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Lipopolysaccharide (LPS) is a powerful stimulator of macrophages and induces apoptosis in these cells. Using primary cultures of bone marrow-derived macrophages, we found that the autocrine production of tumor necrosis factor-α (TNF-α) has a major function in LPS-induced apoptosis. LPS activates PKC and regulates the different mitogen-activated protein kinases (MAPK). We aimed to determine its involvement either in the secretion of TNF-α or in the induction of apoptosis. Using specific inhibitors and mice with the gene for PKCϵ disrupted, we found that LPS-induced TNF-α-dependent apoptosis is mostly mediated by PKCϵ, which is not directly involved in the signaling mechanism of apoptosis but rather in the process of TNF-α secretion. In our cell model, all three MAPKs were involved in the regulation of TNF-α secretion, but at different levels. JNK mainly regulates TNF-α transcription and apoptosis, whereas ERK and p38 contribute to the regulation of TNF-α production, probably through posttranscriptional mechanisms. Only JNK activity is mediated by PKCϵ in response to LPS and so plays a major role in TNF-α secretion and LPS-induced apoptosis. We demonstrated in macrophages that LPS involving PKCϵ regulates JNK activity and produces TNF-α, which induces apoptosis.
- cellular activation
- protein kinases/phosphatases
- signal transduction
lipopolysaccharide, isolated from the outer membrane of Gram-negative bacteria, is one of the strongest stimulators of macrophages and leads to the secretion of nitrogen intermediates, prostaglandins, and cytokines. The secretion of tumor necrosis factor-α (TNF-α), interleukin 1β (IL-1β), IL-6, and IL-12 leads to rapid induction and amplification of the host response to infection (10, 37, 42, 48). Lipopolysaccharide (LPS) has a clearly inflammatory effect and is also thought to induce apoptosis in several cell types, including macrophages (1, 20, 60). Whereas the inflammatory response is mediated through several secreted factors, the cytotoxic effects of LPS on macrophages are ascribed to the production of TNF-α or nitric oxide (NO) (3, 43, 53). Using bone marrow-derived macrophages from mice with disrupted genes for the TNF receptors or the inducible NO synthase, we determined that LPS-induced apoptosis results from two independent mechanisms: first and mainly, through the autocrine secretion of TNF-α (early apoptotic events) and second, through the production of NO (late apoptosis) (69).
LPS interacts with LPS-binding protein (LBP), thus allowing binding to CD14 and association with the Toll-like receptor 4 (Tlr4) containing an intracellular signaling domain (8). Binding of LPS to these receptors results in the activation of a number of signaling cascades, such as PKC and mitogen-activated protein kinase (MAPK). However, the precise mechanism by which LPS triggers apoptosis or the release of cytokines such as TNF-α is unclear (46).
The PKC family consists of several isoforms that fall into three main groups based on their primary structure and activation requirements. In previous studies, we described how macrophages derived from bone marrow express only three PKC isoforms: PKCβ1 (conventional), PKCϵ (novel), and PKCξ (atypical) (63). PKCϵ is involved in the regulation of important aspects of macrophage biology, in particular proliferation and macrophage activation (18, 62). However, LPS also activates the three major MAP kinase cascades in macrophages, namely the ERK, p38, and JNK pathways. All three pathways are linked to activation by LPS and subsequent cytokine gene expression (23, 26, 27, 50, 65).
Because of the importance of TNF-α-dependent apoptosis induced by LPS in macrophage biology, we attempted to identify the signaling pathways involved in this process. Here, we provide evidence that TNF-α-dependent apoptosis induced by LPS in macrophages is mediated by PKCϵ. Moreover, although all three MAP kinases are necessary for correct TNF-α regulation, only the activation of JNK is clearly mediated by PKCϵ. Therefore, JNK is mainly responsible for the TNF-α-dependent apoptosis induced by LPS. Both PKCϵ and MAPK are involved in TNF-α secretion induced by LPS and, thus, in the apoptosis induced by LPS. However, none of the three MAPKs modified the apoptotic events triggered after interaction of TNF-α with its receptor, as we showed by using recombinant TNF-α.
Reagents. LPS was obtained from Sigma Chemical (St. Louis, MO). In several experiments, the results obtained with commercial LPS were compared with those from purified LPS kindly donated by Dr. C. Galanos (Max Planck Institute, Freiburg, Germany) (40), and no differences were found. Murine recombinant TNF-α was purchased from PrepoTech EC (London, UK). Bisindolymaleimide I (GF 109203X), PD98059, SB203580, and curcumin were obtained from Calbiochem (San Diego, CA). Gö6976 was a kind gift from Dr. A. García de Herreros (Institut Municipal d'Investigació Mèdica, Barcelona, Spain). The SP600125 was obtained from Tocris Cookson (Ellisville, MO). The phosphop38 MAP kinase (Thr180/Tyr182) antibody was obtained from Cell Signaling (Beverly, MA). The p38 MAP kinase antibody (sc-535) was purchased from Santa Cruz Biotechnology, (Santa Cruz, CA). Use of all reagents followed the manufacturer's recommendations.
Cell culture. Bone marrow-derived macrophages were isolated as previously described (13). Six-week-old BALB/C mice (Charles River Laboratories, Wilmington, MA) were killed by cervical dislocation, and both femurs were dissected free of adherent tissue. The ends of the bones were cut off, and the marrow tissue was flushed by irrigation with culture medium. The marrow plugs were dispersed by passing a 25-gauge needle through them, and the cells were suspended by vigorous pipetting and washed by centrifugation. Cells were cultured in plastic tissue culture dishes (150 mm) in 40 ml of DMEM containing 20% FBS and 30% L-cell-conditioned medium as a source of macrophage colony-stimulating factor (M-CSF). Once macrophages were 80% confluent (i.e., after 6 days of culture), they were deprived of L-cell-conditioned medium for 16-18 h and treated with LPS (Sigma) in the presence and absence of selective inhibitors. The cells were incubated at 37°C in a humidified 5% CO2 atmosphere. Macrophages from PKCϵ-knockout (KO) mice (11) were obtained in the same way. The use of animals was approved by the Comitè d'experimentació animal of the University of Barcelona with the number 2523.
Analysis of apoptosis. DNA fragmentation due to internucleosomal cleavage was determined as described elsewhere (67). Low molecular weight apoptotic DNA was measured by an ELISA technique (Cell Death Detection ELISA Plus, Boehringer Mannheim), which was directed against cytoplasmic histone-associated DNA fragments. Each stage was performed in triplicate, and the results were expressed as means ± SD. In some cases, apoptosis was determined by flow cytometry with 4',6'-diamidino-2-phenylindole (DAPI) staining or by DNA laddering as previously described (67).
Determination of TNF-α production. TNF-α secretion was measured by using a commercial murine TNF-α ELISA kit (Biosource, Camarillo, CA). Cells (5 × 105) were cultured in 24-well plates and stimulated with LPS (10 ng/ml) in the presence and absence of the indicated specific inhibitors. Supernatant samples were obtained 12 h later and frozen until subjected to ELISA analysis following the manufacturer's protocol.
Protein extraction and Western blot analysis. Western blots were performed as described elsewhere (63). For phosphop38 Western blot analysis, equal amounts of protein (60 μg) were separated on 10% SDS-PAGE. The proteins were then electrotransferred to nitrocellulose membranes (Hybond-ECL; Amersham, Arlington Heights, IL). The membranes were blocked for at least 1 h at room temperature in Tris-buffered saline-0.1% Tween-20 (TBS-T) containing 5% (wt/vol) nonfat dry milk and then incubated with TBS-T containing 5% BSA and the primary antibody (1:1,000) overnight at 4°C. After three washes of 5 min each with TBS-T, the membranes were incubated with peroxidase-conjugated anti-rabbit IgG (Cappel, Durham, NC) antibody for 1 h. After three washes of 5 min with TBS-T, enhanced chemiluminescence (ECL) detection was performed (Amersham), and the membranes were exposed to X-ray films (Amersham). The bands of interest were quantified by densitometry. The Western blot of p38 MAP kinase was performed as described for the phosphorylated form, with a minor modification: the incubation with primary antibody (1:2,000) was performed with TBS-T without 5% BSA.
Northern blot analysis. Total cellular RNA (20 μg), extracted with TRIZOL reagent (Life Technologies, Grand Island, NY), was separated in 1% agarose with 5 mM MOPS [3-(N-morpholino) propanesulfonic acid], pH 7.0/1 M formaldehyde buffer. The RNA was transferred overnight to a Hybond-XL nitrocellulose membrane (Amersham) and fixed by ultraviolet irradiation (150 mJ) (62). For TNF-α mRNA detection, we used the EcoRI/HindIII fragment of pSP65/TNFa kindly supplied by Dr. M. Nabholz (Institut Suisse de Recherches Expérimentales sur le Cancer, Epalinges, Switzerland). To study the expression of IL-1β, we obtained a probe by digesting the construct pGEM/IL-1β provided by Dr. R. Wilson (Glaxo Research and Development, Greenford, UK) with EcoRI/PstI. All probes were labeled with [32Pα]dCTP (Amersham) with the oligolabeling kit method (Pharmacia Biotech, Uppsala, Sweden). To check for differences in RNA loading, the expression of the 18S rRNA transcript was analyzed by using an 18S probe, as previously described (11). After the membranes were incubated for 18 h at 65°C in hybridization solution [5 × standard sodium citrate (SSC), ×5 Denhart's, 1% SDS, and 106 cpm/ml of 32P-labeled probe], they were exposed to Kodak X-AR films (Kodak, Rochester, NY). The pertinent bands were quantified with a molecular analyst system (Bio-Rad).
Determination of MAPK activity. ERK activity was analyzed by in-gel-kinase assay, as described (63). After electrophoresis, the gel was dried, exposed to X-ray films (Kodak), and quantified with a Bio-Rad molecular analyst.
JNK activity was assayed as described elsewhere (61) with minor modifications. Briefly, the cells were washed with PBS and lysed in cold lysis buffer (1% NP-40, 20 mM HEPES-Na, pH 7.5, 10 mM EGTA, 40 mM β-glycerophosphate, 25 mM MgCl2, 2 mM sodium orthovanadate, 1 mM DTT, 0.5 mM PMSF, 1 μg/ml aprotinin, 1 μg/ml leupeptin, and 1 μg/ml iodacetamide). Total protein (150 μg) was mixed with 75 μlof 20% protein A-Sepharose and 1 μl of anti-JNK1 antibody (sc-474, Santa Cruz Biotechnology) in a total volume of 500 μl. The samples were rotated for 2 h at 4°C. The immunocomplexes were washed three times with cold PBS supplemented with 1% NP-40 and 2 mM sodium orthovanadate, once with cold JNK buffer (20 mM HEPES-Na, pH 7.5, 20 mM β-glycerophosphate, 20 mM MgCl2, 0.1 mM sodium orthovanadate, 2 mM DTT) and resuspended in JNK reaction buffer [JNK buffer supplemented with 1 μg GST-c-Jun (1-169) (Calbiochem)] and as a substrate, 20 μM ATP, 1 μCi γ-[32P]ATP. The reaction was run for 30 min at 30°C and was then stopped by adding 12 μl of ×5 Laemmli buffer. The samples were incubated for 3 min at 100°C and separated by 10% SDS-PAGE. After electrophoresis, the gels were fixed in isopropanol:water:acetic acid (25:65:10), dried, and exposed to Kodak X-AR films.
In these studies, we used bone marrow-derived macrophages, which are a homogeneous population of primary and quiescent cells (13). Bone marrow macrophages growing in the presence of M-CSF are unevenly distributed in the different phases of the cell cycle. Upon activation with LPS, macrophages stop at the G0/G1 phase of the cell cycle and die through the induction of apoptosis (11, 25). LPS-induced apoptosis was quantified by using an ELISA kit that measures the presence of histone-associated low molecular weight DNA fragments (Fig. 1). This technique, used for the quantitative measurement of apoptosis in this cellular model, correlates closely with other methods (67-69). In some experiments, apoptosis was also determined by flow cytometry with DAPI staining or by DNA laddering with qualitatively identical results. The induction of macrophage apoptosis by LPS was time and dose dependent (Fig. 1). The kinetics of induction of apoptosis was very fast with a maximal induction as early as 6 h after the start of LPS treatment. The levels of apoptosis did not increase any further after 6 h and up to 24 h of stimulation (Fig. 1A).
Maximal induction of apoptosis was obtained at a concentration of 100 ng/ml of LPS (Fig. 1B), a dose that saturates binding of LPS to its high-affinity receptor, CD14/TLR-4 (46). It has been reported that other molecules may also transfer signals at very high doses of LPS (1-10 μg/ml) (49). To avoid signaling through other LPS-receptors independent of CD14, in this study we used subsaturating amounts of LPS (1-10 ng/ml) that already induce apoptosis in macrophages (Fig. 1B). Moreover, since we were interested in the TNFα-dependent early apoptotic events in response to LPS, we performed all the studies 6-12 h after LPS treatment. Because inducible nitric oxide synthase (iNOS) protein expression and NO production after LPS treatment can only be detected after 12-24 h of treatment, any interference of NO in our results can be excluded (69). This is also confirmed by the fact that an inhibitor of NO production, SMT, does not reduce LPS-induced apoptosis at 12 h of treatment (69). To exclude any possibility of contaminants in the commercial LPS preparation, experiments were repeated using purified LPS (40), and identical results were obtained.
Because a high number of LPS-induced processes are dependent on PKC activation in macrophages (21, 33, 54), we studied the effect of PKC inhibition in macrophages. Our previous studies with specific antibodies had revealed that only PKCβ1, ϵ, and ξ were present in bone marrow-derived macrophages, whereas the rest of the isozymes were not detected (18). Because these cells cannot be transfected efficiently, we used specific chemical inhibitors (12). When the cells were preincubated with the PKC inhibitor calphostin C (10-100 nM) before the addition of LPS, we observed a dose-dependent inhibition of the induction of apoptosis in response to LPS (Fig. 2A). The next step was to establish which of the PKC isoforms present in macrophages was involved in LPS-induced apoptosis. To achieve this, we used Bisindolylmaleimyde I (GF 109203X), which is a specific inhibitor of conventional PKC (β1), and new PKC (ϵ). As shown in Fig. 2B, GF 109203X inhibits LPS-induced apoptosis, which suggests that one GF-sensitive PKC isoform is involved in the regulation of apoptosis by LPS and also excludes the involvement of atypical PKCζ in LPS-induced apoptosis because the GF 109203X doses used in our experiments do not inhibit this isoform (63).
Macrophages were also treated with Gö6976, a selective inhibitor of conventional PKC isoforms, including PKCβ1 (38). Gö6976 did not modify the levels of apoptosis induced by LPS (Fig. 2C). Taken together, these results suggest that PKC regulates LPS-induced apoptosis and that PKCϵ is the main PKC isoform involved in this process.
Finally, to check our results further, we analyzed the apoptosis induced by LPS in bone marrow-derived macrophages obtained from mice in which the PKCϵ gene had been disrupted by homologous recombination (PKCϵ KO). Macrophages from these mice were resistant to LPS induction of apoptosis (Fig. 2D), confirming the results obtained with drug inhibitors.
Because we had found in previous studies that TNF-α secretion induced by LPS is mainly responsible for LPS-induced apoptosis (69), we wanted now to determine whether the effect of PKCϵ on LPS-induced apoptosis is mediated through the induction of TNF-α expression or whether it is a mechanism directly involved with the apoptosis machinery.
Bone marrow macrophages express high levels of TNF-α mRNA after 3 h of LPS stimulation (Fig. 3A). LPS also induces the expression of IL-1β mRNA, partly as a consequence of TNF-α secretion. The inhibition of PKCϵ by pretreatment of cells with GF 109203X before LPS stimulation resulted in almost complete inhibition of the expression of TNF-α and IL-1β mRNA (Fig. 3A). Stimulation with LPS also led to high levels of TNF-α protein secretion in the cell culture supernatant, which rose to concentrations close to 4.5 ng/ml (Fig. 3B). These amounts of TNF-α are clearly sufficient to induce apoptosis in macrophages, because doses between 1 and 10 ng/ml of murine recombinant TNF-α induced significant levels of apoptosis (69). Again, inhibition of PKCϵ activity with GF 109203X inhibited the secretion of TNF-α (Fig. 3B).
Our results suggest that PKCϵ regulates LPS-induced apoptosis and that there is a correlation with TNF-α production. To determine whether LPS induces PKCϵ-dependent TNF-α secretion or TNF-α induces the activation of PKCϵ required for apoptosis, we measured the apoptosis induced by recombinant TNF-α in the presence of GF 109203X. GF 109203X did not affect the apoptosis induced by recombinant TNF-α (Fig. 3C), which suggests that PKCϵ is involved in the apoptosis induced by LPS through the induction of TNF-α production but not in the TNF-α signaling pathway that induces apoptosis.
Of macrophages from wild-type animals, macrophages from PKCϵ KO mice treated with LPS showed lower levels of TNF-α mRNA (Fig. 3D). However, recombinant TNF-α induced apoptosis in macrophages from PKCϵ KO mice at similar levels to control macrophages.
LPS also activated the three major MAP kinase cascades in macrophages, namely ERK, p38, and JNK pathways. All three pathways have been linked to activation by LPS and subsequent cytokine gene expression (24, 57). As well as by LPS, the ERK pathway is activated by growth factors and differentiation signals (51). Activation of the p38 and JNK pathways is often linked to cell stress (5, 25). Because LPS can activate different signal transduction pathways that result in the activation of MAP kinases, we decided to analyze the involvement of these MAP kinases in apoptosis induced by LPS in macrophages.
Although JNK is activated preferentially by cellular stress signals such as irradiation, heat shock, osmotic stress, and protein synthesis inhibitors, stimulation by growth factors has also been reported (28). LPS induced activation of JNK was blocked by the preincubation of macrophages with two JNK inhibitors, SP600125 (25 μM) and curcumin (diferulolymethane, 5 μg/ml), which inhibit JNK activation by different mechanisms: SP600125 acts directly on JNK kinase and curcumin inhibits JNK activation, affecting other components upstream of the JNK kinases (7, 28) (Fig. 4A). These drugs also inhibited the apoptosis induced by LPS (Fig. 4B). Moreover, when we analyzed TNF-α mRNA expression (Fig. 4C) and TNF-α secretion (Fig. 4D), we found that the expression of TNF-α also decreased when cells were incubated with the JNK inhibitors. These results suggest that the JNK pathway can play a role in the expression of TNF-α and the apoptosis induced in response to LPS.
To determine the role of the other two MAP kinases in TNF-α secretion, specific inhibitors of p38 and ERK-1/2 were used. At concentrations of 50 μM of PD98059 and 5 μM of SB203580, activation of ERK and p38, respectively, was blocked in bone marrow macrophages (Fig. 5A). PD98059 slightly decreased the levels of the TNF-α mRNA after LPS treatment, whereas SB203580 did not modify them (Fig. 5B). However, the inhibition of the ERK and p38 MAP kinases produced a minor decrease of TNF-α secretion (Fig. 5C). Because IL-1β expression is induced in response to secreted TNF-α, the SB and PD pretreatments also reduced the levels of LPS-induced IL-1β mRNA (Fig. 5, B and C). These results suggest that ERK and p38 MAPK regulate TNF-α expression mainly through posttranscriptional mechanisms, as has been reported elsewhere (32, 52, 70). Obviously, the lower TNF-α secretion derived from the response to these inhibitors also decreased LPS-induced apoptosis (Fig. 5D).
Taken together, these results suggested an involvement of PKCϵ and MAP kinases in the apoptosis induced by LPS through the regulation of TNF-α. Thus we also wanted to determine whether PKCϵ was involved in the regulation of MAP kinases, as has already been reported in peritoneal macrophages (11). As the differential time course of ERK is crucial in relation to proliferation or differentiation (61), we performed a selected MAPK analysis over time. We observed that JNK activity induced by LPS was clearly inhibited by GF 109203X at all times (Fig. 6A), but not by Gö6976 (Fig. 6B), which suggested that PKCϵ mediates the LPS-induced activation of the JNK pathway. This was confirmed by a large reduction in JNK activity after LPS treatment in macrophages from KO PKCϵ mice (Fig. 6C), whereas ERK and p38 were not inhibited.
We further extended our analysis to determine the involvement of PKCϵ in the activation of ERK and p38 MAP kinase pathways by LPS. GF 109203X slightly decreased the levels of ERK or p38 activity over short time periods but led to a significant extension of activity during the response of macrophages to LPS (Fig. 6, D and E). Again, Gö6976 did not modify the activation of ERK or p38 kinases induced by LPS. Thus PKCϵ inhibition did not block the activation of ERK and p38 kinases but promoted a long activation pattern.
Figure 3 showed that the reduction of apoptosis mediated by the inhibition of PKCϵ by GF 109203X was mediated by inhibition of TNF-α expression and not through a direct effect of PKCϵ on apoptosis signaling machinery. We extended this observation to the three MAP kinases. None of the three inhibitors (i.e., curcumin, PD, or SB) blocked the apoptosis induced by recombinant TNF-α (Fig. 7A). This suggested again that the role of the three MAP kinases in apoptosis induced by LPS is not mediated through modulation of the mechanisms involved in TNF-α-induced apoptosis signaling but through the direct regulation of TNF-α expression induced by LPS.
In this context, GF 109203X did not inhibit JNK activation (Fig. 7B) or TNF-α-induced IL-1β expression (Fig. 7C). TNF-α (10 ng/ml) did not induce the activation of ERK or p38, either. Moreover, the fact that curcumin or SP600125 did not affect apoptosis induced by recombinant TNF-α indicates that JNK activation in macrophages in response to TNF-α did not modulate the signaling cascade leading to apoptosis in response to TNF-α. Our results suggest that in response to LPS, there is a new signaling pathway that regulates TNF-α production in macrophages; LPS activates PKCϵ, which in turn activates the c-jun kinase and induces the production of TNF-α with the subsequent triggering of apoptosis.
In a previous study, we established that bone marrow-derived macrophages die through apoptosis in response to LPS, predominantly through autocrine secretion of TNF-α (69). The present paper reports the signal transduction pathway leading to the secretion of TNF-α and subsequent apoptosis. It describes the role of PKCϵ and JNK in the cascade of events that start after interaction of LPS with the cell membrane and finish with autocrine secretion of TNF-α, which induces apoptosis in macrophages.
Several processes induced by LPS in macrophages are dependent on PKC activation (21, 33, 54), such as the induction of MAPK phosphatase (MKP)-1 (62), NO production (19, 21), or the expression of TNF-α (54). As a cell model for macrophages, we used bone marrow-derived macrophages, which are primary cell cultures that can proliferate, activate, differentiate, or suffer apoptosis when induced with different stimuli. However, transfection efficiency is very low in this cell model (as is the case in other primary cell cultures) (13). Therefore, we had to use specific chemical inhibitors to study the signaling pathways involved in macrophage biology. Our results suggest that LPS induces TNF-α secretion and apoptosis through a pathway that involves the activation of PKC. The activation of this kinase is sufficient to induce apoptosis in several cell types (22, 47, 55). By using two unrelated PKC inhibitors, we showed that PKC is also involved in the induction of apoptosis in macrophages by LPS. Although PKC participates in the control of several LPS-induced events (21, 33, 54), it is still unclear which isoform(s) is involved in each of these effects. In fact, the comparison of different macrophage cell lines, or even different primary monocytic/macrophagic populations, shows significant variations in the expression of PKC isozymes (15, 39, 41), perhaps as a consequence of their specific state of differentiation/maturation. We had previously found that bone marrow macrophages express PKC isoforms β1, ϵ, and ζ (18). Although LPS shows great structural similarity with ceramide (66), a second messenger that activates PKCζ (36), it is unlikely that this isoform mediates the induction of apoptosis by LPS for several reasons. First, the PKC inhibitor GF 109203X that blocks apoptosis induction was used at doses that inhibit conventional and novel PKCs, but not atypical isoforms such as PKCζ (38). Second, we detected no PKCζ translocation in response to LPS (62). Third, although PI-3K is activated by LPS and mediates the synthesis of PIP3, a second messenger that activates PKCζ (44), wortmannin (a specific inhibitor of PI-3K), does not protect macrophages from apoptosis induced by LPS (data not shown). Finally, it has been shown that LPS and ceramide use divergent signaling pathways in macrophages to induce cell death (31). These observations suggest that PKCζ is not involved in the LPS-signaling pathway that leads to apoptosis induction.
Several observations support the involvement of PKCϵ rather than PKCβ1 in the induction of TNF-α and apoptosis by LPS. First, GF 109203X inhibits conventional PKC isoforms better than novel ones (38). Concentrations of GF 109203X up to 1 μM completely inhibit the activation of conventional PKCs, including β1, whereas concentrations up to 5 μM are needed to completely block novel isoforms, including ϵ. In our experiments, maximal inhibition of apoptosis or TNF-α production were reached at concentrations of 3-5 μM of GF 109203X. Second, Gö6976, a selective inhibitor of conventional PKCs, does not block induction of TNF-α or apoptosis by LPS. Finally, LPS does not induce apoptosis or TNF-α gene expression in bone marrow-derived macrophages from mice with an inactivated PKCϵ gene. All these results demonstrate that PKCϵ is specifically involved in the LPS-induction of TNF-α and apoptosis in bone marrow macrophages. Our results corroborate a recent report showing a critical role of PKCϵ for LPS-induced IL-12 synthesis in monocyte-derived dendritic cells (2).
Finally, we have demonstrated using inhibitors that PKCϵ plays a major role in the LPS-induced activation of JNK and in the TNF-α secretion, but in the macrophages of PKCϵ-knockout mice there is a residual JNK activity and TNF-α expression, which is not sufficient to induce apoptosis in these cells. This residual JNK activity and TNF-α expression can be blocked completely by a pretreatment with Gö6976 (data not shown), suggesting that PKCβ1 could play a small compensatory activity in the LPS signaling pathways in this PKCϵ-null mice.
The signaling events that occur after the interaction of LPS with CD14 are not fully understood, but several studies have suggested the involvement of MAP kinase activation (23, 26, 27, 50, 65). Upon receptor engagement, TNF-associated factor (TRAF)2 is recruited to CD40 and transferred to lipid rafts in a RING (really important new gene) finger-dependent process, which enables the activation of downstream signaling cascades, including JNK and nuclear factor-κB (NF-κB) (4). A small inhibition of LPS-induced apoptosis was found when macrophages were incubated with the MAP kinase inhibitors for ERK and p38. However, JNK is the kinase pathway mainly involved in the TNF-α production induced by LPS that involves activation of PKCϵ. JNK is involved in the transcriptional regulation of TNF-α through the regulation of the formation of the AP-1 transcription factor complex and the binding to the TNF-α promoter (56).
The ultimate fate of a cell exposed to TNF-α is determined by signal integration between its different effectors, including IκB kinase (IKK), c-Jun NH2-terminal protein kinase (JNK), and caspases (6). Activation of caspases is required for apoptosis (59), whereas activation of IKK inhibits apoptosis through the transcription factor NF-κB, whose target genes include inhibitors of caspases (64). JNK activates the transcription factor c-Jun/AP-1, as well as other targets (14). However, the function of JNK activation in apoptosis induced by TNF-α is less clear (34, 45).
In our experiments, the fact that PKC and MAPK inhibitors blocked apoptosis induced by LPS and TNF-α expression, but did not inhibit apoptosis induced by recombinant TNF-α treatment, indicates that PKCϵ and the three MAP kinases can modulate LPS-induced apoptosis through the production of TNF-α but not through a direct effect on the apoptotic mechanism. In endothelial cells, JNK was involved in TNF-α-induced apoptosis (30). However, in other cell models, no involvement of MAP kinases or PKCϵ has been described in the TNF-α-induced pathway leading to apoptosis (9, 16, 29). Recently, research conducted on fibroblasts has found that the NF-κB pathway negatively modulates JNK activation mediated by TNF-α and contributes to the inhibition of apoptosis (17, 58). However, our results with curcumin do not confirm these observations. The differences could be due to different responses of macrophages and fibroblasts to TNF-α (35, 69) because TNF-α induces apoptosis in macrophages without inhibiting protein synthesis or the NF-κB pathway.
Our results suggest that only JNK is downstream of the PKCϵ activity. This supports the existence of a new pathway in macrophages where LPS induces PKCϵ, which activates JNK, which in turn modulates TNF-α expression at the transcriptional level and thus mediates apoptosis induced by LPS. Kinase activity experiments support our hypothesis because JNK activity induced by LPS is inhibited when macrophages are pretreated with the PKC inhibitor GF 109203X. Our results were confirmed by the use of mice with a disrupted PKCϵ gene (40). These mice showed lower resistance to infection by Gram-positive or -negative bacteria, and the peritoneal macrophages showed a dramatic reduction of LPS-induced TNF-α production with a partial reduction of LPS-induced ERK or p38 MAP kinase activities (40). Our results confirm and extend these observations. In our experiments, the bone marrow-derived macrophages from these mice were resistant to LPS-induced apoptosis, and both TNF-α secretion and JNK activity were lower. This suggests preferential induction of JNK activity over the other two MAP kinases.
This work was supported by Ministerio de Ciencia y Tecnología Grant BMC2001-3040 (to A. Celada).
We thank Drs. Lisardo Boscá from the Instituto de Bioquimíca, Consejo Superior de Investigaciones Científicas, Madrid, and Peter Parker of the Imperial Cancer Research Fund, London, United Kingdom, for help with PKCϵ KO mice.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
- Copyright © 2003 the American Physiological Society


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We've just finished week two of the 2007 season, let's look at a few misconceptions, or just interesting facts, regarding the NFL.
Sean Payton and the New Orleans Saints have a dynamic offense that can score at will. Really? Through the first two games, the Saints have two offensive touchdowns. In fact, stretching back to last year the Saints have lost five of their last seven games.
The New York Giants are contenders. Really? Including the end of last season, Eli Manning and his Giants pals have lost nine of their last eleven games.
Brett Favre is done. Really? In the first two games of the season Brett Favre has more touchdown passes than interceptions, he has a combined 492 passing yards, and the Packers are 2-0. In fact, going back to the 2006 season, the Packers have won six consecutive games.
No Jerome Bettis and no Bill Cowher, the Steelers are primed for a decline. Really? Through the first two games of the year the Pittsburgh Steelers are 2-0, have given up only 10 points, and Quarterback Ben Roethlisberger has five touchdown passes and only one interception. Not to mention that in game one the Steelers gave up only seven points to the Cleveland Browns, the same Cleveland Browns who scored 51 points this week against the Bengals who were picked by most teams to win that division.
Sometimes all is not what it seems in professional sports, and sometimes those talking heads just plain don't get it… then again, it is only week two.

subset: commoncrawl


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Poll: Should Bella and Jasper get together withing the next chapter? Vote Now!
Author has written 7 stories for Twilight, Sonic the Hedgehog, and Supernatural.
Hi. My name is Tiffani.
When I first joined this site I was only interested in twilight. Well Edward/ Bella to be exact.
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Uriel: How dare you come in this room.. you pussing sore?
Ruby: What?
Dean: That's another question. Why would you fall? Why would you want to be one of us?
Pamela: Sam, is that you?


text: My friend since 6 episodes trying to take part in MTV Roadies contest, but he keeps failing like he will miss audition or he will be held up with important projects. This time I hope he has kept himself free for participating in this competition. Fine, currently we are in search for 2012 MTV Roadies 10 Audition dates, places, timings and more information concerning it..


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Justin O'Heir
Justin is a multi-disciplinary digital producer based out Toronto, Canada. With over 7 years of experience in the industry he has the experience and talent to bring new media projects to life.
His work has been used by industry leaders such as Live Nation, KISS, Deadmau5, Stan Lee, British Academy of Film & Television Arts (BAFTA), & The Honda Indy. He has also secured a Guinness World Record for most countries involved in an online chat.
Over the past year he has been exploring the VR world, working with Lightbox Video producing an immersive experience for music lovers across the world.
- 145 Affection
- 40,487 Photo Views
- 21 Followers
- 41 Following
- Toronto, Ontario, Canada


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how can i teach vocabulary such as " depend " to starters who english is their second language? (we are not supposed to speak in our 1st language in the class. all our talks must be in english)
English Teacher
how can i teach vocabulary such as " depend " to starters who english is their second language? (we are not supposed to speak in our 1st language in the class. all our talks must be in english)
How do you teach all the other vocabulary in your class? Are you specifically having trouble with the word "depend" or was that just an example?
Only speaking in the second language is quite common when teaching a foreign language. It stops both the teacher and the student from constantly falling back on their own language. Everyone needs to work harder.
Please look at my corrections to your post. It is very important to use correct written English on the forum so please follow the rules of written English:]
- Start every sentence with a capital letter.
- End every sentence with a single appropriate punctuation mark.
- Always capitalise the word "I".
- Always capitalise proper nouns (ie English, London, Prime Minister).
- Do not put a space after an opening quotation mark or before a closing quotation mark.
Remember - if you don't use correct capitalisation, punctuation and spacing, anything you write will be incorrect.
That seems too advanced a concept for beginners, in my view.
Last edited by 5jj; 19-Aug-2013 at 09:23. Reason: typo corrected
While teaching vocabulary to starters we use different ways:
1. You can show pictures or situations in pictures.
2. If the first way doesn't help, just use the method of miming.
3. Last of all comes the method of translating it into your first language.
You can do it in English using only one word in your native tongue.
e.g. you can say: the word depend means ......... in our native tongue...
This is really useful. Thank you so much!


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Västerbotten » Ammarnäsfjällens Islandshästar
You start to sense the atmosphere already in Ammarnäs, a small mountain village at the beginning or end of the road, depending on how you look at it. Ammarnäs is a small mountain village in of one of Europe's largest protected areas, Vindelfjällen nature reserve, a wild and exciting part of Swedish Lapland.
Ammarnäsfjällens Islandshästar offers you various rides on Icelandic horses in the magnificent mountain scenery. The ride goes along the river Vindel's unregulated waters, along the banks of the river, into the forest or up into the bare mountain region above the tree line. The speed varies from an easy walking pace to a glorious gallop. Try the rack, that particular gait that makes the Icelandic horse so easy to ride. Horse riding tours from 2 hours up to 4-5 days. You can also try a log-fired sauna and/or bathe in the wooden bathtub at the company´s base "Hedebo farm". Eco-tourism operator since 2003 ("Nature´s best").


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Crop Protection
From Horticulture
Whereas in the past we controlled pests by applying a crop protection product, nowadays along with the grower we concentrate on supervising and managing the crop as a whole. This requires from us, as supplier, a broad, pro-active and knowledge-intensive approach to the whole crop.
The basis of crop protection is monitoring the gro...
The basis of crop protection is monitoring the growth of the crop, the pressure of diseases and pests, and factors regarding the soil and the environment. On this basis, sustainable choices are made. The deployment of crop protection products takes place within an integrated approach. Organic crop protection products and natural enemies therefore form a vital part of the integrated crop protection. Many flower, plant and vegetable growers are successfully using this approach.
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Public Discussion and Q&A " 'The Great War': Screening and Reflections"
4/1/17Saturday @ 10:30 AM
The Institute on World War II and the Human Experience, Department of History, Florida State University invites the general public to attend a free screening of a preview of "The Great War" a three-part documentary featured in the PBS American Experience series, and a Q&A session following with the film's writer, director, and producer Stephen Ives and historian Michael Neiberg on Saturday, April 1, 2017 at 10:30 a.m. at the Hyatt Regency Jacksonville Riverfront, Jacksonville, Florida. Stephen Ives is a groundbreaking documentarian with over 20 years of experience in television, and is well known for his work on the American Experience, PBS, HBO and THE WEST, one of the most watched PBS programs of all time. Michael Neiberg is a leading historian of the First World War; he is currently a professor of history at the U.S. Army War College and has written prominent books such as Dance of the Furies: Europe and the Outbreak of World War I (Harvard University Press, 2013), which the Wall Street Journal recently named one of the five best books ever written about the First World War.
"The Great War" tells the diverse story of World War I through the voices of nurses, journalists, aviators and American troops. The series explores the experiences of African-American and Latino soldiers, suffragists, Native American "code talkers" and others whose participation in the war to "make the world safe for democracy" has been largely forgotten. The series will premiere Monday April 10 through Wednesday, April 12, 9-11pm on WJCT and PBS stations nationwide.
The public screening of "The Great War" is part of series of programs open to the general public as part of the 84th Annual Meeting of the Society for Military History (March 30-April 2, 2017). To learn more about these events and the 84th Annual Meeting hosted by the Institute on World War II and the Human Experience, please visit ww2.fsu.edu/smh-conference or contact the Institute Director, G. Kurt Piehler at email@example.com or (850) 644-9541. The Society for Military History (SMH), founded in 1933, strives to advance the study of military history and stimulate interest in the subject.


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revealing journal, entice her to stay and discover more, including a way to save the gallery from financial ruin. And that means remaining in England.When her childhood sweetheart, Adam Spencer, begins work on a restoration project in Noelle's village, their friendship blossoms. But as her feelings for Adam deepen, she struggles with memories of what might have been and yearns for a future once thought lost. Faced with a life-altering revelation Aunt Joy took to her grave and a wrenching choice regarding the man she loves, Noelle could lose far more than her heart.
happy that this book was able to remind me of that.
Not only is she re-discovering things about herself that she had long since forgotten, but she runs into her childhood friend Adam who is no longer a child, but a smokin' hot man. A smokin' hot man that is engaged to be married.
As things between she and Adam grow and a friendship is formed, she also discovers letters and diaries left behind her great aunt's house that reveal many secrets.
can't believe I just typed that.
The language is so descriptive, I think it takes true talent to paint such a complete picture and I loved it.
detractor for me in this instance. Color me shocked! I loved the setting, the characters, the storyline(s)…just a fabulous book all around. 5 stars.
Traci Borum is a writing teacher and native Texan. She's also an avid reader of women's fiction, most especially Elin Hilderbrand and Rosamunde Pilcher novels. Since the age of 12, she's written poetry, short stories, magazine articles, and novels.
Traci also adores all things British. She even owns a British dog (Corgi) and is completely addicted to Masterpiece Theater–must be all those dreamy accents! Aside from having big dreams of getting a book published, it's the little things that make her the happiest: deep talks with friends, a strong cup of hot chocolate, a hearty game of fetch with her Corgi, and puffy white Texas clouds always reminding her to "look up, slow down, enjoy your life."


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Heating & Air Conditioning Sales, Service
and Installation Sevierville, TN
Cox & Webb Inc
Cox & Webb Inc has been serving Sevier County's heating and air conditioning sales, service, and installation needs since 1965. Locally owned and operated by Jim Cox & Johnny Cheek, our company specializes in residential and light commercial heating and air conditioning installation. With licensed and insured technicians, we can assure high quality service.
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Call Cox & Webb today at 865-366-5044.
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Embracing cooking that's satisfying to the senses: In the kitchen of Aru Reddy
Like her mother and those before her, Aru Reddy said she cooks by feel, smell and taste.
John Curry/Staff
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My new favorite beverage…
I've been wanting to add more probiotics into my life without yogurt or additional supplements. And I'd heard of Kombucha and its benefits extensively, but the name really deterred me into thinking it was some ancient pagan herb. I've read so many people, though, of a similar school of thought when it comes to nutrition and health, who swear by this stuff.
So I bought it…for $3.49 a bottle! At Whole Foods, of course. Which is just outrageous. But I did it. Because I was curious.
I'm pretty sure it's not whole 30 compliant, though, because there's a bit of alcohol and its made with a bunch of sugar at the start of the brewing process. Eh. Can't win 'em all.
I brought it home to Kevin, who loved it as well (mind you we had the ginger flavor and I LOVE ginger). I think we'd like to brew our own! So we'll add it to our list of DIY to-do's, but if I can get all those benefits for the fraction of the cost – I'm all in!
Also, does anyone know of a cheaper brand in the meantime? :)


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Blonde TV presenter fails to notice EYE-POPPING wardrobe malfunction live on air
A STUNNING TV host who failed to notice a humiliating wardrobe gaffe has become a viral sensation.
The woman is seen donning a black leather dress and a sheer top that leaves very little to the imagination.
Her ample cleavage struggles to contain itself as she chats to her colleagues.
At first – barring the skimpy outfit – nothing seems to be wrong.
But when she sits down to talk to another brunette beauty, disaster strikes.
The busty babe's assets finally burst out and all is revealed.
She continues to smile and chat away – completely oblivious to how much flesh she is flashing.
Unfortunately for her, footage of the clip has since gone viral on YouTube, with more than 900,000 views.
One user commented: "Wonder if anyone told her later about it."
Another wrote: "She was tempting fate with that top."
A third added: "That is so embarrassing…how did she not notice!?"
It comes after a gameshow host flashed her boobs when her top fell down midway through the programme.
A similarly shocking video of Korean pop princess recently went viral, after she performed a raunchy routine in a scandalously revealing outfit.


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A World at Risk
♦ The pace of global economic growth started to slow down at the beginning of the year and is now close to zero.
♦ The main cause of this slowdown in growth is an almost universal tightening of economic policy. In the developing world, interest rates were raised to stop inflation. In the developed world, government spending was cut in order to reduce the debt. Thus, irrespective of its motives, tightening of monetary or fiscal policy has caused a slowdown around the world.
♦ The immense power of a uniform policy (tightening in this case) can be appreciated by the fast exit from the large recession once all governments around the globe eased policy. But despite the already obvious result of the tightening measures, policy makers are still tightening in the developing world (China, India) and the developed world (U.S., Italy). Therefore, steps in this direction could guarantee a doubledip in the near future.
♦ The US: Total government spending – – including the federal government and the much larger sector of state & local governments – – has been declining steadily from the beginning of the year. This tightening of fiscal policy, with the added effects of the tsunami in Japan and the slowdown in the rest of the world, slowed growth to a pace that historically ended in a recession. The reaction of Congress was a decision to cutback federal spending in the next ten years. Even worse, this was done in a way that made it clear to everybody that the president is weak and most members of Congress are willing to cause any damage to the economy for their political gain. Not surprisingly this horror show in Washington caused a collapse in consumer confidence, producer confidence, economic growth and the stock market, despite the sharp rise in corporate profits. The danger of doubledip has thus increased considerably in the past few weeks.
♦ Europe: In the Eurozone the rate of growth of the economy also slowed to zero for similar reasons. When the attack on the peripheral economies got started the Eurozone forced them to cut spending sharply in return for the IMF's help. At the same opportunity it also demanded a budget cut from all other members of the Eurozone, immediately and in coming years. England too joined this move. Lately the bonds of Spain that is already in a recession and Italy that has one of the highest debts/GDP ratio in the world, came under attack and prices of these bonds fell so that the yield increased to over 6.25%. This means a tightening of monetary policy as well as fiscal policy because cost of interest on the debt increased. The automatic reaction of policy makers was to declare a further cutback of the budget. And now when even the strong economy of Germany is slowing down it is doubtful if Europe can escape a doubledip.
♦ China: In many of the developing economies and especially in China and India interest rates were increased and other monetary measures were implemented in order to cool off the economies that suffered from overheating. In China growth of the industrial sector has already stopped while the services sector is still growing but at a slower pace. It appears that policy makers in China still haven't grasped the relative size of the Chinese economy and the importance of growth there to the rest of the world and vice versa. Therefore it is reasonable to assume that tightening of monetary policy will finally prove to be overdone, as was the case with the easing policy in the big recession.
♦ Japan: In Japan as in Japan. The economy recovered quickly at, least partially, from the impact of the tsunami, but lately it has stopped growing. The big budgets that were promised for the rehabilitation of the areas impacted by the tsunami haven't been spent yet, but policy makers are already talking about budget cuts and tax increases in order to reduce the deficit.
♦ The banking system: In the large recession the banking system failed in many parts of the world. In the US government was forced to inject capital to several banks and even now banks are still forced to write off loans that were made before the recession, especially mortgages. Their share prices are still about a third of what they were on average before the recession. In Europe too banks were impacted the same way but losses are now increasing on bonds of their governments and other governments in the Eurozone. In China too the banking system is unstable mainly because of off budget loans, loans to foreign entities and to government owned companies, mainly those owned by regional governments. Although the large banks have allocated meaningful reserves to cover such losses it is doubtful if those will suffice in case of recession.
♦ Money & exchange rates: The dollar fell in recent months to new all times lows. This is a result – – maybe intentional – – of massive printing of dollars by the Fed in QE2 and recently of the "horror show" in Washington. The dollar is recently getting some support mainly due to the fact that there is no alternative. The Euro of course is not more stable than the dollar. Scared investors have been moving their money into non-reserve currencies such as the Japanese Yen or the Swiss Franc, or into gold. But all of these have already made sharp new highs so that the risk is high in case the world economy recovers. The gravest risk to the world economy is therefore loss of confidence in fiat money in general, and in such a case the Swiss franc does not provide any protection.


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Hay is for horses! Everyone knows that, but do you know how much hay they need? Or how much water? Did you know your horse can talk to you with his ears? Or that he can sleep standing up? Follow along with Athena the horse as she explains the basics of keeping a horse in your backyard. She'll teach you the basic necessities of caring for your equine friend, from what kind of fencing you can use, to tips and tricks for keeping water thawed in winter weather, and even fun stuff you can do with your horse. Keeping a backyard horse can be fairly simple, and even if you have a tight budget, it can be done!
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Doesn't Everyone Want a Horse?
I absolutely loved learning how to take care of your first horse through the perspective of the horse. The excitement in the performance made it a very easy read to listen to.
This is the first book I've read/listened to on the care of horses. It's definitely one I would recommend to anyone thinking of purchasing their first horse as the tips and insights are presented in a manner that is easily understandable...even for those who have never been around horses. I would have to do some research to find other books about horse care,.
I loved her strong presentation. The humor came through perfectly, and her delivery was never weak or unsure. I would certainly recommend her to produce your next audio project.
The tips on things to watch for to determine a horse's current attitude are excellent. Listen carefully here so you don't get kicked and so you'll know if something's wrong with your horse.
This was an excellent primer for people who have recently purchased a horse, and even more important for those who are contemplating purchasing a horse. It was a delightful listen and jam-packed with the information you will need to know from helping your new horse become familiar with their pen, to how to properly give an injection and check for hoof problems. I strongly recommend this book.
- Peggy M.
Informative, Engaging, and Entertaining!
- Valerie


text: Only DanB could come up with a crazy idea like this. That's one of our Item#60 2 inch dia. x 1/2 inch discs supporting an old gasoline Maytag washing machine engine, which we started before lifting! It made the engine swing around a bit, but it hung there running with no problem.


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The idea for this post came to me after seeing my friend Cara's review of her first episode Rose that she watched about 9 years ago. I recommend that you give it a read.
The first time I was even introduced to Doctor Who (and I remember this very vaguely) was 2 years ago, but at the time I could not have cared less for it because I looked up its Wikipedia page, and going through 11 Doctors seemed a bit of a work. Fast forward a year later since that and I am thinking to myself, "Lets give Doctor Who a try and see what it is. It seems to be all over my Facebook wall". And to this date, that is the best sentence I've thought to myself. So, I decide to look up Doctor Who's wiki again, but this time only to see where to begin. I figured starting up with the latest Doctor should be fine, and I can look up things that I do not seem to understand, because well, Youtube. That's when I found this, by The Fine Bros. This got my psyched a little and there began the wibbily wobbly timey wimey.
Being only slightly educated about Doctor Who, I started watching The Eleventh Hour. This is probably the most trivial of all the things but the first time I saw the TARDIS falling through space, I assumed the title sequence was a part of the episode and it took me over a minute to realize that I was wrong. The transition from the scene to the title sequence was very well done for a first timer.
I then saw this little girl praying to Santa to send someone to fix the crack in her wall, which meant that her house would be the crash site, obviously, but I did not expect what I saw next.
A man, dressed all raggedy, in soaking wet clothes and his witty thinking, confusing that little girl and me alike, claims to be The Doctor, bumps into a tree, and all he asked for was an apple. The irony in that. But guess what? He doesn't like apples. He has a craving, but does not like apples. Then came his favorite yogurt which he spits out, then the poisonous Bacon, the evil beans and lastly, my favorite, bread and butter. But all he ever really wanted was Fish Fingers and Custard, who knew?
Amelia Pond, like a name in a fairy tale, the girl who waited 12 years to meet the man who promised to be back in five minutes. 12 years and four psychiatrists. The girl without a mom and dad, and possibly a very rude aunt. Heart breaking, isn't it?
And guess what you get for making Amelia Pond wait for 12 years? A hit on the head with a cricket bat. But being hit in the head is not the only thing the Doctor has to worry about - "Turn back and look at the corner of your eye", because guess what?
Prisoner Zero will vacate the human residence, or the human residence will be incinerated.
Being through all of this, The Doctor decides to make a run for but apparently seems to be up to something with that blue thing in his hand. Oh wait, that's the Sonic Screwdriver the Fine Bros mentioned, yes. The episode then starts to become a huge adventure of solving a big puzzle, because lets be honest, that's what it all is for the Doctor - One Big Puzzle.
We also get to see that Amy apparently made cartoons of The Raggedy Doctor, the man from her dreams. These cartoons were how everyone knew of The Doctor which was surprising for him because he just got a new face.
Soon, we see Amy's authoritative nature when she traps The Doctor in a car door without even so much as bothering about the fact that she has merely 20 minutes of survival. No, this is Amy, she demands an explanation. We start to see so much of this behaviour of hers in later episodes, and one such scene was in A Town Called Mercy when she lectures the Doctor against sacrificing Kahler-Jex.
But this is not all, we were yet to meet Rory - Amy's boyfriend and the soon to be 'Last Centurion'. He was this really skinny guy in a nurse's uniform and looked so adorably like a kid. It was not until later in season 5 did we actually see Rory maturing. But it was only in season 7 that we realized how this kid grew up so fast into a man so mature and strong-willed.
The Doctor then makes a brilliant entrance into the hospital, where Prisoner Zero is hiding, with a fire truck. Soon Prisoner Zero is taken back by the Atraxi but not before it warned The Doctor about the Pandorica and the crack in the wall.
The universe is cracked. The Pandorica will open. The Silence will fall.
The minute I heard the word, my mind turned towards Pandora's box. Only later in the season do we start to gather that it is hardly anything like the Pandora's box.
The Doctor then decides this is all too easy and decides to call back the very aliens that were willing to destroy the planet. At this time, I was Rory, wondering why did he do that? But as he says, "Going away is good. Never returning is better." Towards the end of the episode we see a glimpse of eleven's ability to give a spectacular speech and scaring away the Atraxi.
The Doctor then dashes away when his TARDIS is ready, and the girl who waited has to waits some more. Two years to be precise, and just the night before her wedding and takes her away, which to me was rather anti-climatic.
This episode is rather close to my heart since it was my first stepping stone into being an obsessive whovian for over a year today. It is and forever shall be one of my favorites simply because there isn't a scene in the entire duration of this episode that I can say is not funny or entertaining. From the time the anchor fell out of the blue box, till the time we see Amy's wedding gown, every scene counted to be just as good, if not better than the last scene.


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ThemeForest Hostium Responsive Hosting Theme 5106172
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Paws Trailer (1997)
25 September 1997 Adventure, Fantasy, Family 83 mins
Paws is an independent 1997 Australian family film that was released on 25 September 1997 in Australia and filmed in Sydney, New South Wales. The film stars 15-year-old guitarist Nathan Cavaleri who has adventures with PC - a talking Jack Russell Terrier (voiced by comedian Billy Connolly). The dog is computer literate and the pair must a stop a valuable disk from falling into bad hands
Watch the official Paws 1997 trailer in HD below or find more Paws videos on Vidimovie.


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About Pat
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Providing Excellent Constituent Services
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Serving my constituents is one of my priorities as a Member of Congress. Whether you need help obtaining a Social Security benefit, would like to tour the U.S. Capitol Building or would like to purchase an American flag, my staff and I would like to help.
You may always contact my offices at (614) 523-2555 or (202) 225-5355. We also have a toll free number for your convenience 1-855-PAT-OH12 (855-728-6412).
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: Ask for assistance with federal agencies like the VA or Social Security Administration.
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: Access resources for grant applications.
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This webinar will introduce clinicians to the Pathways 4th Edition workbook. The author will review what he has learned in 26 years of clinical use with Pathways, and he will provide helpful ideas and tools that will help clinicians use the workbook effectively in their programs.
You can access the PowerPoint slides from this presentation here.
You can access the handouts from this presentation here, here, here, here, here and here.
You can access the YouTube recording of the webinar here.
For our March webinar, NEARI Press has partnered with Global Institute for Forensic Research (GIFR) to provide FREE CE Credits for our live webinars. If you want the opportunity to be automatically registered for every webinar this season and earn FREE CE credits for March, consider becoming a sponsor! Here is the process:
- When you register, you will be asked if you want CE credits -- just respond yes or no*.
- Whatever you choose, you will then be directed to register for the webinar.
- Within a week after you complete the live webinar, GIFR will send you a certificate of completion.
*If you do request credits, we will send over your name and email to GIFR. It will not be given or sold to any other vendor. We will be in touch directly after the webinar with instructions and a link to a short quiz you must complete within 7 days of the webinar to qualify for your CE credits. Please watch your email. To view which CE credits are offered, go to:https://www.gifrinc.com/continuing-education-approvals/.
Please note: We will NOT be able to provide CE credits yet for those who listen to past recordings of the 2016-2017 webinar series, only for current LIVE webinars.


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We wrote about this the other day. It bears repeating. Click here to read what author Ken Davis has to say about the closing of libraries, and check out our original post.
Posted at 03:13 PM in Current Affairs, Free Library of Philadelphia, Kenneth C. Davis, Libraries | Permalink
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In less than a month the Free Library of Philadelphia will be forced to close its neighborhood branch libraries, regional libraries and the Parkway Central Library. Click here for more information.
Posted at 01:48 PM in Books, Current Affairs, Free Library of Philadelphia, Libraries | Permalink
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Anthony Horowitz: Magpie Murders: A Novel
Bryn Chancellor: Sycamore: A Novel
Dennis Lehane: Since We Fell: A Novel
Kate Quinn: The Alice Network: A Novel
Matthew Quick: The Reason You're Alive: A Novel
Michael Crichton: Dragon Teeth: A Novel
Neal Stephenson: The Rise and Fall of D.O.D.O.: A Novel
Roxane Gay: Hunger: A Memoir of (My) Body


text: Since the arrival of sporty Tiburon and now the Genesis, Hyundai has taken a large step towards success. With the best warranty in the industry combined with up-to-date sporty appearance and most important of all, a low price tag makes Hyundai models like Genesis, Tiburon, Sonata and Tucson very popular. Dress up your Hyundai with body styling accessories like DuraFlex body kit, ViS OEM carbon fiber hood or Seibon carbon fiber trunk. For the performance-getters, we got quite a collection of performance and suspension parts like AEM cold air intake, B&G RS2 coilover suspension system, Tein S.Tech lowering springs, Tanabe catback exhaust, DC Sports exhaust header and much more. Select your model below to start.


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Creative Needle Lot 5 1996 Smocking Heirloom Sewing Christening gown Holiday
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These are very beautiful magazines for the creative seamstress. Jan/Feb 1996 Southern Classics, Ribbons and Bows, Doll-sized smockingMar/April 1996 Sewing Bishop Dresses for Dolls, Easter Elegance, Shadow work Embroidery July/Aug 1996 Big Sky Country, Smocked Backpack, Shades of Autumn Sep/Oct 1996 Holiday Issue Nov/Dec 1996 Heirloom Christening Gowns All the patterns, instructions, pictures and pages inside are in perfect condition. I tried to explain what is worn. Check the pictures out also. SMOKE FREE HOME Please contact me with any questions. I strive to describe all my items accurately. However, if you feel I made an error in my description, please do not hesitate to contact me.


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Some people don't understand that my love for this band is way past obsession. Most screamo music will drive most normal people crazy. But, I've learned that I am not normal. But this band settles me down better than anything. Also for them being a christian band, it's the closet thing I have to church. I'm serious about this, some of theyre lyrics keep me going at my worst. The most recent that has kept me going is a song called "Rosemary Had An Accident". There are many people that have never seen the lyrics to this, and I am glad because it is one of the very few things that keeps me going.
You were nothing but an obstacle in my pathway tripping me with my own trust. Without your hood of falsification, your beauty is found to be nothing but true revulsion and that's the only true thing about your. The moon and stars still retain these eyes. you're falling now as I move forward; fueled by the hope you couldn't take me and scarred by knowlege of painful winters. Christ still stands perfect in my mind. Christ still stands perfect in my heard. Jesus is the beautiful structure of love. Repent me to hope and love. Beware Nihilists. You shall fall by the sword. Oh God, bringeth the day of judgment.
Seriously, when me and my best friend went through a fight, this is what I listened to constantly. It kills me now that we aren't friends anymore, but they keep me going.
I used to laugh at the people that say this band saved my life, but now I truely understand stand that, and I am sorry even though I never said anything to you.
I am sorry.
- Current Music:And the Sentence Trails Off...-The Devil Wears Prada


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Every Tuesday, the Going Out Guide highlights the week's best DJs, bands, dance nights and parties. Find a longer list of events at www.goingoutguide.com.
Wall Street Journal drama critic Terry Teachout is a writer of the first order and longtime jazz fan. His Louis Armstrong biography, "Pops: A Life of Louis Armstrong," was one of The Post's 10 best books of 2009. Teachout's latest work covers the life of Washington's own Duke Ellington. "Duke: A Life of Duke Ellington" was released last month and was on the longlist for the 2013 National Book Award for nonfiction. Ellington probably would have felt right at home at the elegant art-deco Bethesda Blues and Jazz Supper Club, which makes it the perfect place for Teachout to discuss Ellington's life and legacy. And since you can't talk about the Duke without listening to the Duke, a jazz quartet will be on hand to perform Ellington's swinging hits.
Tuesday at 7 p.m. Bethesda Blues & Jazz Supper Club, 7719 Wisconsin Ave., Bethesda. 240-330-4500. www.bethesdabluesjazz.com. $25.
Washington has had a beer boom in recent years, but our next big spirit might just be gin. Sperryville's Copper Fox Distillery is introducing its new gin — "Vir Gin," after its home state — to D.C. this week, joining the city's own Green Hat. You can get a first taste of Vir Gin at Vinoteca. Distiller Rick Wasmund will be at the 11th Street NW wine bar, pouring free samples and chatting about the gin. Mixologist Horus Alvarez has come up with four cocktails to play off the gin's full body and bold anise and juniper notes, including variations on the Rickey and Hanky Panky, which will sell for $7. No reservations are required.
Thursday from 6 to 8 p.m. Vinoteca, 1940 11th St. NW. 202-332-9463. www.vinotecadc.com. Free.
"Overnight Scenario." "Work the Walls." "Pump Me Up." "Drop the Bomb." Those songs are part of the essential heartbeat of Washington, the city where Rare Essence and Trouble Funk have been packing clubs and spreading the gospel of go-go since the 1970s. That longevity will be celebrated at the Veterans Old School Jam on Sunday, where the two groups will be joined by other vets of the scene, including one-time Rare Essence members Jas Funk and Mike Muse and the Chuck Brown Horn Section. Proper attire is required, but make sure you're wearing comfortable shoes.
Sunday at 7 p.m. Howard Theatre, 620 T St. NW. 202-803-2899. www.thehowardtheatre.com. $17.50 in advance, $22.50 on the day of the show.
Over the past decade, Wayna's flawless soprano and vocal arrangements have solidified her as one of the most fully realized artists of the indie soul scene. Her cover of Minnie Riperton's "Loving You" earned her a 2009 Grammy nomination; she has been a vocal muse for acclaimed local beatsmiths Roddy Rod and Kev Brown; and she earned a co-sign from Stevie Wonder. On "The Expats," her third full-length release, Wayna has forged a new creative path. Hunkering down with a collective of musicians in Toronto, she has crafted a record with a much broader spectrum of styles, including touches of Afrobeat, psych rock and Ethiopian rhythms, and the result is brilliant. "The Expats" puts Wayna among the cadre of young world music artists forging new ground. For the official D.C. release of her new album, Wayna will be joined by another longtime D.C. peer, Wes Felton, whose new project, Imagine the Future, was produced Transatlantically with local singer Kenny Allen, who is now based in Ethiopia.
Sunday at 7 p.m. U Street Music Hall, 1115 U St. NW. 202-588-1880. www.ustreetmusichall.com. $10 in advance, $20 at the door.


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GraphicRiver Abstract Round Banner with Small Fairy Town 4671642
Stock Vector \ Objects \ Buildings
Banner \ Background \ Scaled \ Illustration \ Vector \ Without \ Editable
Stock Vector - Abstract Round Banner with Small Fairy Town 4671642 by lian_2011 @ GraphicRiver
Stock Vector \ Objects \ Buildings
Abstract round banner with small fairy colorful town on light blue sky background. This image is a vector illustration and can be scaled to any size without loss of resolution. All parts of the image are editable. EPS file included.
Layered: Yes; Graphics Files Included: Vector EPS; Minimum Adobe CS Version: CS.
Cat on Moon; Frog Prince with Crown; Set of Girls Silhouettes with Umbrellas; Girl with Umbrella on Blue Sky Background; Back to School Card; Set of Seamless Patterns in Pink and Blue; Fairy Tale Magic Princess Castle.
Keywords: apartment, architecture, background, banner, city, colourful, construction, design, downtown, element, frame, green, home, house, little, object, poster, rainbow, round, shape, small, street, stylized, summer, town, tree, urban, vintage, wallpaper.


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8 "Grate" Cheese Hacks!
HouseholdHackerPublished: June 21, 2016Updated: June 23, 201620,149 views
Published: June 21, 2016Updated: June 23, 2016
We'll show you how to make the best omelette, deep fry your mac and cheese, slice cheese like a pro and even store cheese longer.
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Viktor Build Guide by Sir Wargasm
Not Updated For Current Season
This guide has not yet been updated for the current season. Please keep this in mind while reading. You can see the most recently updated guides on the browse guides page.
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Viktor Build
Ability Sequence
Not Updated For Current Season
The masteries shown here are not yet updated for the current season, the guide author needs to set up the new masteries. As such, they will be different than the masteries you see in-game.
Version: 1.05A
Last Updated: 31-12-11
Welcome Summoners,
Who am I?
Good day, I am Sir Wargasm. I have been playing LoL for some time now. want to know more about the oh so interesting me? No? thought so.
If you have some constructive criticism please do not be shy and comment on my build, I might even listen.... once in a while. If you want to flame or troll that is fine too, just kneel, close your eyes, clasp your hands and talk to your god / oblivion / dentist.
This build is updated on a regular basis and will be expanded to encompass more depth.
I apologise for any grammar or spelling errors as English is not my first language.
PRO's / CONS
* Viktor is extremely versatile
* Has a high impact in a match
* can surpress enemy champions from advancing
* has a shield
* Quite survivable
* does not have an instakill burst
* has a real hard skillshot to master
* has only 5 free inventory spots
* can't dance.
Power transfer
This skill is quite useful due to the shield that you get (delayed, the projectile needs to return to you) The damage is ok and the CD is fine, cast range is rather short though.
Gravity Field
His teamfight skill, this skill allows for guiding your opponents into the traps or stunning them. don't expect to stun that often though but use it to seperate the weak from the herd.
Death Ray
This Is his bread-and-butter skill. Great range, decent CD, Acceptable mana costs, Great damage. It's biggest con is how hard it is to use though. It is a dual step skill shot, first pick a castingplace then hold down your mousebutton to pick the direction.
Chaos Storm
Decent ultimate, it is a bit harsh on the normal cooldown and lacks single-target damage but is great for doing that bit of extra damage when an enemy runs away. Doesn't have a large impact in teamfights though can be used (due to its awesome animation) for a psychological advantage
I have chosen for this setup of runes because you need the Magic penetration more than anything else. To slightly offset viktor's early lack of mana regeneration I have picked the greater seals of replenishment.
Masteries are pretty straightforward with 9-0-21. These are not set in stone. Some things that you need to keep in the back of your head when picking your own masteries is that viktor needs mana regen and for his defence he needs Cooldown Reduction.
At start buy a sapphire crystal. This is to make sure you will not run out of mana before your first recall and thus have some more sustainability. Logically you ahould buy 2 health pots as well. This allows you to stay in the lane longer and survive DPS-ganks.
About those health pots; during the game try to have around 2 with you right up until the late-game. This gives you a bit more flexibility instead of having to recall all the time.
Next up you need to buy a tear of the goddess, the earlier you can buy it the higher your mana will be (and the more you get from the later archangel's staff bonus)
if possible buy the Boots of Speed at the same time, if it isn't possible then you need to buy it when getting the other parts of the archangels staff.
Next focus on getting the archangels staff for your AP bonus. Usually your mana bonus will be around 400-500 mana (if you spammed E) which will grow into 1000 during the game. and combined with your own mana should give you a 80-100 AP bonus (at level 18).
Now you need to upgrade your Hex core to the Augment: Gravity variant. The range increase will give you more zoning power and will help your team a lot in teamfights. The added stats bring should give you that little boost in mana regen and CDR that you needed to keep (most) enemies from running away all the time at low health.
Next build the sorcerers shoes for the added speed boost and the Magic penetration.
Next up you need more AP and a bit of CDR to help keep your Shield with Q up. Therefore you buy Morellos Evil Tome. The added mana regen should help you keep your mana around 70% of max outside of teamfights. during teamfights this should help you keep the enemy where you want them.
Next up you build the mage's staple, Rabadons Deathcap; Rushing it would have put you behind other mages and decreased your impact throughout the game but it is still one of the best items a mage can get.
optional finishers
That concludes the fairly mandatory basic build and brings us to the optional finishers, now a great help whilst saving for your finisher is buying the stat-improving potions because it boosts your utility and keeps you on par with other people that have bought finishers already.
Buy a deathfire grasp if somehow you can't kill their off-tanks in ganks or non-team fights. Its active delivers a whopping 30% of max health punch with a 1 minute base cooldown. This is more single-target damage than your ulti does. Great item and if you aren't getting targeted I can really recommend it
if you are getting ganked or targeted too much you finish with Zhonyas hourglass. It's active shields you for 3 seconds (in which you can still control your storm cloud) AND is just long enough to get your Q off cooldown most of the time(after first shield is depleted).
These last two are based on personal preference and are quite open to debate.
Skill Sequence
Levelling sequence
Start of with your Q, followed by your E and then your W for your first three levels. After this you max your E asap, taking Q whenever your R and E aren't available. Max your W last.
1v1 Fights
1) (Use your Deathfire Grasp on your target)
2) Immediately follow up with your Q for the shield
3) Try and land your W right behind them if they are squishy and right in front of them if they are tanky.
4) Use your E in a line with their direction hitting them as much as possible. This is tricky and requires some skill before mastered
5) If still needed pop your R on them to finish them of, after they die send your R into minions or other champs.
1) Wait for iniate
2) Use your W right behind them
3) send a line of E through at least 2 or 3 (usually horizontal)
4) use your Q to survive their focus for now
5) use your R to keep them surpressed and allow yourself or your carries to get the kills.
Summoner Spells
Flash Allows you to escape from ganks and increases your flight potential. A genre savvy enemy will have looked at your Summoner Spells and know that you have the potential to flash away. If your opponent has flash as well try to force him to use his before you use yours.
Ignite is to stop their healing and hopefully deal that last bit of damage needed to secure the kill. Using ignite for a psychological advantage is always a good idea.
If you come into a situation where you are fighting a carry and someone else, you try and get the carry to half health and throw ignite on him. in most cases the carry will think twice about chasing you and in some he might even start running away. This helps you survive their gank.
Early Game
If possible take mid, if there are better options for mid, then you go bottom. Start with a little bit of harassing but wait for your E and W to start going for kills and zoning. One of his specialties is zoning an enemy. Just use your Q and autoattack them, try to send them backwards with your W and follow it up with an E.
Getting Tear of the goddess is your main focus during early game. This item allows you to build your 300 starting mana up to something more respectable. Follow it up with boots because the faster you get the more control you have over the battlefield.
Mid Game
Your next purchase should be the Augment: Gravity upgrade which will allow you to zone your laning enemy better and help you defend yourself against ganks.
During this phase it is imperative that you keep your map awareness high and your health up. A dual-enemy gank is not something you'd like to run into with viktor.
Try luring your enemy near the middle of your lane and then hit him with your W followed by Q and then the E going towards his tower. Ignite him if he still has health left or hit him with your R followed by the ignite.
Late Game
During teamfights you should always try to come from the right (if you are right-handed, if you are lefthanded it is the other way around) this is because moving your hand towards your body is slightly more precise and just a bit quicker than moving your hand away from your body. (this has actually been proven). Your main task during the teamfight is slowing their carries with your W and if possible hitting them with your E. Use your Q on one of their tanks First and try hitting their carries later. This is because you need your shield up. (This helps with being focussed in the first "round")
Your pushing abilities are grand, Use your E to kill the caster minions and your Q on a minion if you need to last hit their tower. Always be prepared to throw your W between you and the nearest lane entrance against ganks.
If somehow your team is being outpushed and you are forced on the defensive inside the base it is very important to throw your W a bit behind the enemy champions because the usual reaction to a champion coming into view is moving back slightly, you can even force this reaction by casting your E in front of them in a line. This might seem like a waste of mana and a round but 9 times out of 10 the enemy will move back slightly and run into your W.
Fight or flight
When is it a good time to fight and when is it a good time for flight? It should be understood that discretion is the better part of valor. And sometimes hanging to the sides slightly will help you escape or flank the enemy champions. If you manage to flank them you should try zoning their tanks away from the rest of their team and let your own team make the kill.
To conclude; this build should give you enough insights to make you a decent viktor player.
50% of being good is knowledge;
25% of being good is skill;
15% of being good is luck;
10% of being good is bragging.
I wish you the best of luck and hope that you found my build / insights useful. Comment if you think the build is lacking in any department and vote once possible.
Version 1.05C
(Anything in this build may be reproduced as far as MOBAFIRE's (E)ULA allows it) and with a clear mention of the original creator: Sir Wargasm.
Kind Regards,
Sir Wargasm