 nM to 5 μM wortmannin, and 20 μM MG132, all from Calbiochem (San Diego, Calif.), and 2 μg of cycloheximide (CHX; Sigma)/ml. Cells were irradiated with 80 Gy with a MARK I gamma irradiator.
Transfection conditions.For transient transfection, HEK 293 and GM09607 cells were plated at low confluence in 96-well plates and transfected in triplicate with a solution of DNA and Lipofectamine reagent according to manufacturer's instructions (Gibco/BRL). Sixteen hours after transfection, cells were treated with 5 μM DOX or DMSO carrier solution for 5 h. Cells were harvested after treatment according to the manufacturer's protocol in the dual-luciferase reporter assay system (Promega, Madison, Wis.), and lysates were analyzed with a Labsystems Luminoskan 96-well plate luminometer (Thermolab Systems, Needham Heights, Mass.). Firefly luciferase activity was normalized for Renilla luciferase activity, and results were expressed as levels of induction over vehicle-treated cells. Means and standard deviations reflect the results of at least three experiments, each carried out in triplicate.
Plasmids, small interfering RNAs (siRNAs), adenoviruses, and herpes simplex virus (HSV) amplicon vectors.The wild-type IκB-α-glutathione S-transferase (GST) was a generous gift from J. Hiscott (Institut Lady Davis de Recherches Medicales, Montreal, Quebec, Canada) (27). N-terminally Flag-tagged ATM (pcDNA3-Flag-ATM) and its kinase-dead mutant version (pcDNA3-Flag-KD-ATM) were kind gifts from M. Kastan (St. Jude Children's Research Hospital, Memphis, Tenn.). N-terminally hemagglutinin (HA)-tagged p90rsk (pcDNA3-RSK1) and its kinase-dead mutant version (p90rsk-D205N) were generous gifts from A. Zantema (Leiden Univiversity, Leiden, The Netherlands) (45). dnMEK1, the dominant negative mutant version of MEK, was a generous gift from J. Xie (University of Texas, Galveston) (54). Vectors expressing constitutively active mutant IKK-2 (IKK SS/EE) and the dominant negative mutant IKKAP1 (IKKAPΔC) have been described previously (34).
The SMARTpool siRNAs specific for p90rsk and MAPK1 (ERK2) were purchased from Upstate Biotechnology (Lake Placid, N.Y.) and were lipofected into cells according to the manufacturer's specifications with Lipofectamine 2000 (Invitrogen, Carlsbad, Calif.). As a negative control, we employed four pooled nonspecific siRNA duplexes that were purchased from Upstate.
The adenovirus vectors expressing wild-type IKK-1 and IKK-2 and dominant negative forms of IKK-1 (IKK-1 K>M) and IKK-2 (IKK-2 K>M) have been described previously (2). Virus stocks were amplified to high titer (Quantum Biotechnologies, Montreal, Canada). The concentration of viral particles was determined as described previously (2).
The basic amplicon vector HGCX, containing a reporter cassette consisting of the enhanced green fluorescent protein (EGFP) gene under the control of the HSV type 1 (HSV-1) immediate-early promoter, IE4/5, has been previously described (42). The HSV-1 amplicon vector carrying the full-length cDNA encoding NH2-terminal Flag-tagged wild-type ATM (pHGC-ATM) was obtained by subcloning the Flag-ATM into the HGCX amplicon under the control of the cytomegalovirus promoter (9). The efficiency of gene delivery was determined as described previously (9).
Electrophoretic mobility shift assay (EMSA).Nuclear extracts were prepared as described previously (1). Protein concentration was determined with the Bio-Rad Laboratories (Hercules, Calif.) protein assay. The sequences of the URE-κB- and Octamer-1 (Oct-1)-containing oligonucleotides are 5′-AAGTCCGGGTTTTCCCCAACC-3′ and 5′-TGTCGAATGCAAATCACTAGAA-3′, respectively.
EMSA was performed as described previously (1) using approximately 2 ng of labeled oligonucleotide (20,000 dpm) and 5 μg of nuclear extract, and complexes were resolved in 4.5% polyacrylamide gels.
Immunoblot analysis and IKK kinase assay.For isolation of whole-cell extracts (WCEs), cells were resuspended in cold PD buffer (40 mM Tris [pH 8], 500 mM NaCl, 6 mM EDTA, 6 mM EGTA, 10 mM glycerophosphate, 10 mM NaF, 10 mM p-nitrophenyl phosphate, 300 μM Na3V04, 1 mM benzamidine, 2 μM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol, 1 μg of leupeptin/ml, 10 μg of aprotinin/ml, 1 μg of pepstatin/ml, and 0.5% NP-40), sonicated briefly, and incubated at room temperature for 30 min. Extracts were then cleared by centrifugation at 40,000 rpm (Beckman TLA-100.3 fixed-angle rotor) for 30 min at 4°C. The IKK and p90rsk kinase assay was performed using GST-IκB-α or GST-IKK-2 as described previously (1). Samples (20 to 40 μg) were subjected to electrophoresis on a sodium dodecyl sulfate-10% polyacrylamide gel, and immunoblotting was performed as previously described (3). The antibody preparations for IκB-α (sc-371), IKK-1 and -2 (sc-7607), ERK (Sc-93-G), phospho-ERK (E-4), actin (sc-1615), ribosomal S6 kinase (sc-231), and HA (sc-805) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, Calif.). The anti-Flag (F4042) and anti-phospho-MEK1 and -2 (M7683) antibodies were purchased from Sigma. The anti-rabbit immunoglobulin G (IgG; 12-370) was purchased from Upstate Biotechnology. The antibody preparations against ATM (MS-ATM10-PX1) and phospho-ATM (pS1981) were purchased from GeneTex (San Antonio, Tex.) and Rockland (Gilbertsville, Pa.), respectively. The antibody preparation specific for phospho-IKK-1 and -IKK-2 was obtained from Cell Signaling Technology Inc. (Beverly, Mass.).
Activation of the prosurvival IKK/NF-κB signaling pathway by anthracyclines is independent of p53 and DNA DSBs.Because ectopic expression of p53 has been shown to promote activation of NF-κB (41), we sought to determine whether NF-κB activation by DNA-damaging agents would be impaired in p53 null cells. Surprisingly, EMSA of p53 null 10(1) cells treated with 5 μM DOX showed an activation of NF-κB DNA binding activity at the 3-h time point that remained elevated up to 12 h (Fig. 1A, left). Next, we assessed NF-κB activation by the anthracycline congener AD 288, which inhibits topo II without causing DNA DSBs (28). (10)1 cells treated with 5 μM AD 288 displayed a more pronounced induction of NF-κB DNA binding activity at the 3-h time point than DOX-treated cells, and induction started to decrease at the 12-h time point (Fig. 1A, right). Similar results were obtained with DOX- or AD 288-treated p53 null SAOS cells (data not shown). Thus, p53 activity is not required for NF-κB activation by topo II inhibitors. In addition, NF-κB can be activated in response to catalytic inhibition of topo II independent of DNA DSBs.
Because the slow kinetics of activation of NF-κB by DOX suggested that activation might require prior protein synthesis, we assessed the levels of NF-κB activity in (10)1 cells treated in the presence or absence of the protein synthesis inhibitor CHX (2 μg/ml) for 10 min, followed by treatment with 5 μM DOX for 3 h. CHX or DOX treatment alone led to an approximately fivefold induction of NF-κB DNA binding activity (Fig. 1B). Importantly, treatment with CHX plus DOX resulted in an eightfold induction of NF-κB activity. Thus, these data rule out the possibility that a cytokine secreted in response to DNA damage mediates NF-κB activation.
To assess the ability of IKK-1 and IKK-2 to phosphorylate IκB-α in response to DOX and AD 288 treatment, we carried out a kinase assay using GST-wild-type IκB-α fusion proteins. Extracts were prepared from DOX-treated (10)1 cells, immunoprecipitated with antibodies against IKK-1 and -2, and subjected to kinase assays as described previously (1). We detected maximal induction of IKK kinase activity after 2 to 3 h of DOX treatment (Fig. 1C), which indicates conformity with the kinetics of NF-κB DNA binding activity observed previously (Fig. 1A). In contrast, we did not detect IKK kinase activity using IκB-α with mutations at serines 32 and 36 (data not shown). Thus, the IKK complex mediates IκB-α phosphorylation in response to AD 288 and DOX treatment.
To further confirm these results, (10)1 cells were infected with adenovirus constructs expressing wild-type IKK-2 or the dominant negative IKK-2 K>M, a potent repressor of NF-κB activity (34). After 24 h of infection, nuclear extracts from cells treated with DOX, AD 288, or TNF-α were isolated and subjected to EMSA as described above. Cells expressing green fluorescent protein (GFP) or wild-type IKK-2 displayed increased levels of NF-κB binding in response to DOX or AD 288 treatment (Fig. 1D). In contrast, NF-κB activation by DOX or AD 288 was inhibited in cells expressing IKK-2 K>M. As expected, IKK-2 K>M, but not wild-type IKK-2, attenuated NF-κB activation in response to TNF-α treatment. Similarly, ectopic expression of the dominant negative mutant IKKAPΔC, which had been shown previously to block IKK complex activation by TNF-α and Ras (1, 33) but not that of its wild-type counterpart, wt IKKAP, inhibited DOX- and AD288-mediated induction of NF-κB luciferase activity (Fig. 1D). Thus, activation of the IKK complex is required for induction of NF-κB by anthracycline congeners.
To determine the role of NF-κB during anthracycline-mediated cell killing, cultures of wild-type and p65 (RelA) null NIH 3T3 cells were incubated in the presence of DOX, AD 288, or DMSO carrier alone for 24 and 48 h and cell viability was monitored by MTS assay. DOX or AD288 treatment for 48 h resulted in approximately 40% cell death of wild-type NIH 3T3 cells. Interestingly, similarly treated RelA null cells displayed 75 and 95% cell death, respectively (Fig. 1F). Furthermore, ectopic expression of RelA in RelA null NIH 3T3 cells rescued cell killing by DOX (data not shown). Thus, the results from this genetic analysis support a model in which inhibition of NF-κB activity potentiates anthracycline-mediated cell killing.
A p53-independent MEK/ERK signaling pathway mediates IKK complex and NF-κB activation in response to anthracycline treatment.MAPKs have been implicated in NF-κB activation in response to Ras activation (1, 13, 45, 50) or ectopic expression of p53 (41). Thus, we sought to determine whether anthracycline drug treatment also promoted activation of NF-κB through the MEK/ERK signaling pathway. Indeed, we detected induction of ERK and MEK phosphorylation after 2 h of DOX or AD 288 treatment (Fig. 2A and B). Consistent with our previous results in Fig. 1B, CHX treatment did not prevent induction of ERK phosphorylation by DOX or AD 288 (data not shown), excluding the possibility that the autocrine secretion of a cytokine in response to DNA damage promoted MAPK activation.
Given the coordinated induction of ERK phosphorylation and NF-κB DNA binding activity in response to either DOX or AD 288 treatment, we sought to determine the effects of inhibition of MEK on NF-κB activation. Treatment of (10)1 cells with the MEK inhibitors PD98059 and UO126 resulted in downregulation of DOX-mediated activation of NF-κB (Fig. 2C). Similar results were obtained for AD 288-treated cells (data not shown). Likewise, PD98059 treatment of (10)1 val cells at the nonpermissive temperature led to complete inhibition of NF-κB induction by DOX (Fig. 2D). Importantly, treatment with 50 to 100 μM PD98059 or 10 to 20 μM UO126 did not inhibit the activation of NF-κB elicited by TNF-α treatment (Fig. 2E), indicating that these compounds selectively target the MEK/ERK/NF-κB pathway at these concentrations. As predicted by our previous results, DOX- and AD 288-mediated induction of endogenous IKK-1 and IKK-2 kinase activity was inhibited by PD98059 treatment (Fig. 2F). Furthermore, ectopic expression of a dominant negative mutant MEK (54) in HEK 293 cells reduced DOX-mediated induction of an NF-κB-driven luciferase construct in a dose-dependent manner (Fig. 2G).
To further assess the role of ERKs during activation of NF-κB in response to DOX treatment, we used RNA interference to disrupt ERK2 gene expression and measured the levels of activation of the IKK complex in response to DOX treatment. For this purpose, we used an antibody specific for the phosphorylated forms of IKK-1 and IKK-2 raised against serines 180 and 181, respectively, within their activation loop. We detected a significant enhancement of IKK phosphorylation levels following 2 h of DOX in untreated or siRNA control-treated cells. In contrast, silencing the expression of ERK2 attenuated the phosphorylation levels of the IKK complex following DOX treatment (Fig. 2H). Thus, ERK2 is required for DOX-mediated activation of NF-κB.
We next determined the levels of NF-κB activity in gamma-irradiated cells treated in the presence or absence of PD98059. Exposure of (10)1 cells to 80 Gy, a dose that has been previously shown to induce NF-κB activity (16), resulted in upregulation of NF-κB DNA binding levels, which was inhibited by pretreatment with PD98059 (Fig. 2I). Thus, the MEK/ERK pathway mediates NF-κB activation in response to another DNA DSB-inducing agent.
In conclusion, the results from the EMSA, kinase assay, transient transfection, and RNA interference assay indicate an essential role of the MEK/ERK signaling pathway during NF-κB activation by DNA-damaging agents.
Inducible association of p90rsk and IKK-2 mediates NF-κB activation in response to DOX treatment.Given the ability of ribosomal S6 kinase (p90rsk), a downstream target of ERK, to mediate NF-κB activation in response to phorbol ester treatment (12, 50), we sought to determine its role during anthracycline-mediated activation of NF-κB. To address this issue, WCEs of untreated or DOX-treated (10)1 cells were isolated, immunoprecipitated with antibodies against p90rsk, and subjected to kinase assays using GST-wild-type IκB-α fusion proteins. As depicted in Fig. 3A, immunoprecipitated p90rsk from DOX-treated cells displayed three- to fourfold induction of IκB-α kinase activity over control values. Consistent with our previous data, IκB-α phosphorylation by p90rsk activity was inhibited by treatment with PD98059 (Fig. 3A). To assess the functional relevance of p90rsk IκB-α kinase activity, we next determined the effects of ectopic expression of a dominant negative mutant p90rsk (p90rsk-D205N) on DOX-mediated NF-κB activation in HEK 293 cells. In p90rsk-D205N, the aspartic acid at position 205 in the ATP-binding site of the amino-terminal kinase domain is replaced by asparagine (45). Expression of p90rsk-D205N, but not of its wild-type counterpart, abolished DOX-mediated transcriptional activation of NF-κB (Fig. 3B). Thus, both IKK and p90rsk kinase activities are required for efficient NF-κB activation in response to anthracycline treatment.
To examine whether IKKs and p90rsk form a complex in vivo, we transfected an expression vector for N-terminally HA-tagged p90rsk into HEK 293 cells, together with an expression vector for Flag-tagged IKK-2. After 24 h of transfection, cells were incubated for 2 h with DMSO alone or DOX. Subsequently, WCEs were immunoprecipitated with an anti-Flag antibody and immunoblotted with an anti-HA antibody. Immunoprecipitation of Flag-IKK-2 with the anti-Flag but not the anti-IgG antibody resulted in coprecipitation of HA-p90rsk (Fig. 3C, i). Interestingly, the interaction between HA-p90rsk and Flag-IKK-2 increased approximately threefold in response to DOX treatment. Similar results were obtained when WCEs were immunoprecipitated with an anti-HA antibody (Fig. 3C, ii). Thus, p90rsk can associate with the IKK complex and this interaction is induced following DOX treatment.
To assess whether endogenous p90rsk and IKK-2 proteins form a complex in vivo following DOX stimulation, we immunoprecipitated WCEs of (10)1 cells treated for 2 h with 5 μM DOX or DMSO vehicle using an anti-p90rsk antibody (Fig. 3D, i) or an antibody specific for IKK-1 and -2 (Fig. 3D, ii). We detected association of IKK-2 with p90rsk in DMSO-treated cells, better seen on prolonged exposure (Fig. 3D and data not shown). However, as predicted by our previous results, DOX treatment led to a significant enhancement of the interaction between p90rsk and IKK-2 (Fig. 3D). Furthermore, p90rsk and IKK-2 protein expression levels remained unchanged following DOX treatment (Fig. 3D), indicating that their inducible association is not simply due to augmented expression of p90rsk or the IKK-2 protein. Similar results were obtained with AD 288-treated cells (data not shown). To assess whether p90rsk kinase activity was required for enhanced p90rsk association with the IKK complex, we transfected HA-p90rsk and HA-p90rsk-D205N with Flag-tagged IKK-2 in HEK 293 cells. Immunoprecipitation of HA-p90rsk or HA-p90rsk-D205N with the anti-HA antibody resulted in coprecipitation of Flag-IKK-2 (Fig. 3E). As we observed previously (Fig. 3D), the association between HA-p90rsk and Flag-IKK-2 was increased by DOX treatment. In contrast, DOX treatment did not increase further the interaction of HA-p90rsk-D205N with Flag-IKK-2. Importantly, expression of HA-p90rsk-D205N did not inhibit the expression of the cotransfected wild-type IKK-2 (Fig. 3D). Thus, the inducible association between p90rsk and IKK-2 is dependent on an intact ATP-binding site in the amino-terminal kinase domain of p90rsk.
To determine the effect of the inducible association of p90rsk with IKK-2 on IκB-α degradation, we transfected 293 cells with the HA-p90rsk or the HA-p90rsk-D205N construct and assessed IκB-α protein expression levels following DOX treatment. We observed a significant delay in IκB-α degradation in cells expressing HA-p90rsk-D205N compared to cells transfected with the HA-p90rsk vector (Fig. 4A). Consistent with the inducible association between p90rsk and IKK-2 being required for IKK complex activation by DOX, cotransfection of the D205N dominant negative mutant, but not the wild-type p90rsk, abolished the induction of IKK complex phosphorylation in response to DOX treatment of HEK 293 cells (Fig. 4B). To determine whether p90rsk phosphorylated IKK-2 directly, aliquots of the same extract described in the legend for Fig. 4B were immunoprecipitated with an anti-HA antibody and subjected to kinase assays using GST-IKK-2 as the substrate. DOX treatment resulted in a significant induction of p90rsk phosphorylation levels (Fig. 4C), presumably due to autophosphorylation. Importantly, we did not observe a significant increase of p90rsk-mediated phosphorylation of GST-IKK-2 in response to DOX treatment (Fig. 4C). Consistent with this finding, we did not measure a significant phosphorylation of GST-IKK-2 by the endogenous p90rsk in DOX-treated (10)1 cells (Fig. 4D). Thus, p90rsk does not phosphorylate directly the IKK-2 subunit.
To provide genetic evidence of the requirement of p90rsk for DOX-mediated activation of NF-κB, we used RNA interference to disrupt p90rsk gene expression and measured the levels of activation of the IKK complex in response to DOX treatment. We observed a significant enhancement of IKK phosphorylation levels following 2 h of DOX in siRNA control-treated cells. In contrast, silencing the expression of p90rsk attenuated the phosphorylation levels of the IKK complex following DOX treatment (Fig. 4E). Thus, p90rsk is essential for DOX-mediated activation of NF-κB.
Collectively, our results indicate that inducible association between the endogenous p90rsk and IKK-2 molecules is essential for anthracycline-mediated activation of NF-κB.
ATM is required for NF-κB activation in response to DOX, but not AD 288, treatment.Because Rotman and coworkers reported an essential role of the ATM gene during NF-κB activation in response to CPT and ionizing radiation (26), we sought to determine the levels of NF-κB activity in response to DOX treatment of wild-type (GM00637) and A-T (GM09607) fibroblasts (35). Treatment of GM00637 cells with 5 μM DOX for the indicated times (Fig. 5A) resulted in activation of NF-κB DNA binding activity with kinetics similar to those seen in (10)1 cells (Fig. 1A). In contrast, GM09607 fibroblasts failed to display NF-κB activation in response to DOX treatment (Fig. 5A, right). Importantly, TNF-α treatment of both cell lines resulted in efficient activation of NF-κB, indicating the specificity of the signaling impairment. Thus, ATM is required for DNA DSB-mediated induction of NF-κB activity in response to topo II (DOX) as well as topo I (CPT) poisons (26).
To further confirm the involvement of the IKK complex during anthracycline-mediated NF-κB activation, we infected wild-type fibroblasts with adenovirus constructs directing expression of IKK-2 K>M or GFP and measured NF-κB DNA binding activity following DOX or AD 288 treatment. As shown in Fig. 5B, both uninfected cells and cells expressing GFP treated for 5 h with 5 μM DOX or 5 μM AD 288 displayed increased levels of NF-κB DNA binding activity. In contrast, NF-κB activation by DOX or AD 288 in cells expressing IKK-2 K>M was dramatically reduced. Thus, the IKK complex is required for ATM-mediated activation of NF-κB in response to anthracycline treatment of GM00637 fibroblasts.
Next, we assessed the role of ATM during NF-κB activation in response to AD 288 treatment that does not cause DNA DSBs. Once again, DOX treatment of A-T GM09607 fibroblasts failed to activate NF-κB (Fig. 5C). In contrast, AD 288 treatment of A-T fibroblasts led to induction of NF-κB DNA binding levels to an extent comparable to that seen in their wild-type counterpart (Fig. 5C, right), indicating that ATM is dispensable for AD 288-mediated induction of NF-κB.
Because the MEK/ERK pathway is required for NF-κB activation in response to DOX treatment and ATM is essential for NF-κB activation by DOX, we sought to determine whether ERK activation by DOX in A-T fibroblasts is impaired. As shown in Fig. 5D, either DOX or AD 288 treatment of wild-type GM00637 fibroblasts led to induction of ERK phosphorylation at the 3-h time point, which indicated conformity with the kinetics of NF-κB activation seen previously (Fig. 5C). In contrast, DOX treatment of A-T fibroblasts failed to induce ERK phosphorylation (Fig. 5D), as well as NF-κB activation (Fig. 5A and C). Interestingly, AD 288 treatment of A-T fibroblasts led to efficient induction of both ERK phosphorylation (Fig. 5D) and NF-κB DNA binding activity (Fig. 5C), again indicating that AD 288 stimulates ERK and NF-κB activities through different pathways.
ATM promotes NF-κB activation through activation of the MEK/ERK signaling pathway.To further establish the role of ATM during activation of the MEK/ERK signaling pathway in response to DOX treatment, we sought to determine the effect of inhibitors of ATM kinase activity on NF-κB activation and ERK phosphorylation. For this purpose, we treated (10)1 cells with DOX or AD 288 in the presence or absence of caffeine (5 mM), which, at this concentration, has been shown to inhibit ATM and ATR kinases but not DNA-PKcs (43). As shown in Fig. 6A, caffeine reversed NF-κB activation only by DOX, not by AD 288, again suggesting that ATM and ATR do not mediate NF-κB activation in response to AD 288 treatment. Likewise, treatment of GM00637 cells with wortmannin, another well-established ATM inhibitor (4), inhibited NF-κB activation by DOX in a dose-dependent fashion (Fig. 6B), suggesting that ATM mediates NF-κB activation following DOX treatment also in GM00637 fibroblasts. As predicted by our previous data, treatment of (10)1 cells with 20 μM U0126, 5 mM caffeine, or 5 μM wortmannin reduced phosphorylation of ERK as well as activation of NF-κB following DOX treatment (Fig. 6A and C).
To demonstrate directly that ATM regulates NF-κB activation in response to DOX treatment, we infected A-T fibroblasts with the amplicon vector HGCX (expressing EGFP) or HGC-ATM (expressing EGFP and Flag-ATM), as described previously (9), and measured NF-κB DNA binding activity and ERK phosphorylation levels following DOX treatment. To assess the kinase activity of ectopic Flag-ATM, we immunoprecipitated WCEs of DOX-treated HGC-ATM-infected cells with an anti-Flag antibody. Equal aliquots of the immunoprecipitants were then subjected to immunoblot analysis using an antibody that recognizes the autophosphorylated form of ATM (4). We detected a significant induction of phosphorylated ATM at serine 1981 in response to 1 or 3 h of DOX treatment (Fig. 6D). Thus, ectopic Flag-ATM is activated in response to DOX treatment. We next assessed the levels of NF-κB and ERK activity in HGCX- and HGC-ATM-infected GM09607 (A-T) cells following DOX treatment. As shown in Fig. 6E, we did not observe induction of NF-κB DNA binding activity or enhancement of ERK phosphorylation in either uninfected cells or cells expressing GFP treated for 5 h with 5 μM DOX. In contrast, DOX-treated HGC-ATM-infected cells displayed a three- to fourfold induction of NF-κB DNA binding activity and ERK phosphorylation levels. Thus, ectopic expression of ATM is sufficient to restore both NF-κB and ERK activation by DOX in A-T cells.
To demonstrate the requirement of ATM and MEK/ERK for the induction of NF-κB transcriptional activity following DOX treatment, we transfected A-T fibroblasts with pcDNA3 mammalian vectors directing expression of N-terminally Flag-tagged ATM (pcDNA3-Flag-ATM) or its kinase-dead mutant version (pcDNA3-Flag-KD-ATM) and measured its effect on NF-κB-luciferase activity in the presence or absence of the MEK inhibitor PD98059. As expected, in A-T fibroblasts we detected only marginal activation of NF-κB-luciferase activity in response to DOX treatment (Fig. 6F). In contrast, ectopic expression of Flag-ATM resulted in an approximately sevenfold induction of NF-κB-luciferase activity following stimulation with DOX whereas ectopic expression of Flag-KD-ATM led to a more modest threefold increase of NF-κB-luciferase activity. Importantly, treatment with 50 μM PD98059 resulted in complete inhibition of DOX-mediated induction of NF-κB luciferase activity in Flag-ATM-transfected cells. Thus, ATM regulates NF-κB activation through the MEK/ERK signaling pathway.
DNA-PKcs is required for activation of the MEK/ERK signaling pathway by AD 288.Because treatment with 5 mM caffeine did not inhibit NF-κB activation by AD 288 (Fig. 6A), we asked whether another PIK-related family member was required for AD 288 activation of NF-κB. To explore this issue, we treated A-T fibroblasts with wortmannin, a broad PIK inhibitor, and assessed NF-κB DNA binding activity following incubation with AD 288 (44). Wortmannin treatment reversed NF-κB activation by AD 288 (Fig. 7A). Likewise, treatment with PD98059 or with the proteasome inhibitor MG132 inhibited the induction of NF-κB in response to AD 288 treatment (Fig. 7B), indicating that the MEK-to-IKK pathway regulates AD 288-mediated NF-κB activation also in GM09607 (A-T) cells.
Previously, it has been shown that DNA-PKcs, a member of the PIK-related family involved in DSB repair or V(D)J recombination, could be inhibited by wortmannin but was not sensitive to 5 mM caffeine treatment (44). Thus, we sought to determine whether DNA-PKcs mediates activation of NF-κB by AD 288. For this purpose, we assessed the levels of NF-κB DNA binding activity following AD 288 treatment of DNA-PKcs−/− cells. As shown in Fig. 7C, left, treatment of wild-type Chinese hamster ovary (CHO) AA8 cells with 5 μM AD 288 resulted in induction of NF-κB DNA binding activity with kinetics similar to those seen in GM00637 fibroblasts (Fig. 5C). In contrast, induction of NF-κB activity in AD 288-treated DNA PKcs−/− CHO V-3 cells was impaired (22, 36) (Fig. 7B, right). Importantly, stable expression of DNA-PKcs in the DNA-PKcs−/− CHO V-3F18 cells restored induction of NF-κB DNA binding activity following AD 288 treatment (Fig. 7D). Consistent with ectopic DNA-PKcs being the mediator of NF-κB activation, treatment of CHO V-3F18 cells with PD98059 or wortmannin, but not caffeine, inhibited NF-κB activation in response to AD 288 (Fig. 7E).
Collectively, the results both from the genetic and the pharmacological analyses are consistent with a model in which DNA-PKcs is required for NF-κB activation in response to AD 288-mediated catalytic inactivation of topo II in the absence of DNA DSBs.
Here, we have identified a novel pathway of ATM signaling in response to DNA damage by anthracyclines involving sequential activation of MEK/ERK and p90rsk kinase activities that results in induction of IKK/NF-κB activity and cell survival. Our findings can be summarized in the following scenario. Upon DOX treatment, MEK is phosphorylated at Ser218 and Ser222 in an ATM-dependent fashion. This event results in sequential phosphorylation of ERK and of its downstream target p90rsk, which in turn associates with the IKK complex, thereby inducing IκB-α kinase activity and NF-κB activation. Remarkably, we report that DNA-PKcs, but not ATM, can activate the same MEK-to-IKK pathway in response to catalytic inhibition of topo II by AD 288 in the absence of DNA DSBs.
Previously, the ERK/MEK pathway has been implicated in NF-κB induction upon ectopic expression of p53 in SAOS cells (41). Since DOX treatment results in ATM-mediated phosphorylation of p53 and since ATM is required for NF-κB activation by DNA DSBs (17), we sought to determine whether p53 activation by ATM would induce NF-κB activity through the MEK/ERK pathway. Surprisingly, we report that p53 null (10)1 cells, as well as SAOS osteoblasts (data not shown), display efficient NF-κB activation in response to DOX treatment, indicating that NF-κB activation by DOX does not rely on p53 activity. This conclusion is further supported by our data indicating that DOX treatment can induce NF-κB activity in simian virus 40-transformed GM00637 fibroblasts in which p53 is inactivated by the presence of the large T antigen (35). Although we cannot rule out the possibility that other p53-related proteins might compensate for p53 deficiency, the observation that the kinetics of NF-κB activation by DOX were not affected by the inducible expression of a temperature-sensitive p53 protein in the derivative (10)1 val cell line at the nonpermissive temperature (data not shown) strongly argues against a role for p53 during activation of NF-κB in response to DNA DSBs caused by anthracycline treatment.
Furthermore, our data together with a recent report by Tergaonkar et al., showing that IKK-1 and -2 null fibroblasts display increased cell death and p53 induction in response to DOX treatment due to downregulation of Mdm2 expression levels (49), imply that, under physiological conditions, p53 does not regulate IKK/NF-κB activity but rather lies downstream of the IKK complex.
In addition, here we have characterized the functional mechanism underlying NF-κB activation by the MEK/ERK/p90rsk signaling pathway in response to anthracycline treatment. We show that disruption of this signaling pathway at multiple steps abolishes NF-κB activation by DOX or AD 288. Furthermore, we report for the first time that the ERK target p90rsk associates efficiently with the IKK complex, promoting IκB-α phosphorylation and NF-κB activation. From previous data, it remained unclear how p90rsk-mediated phosphorylation of IκB-α at the Ser32 residue in response to phorbol ester treatment (13, 45, 50) or Ras transformation (1, 25) would promote NF-κB activation, when efficient IκB-α degradation requires dual Ser32 and Ser36 phosphorylation (7). Here, we show that the inducible association of IKK and p90rsk kinases is required for NF-κB activation by DOX. Thus, we propose that ERK-mediated phosphorylation of p90rsk, which promotes enhanced interaction of p90rsk with the IKK complex, is essential for NF-κB-induced chemoresistance. In this context, it will be crucial to determine whether IR-mediated activation of the ERK/p90rsk pathway (19), which in this report we have shown to promote NF-κB activation, likewise induces the association between the IKK complex and the p90rsk kinase.
Very recently, Hur et al. has reported that the death domain kinase RIP forms a complex with IKKs and that this interaction is essential for DOX-mediated activation of NF-κB (16). Although neither TNF receptor signaling nor RIP kinase activity seems to play a role in this pathway, it appears that the inducible association of more than one factor with the IKK complex (e.g., p90rsk and RIP) is required for efficient NF-κB activation by DOX.
In addition, we report a novel mechanism of NF-κB activation by the anthracycline congener AD 288 based on sequential activation by DNA-PKcs of the downstream MEK/ERK/p90rsk/IKK signaling pathway.
Previously, DOX has been shown to stabilize DNA/topo II complexes generating DNA DSBs (39) whereas AD 288 was found to inhibit cleavable complex formation by reducing the binding of topo II to target DNA (28). Thus, the observation that DNA-PKcs mediates NF-κB activation in response to catalytic inhibition of topo II in the absence of DNA DSBs implies that DNA-PKcs is activated in response to DNA insults through a novel mechanism distinct from DNA DSB repair or V(D)J recombination. In support of this hypothesis we did not detect phosphorylation of DNA-PKcs at Thr 2609 following AD 288 treatment (data not shown), suggesting that the mechanism of DNA-PKcs activation in response to catalytic inhibition of topo II differs from that promoted by IR (8).
Last, we have identified the MEK/ERK pathway as a novel downstream signaling pathway that is dependent on ATM kinase activity. Our findings expand a recent study by Tang et al. that reported an essential role of ATM for etoposide-mediated activation of MAPKs (48). Intriguingly, the sequence surrounding Ser218 and Ser222 within the activation loop domain of MEK does not satisfy the criteria of the ATM consensus motif (21), indicating that intermediate kinases might be responsible for phosphorylation of MEK in response to ATM activation, as shown for c-Abl during induction of the JNK/stress-activated protein kinase pathway (20). Studies to determine the mechanism of MEK activation by ATM are under way.
Overall, our findings provide the first example of selective activation by topo-interactive agents of distinct DNA damage checkpoints, which then results in the activation of the common MEK/ERK/IKK signaling pathway, resulting in cell survival. Given the ability of ATM and DNA-PK to signal cell growth arrest and apoptosis through regulation of p53 activity in response to DNA damage (17), we propose that selective activation of ATM and DNA-PKcs by DNA-damaging stimuli results in activation of common opposing signaling pathways whose relative balance determines the ultimate fate of a cell.
This work was supported by the ACS grant RSG-02-255-01-TBE (M.A.) and in part by NIH grant CA78616 (M.A.). G. R. Panta was sponsored in part by the American Liver Foundation Irwin M. Arias, MD, Postdoctoral Research Fellowship. L. G. Cavin was supported by the Research Supplement for Under Represented Minorities Program, CA78616-S1. M. L. Cortés was sponsored by a fellowship from the A-T Children's Project.
We gratefully acknowledge Michael Kastan, Xandra Breakefield, Frederick Alt, John Hiscott, David Chen, Alt Zantema, Carlos Paya, David Sasson, Parker Suttle, Thomas Wirth, Amer Beg, and Jingwu Xie for kindly providing cloned DNAs and cell lines. We thank Steve Tavalin for helpful comments on the manuscript. We thank John Stuart and Mike Kaiser for the use of the gamma irradiator.
- Received 2 July 2003.
- Returned for modification 21 August 2003.
- Accepted 2 December 2003.
- Copyright © 2004 American Society for Microbiology


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Christine's Blog
--Guidance, Clarity, & Healing
Medical Intuitive Readings
Are you wondering what Medical Intuitive Readings are? Hi, this is Christine McClendon I offer Medical Intuitive Readings by phone or in person in the Midland, TX. and Odessa, TX. area for you or your pet. In addition, I offer other types of Clairvoyant, Medium, Pet Readings, and energy healing. However, Today I want to share what Medical Intuitive Readings are and are not. Also, how they can help you or your pet.
How to prepare for Pet Communication Readings can vary. Each Pet Communicator varies as to what they may need from you. As well as, the type of Pet Communication Reading you will be receiving. Hi, this is Christine McClendon. Certified Animal Communicator in Midland, TX. and Odessa, TX. area since 2001. I offer in person and by phone Animal Communication readings, Pet Medical Intuitive Readings, Pet Medium Readings, Pet behavior readings, and Energy Healing for your pet. As well as, different types Readings for you. For now, I want to share with you how to prepare for Pet Psychic Readings. Also known as, Animal Communication Readings. I want you and your pet to get the very best experience possible. And knowing how to prepare for Pet Psychic Readings properly will help you get the best experience possible.
Christine McClendon is a Professional Certified Clairvoyant Psychic & Intuitive Coach, offering information to help you create the life you love to live.
September 2019

subset: commoncrawl


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Aura Sensing Exercise
Ever wondered what an aura is? The term is widely used to describe the energy field around our bodies which we all have. This is part of our divine connection or part of our own spirit, because we are all spiritual beings with a physical body. If you have not experienced this, its a bit like the advert for the ready break advert and the glow around the body.
I will go into more detail about our Aura's and Chakras in other posts but for now why not try out the simple mediation below. This is an audio file so you will need speakers or earphones for this.
Its best enjoyed without any distractions from phones or people, sitting comfortably, preferably in a chair, with your back straight. its only 15 minutes long and is really simple for anyone, regardless of spiritual experience etc. Its a bit of fun and not to be taken too seriously so dont worry if you dont get it first time, meditation and spiritual stuff is like anything else in life, the more we practice, the better it gets! Please enjoy this free of charge with my love and blessings.
I have included a picture to give you an idea of the later part of the meditation but to start with, just sit comfortably with your body and arms relaxed, take a few deep breaths….
Are you sitting comfortably, then I will begin….


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Each week Ball So Hard, in association with Fiasco Sports, will be bringing you a comprehensive wrap of the past week's best performers from the NBA. Did your favourite player have a dominant week? Did he make the cut for the Team of the Week? Check out our selections below and let us know your thoughts in the comment section.
Note: The Team of the Week has a strict positional structure – two guards, two forwards and a centre in the starting five, plus a three-guard, three-forward and two-centre bench. Selections are strictly position dependant. Players must also play a minimum of three games over the seven days.
In a crucial week for final playoff seeding, Miami Heat faultered slightly in their attempt to take hold of the first seed in the Eastern Conference, going 2-3 for the week. However, superstar LeBron James did his best to push the Heat over the top, averaging 33.4 points (most in the league), 7.0 rebounds, 4.6 steals and 1.2 steals per game, while shooting 55.3% from the floor and 42.3% from beyond the arc.
Making up the backcourt in this week's Team of the Week is a pair of free-scoring guards.
Golden State Warriors sharpshooter Steph Curry was on fire from the field over his three-game schedule, averaging 28.3 points (fifth in the league) on 56.3% field goal, 50.0% three-point and 100.0% free throw shooting. Curry was also averaged the third-most assist per game for the week with 11.3 per contest.
Meanwhile, James Harden of the Houston Rockets helped his squad to a 3-2 record over the week, averaging 28.2 points (sixth in the league), 10.2 assists (fifth), 4.6 rebounds and 2.6 steals per game.
Curry and Harden were the only players to hand out more than 10 assists while scoring more than 20 points per game over the week.
Rounding out the starting five is a couple of the league's most energetic hustle players: Kenneth Faried and Joakim Noah.
Faried was his usual frantic-rebounding self, grabbing 15.0 per game, but was also impressive scoring the ball, putting in 20.3 points per game (at 52.1% from the field) over his four-game week. Faried's Nuggets also went 3-1 over the week.
But perhaps the most impressive stat line of the week goes to Noah, who almost averaged a triple-double through three games – 11.3 points, 14.0 rebounds and 9.7 assists per game– while also continuing to be a menace on the defensive end – 1.3 steals and 2.0 blocks per night.
Phoenix Suns guard Eric Bledsoe came extremely close to making the starting five thanks to his exceptional all-around performance through a four-game week. The former Clipper averaged 23.3 points (on 61.4% FG and 50.0% 3FG), 6.3 rebounds, 6.3 assists and 3.0 steals (tied third in the league) for the Suns.
Also unlucky to find himself on the bench, Thunder guard Russell Westbrook was similarly impressive across the box score, averaging 29.0 points (50.7% FG), 6.7 rebounds and 7.0 assists through three games – a stretch that Oklahoma City went 2-1.
Rounding out the bench backcourt, Chris Paul (21.7 points, 8.0 assists, 5.3 rebounds and 1.7 steals per game) continued to ready his Los Angeles Clippers team for the playoffs, going 2-1 for the week. He also enjoyed an efficient week shooting the ball, going 53.7% from the field and 53.3% from deep.
Paul's teammate Blake Griffin was also impressive for the third-seeded Clippers, averaging 26.7 points, 7.7 rebounds, 5.3 assists and 1.7 steals per game. Like Paul, he also shot the ball extremely well, hitting 51.9% from the field and 33.3% from three – which is impressive considering Griffin's history of poor outside shooting.
Also representing the Western Conference, Kevin Durant (28.8 points, 6.0 rebounds, 4.0 assists and 1.3 steals) continued his run towards the MVP, as he carried the Thunder to a 3-1 record over the past seven days.
However, the Eastern Conference is not without a representative. Atlanta Hawks forward Paul Millsap was in fantastic form (18.6 points [50.7% FG), 11.0 rebounds, 3.4 assists, 1.2 steals and 1.2 blocks through five games) as he led his team to a 4-1 record, as they clinched the final postseason spot out East.
The Eastern Conference also enjoyed a pair of sensational performances from the centre position.
The Detroit Pistons may have gone 0-3 for the week, but big man Andre Drummond manned the pivot exceptionally. The 20-year-old former UConn Huskie averaging a monster double-double of 18.7 points and 19.0 rebounds per game while shooting 64.2% from the field.
Likewise, the Sacramento Kings continued to struggle, going 1-3 last week, but the play of DeMarcus Cousins continued to impress. The enigmatic pivot notching 28.5 points, 12.0 rebounds, 2.5 steals and 1.5 blocks over his four-game week, making him one of only five players to average at least 20 points and 10 rebounds for the week.
Honourable Mentions:
Al Jefferson – 27.0 PPG, 13.3 RPG, 3.0 APG, 58.5% FG, 2-1 record
Marcin Gortat – 17.3 PPG, 11.0 RPG, 52.4% FG, 2-1 record
Timofey Mozgov – 17.8 PPG, 13.0 RPG, 1.5 BPG, 60.4% FG, 3-1 record
LaMarcus Aldridge – 21.7 PPG, 13.3 RPG, 1.7 BPG, 3-0 record
Monta Ellis – 25.0 PPG, 4.0 RPG, 4.5 APG, 50.6% FG, 46.2% 3FG, 3-1 record
Omer Asik – 11.6 PPG, 14.6 RPG, 1.6 BPG, 57.1% FG, 3-1 record
Gorgui Dieng – 14.0 PPG, 14.0 RPG, 2.3 BPG, 50.0% FG
Follow Ball So Hard on Twitter and Facebook.
Check out our Team of the Week archives here.


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Go Penguins
Due to the popularity of the 'Superlambanana' project in Liverpool, the Community group Wild in Art developed a new project for the region called 'Go Penguins' designed to dazzle the world with creativity as colonies of beautifully created penguins went on display around Liverpool, highlighting the city's cultural and seasonal offerings. On my own initiative and remembering the success of the 'superlambananas', I entered my designs for submission, representing Wirral Metropolitan College. Being successful in my application I was chosen as the artist to represent WMC for the 'Go Penguins' winter exhibition.
As well as having an educational theme, the penguin design also has a nautical touch, featuring portholes on the front & reverse of the penguin with views of the Mersey, the Liverpool skyline and the College campuses.


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Advertisments, Illustration, St. Clair College, Stop the press, The Work
St. Clair College Tecumseh Mall Murals
In 2012, We were asked to design and illustrate two hording murals for St. Clair College to be displayed inside the Tecumseh Mall. The purpose was to create an urban feel while promoting both full and part time programs being offered. The murals remained there for many years inside the mall with a lot of foot traffic increasing awareness of the College. Check out more of our work.


text: Bozo criminal for today comes from Boonville, Indiana, where bozo Jesse Gerard broke into a food co-op with big plans for stealing the anhydrous ammonia used for refrigeration. Unfortunately, during the break-in he accidentally hit the speed dial button on his phone. And even more unfortunately, the speed dial button he hit was for 911. The emergency operator could hear what was going on and, thinking something sounded suspicious, dispatched police to the scene where our bozo was caught in the act. He's under arrest.


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Jungle Juice Eliquids
-50ml Short Fill
-60ml bottle with room for a nicotine shot if required
-Made in the UK
-10 fantastic flavours available
Banana Custard:
Slip back in time to those relaxing times with you curled up on a chair, enjoying your favourite shows on the TV, with a bowl of delicious smooth creamy custard and chunky pieces of banana in it. Each vape of this delicious, all-time favourite will give you the same unmistakable taste.
Banoffee Pie:
This family favourite can never go out of fashion- taste the tropical sun-warmed perfectly yellow bananas layered over the sweet, sticky toffee and topped with light as air cream with the Jungle Juice Banofee Pie.
Blue Lagoon:
Take a plunge in the Jungle Juice Blue Lagoon. A refreshing and revitalising Blue Slush E liquid with a twist. Your taste buds will crave for more on every vape as you taste Raspberry followed closely with a hint sour but nothing too overpowering. It's the perfect blend to put a smile on your face.
Classic Bubblegum:
Relive the moments of wild abandon, of fun in the sun and sharing secrets with friends sharing a stick of bubble gum.
Fruit Salad Chews:
Separating the Black Jacks from the Fruit Salads was always a challenge but met with the reward of a fruity berry and tropical fruit sweet chew is worth it. The combination is a side of sweetness yet curiously manages to be both invigorating in its fruitiness as well as relaxing in the mellow sunshine-like flavours.
Pink Lemonade:
Close your eyes and be transported back to the cool poolside partying with friends and sipping on an ice-cold drink with a punch of deep red raspberry, cloudy Sicilian lemons and wild field Strawberry's.
Rhubarb & Custard:
The Sweetshop classic that everyone adores! Bursting with flavour to make your tastebuds scream out for more. The sharpness of Rhubarb is balanced with the creamy custard making the perfect panrtnership.
The RY4 flavour has heritage, it claims the throne on one of the original and oldest flavour profiles as a vaping E liquid. Jungle Juice has evolved RY4 to perfection! The perfect marriage of a traditional tobacco flavour carefully blended with smooth, silky caramel and Vanilla pods.
Sumptuous Strawberry:
What is summer without luscious fat juicy deep red strawberries? Bring in the bounties of summer anytime of the year with the Jungle Juice Sumptuous Strawberry E Liquid. Jungle Juice captures the taste and tactility of the strawberries to perfection to our divine celestial VG PG mix and enjoy summer anytime anywhere.
The Snowman:
He is the Friend that everyone wishes that they had. Creamy White Chocolate and light fluffy Marshmallows chunked on a bed of soft baked Cheesecake with the light aftertaste of buttery biscuit.


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I'm gratified by the peace and joy that I see in my Mindfulness Practice students as they learn to work with stress and their emotions. Through Mindfulness techniques they're able to decrease their reactions to the stress and achieve tranquility in their lives. Mindfulness is gaining wide adoption in our society, as shown by many recent articles in the mainstream media. Why this growing focus on mindfulness?
Some supporters note the hectic pace of our daily lives, which tend to be overscheduled and rife with distractions and information overload. More than ever before, it seems, we look for tools and skills to maintain our sense of self amidst the noise and demands for our attention. Ironically, the "time saving" devices we use can be part of the problem. As Kate Pickert says in the February 3, 2014 issue of Time Magazine (paywall):
"In a time when no one seems to have enough time, our devices allow us to be many places at once–but at the cost of being unable to fully inhabit the place where we actually want to be."
That constant pull away from the place where we actually want to be can take a toll on our sense of wellbeing, draining our resilience and frustrating our intentions to live life fully. Mindfulness practice can help! As Pickert observes,
"If distraction is the pre-eminent condition of our age, then mindfulness, in the eyes of its enthusiasts, is the most logical response. Its strength lies in its universality. Though meditation is considered an essential means to achieving mindfulness, the ultimate goal is simply to give your attention fully to what you're doing. One can work mindfully, parent mindfully and learn mindfully. One can exercise and even eat mindfully. The banking giant Chase now advises customers on how to spend mindfully."
It's true that whatever we're doing, we can do it with intentional awareness. Even when we're not "doing" something (maybe especially when we're simply "being"), we can be intentionally aware of our own thoughts and feelings.
I like to describe Mindfulness as "waking up to what is." It's a non-judging awareness in the present moment. It's possible to bring mindfulness into all activities of daily life, both internal and external. Practitioners enjoy lower stress and greater calmness, in the mind and the body. We gain insight and wisdom into the habits of judging, thinking and reacting that are at the core of stress reactions.
If the promises of improved wellness for mind and body seem too good to be true, consider the medical evidence that support these claims. In 1979 Jon Kabat-Zinn, the best-known proponent of mindfulness practice in this country, joined with three physicians to open a stress-reduction clinic at the University of Massachusetts that was based on meditation and mindfulness.
"Almost immediately, some of the clinic's patients reported that their pain levels diminished. For others, the pain remained the same, but the mindfulness training made them better able to handle the stress of living with illness. They were able to separate their day-to-day experiences from their identity as pain patients."[i]
Kabat-Zinn's program eventually became the curriculum that's now used by hundreds of Mindfulness teachers. Numerous studies have been funded by Congress and the NIH to evaluate the effectiveness of MBSR [Mindfulness Based Stress Reduction]:
"… scientists have been able to prove that meditation and rigorous mindfulness training can lower cortisol levels and blood pressure, increase immune response and possibly even affect gene expression. Scientific study is also showing that meditation can have an impact on the structure of the brain itself. Building on the discovery that brains can change based on experiences and are not, as previously believed, static masses that are set by the time a person reaches adulthood, a growing field of neuroscientists are now studying whether meditation–and the mindfulness that results from it–can counteract what happens to our minds because of stress, trauma and constant distraction. The research has fueled the rapid growth of MBSR and other mindfulness programs inside corporations and public institutions."[ii]
Whether you're seeking tools to cope with chronic pain, or to find clarity, or to restore your sense of balance and inner peace, I urge you to consider Mindfulness practice. There are many resources available to help you get started:
- Jon Kabat-Zinn's landmark book Full Catastrophe Living: Using the Wisdom of Your Body and Mind to Face Stress, Pain, and Illness was recently revised.
- My Mindfulness CDs are available for sale on this web site.
- I'd love to work with you one-on-one or in a Mindfulness Group – please contact me for more information.
Dr. Pamm
[i] Pickert, Kate. "The Mindful Revolution," TIME, Feb. 3, 2014.


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Bell Biv DeVoe
August 18, 2011
@ Yoshi's SF
Better than: The last time we saw BBD (which was pretty darned good)
America typically eats its child stars, or at least chews 'em up and spits 'em out. But after decades together in show business, the members of Bell Biv DeVoe are still polished and compelling performers possessing the verve of newcomers.
Ricky Bell, Michael Bivins, and Ronnie DeVoe, all 43-years-old, first began singing together at age 11 in New Edition and have been recording for 28 years. New Edition was the '80s answer to the Jackson 5, but proved to have more active longevity than the group its members idolized.With all due respect to Ralph Tresvant, Johnny Gill, and the wayward Bobby Brown, going to see BBD in concert is also like going to see New Edition, since the members perform (or at least dance to) several of that group's big tunes ("Mr. Telephone Man," "Cool It Now," and "Candy Girl") in addition to the saucy BBD jams like "Do Me" and "Something in Your Eyes." At one point, DeVoe asked the crowd if we'd like to see all of New Edition on stage again together, and hinted that it might happen some time around the new year. Only one song, the recently released, bouncy little number "Hello," was unrecognizable. Instead, most of the hour-long set was punctuated by a room full of amateur backup singers, mostly over 30, happily crooning their hearts out to hits from their childhood. Rather than feeling like a nostalgia act, however, the group's energy while singing and dancing blows away the pace of what's accepted on stage even today. Backed by a keyboardist, bassist, drummer, and an impressively precise DJ (who cut in snippets of old-school hip-hop riffs and local Bay Area cuts like E-40's "Tell Me When to Go" and Too $hort's "Blow the Whistle,") the group kept the energy level high and the lulls to a mininum. Only a faulty microphone situation stalled the show for a moment, but before that, it was impressive to watch the three members smoothly trade-off between the two working mics. All three were completely at ease on stage, but DeVoe was the one eliciting lots of laughs throughout, including when he pretended to throw his back out while dancing and repeated his ad lib outro from "Candy Girl" a few times, joking, "I have to do that part a couple times because, back in the day, that was the only damn part I had!" The rapport with the crowd felt natural and genuine. And because these are consummate crowd pleasers, BBD saved its biggest hit, "Poison," for last, inviting up a motley crew of ladies (and one really enthusiastic dude) to join them in one last round of the Running Man and the Roger Rabbit. Critic's Notebook Personal bias: Been unsuccessfully trying to perfect the New Edition choreography for 28 years. Random detail: The stage was too small to bring their female backup dancers along. By the way: BBD takes the Yoshi's stage again tonight at 8 p.m. and 10 p.m.
Follow us on Twitter @SFAllShookDown, follow Tamara Palmer @teemoney415, and like us at Facebook.com/SFAllShookDown.


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People's Parking, the accreditation scheme designed by Helen Dophin MBE, has just achieved an industry first. People's Parking, which primarily aims to improve parking for customers by enabling them to find a car park which meets their needs, recently launched a dual electric charging/disabled bay at a car park in Salford.
UK's First
Alongside co-founder Libbie Bilyard, Helen proposed the idea for the dual bay when making improvements to the People's Parking accreditation.
Helen said: "Within the accreditation we look at provision for electric vehicle charging. I'm only too aware of the increasing growth of electric vehicles and although I don't have an electric vehicle myself, I knew that as a disabled person I would struggle to get out of my car in a standard sized bay."
Dual Design
The dual electric charging/disabled bays can only be used by Blue Badge holders who are charging their electric vehicles and are a brilliant and well thought-out addition to Helen and Libbie's scheme.
Alongside the new dual accreditation, the Salford car park also achieved the People's Parking accreditation. The accreditation aims to improve car parks for customers by highlighting those with facilities for disabled people, parents, commuters, cyclists, motorcyclists, EVs and those who drive wheelchair accessible vehicles or camper vans. People's Parking also strives to drive up standards in the parking industry as all car parks achieving the accreditation will need to demonstrate that they have good signage and pedestrian routes, are well lit, clean and managed. The location of the car park is also noted to make parking easier for people who are new to an area.
Sean Fenney, NCP Head of Operations for Manchester, commented:
"At NCP we work very closely with Helen to really understand the needs of our disabled customers and to try to make our sites the best we possibly can. We were really pleased to be able to add our first dual electric charging bay for our disabled customers, as we expect that to be a very real need in the near future."
There are hundreds of car parks in the scheme which have met the accreditation's high standards all over the UK. People looking to find their perfect car park should visit the website www.peoplesparking.org.


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To commence the start of the 69th Berlinale, we headed to the International Jury Press Conference to discuss this year's selection of films, contextualisation about the festival and what to expect in the coming week. Dieter Kosslick enters his final year as director of Berlinale, bringing with him this year's theme: The Private is Political, which is extracted from the 1968's women's movement; a fitting tribute in the ode of the Time's Up movement. As Berlinale is known for premiering many avant-garde, indie and politically/socially charged pieces, this year should prove to be no exception.
Below we cover the highlight discussions from the panel which consisted of:
Juliette Binoche (France) – Jury President
Justin Chang (USA)
Sandra Hüller (Germany)
Sebastián Lelio (Chile)
Rajendra Roy (USA)
Trudie Styler (United Kingdom)
The conference commenced with Juliette speaking about the fact that over 40% of the films in the competition this year were made by female filmmakers.
"I think it's a good step forward […] ten years ago it was not like this, so now it seems we are opening our hearts, minds and eventually our bodies – but I think it's a good sign".
Rajendra went on to discuss the importance of his shirt that he donned with a blazer, proudly noting that it was created by a friend in Brooklyn in the wake of the movement.
"She decided to start a line of items such as t-shirts and buttons, the proceeds go to young women filmmakers to help with their short films […] As it says here, the future of film is female".
A journalist from Kurdistan took to the mic next, reflecting on the political status of cinema and asking about the political status of Berlinale this year in reflection of its ongoing traditional history. Juliette replied to this:
"It needs to be human first of all. If it is human then it is political. We use our knowledge, our experience, our minds as awareness. It is necessary for the present time and the future. I think that what feels human is political as consequence […] The world is quite egotistical at the moment. Many countries are closing their borders, and with climate change increasing there is a lot for questioning happenings and urgencies. I don't know what the discussions will be, but in my mind we have to do this for future generations. Governments are not working as seriously as they should be."
The following question was in reference and reflection to the whole history of German cinemas. Justin followed in response, giving reference to writing on databases and sites where your opinion is extensively criticised and people tell you not to comment:
"This is my first time back at Berlinale in seven years, and as a critic in the US when there is such a ameri-centric view of cinema, what I love about coming to international festivals is opening up that mind-set. […] The personal being political, and all cinema is political. I'm very happy to be here. I'm especially looking forward to criticism and its about having a conversation. I look forward to having these conversations with new friends and colleagues".
This then passed onto Rajendra, who compares the strands and elements of German cinema history:
"It's impossible to think about contemporary cinema without German cinema. I'm not even speaking about the popular cinema being produced in Berlin and across Germany at the moment. […] It's great to have thriving cinema in Germany again, when you look back at the years of Weimar cinema in particular".
Up next was a question that went on to look at the ability to choose roles and pick films that cover important societal issues. Trudie began this discussion:
"I would like to speak from personal experience here. Berlin has always been very courageous in looking at issues that would not always be considered by other film festivals. I am deeply honoured after coming here in 2017 with an LGBTQ film. SKIN (2018) is a redemption story of a former white supremacist. […] This is not only great, but also having 41% of women directors in the final competition. Hats off!"
Juliette then continued in reflection of what it takes to be taken by a piece during selection:
"When you start a movie, you never know if it will really happen. You have to take a risk and jump into the unknown. That's where I want to go, I like the adventure. I like not knowing. It's the game of life. You know when the film belongs to an actor. There is a link beyond rational knowledge. There is a thread in the heart between knowledge and people making it."
Sandra then gave her perspective from an actress's point of view in terms of roles:
"We are in a lucky position to choose what we want to do, and I'm aware of the responsibility that comes along with it".
The next question was directed at the mogul giant of Netflix acquiring films and the impact this has on the industry, how they felt about its aspirations. Up first was Juliette:
"It does franchise the independent films; we can bring that into question. […] Netflix is a new way of producing, it's exciting as it's something new".
Sebastian then gave his take on cinema's relationship with the streaming service:
"The death of cinema has been declared many, many times. Film is not just 35mm, it is not digital; it is a language. If it keeps being spoken about and being expanded, then it will be alive. I tend to be more integrated than apocalyptical about it. Real cultural relevance and potential of a film, however, can it happen without theatre and theatrical experience? […] I don't think film will die, but instead the collective experience".
The final questions were directed around Harvey Weinstein, with whom Juliette had worked on previous projects:
"I almost want to say peace to his mind and heart, that's all. […] I'm trying to put my feet in his shoes. He's had enough, I think. A lot of people have expressed themselves. Now justice has to do its work".


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House passes mobile workforce tax simplification bill
The House passed legislation Tuesday to simplify state income tax rules for employees who travel across state lines.
The Mobile Workforce State Income Tax Simplification Act of 2017 passed by voice vote in the House, but still awaits action in the Senate, where it has also been introduced.
The bipartisan bill, sponsored in the House by Rep. Mike Bishop, R-Mich., and Hank Johnson, D-Ga., has been a longtime priority of the American Institute of CPAs. The 2017 version of the bill attracted 57 Republican and Democratic cosponsors. It has been introduced and reintroduced in Congress ever since 2006. Last year, it passed the House, but failed to advance in the Senate.
A companion bill, S. 540, has been introduced in the Senate by Sen. John Thune, R-S.D., and Sherrod Brown, D-Ohio. Under the bill, employees would only be subject to another state's taxes if they worked there more than 30 days per calendar year. An employee's earnings would still be subject to full tax in their state of residence, however.
"The House has taken a major step toward streamlining the state income tax reporting and filing process today," Bishop said in a statement Tuesday. "Currently, employers and their traveling employees must navigate their way through a complicated maze of varying state income tax laws when they leave their home state for work. By simplifying these requirements across America, we can reduce compliance costs and confusing paperwork for everyone involved. I applaud the House for passing our bipartisan legislation and urge the Senate to join us in supporting our mobile workforce and economy."
The AICPA praised lawmakers for once again passing the bill. "The House's passage of the Mobile Workforce State Income Tax Simplification Act of 2017 is a victory for taxpayers and their employers," said AICPA president and CEO Barry C. Melancon in a statement. "Enactment of H.R. 1393 would eliminate the need for much of the complex recordkeeping that employers face when their employees cross state lines to work. It also would relieve many workers of the burden of filing state income tax returns for states in which they worked only a few days during the year."
He noted that the bill would create a uniform national standard, eliminating a compliance maze faced by many employers and employees, who need to keep track of numerous state income tax withholding laws and varying de minimis exemption periods. Employee earnings wouldn't be subject to state income tax and withholding outside their home state unless the employee worked in a state for more than 30 days during the calendar year. However, professional athletes, professional entertainers and other public figures wouldn't qualify for the 30-day de minimis exemption. They would still need to pay tax to the state where they are appearing. Non-headline performers, including dancers and musicians, would be covered by the 30-day national standard.
"The AICPA has strongly supported the passage of mobile workforce legislation for many years, and we appreciate the leadership shown by Representatives Bishop and Johnson," said Melancon. "We urge the Senate to pass its companion bill soon so that thousands of employers and employees can be relieved of the burden imposed by inconsistent state tax laws."


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Authentic and friendly meals at the foot of the Jura mountains, elegant and refreshing dishes at the lakeshores: this region's art de vivre adapts itself to the surroundings.
How pleasant to change from one atmosphere, natural setting or altitude to another, all within one region. Nyon and its region offer this diversity, be it in terms of landscapes or dishes. Within one day, you can enjoy fish from Lake Geneva on a terrace at the waterfront at lunchtime and a traditional meal in a typically Swiss setting in the evening. The only thing they have in common: the delicious wines from the La Côte AOC vineyards, the canton's largest.


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Come dine with the Sno-King Community Chorale at the Sno-King Chorale Annual Fundraiser: Music is Magic Benefit Gala
When: November 8, 2020
Where: Edmonds Yacht Club 326 Admiral Way, Edmonds, WA 98020
Who: Family, Friends, Students. Anyone looking to enjoy good food, good company, some singing and a wonderful time
Admission: $25 per person
$5 Children ages 6 -12
Children under 6 – Free
Full meal
Live music
Live Auction
Dessert Dash
Coin Toss
But most of all there will be lots of laughter and fun, fun, fun.
Purchase your tickets for the Music is Magic Benefit Gala using paypal below. Convenience fee apply.
Music is Magic Benefit Gala


text: TriSpiral logo design By Sarah Slattery | Published May 24, 2018 | Full size is 286 × 289 pixels TriSpiral logos and tagline TriSpiral corporate collateral TriSpiral logo design. Sweet! are a experienced design agency with a passion for new identities, branding and strategies with a particular love of designing food packaging.


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The Magpies made the short journey to Cae Carwyn James on Saturday afternoon for the first Cwm Gwendraeth Derby of the season against Cefnethin RFC. To allow squad rotation Tumble made a number of changes from the team that had played Betws RFC the previous Saturday and started the game on the back foot against a Cefen side who looked determined to get one over their local rivals. Under pressure the Magpies found themselves down to 14 men after 12 minutes when experienced lock forward Gareth Beynon was sent to the bin after a transgression at a ruck. It was while a man down that the Magpies put together their first real attacking move of the game when an arching run by outside half Scott Jones saw him release full back Steve Hewitt who beat the covering defence to score in the corner. From a wide angle on the left hand touchline Hewitt's conversation attempt went wide, leaving the score 0-5 to the visitors. Unfortunately this was not to signal the Magpies getting into their rhythm and establishing themselves in the game. Again Cefen seemed to have greater urgency and aggression at the break down and took the lead after 27 minutes when their left wing broke away to score a try under the posts which was converted by veteran former Magpie Richard Watts. Watts was to have a telling contribution and he did so again just before halftime when he broke through a Tumble tackle to score under the posts. He added the conversion to his own try to make the score 14-5 to the Green and Golds which is how it remained at the break.
The second half started similarly to the first for the Magpies with them absorbing Cefnethin pressure and the Green and Golds nearly scoring after three minutes but for a dropped pass. Tumble started to re-establish themselves in the game and could have scored themselves six minutes later but for a bad pass and a knock on when the try looked likely. Possession now went back and forth between the two sides and the Magpies had opportunities to create something but were too lateral in their running and displayed poor handling at key moments. The second half progressed with no additions to the scoring and it looked like the game may finish at 14-5. Late in the game it appeared that Tumble's chances of getting a score to claw Cefen's lead back had been given a boost when a Cefnethin replacement was shown the yellow card by the referee after 70 minutes. The game was now in injury time and with the Magpies deep in Cefnethin territory a loose ball saw Cefen launch a 70 metre counterattack which ultimately resulted in their wing scoring under posts. Watts slotted over the easy conversation to make the score 21-5 to the home side which is how it remained despite late pressure from Tumble that saw Gareth Beynon held up over the try line.
The boys now have a week to get over this disappointment and refocus on Round 1 of the Specsavers National Bowl where they travel to Llanelli side New Dock Stars RFC, kick off 2.30pm.
Ymlaen Y Piod
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SAN LEANDRO — A proposed law that would have required some San Leandro landlords to pay up to $10,000 in relocation costs tor their tenants is heading back to the drawing board.
The 5-2 decision by the San Leandro City Council on July 3 sends the draft ordinance to their Rules Committee, where the proposal will be revisited and revised before it is resubmitted for the full seven-member board to reconsider. That three-member Rules Committee consists of Mayor Pauline Cutter, Vice Mayor Lee Thomas and Councilman Pete Ballew.
"I think we oversimplified this tenant relocation assistance (law)," Councilman Benny Lee said at the meeting.
"We have to look at how complex this really is, and … I think we need to revisit this. In the background of the stuff that I've worked on, we did a lot of computer simulations to simulate different activities, and I think, particularly with this, we have to basically look a lot more scenarios," he said.
The two dissenting council members, Cutter and Ballew, agreed that the proposed law may require some changes but insisted that the additions could be made independently after the law's passage.
"We have to do something to protect our tenants," Ballew said at the meeting.
"We can go back to (the Rules Committee) to tighten up the ordinance and look at what we have to look at, but I think we have a moral and human obligation as representatives of this community to do something for the underserved, and I'm really disappointed in the direction that this has gone," he said.
The council made several changes to the proposed ordinance last month, including a provision requiring landlords to pay relocation costs for their tenants if rents are raised 12 percent or more within a year and a tenant chooses to move out. An earlier version from city staff set the rent increase threshold at 15 percent.
The council also opted to exempt from the law landlords who own up to four units on a single parcel. They also agreed that landlords can deduct for damages not covered by security deposits.
The proposed ordinance would currently set relocation costs at either three times the most current fair market rents or three times the monthly rent that tenants paid when they received an eviction notice, whichever is higher. Tenants with disabilities, who are seniors or have children younger than 18 years old would receive an additional $1,000.
Landlords would be entitled to deduct any past due rent incurred over one year from the relocation costs.
The law would exclude cases where tenants fail to pay rent, violate rental contracts, use rented units for illegal activities, lose their job or are given a one-year notice for planned renovations or are displaced for temporary repairs or renovations but offered alternative housing options by their landlord.
Half of the relocation costs would be paid no later than five business days after notices are delivered or tenants deliver their move-out notice. The remaining half would be paid no later than the last day for which a tenant has paid rent.
Renters and tenant advocates criticized the city's proposed law, and the changes made to it over time, as unfairly benefiting landlords. Landlords and real estate organizations, meanwhile, say the law includes a number of wrinkles that need to be worked out.
"I have really not heard anybody say, 'I like this,' not from the renters and not from the landlords, and that should say something," Councilwoman Corina Lopez said.
"We've got to find common ground and balance for this diverse community," she said.
Southern Alameda County Rental Housing Association Executive Director Bill Mulgew said the law contains "a number of landmines for both tenants and landlords that, I think, requires further work," such as having the law apply to increases in fair market rate rents, rather than base rents, which can sometimes be lower.
Landlords should also be given time to assess any possible damages to rented units before paying the second relocation assistance installment to tenants who are moving out, Mulgrew said. The law also should require that tenants give up their legal right to fight against legal eviction proceedings, if they initially accept relocation assistance, return it and do not move out.
San Leandro resident Jeromey Shafer said the proposed law should be clarified to only permit exemptions for an owner-occupied properties with up to four rental units on it.
"We can't clump everyone in the same basket," Shafer said.
"We're trying to help people whose landlords own multiple buildings, so that would help clarify that situation," he said.
Pleasanton residents Phoebe and Gary Schwaegerle said they own a 12-unit apartment complex in San Leandro and consideration should be made for longtime landlords of rental housing properties.
"We're not these greedy, corporate people who come in, like they do in Pleasanton, build a big apartment building and put the rent out there at $1,100 a month and then raise it $500 to $800 after the one-year incentive to move in," Gary Schwaegerle said.
"We're small mom-and-pop owners and we give people affordable housing for 20 years, with minuscule rent increases, so it does put a burden on us," he said.
Contact Darin Moriki at 510-293-2480 or follow him at Twitter.com/darinmoriki.


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River Description:General
The Codorus Creek is also called the inky stinky Codorus, because there is definitiely an unpleasant smell and taste to the water. For one, PH Gladfelter (pulp and paper company) dumps "treated process" water into the Codorus Creek miles upstream. This gives the water the light chocolate color. In addition, directly below the PA-24 bridge, a waste water treatment plant dumps treated water into the Codorus from river right. The water quality is acceptable for plant and fish (according to PA DEP), however a few people have gotten sick from taking in two much water on this creek. At lower levels, the Creek definitely smells, at higher levels it is not as noticeable.
With all that said the creek is still a fun beginner to intermediate level creek.Put In
From the South, on I-83 North - Take exit 22 to PA-181 North George Street. Go 1.8 miles North on PA-181, then turn right onto Emig Road. From the North, on I-83 South - Take exit 24 to PA-238 East toward Emigsville and go one mile. Turn right onto PA-181 and follow it for 0.4 miles then turn left onto Emig Road. Both: Follow Emig Road for 0.6 miles, then turn left onto Mundis Race Road. After about 0.8 miles arrive at John Rudy Park. Look for parking at the Administrative area of the park and walk your boat to Codorus creek on the other side of Mundis Race Road. The old put-in at the Sewage Treatment plant has been closed and marked "No Parking" by the plant operators.
From the Put In at John Rudy Park, go east on Mundis Race Road for about 0.5 miles. Turn left (North) onto PA Rt 24 (Sherman Street Extended) and go for approximately 4 miles. You will come to a tee in the road. Turn Right onto Codorus Furnace Road. Follow this road for approx. 3 miles. You will go down a hill and at the bottom you will cross over the Codorus Creek at the take out bridge. After you cross over the take out bridge turn right, continuing on Codorus Furnace Road and travel another 50 yards. Parking is available on your left at the stone Codorus Furnace.Major Rapids:
The first mile is mostly flat water.Entrance (II)
The first rapid has no name, but lets call it "Entrance" (If you know another name comment below). It starts out with a nice little surfing hole at the top (on river left) of the sloping rapid. at the bottom of the rapid, catch the river right eddy and try your luck at the surfing wave/hole attached to this eddy.Buckshot Rapid (III-)
This Rapid can be run straight down the center, boofing off the rock in the middle. At higher levels (>York, 4.3 ft; Pleasantville, 840 cfs), there is a slightly sticky hole below the boof rock. You can also run a fun slot move on river right from the top and then work back into the center flow and catch the boof rock. This rapid got its name because shotgun pellets from above can rain down on the river. There is a shooting range (clay pigeons) on top of the canyon wall. It is a little unnerving when it happens and you don't know why. Don't worry, no one has been hurt by the pellets.Upper Dees Rapid(III)
Start this rapid in the center, drop over the first ledge and try to catch the left or right eddy. In the next drop is a rock that has water rooster tailing off of it. This rock is not a big concern and at or above 400 cfs can be paddled over; but to avoid it set right angle and go to river right. Below the first set of right and left eddies is a second set of right and left eddies that allows you to surf a river wide wave. Below this is a large rock with a channel (slot) running through it. Most paddlers go to the right of the rock, but going left or through the center slot are options. The hardest move is going through the rock. A slightly sticky hole is on the other side of the center slot. On the river right side of the rock , you can try your luck with stern squirts and cartwheels of the edge of the rock. Lower Dees Rapid(III)
Lower Dees is separated from Upper Dees by a moving water pool of about 100 yards, in which Dees Run comes in from river right. Start this rapid in the center and work right. You can catch the river left eddy in front of the huge rock in the center of the river and from this eddy, you can surf a small dynamic wave. When you peel out of this eddy, you need to work towards river right so you do not get hung up on the rock. Below this is a pool. This a neat little falls.Trout Run Rapid or Dead Dog(II-)
This is a minor rapid, but it has an awesome squirt line on river left behind a sloping rock. You will know you are there because on river right there is a huge rock sticking out of the bank and Trout Run comes in from the right behind this rock.
For more information, see Keystone Canoeing by Gertler, E.
8 years ago
by bret goering
York Gauge in feet: 2.3 feet on York Gauge correlates to approximately 0 ft on the put in gauge located on the river left downstream adbument of the bridge. 2.3 ft is my minimum, some people will run it lower than that, but not much lower. 4.3 feet on York Gauge correlates to approximately 2.0 ft on the put in gauge. Above 5.0 feet on York Gauge would increase the overall river difficulty to Class III. I'm not sure the highest level ever run, I've run it as high as 6.2 ft on York Gauge. At that level I would rate the river difficulty as Class IV.
Because the Pleasureville gauge in cfs is more used by paddlers for this run and because York 4.3 seemed to be the top of medium runnable rather than the top of high runnable, the gauge has been changed to Pleasureville in cfs 280 - 400 low runnable; 400-840 cfs medium runnable; and 840-1280 cfs high runnable.
Permits are not required for this reach.
We have no additional detail on this route.
Use the map below to calculate how
to arrive to the main town from your zipcode.
on Codorus Creek @John Rudy Park to Codorus Furnace Road
4.71 lower dees
Low level Lower Dees
Low level Upper Dees
Surfing above SchoolHouse
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American Whitewater. Don't worry about multiple submissions from other witnesses, as our safety
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Log into the American Whitewater website and you can contribute to river descriptions,
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runs to the inventory!


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CHICAGO (CBS) — Two teenagers face charges after they stole a minivan Monday from the Galewood neighborhood and later abandoned a toddler they found inside in Oak Park.
A 17-year-old boy was charged with a felony count of unlawful possession of a stolen vehicle and a misdemeanor count of causing a child to be endangered, according to Chicago Police.
A 16-year-old boy faces misdemeanor counts of causing a child to be endangered and criminal trespass to a vehicle, police said.
Their identities were not released because they are juveniles.
At 6:18 p.m. Monday, a woman left her 1999 Pontiac minivan running with the 2-year-old inside in the 6500 block of West North Avenue and the two boys got in and drove off, police said.
The toddler was found unharmed after being abandoned by the pair in west suburban Oak Park, according to police.
The two boys were taken into custody after crashing the minivan at Central Avenue and Jackson Boulevard in Chicago's Austin neighborhood, police said.
(Source: Sun-Times Media Wire © Chicago Sun-Times 2017. All Rights Reserved. This material may not be published, broadcast, rewritten, or redistributed.)


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Ferrari Displays 'Tailor Made' Lussos in Opulent Display at Italian Embassy in Tokyo
These custom shooting brakes were centerpieces for an exclusive party held for government officials and national celebrities.
Ferrari recently put up a special one-night exhibit known as "The Art of Ferrari Tailor Made," held at the Italian embassy in Tokyo. For this event, the automaker invited 300-plus guests to take a look at nine custom renditions of the GTC4Lusso T shooting brake.
These nine cars were created as part of Ferrari's "Tailor Made" program, the goal of which is to personalize each vehicle to a specific buyer's taste. Tailor Made customers are able to select from a variety of options, such as unique paint colors, racing stripe graphics, upholstery materials, and colored interior accents. The company officially started the program in September, when it opened up a specific Tailor Made branch of its factory in Maranello.
In its release, Ferrari says that the program is "for those customers who want to customize each element of their car for a perfect fit, creating an unmistakable, one-of-a-kind vehicle which reflects their personality and tastes."
At the Art of Ferrari party, the bespoke Lussos were displayed alongside the embassy's Italian architecture and Tokyo's beautiful cityscapes. In attendance of the exhibit were guests from both countries, most noteworthy being Italian ambassador to Japan Giorgio Starace and Japanese actor Yūseke Iseya.
The GTC4Lusso T, introduced last year, forgoes the standard Lusso's naturally aspirated V12 and all-wheel-drive system for a 3.9-liter turbocharged V8 that sends its 600 horsepower exclusively to the rear wheels. Like the FF before it, the Lusso adopts a 2+2 shooting brake design able to carry four occupants quickly and comfortably. These nine Lussos were built specially for Japan, which Ferrari calls "the land where innovation, tradition, and aesthetics were dedicated."
- RELATEDFive Injured, Ferrari 812 Superfast Wrecked in Separate Crashes at Robb Report Event [UPDATED]What police are calling a "mass casualty" incident mars the luxury publication's Car of the Year event.READ NOW
- RELATEDFerrari Profits Double in Q3 2018 Thanks to Dubious, One-Off Tax BreakThe Prancing Horse wowed with a profit of $327 million, which definitely sounds better than just $161 million.READ NOW
- RELATEDA Horse-Loving Madman Stuffed a Ferrari V8 Engine Into a 1968 Ford MustangWhen you just have to have the best of two different worlds, you take matters into your own hands.READ NOW


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Always there for you
Please call our 24h service hotline in case of ALL emergencies: +49 30 403 907 900Read more
An emergency is any situation of impending danger which the tenant is unable to resolve and which needs to be resolved before the next working day.
Normally, such emergencies are power cuts in the tenants' apartment or building, burst pipes, broken down central heating systems in winter or emergency lockouts.
Please contact our service hotline in case of an emergency
Otherwise, any costs incurred by you for substitute performance (Ersatzmaßnahme) may not be reimbursable. In addition, claims for damages pursuant to § 536c BGB (German Civil Code) may arise in the event of continued damage resulting from a failure to notify us of the damage.
Emergency number
Please call our 24h service hotline in case of all emergencies:
+49 30 403 907 900