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Tom Arnold
Guest Star
G. Love and Special Sauce
Guest Star
Dannah Feinglass
Guest Star
Mary Mae Atwill
Spike's Wife
Recurring Role
The music that was playing at Spike's house during the "Trading Spouses" sketch was Sister Christian by "Night Ranger" and Jessie's Girl by "Rick Springfield".
The actress who played the wife in the "Trading Spouses" sketch, Dannah Feinglass, also played the "Mother" in the "Nanny 911" sketch which aired in the "Jeff Garlin" episode.
(In the "Trading Spouses" sketch)
Spike's Wife: Okay these are my rules: No one touches my smokes. Your Dad and I are going to sleep together to save fuel. When I say the TV is on, it stays on. You got that... huh?
Husband: Yes. Absolutely, Yes Miss.
Spike's Wife: I'm going to the crapper.
(In the "Trading Spouses" sketch)
Spike: It's a very touching story, Choi, who I found in Cambodia, blew his own testicles off in a firecracker accident when he was only nine years old. After that he really took his "firecrackering" seriously. We're really proud of him.
(The husband enters the bedroom and climbs into bed with Spike's wife... there is an unmistakable buzzing sound emanating from the bed)
Husband: How does this save fuel again?
Spike's Wife: For one thing I can turn this off.
(There is a click and then the buzzing stops)
Spike's Firecracker Boy: Before I met Spike I was Adrian Brody's "firecracker boy".
(Tom Arnold apologizes to Spike)
Tom Arnold: Most of all, sincerely, I want to apologize to you Spike.
Spike: To me? Why do you want to apologize to me?
Tom Arnold: Because, I think I just "sharded". You're probably going to have to burn this chair.
(Tom Arnold making apologies)
Tom Arnold: I'm sorry about the book, I didn't even write that book... it was written by college kids living in my shed and I paid them in dope and funions.
(Talking about a woman's prison)
Tom Arnold: It was like KD ranch, except for all women and they are so frickin horny... (slurred words) ...they haven't been with a man for a long time, they're just happy to see me.
Spike: Did you get some?
Tom Arnold: I gave some!
In the "Trading Spouses" sketch the scene at Spike's house is an allusion to a scene in the movie Boogie Nights at a house where the stoned host wears a bathrobe, while a young Asian boy continually flings small firecrackers around. Also the scene also features some of the same music such as Sister Christian and Jesse's Girl.
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Simian virus 40 (SV40) is a nonenveloped virus that has been shown to pass from surface caveolae to the endoplasmic reticulum in an apparently novel infectious entry pathway. We now show that the initial entry step is blocked by brefeldin A and by incubation at 20°C. Subsequent to the entry step, the virus reaches a domain of the rough endoplasmic reticulum by an unknown pathway. This intracellular trafficking pathway is also brefeldin A sensitive. Infection is strongly inhibited by expression of GTP-restricted ADP-ribosylation factor 1 (Arf1) and Sar1 mutants and by microinjection of antibodies to βCOP. In addition, we demonstrate a potent inhibition of SV40 infection by the dipeptide N-benzoyl-oxycarbonyl-Gly-Phe-amide, which also inhibits late events in cholera toxin action. Our results identify novel inhibitors of SV40 infection and show that SV40 requires COPI- and COPII-dependent transport steps for successful infection.
Viruses have exploited endocytic pathways to overcome the barrier presented by the plasma membrane and enter animal cells. Although the entry of viruses via clathrin-coated pits has been extensively studied and is well understood (Marsh et al., 1984 ), entry of viruses by alternative pathways has been less well characterized. Simian virus 40 (SV40) is a nonenveloped virus that uses an apparently unique entry route to reach the nucleus of animal cells. SV40 binds to major histocompatibility complex (MHC) class I molecules on the cell surface (Breau et al., 1992 ) and then is observed in tight-fitting, nonclathrin-coated pits (Kartenbeck et al., 1989 ; Anderson et al., 1996 ; Stang et al., 1997 ; Norkin, 1999 ; Parton and Lindsay, 1999 ). These pits are enriched in caveolin-1 but are smaller than caveolae, raising the possibility that caveolin is recruited around the virus (Stang et al., 1997 ). Consistent with this model, the levels of caveolin-1 associated with virus-containing membranes were shown to increase with time (Stang et al., 1997 ). Subsequently, the virus is internalized in a process that is sensitive to cholesterol-perturbing agents (Anderson et al., 1996 ). The role of caveolin in this process is suggested by the finding that viral infection is also inhibited by dominant negative mutants of caveolin (Roy et al., 1999 ). Viral entry apparently occurs without concomitant internalization of MHC class I (Anderson et al., 1998 ), in a process dependent on tyrosine kinase activity (Dangoria et al., 1996 ; Chen and Norkin, 1999 ; Norkin, 1999 ). Early electron microscopic studies suggested that the virus is then transported directly to the endoplasmic reticulum (ER) because no intermediate stations were identified (Kartenbeck et al., 1989 ). A direct pathway between the cell surface and ER would be a novel finding, although parallels can be made to the cholesterol oxidase-induced redistribution of caveolin-1 from caveolae to the lumen of the ER (Smart et al., 1994 ).
The virus eventually reaches the nucleus where uncoating and replication occur. At some stage in the entry pathway the SV40 virions appear to translocate across a membrane into the cytosol and then enter the nucleus via the nuclear pore complexes (Clever et al., 1991 ; Yamada and Kasamatsu, 1993 ). Cytosolic microinjection of antibodies against viral proteins blocks the infection process (Nakanishi et al., 1996 ), suggesting that the virus is actually free in the cytosol at some stage in infection but the mechanism by which translocation into the cytosol occurs, and the compartment involved in this process, are unknown.
Many toxins are retrogradely transported from the cell surface to the ER where translocation into the cytosol occurs. This has provided important insights into the retrograde transport process and the mechanisms involved in translocation. This process has been particularly well characterized for cholera toxin (CT). CT binds via its binding subunits to the ganglioside GM1 at the cell surface. GM1 is slightly enriched in caveolae but is present over the entire cell surface, including clathrin-coated pits (Parton, 1994 ). Inhibitor studies, using cholesterol-disrupting agents, have suggested that CT is internalized via caveolae (Orlandi and Fishman, 1998 ). However, CT is also efficiently internalized, in a cholesterol-dependent process, in cells lacking caveolae (Orlandi and Fishman, 1998 ). Elegant studies using chimeric toxins have shown that association with raft domains is critically important for toxic entry of CT (Wolf et al., 1998 ). Internalization of CT and budding of caveolae have both been proposed to be dynamin dependent (Henley et al., 1998 ; Orlandi and Fishman, 1998 ). After internalization CT passes to endosomes and then the trans-Golgi network. The toxin then arrives at the ER in a process that can be blocked experimentally by brefeldin A (BFA) and by microinjection of coatomer antibodies (Lencer et al., 1993 ; Nambiar et al., 1993 ; Majoul et al., 1998 ; Girod et al., 1999 ). This process is facilitated by a KDEL sequence in the A subunit and retrieval by the KDEL receptor (Majoul et al., 1998 ). On reaching the ER, redox-dependent unfolding of the toxin by protein disulfide isomerase occurs (Tsai et al., 2001 ). The A subunit is then reduced to produce the enzymatically active A1 peptide, which is translocated to the cytosol via the Sec61 channel, identifying the rough ER as the compartment from which translocation occurs (Schmitz et al., 2000 ).
Although recent morphological studies have suggested the involvement of a novel endosomal intermediate, termed a caveosome, in the SV40 entry pathway (Pelkmans et al., 2001 ), the molecular mechanisms directing SV40 along this pathway and the similarities to known trafficking pathways are still unknown. Possible parallels between the SV40 pathway and that of CT are evident with both suggested to involve caveolae, the ER, and then translocation to the cytosol (Parton and Lindsay, 1999 ). This prompted us to examine SV40 infectious entry by using a range of manipulations previously shown to inhibit CT toxicity. We now show that SV40 infection is blocked by BFA, with inhibition at two distinct steps: the initial virus entry step and an intracellular step. Infection is also blocked by a dominant negative ADP-ribosylation factor 1 (Arf1) mutant, by a dominant negative Sar1 mutant, and by microinjection of antibodies to βCOP. These results indicate that SV40 infection either proceeds via a membrane trafficking pathway involving Golgi intermediates or requires a functional exocytic pathway. We compared the effect of these inhibitors of SV40 infection on CT entry and show that CT transport from early endosomes to the Golgi complex is inhibited by both Arf1 and Sar1 mutants. We also show that a novel inhibitor of late events in CT toxicity, the dipeptide N-benzoyl-oxycarbonyl-Gly-Phe-amide (Cbz-gly-phe-NH2), blocks late intracellular steps in SV40 infection, providing new insights into this novel virus entry pathway.
The expression plasmids encoding Arf1(Q71L) (Pepperkok et al., 1998 ; Girod et al., 1999 ), Sar1(H79G) (Aridor et al., 1995 ; Pepperkok et al., 1998 ), and green fluorescent protein (GFP)-tagged temperature-sensitive vesicular stomatitis virus glycoprotein (ts-045-G) (Scales et al., 1997 ), and affinity-purified anti-βCOP (EAGE) (Pepperkok et al., 1993 ) have been described previously. Anti-βCOP (EAGE) antiserum (used for immunofluorescence labeling) and mouse monoclonal antibodies to protein disulfide isomerase (PDI) (Pizarro-Cerda et al., 1998 ) were kind gifts of Drs. Rohan Teasdale and Jenny Stow, respectively (Institute for Molecular Bioscience, Queensland, Australia). Rabbit neutralizing antiserum to SV40 (Butel et al., 1984 ) from Dr. Janet Butel (Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, TX), affinity-purified anti-p23 (Rojo et al., 1997 ) from Dr. Manuel Rojo (Department of Biochemistry, University of Geneva, Switzerland), human antiserum to EEA1 from Dr. Ban-Hock Toh (Melbourne, Australia), and murine antigiantin (Linstedt and Hauri, 1993 ) from Dr. Hans-Dieter Soeling (Gottingen, Germany) were also generous gifts. Murine monoclonals against caveolin-3 (Transduction Laboratories, Lexington, KY), GM130 (Transduction Laboratories), and SV40 T-antigen (PharMingen, San Diego, CA) were commercially obtained. Brefeldin A, Cbz-gly-phe-NH2, Cbz-gly-gly-NH2, fluorescein isothiocyanate (FITC)-conjugated CT-B subunit, unlabeled holotoxin, and antiserum to CT were obtained from Sigma-Aldrich (St. Louis, MO). 4,6-Diamidino-2-phenylindole (DAPI) and Texas Red-conjugated transferrin were from Molecular Probes (Eugene, OR). Media and cell culture reagents were purchased from Invitrogen (Carlsbad, CA) or BioWhittaker (Walkersville, MD).
Vero cells (African green monkey kidney cells, ATCC CCL 81) and CV1 cells (African green monkey kidney cells, ATCC CCL 70) were maintained in DMEM supplemented with 10% (vol/vol) serum supreme (BioWhittaker) and 2 mM l-glutamine plus or minus penicillin (100 U/ml) and streptomycin (100 μg/ml). Stocks of recombinant Semliki Forest virus (SFV) encoding caveolin-3 (from Dr. Elina Ikonen, National Public Health Institute, Helsinki, Finland) were diluted into serum-free medium containing 2 mM l-glutamine and 0.2% bovine serum albumin before addition to cells. After 1 h of infection at 37°C, cells were washed and infection allowed to proceed for 5 h or more before fixation and immunolabeling for caveolin-3 to detect successfully infected cells. Preparation of SV40-containing supernatant from infected CV1 and Vero cultures and subsequent use of these viral stocks in infection experiments was as described previously (Stang et al., 1997 ). Stocks used to generate SV40 supernatants were a generous gift from Dr. Jurgen Kartenbeck (Division of Cell Biology, German Cancer Research Center, Heidelberg, Germany).
Antibody samples for microinjection were each prepared as previously described in the cited references. Purified rabbit IgG (Sigma-Aldrich) solubilized in 10 mM KH2PO4, pH 7.2, and 75 mM KCl microinjection buffer (Henley et al., 1998 ) was injected at 10 mg/ml as a control. Plasmid DNA for microinjection was purified on a cesium chloride gradient and injected into the nucleus of cells at 50–100 μg/ml. Capillary microinjection of antibodies and DNA into cells was performed using a computer-assisted, automated microinjection system (CompiC INJECT, AIS2; Cellbiology Trading, Hamburg, Germany) by using microcapillaries pulled from thin-wall borosilicate glass capillaries (1.2-mm outer diameter; 0.94-mm inner diameter) (Clark Electromedical Instruments, Pangbourne Reading, England) by a Flaming/Brown P-97 micropipette puller (Sutter, Novato, CA). Cells to be injected were plated onto small glass coverslips and injected at room temperature (RT) in Hanks' medium containing 0.75 mg/ml bicarbonate and 10 mM HEPES, pH 7.4. Cells were allowed to recover for 0.5–4 h postinjection (or 5 h for expression of injected cDNA) in penicillin-streptomycin–containing medium at 37°C before subsequent manipulations.
Experiments requiring immunolabeling with the monoclonal antibody to SV40 T-antigen were fixed in 100% methanol for 5 min at −20°C. Microinjection experiments requiring this fixation were often first fixed with 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS) at RT for 20 min and subsequently fixed with 100% methanol as described above. This reduced the washing out of the injected antibody by methanol fixation. All other experiments were fixed only with 4% PFA in PBS at RT for 20 min. Processing of cells for immunofluorescence analysis was as follows. PFA-fixed cells were permeabilized in 0.1% saponin in PBS for 10 min before subsequent incubation in 50 mM NH4Cl in PBS for 10 min (to quench aldehyde groups) and in blocking solution (composed of 0.2% bovine serum albumin and 0.2% fish skin gelatin in PBS) for 20 min at RT. A 30-min incubation in blocking solution containing primary antibodies was followed by 4 × 5-min PBS washes before incubation in blocking solution containing fluorescently tagged secondary antibodies for 20 min. After further 4 × 5-min PBS washes, the cells were rinsed in water and mounted in Mowiol mounting medium. In experiments requiring nuclear staining, the cells were incubated in 1 μg/ml DAPI in PBS for 2 min just before rinsing and mounting. Cells fixed with methanol did not require saponin permeabilization. Experiments were viewed and photographed with an Olympus BX60 microscope connected to a Daige charge-couple device camera image capture system. Immunofluorescence images were prepared using Adobe Photoshop 5.0 (Adobe Systems, Mountain View, CA). Cells were processed for electron microscopy according to Stang et al. (1997) .
Infection efficiencies of 20 or more antibody-injected cells and 40 or more transfected cells per experiment were quantified. For drug treatment and temperature-sensitivity experiments, efficiency of infection was quantified from 100 or more cells from each treatment.
SV40 has been shown to be internalized by caveolae and then to reach a subdomain of the ER. The molecular machinery and sorting mechanisms by which the virus reaches the compartment are presently unknown.
We first examined the nature of the SV40-containing compartment by using both plastic sections and immunolabeling. Epon sections of cells incubated with SV40 for 21 h revealed the virus in reticular, smooth-membraned areas of the ER connected to ribosome-studded rough ER membranes (Figure (Figure1,1, A and B). The membrane is closely apposed to the surface of the viral particles, suggesting that the virus remains bound to the lumenal surface of the membrane. Ultrathin frozen sections of these cells were labeled with antibodies to the virus together with antibodies to a cis-Golgi marker, p23, or to a marker of the ER, PDI. The virus-containing membranes were negative for the cis-Golgi/intermediate compartment labeled by antibodies to p23 (our unpublished data) but were labeled by antibodies to PDI (Figure (Figure1C).1C). Although the majority of the virus particles was observed in these very prominent enlarged ER domains, virus particles were occasionally observed in proximity to the Golgi complex, and possibly in Golgi-associated membranes (our unpublished data), raising the possibility that virus may transiently associate with these compartments during infection.
The above-described observations prompted us to further examine the possibility that SV40 passes transiently through the Golgi compartment in a similar manner to many bacterial toxins, which also follow an endocytic route to the ER. Because transient intermediates in infection may be difficult to identify morphologically and the viral infection process is relatively unsynchronized, we chose to use defined membrane trafficking inhibitors together with assays of infection to further delineate the trafficking pathway.
We first examined the possible involvement of the Golgi complex in the SV40 trafficking pathway by using the fungal metabolite BFA. This drug inhibits guanine nucleotide exchange on the small GTPase Arf1 (Jackson and Casanova, 2000 ), an adapter protein responsible for recruitment of COPI coats onto endosomal and biosynthetic membranes. The result is an inhibition of anterograde transport from ER to Golgi complex (Misumi et al., 1986 ) and a concomitant tubulation and fusion of the Golgi with the ER (Lippincott-Schwartz et al., 1989 ; Scheel et al., 1997 ). Consequently, BFA has been widely used to inhibit both anterograde and retrograde transport between the Golgi complex and ER. Vero cells were pretreated for 3 h with BFA and then incubated with SV40 plus BFA for 21 h before fixation and immunolabeling for the virus-encoded T-antigen (T-ag). As shown in Figure Figure2,2, BFA is a potent inhibitor of SV40 infection. With concentrations as low as 0.1 μg/ml we observed 99% inhibition of infection (Figure (Figure2E).2E). These relatively low doses of 0.1 and 0.5 μg/ml BFA were found to be effective in disrupting Golgi morphology within 1 and 3 h of treatment, respectively (Figure (Figure2,2, A and B).
To determine the stage of the infectious entry pathway that was affected by BFA, Vero cells were exposed to a 3-h pulse treatment of 0.1 μg/ml BFA at various time points into SV40 infection at 37°C. Fixation and immunolabeling for T-ag after the 21-h infection period revealed a clear inhibition of SV40 infection when BFA was applied at early stages of viral infection (Figure (Figure2E).2E). To examine the possibility that BFA blocks viral entry, we made use of a neutralizing anti-SV40 antibody, which inactivates surface virus. Addition of the neutralizing antibody to cells incubated with the virus at 4°C with no warming step to allow internalization caused a complete block of infection (our unpublished data). Vero cells cultured in the presence or absence of 0.5 μg/ml BFA for 1 h at 37°C were incubated with SV40 plus or minus BFA on ice for 1 h and then at 37°C for 2 h to allow infection to occur. The cells were then incubated in SV40 neutralizing antibody for 30 min and washed to remove BFA. A further 46 h of infection at 37°C was followed by fixation and immunofluorescence labeling for T-ag. Quantitation of SV40 infection efficiency revealed a substantially greater sensitivity of SV40 infection to the neutralizing antibody in BFA-treated cells compared with untreated cells, indicating a trapping of the virus at the cell surface by BFA (Figure (Figure2F).2F). Thus, it was apparent that the first effect of BFA on SV40 infection was the inhibition of initial viral entry. Consistent with this, treatment with 0.3 μg/ml BFA and incubation with the virus for 21 h showed SV40 immunolabeling only at the periphery of cells, unlike the pattern observed in untreated controls (Figure (Figure2,2, C and D). Dual immunolabeling for caveolin-1 and SV40 revealed no significant colocalization (our unpublished data). Similar results were obtained in cells transiently transfected with caveolin-1-YFP with no colocalization of peripheral SV40 labeling with caveolin-1-YFP (our unpublished data). Epon sections of these cells revealed virus particles in tight-fitting, surface-connected invaginations indistinguishable from those seen in untreated cells at early time points. In accordance with the immunofluorescence data, few virus particles were observed intracellularly (Figure (Figure22G).
To investigate whether BFA also inhibits postsurface trafficking steps, Vero cells were incubated with SV40 for 2 h at 37°C in the absence of BFA to allow viral infection to proceed past the initial internalization step. Any virus remaining at the cell surface was then inactivated with neutralizing antibody and the cells incubated in either the presence or absence of 0.5 μg/ml BFA for the remaining 20 h of infection. Quantitation revealed a strong inhibition of infection in BFA-treated cells compared with untreated controls, showing inhibition of an internal step in SV40 infection (Figure (Figure22H).
We conclude that the initial entry step of SV40 entry is BFA sensitive. In addition, SV40 infection involves a second postentry BFA-sensitive step, possibly involving endosomes and/or the Golgi complex.
Although BFA is best known for its inhibitory effect on transport between ER and Golgi complex, it has also been shown to inhibit early endosome-to-Golgi complex traffic (e.g., of endocytosed Shiga toxin; Mallard et al., 1998 ). To determine whether SV40 is internalized to early endosomes, we made use of the observation that exit from the early endosome is blocked at 20°C (Griffiths et al., 1988 ). Vero cells were infected with SV40 at 20°C for 4 h. Fixation and immunolabeling for the virus revealed a striking loss of staining for the virus (our unpublished data). To confirm this apparent failure of the virus to be internalized at 20°C, we again used the SV40 neutralizing antibody assay. Vero cells were infected with the virus either at 20 or at 37°C for 4 h. Subsequent exposure of the cells to the neutralizing antibody for 30 min was followed by incubation at 37°C for a further 24 h before fixation and immunolabeling for T-ag. Quantitation of infection efficiency revealed a much greater sensitivity of the virus to the neutralizing antibody after a 4-h infection at 20°C than after infection at 37°C (Figure (Figure3).3). We conclude that initial entry of SV40 is sensitive to both BFA and incubation at 20°C.
We next examined the effects of these treatments on a well-characterized retrograde transport pathway. CT is efficiently transported to the ER via the Golgi complex (see INTRODUCTION). We have used either FITC-labeled CT-B (CT-B-FITC), which is transported to the Golgi, or immunolabeling for the holotoxin, which is transported to the ER (Lencer, 2001 ). Vero cells allowed to bind and internalize the FITC-labeled B subunit of the toxin at 20°C showed no inhibition of toxin accumulation in early endosomes (Figure (Figure4,4, A–C). Similarly, Vero cells pretreated for 1 h with 0.5 μg/ml BFA also internalized the toxin to early endosomes (Figure (Figure4,4, D–F). In cells exposed to BFA for a 21-h period comparable with that used in SV40 infection experiments, internalization of CT was still observed, although a greater intensity of plasma membrane fluorescence, possibly indicating a decrease in uptake, was detected (our unpublished data).
Although BFA and a 20°C incubation had little effect on internalization of CT-B, subsequent transport to the Golgi was completely inhibited by both treatments. Even after a 1-h incubation at 20 or 37°C in the presence of BFA, the toxin failed to reach the Golgi (Figure (Figure4,4, A–F). In cells exposed to BFA (Figure (Figure4,4, D–I) the toxin accumulated in sorting endosomes and tubulated recycling endosomes identified by colocalization of cointernalized Texas Red-labeled transferrin with CT-B-FITC (Figure (Figure4,4, G–I). We conclude that the sensitivity of SV40 entry to both BFA and incubation at 20°C is in contrast to other known endocytic pathways, including that of the putative caveolae marker CT (Lencer et al., 1993 ; Nambiar et al., 1993 ) and suggests that SV40 uses a novel entry pathway.
The sensitivity of a postsurface SV40 trafficking step to BFA treatment raised the possibility of Golgi complex involvement in the viral entry pathway. Inhibition of retrograde traffic between the Golgi complex and ER has been convincingly demonstrated by microinjection of an antibody to βCOP, a component of the coatomer complex of COPI-coated vesicles. Microinjection of anti-βCOP (EAGE) (Pepperkok et al., 1993 ) was found to inhibit COPI-mediated retrograde transport of the KDEL receptor and of ERGIC-53, both molecules that constitutively cycle between the ER and Golgi complex (Girod et al., 1999 ). Retrograde traffic of endocytosed CT-A subunit has also been found to be inhibited by anti-βCOP injection (Majoul et al., 1998 ; Girod et al., 1999 ). Vero cells were microinjected with anti-βCOP antibodies. Three to four hours later the cells were infected with SV40 for 21 h before fixation and immunolabeling for the microinjected antibody and T-ag. The microinjected antibodies showed cytosolic labeling and strong Golgi staining (Figure (Figure5A).5A). Quantitation of infection efficiency revealed a strong inhibition of viral infection in microinjected cells, compared with surrounding uninjected cells (Figure (Figure5B),5B), showing that the anti-βCOP antibody is a potent inhibitor of SV40 infection.
As a second independent method to inhibit COPI-mediated transport we used overexpression of a GTPase-deficient Arf1 mutant (Q71L). This mutant has previously been shown to inhibit COPI-dependent transport in the early secretory pathway (Dascher and Balch, 1994 ) and also to inhibit sorting and concentration of cargo molecules into COPI-coated vesicles in vitro (Lanoix et al., 1999 ). Vero cells were microinjected with either a mixture of Arf1(Q71L) and GFP expression plasmids or the GFP plasmid alone (as a control). After 5 h to allow expression of proteins the cells were incubated with SV40. Expression of Arf1(Q71L) in GFP-expressing cells injected with both plasmids was verified by immunolabeling the cells for βCOP. A clear disruption or complete disappearance of the Golgi βCOP labeling pattern in cells showing high levels of GFP expression was observed in cells injected with both plasmids but not in cells injected only with the GFP construct (Figure (Figure6,6, A–D). Quantitation of infection efficiency revealed a potent inhibition of SV40 infection in Arf1(Q71L)-expressing cells compared with neighboring nonexpressing cells. No such inhibition was observed in cells expressing GFP alone (Figure (Figure6E).6E).
In addition to their effects on COPI-mediated retrograde transport between the Golgi and ER, the dominant negative Arf1 mutant and microinjected βCOP antibody could also be affecting events early in endocytosis because Arf1 and COP proteins have been implicated in endosomal trafficking (Aniento et al., 1996 ; Daro et al., 1997 ; Gu et al., 1997 ; Gu and Gruenberg, 2000 ; Jackson and Casanova, 2000 ). To determine whether disruption of ER/Golgi transport could be responsible for inhibiting viral infection, we examined the effect of a selective inhibitor of ER/Golgi transport on SV40 infection by expression of the GTP-restricted mutant of Sar1, Sar1(H79G) (Aridor et al., 1995 ). We observed that cells coinjected with expression plasmids for GFP and Sar1(H79G) showed significant inhibition of SV40 infection (Figure (Figure6F).6F). Use of a temperature sensitive form of vesicular stomatitis virus glycoprotein (ts-045-G) confirmed that Sar1(H79G) and also Arf1(Q71L) were potently inhibiting anterograde transport in this system (Figure (Figure7).7). These results show a direct or indirect role for Arf1, Sar1, and COP-dependent trafficking steps in SV40 infection.
To further compare the CT and SV40 trafficking pathways, we examined the effect of the above-mentioned inhibitors on trafficking of CT-B to the Golgi complex or of CT holotoxin to the ER. Cells microinjected with plasmids encoding Arf1(Q71L) and Sar1(H79G) were incubated for 5 h and then allowed to bind and internalize CT-B-FITC for 40 min. A clear inhibition of toxin arrival at the Golgi was observed. The toxin was, however, internalized to early endosomes, although an obvious increase in intensity of cell-surface signal indicated a slight inhibition of entry (Figure (Figure8).8). A similar inhibition of CT internalization was elicited by microinjection of the anti-βCOP antibody (EAGE) (our unpublished data).
We then examined trafficking of CT holotoxin to the ER. Untreated cells showed that CT reached the ER in 2 h as judged by immunofluorescence (Figure (Figure9).9). Vero cells were injected with the above-mentioned constructs and protein expression was prevented by incubation with cycloheximide for 4 h. The cells were then allowed to bind and internalize CT holotoxin at 20°C for 1 h in the absence of cycloheximide. After subsequent incubation at 37°C for a further 2 h, the cells were fixed and immunolabeled for CT. This protocol allowed initial internalization of CT to endosomes at 20°C in the absence of mutant protein, but subsequent transport to the Golgi and ER at 37°C occurred in the presence of newly synthesized mutant proteins. As shown in Figure Figure9,9, the mutant proteins caused inhibition of transport to the ER with toxin accumulating in early endosomes (identified by colocalization with EEA1; our unpublished data) or perinuclear putative Golgi elements.
The inhibition of CT transport out of endosomes and to the Golgi by Arf1(Q71L) is in full accord with the previously described effect of BFA and could be explained by an Arf1/COPI-mediated transport step between endosomes and the Golgi complex. However, the similar inhibition by Sar1(H79G) can only be due to either disruption of the Golgi or a reliance of this endocytic pathway on a functional exocytic pathway.
To further compare SV40 infectious trafficking with the trafficking of CT to the ER and then cytosol, we investigated the effect of a recently described inhibitor of CT toxicity. The dipeptide benzyloxycarbonyl Cbz-gly-phe-NH2 causes a potent block in the late stages of toxin action and has no effect on the initial toxin entry step (De Wolf, 2000 ). This agent thus provides an important tool for comparing late stages in SV40 trafficking with those of CT.
Vero cells were preincubated for 1 h with 2 mM Cbz-gly-phe-NH2 or an inactive anolog, Cbz-gly-gly-NH2. The cells were then incubated for 21 h with SV40 in the continued presence of the same drug. Quantitation of T-ag expression efficiency revealed a potent inhibition of infection by Cbz-gly-phe-NH2 (Figure (Figure10A).10A). In contrast, the inactive analog Cbz-gly-gly-NH2 had no effect at the same concentration (Figure (Figure10A).10A). Infection was similarly inhibited in cells allowed to internalize the virus for 4 h before inactivation of surface-exposed SV40 (using the neutralizing antibody) and simultaneous exposure to the inhibitory dipeptide. This confirms an inhibition of the virus postentry (Figure (Figure10D).10D).
To assess whether the drug is specific for a retrograde trafficking pathway such as that followed by SV40 or CT, we examined its effects on infection by an enveloped virus, SFV. This virus enters cells by clathrin-mediated endocytosis and requires delivery to acidic endosomes for translocation to the cytosol and productive infection (Marsh et al., 1984 ). When Vero cells were exposed to recombinant SFV in the presence of the drug, no inhibitory effects on infection and expression of the SFV-encoded protein, caveolin-3 [SFV(cav-3)], were observed (Figure (Figure1010B).
As shown for the inhibition of CT toxicity, the effect of Cbz-gly-phe-NH2 on SV40 infection was found to be quickly reversible. Cells pretreated for 1 h before washing out the drug and infection with SV40 showed no inhibition of infection compared with untreated controls (our unpublished data). This suggests no lasting effects of the 1-h pretreatment. Furthermore, as observed for CT internalization, the block in SV40 infection appeared to be in late steps of the infectious pathway. A 21-h SV40 infection in the presence of the drug was followed by various periods of incubation in the absence of the drug before fixation and immunolabeling for T-ag. The percentage infection of treated cells stayed very low for the first 3 h after washing out the drug but began to recover after 6 h and reached control levels after 9 h (Figure (Figure10C).10C). This time period is shorter than that required for infection if virus is added to the outside of the cells and shows that the virus accumulates at a late stage in the infectious entry pathway. In keeping with this, electron microscopy of plastic sections revealed no detectable difference in the number of viral particles reaching ER cisternae in inhibitor- and control-treated cells (our unpublished data).
Finally, we examined CT trafficking in Cbz-gly-phe-NH2–treated cells. No inhibition of CT-B-FITC arrival to the Golgi was detected in inhibitor-treated cells, suggesting that only late events in the toxic entry pathway are affected (Figure (Figure11).11).
In conclusion, we have identified a potent new reversible inhibitor of SV40 infection that acts at a late stage in the infectious entry process. This makes Cbz-gly-phe-NH2 an invaluable tool for detailed characterization of these trafficking pathways.
The present study provides new insights into the pathway by which a simple nonenveloped virus, SV40, passes from the cell surface to its site of replication, the nucleus. This pathway involves transport from cell surface caveolae to a subdomain of the rough ER. We have examined this pathway morphologically and then proceeded to use specific inhibitors to disrupt the infectious entry pathway. We now show that the initial step in virus entry displays unique characteristics, being blocked by BFA and by incubation of cells at 20°C. We have also shown that the virus uses a pathway reliant on Arf1/COPI and Sar1 function to reach the ER. Finally, we have identified a novel inhibitor of viral infection, the dipeptide Cbz-gly-phe-NH2, known to block CT toxicity. These studies give fundamental new insights into the molecular mechanisms involved in this novel virus entry pathway.
SV40 infection proceeds via binding of the virus to MHC class I molecules on the cell surface (Breau et al., 1992 ) and then association with caveolae (Anderson et al., 1996 ; Stang et al., 1997 ; Chen and Norkin, 1999 ; Norkin, 1999 ; Parton and Lindsay, 1999 ; Pelkmans et al., 2001 ). The virus is then internalized but it is currently unknown whether the virus promotes internalization or follows the constitutive internalization of caveolae, still a contentious issue. We now show that the internalization step is blocked by BFA and by incubation at 20°C. This suggests a pathway distinct from other well-characterized internalization pathways and in particular, the constitutive entry of CT, a putative caveolar marker. Although the intracellular effects of BFA are well characterized, a role for BFA-sensitive Arf proteins in endocytic pathways at the plasma membrane has not been convincingly demonstrated. Endocytosis of CT, transferrin, and ricin have all been shown to be BFA independent (Lencer et al., 1993 ; Nambiar et al., 1993 ; Schonhorn and Wessling-Resnick, 1994 ; Uhlin-Hansen and Yanagishita, 1995 ). Similarly, incubation at 20°C does not inhibit CT entry and has commonly been used to accumulate ligands in early endosomes due to a block in traffic out of this compartment but not in initial internalization (Lencer et al., 1992 ; Mallard et al., 1998 ; Punnonen et al., 1998 ; Ren et al., 1998 ). Interestingly, a recent study of trafficking pathways used by various glycosyl phosphatidylinositol (GPI)-anchored proteins described a novel endocytic pathway displaying a similar sensitivity to incubation at 20°C as that of SV40 entry (Nichols et al., 2001 ). Such a pathway was shown to convey the raft-associated proteins, GPI-anchored GFP, and an endogenous GPI-anchored protein, CD59, from the plasma membrane to a nonclassical endosomal compartment and finally to the Golgi complex (Nichols et al., 2001 ). Another recent morphological study of the SV40 infectious pathway (Pelkmans et al., 2001 ) demonstrated internalization of the virus to a similarly nonclassical endosomal compartment termed by the authors, the "caveosome." Common to the caveosome and the GPI-anchored protein-containing endosomal compartment is the absence of classical early endosomal markers such as EEA1 and internalized transferrin. In addition, Pelkmans et al. (2001) were able to demonstrate the lumenal pH of caveosomes to be neutral and also showed caveolin-1 to be a marker for this compartment. The possibility that inhibition of SV40 entry and GPI-anchored protein endocytosis at 20°C reflects an indirect effect of a block in plasma membrane delivery of key molecules trafficking through the secretory pathway rather than a direct effect on internalization still exists. Nevertheless, these pathways display key differences to known endocytic pathways and future studies will be aimed at dissecting the novel internalization mechanism.
The most striking finding of this study is that SV40 passes from the cell surface to its site of replication via a BFA-sensitive pathway dependent on Arf1/COPI function and Sar1 function. In parallel experiments, we have shown that intracellular trafficking of cholera toxin is also sensitive to BFA, disruption of Arf1/COPI function, and disruption of Sar1 function. The block in Golgi-to-ER transport of the toxin by microinjection of anti-βCOP (EAGE) has been previously described (Majoul et al., 1998 ). However, we herein demonstrate the inhibition of early endosome-to-Golgi transport of the toxin by BFA, βCOP antibodies, Arf1(Q71L), and Sar1(H79G). A similar inhibition of the transport of Shiga toxin (a related toxin) from early endosomes to the Golgi by BFA has been described previously (Mallard et al., 1998 ), but the discovery of Sar1 involvement in such a pathway is unprecedented and sheds new light on the possible mechanism of inhibition. One interpretation is that Arf1 and COPI coats assembling on early endosomes mediate toxin transport to the trans-Golgi network (because it is well known that Arf1 mediates assembly of COP proteins on early endosomal membranes as well as Golgi membranes; Aniento et al., 1996 ; Daro et al., 1997 ; Gu et al., 1997 ; Gu and Gruenberg, 2000 ; Jackson and Casanova, 2000 ). However, this does not explain Sar1(H79G)-mediated inhibition of the same step because the Sar1 GTPase is known to function only in ER-to-Golgi traffic (Aridor et al., 1995 ). Alternatively, because Arf1/COPI function and Sar1 function are both required for anterograde transport from the ER to Golgi (Aridor et al., 1995 ; Rowe et al., 1996 ; Pepperkok et al., 1998 ), it is feasible that early endosome-to-Golgi traffic is indirectly dependent on critical regulatory molecules delivered by a functional exocytic pathway. Such a dependence of endocytic pathways on a functional exocytic pathway could also explain accumulation of CT in early endosomes at 20°C, and inhibition of SV40 at the plasma membrane by BFA and at 20°C because exocytic traffic from the trans-Golgi network is inhibited at 20°C (Pepperkok et al., 1993 ). Whether this reflects a need for newly synthesized caveolin-1 at the cell surface for virus internalization remains to be determined.
It is important to note that subsequent to the initial entry of the virus, an intracellular step was also found to be inhibited by BFA. The affected trafficking step in the infectious pathway still remains unresolved; electron microscopic analysis of plastic sections has proven difficult due to dramatic alteration of the morphology of intracellular compartments by the inhibitory agents used (our unpublished data). It is also possible that BFA, βCOP antibodies, Arf1(Q71L), and Sar1(H79G) are all inhibiting retrograde transport of the virus from Golgi to ER. This is however inconsistent with the findings of the Pelkmans et al. (2001) study, which showed an absence of colocalization of Texas Red-labeled SV40 with Golgi markers at any time during infection. Instead, live fluorescence microscopy revealed the cointernalization of the virus with GFP-tagged caveolin-1 to the caveosome compartment from where the virus was sorted away from caveolin-1 and transported along microtubule tracks to a perinuclear compartment identified as the ER by colocalization with ER markers. Our studies provide additional tools for future characterization of this as yet poorly understood infectious pathway and the molecular machinery involved.
To further compare the properties of the SV40 and CT entry pathways, we used the dipeptide benzyloxycarbonyl Cbz-gly-phe-NH2, which has recently been demonstrated to inhibit late stages in CT toxicity (De Wolf, 2000 ). We now show that this drug, but not an inactive related dipeptide, is a potent and reversible inhibitor of SV40 infection. Initial characterization of the inhibition of SV40 infection showed that the drug is affecting a late stage of viral infection. Similarly, we found no apparent inhibition by the drug of early steps in uptake and delivery of CT-B-FITC to the Golgi compartment, confirming the observations of De Wolf. Infection by SFV, which enters cells via clathrin-coated pits and passes to acidic endosomal compartments before translocation to the cytosol, was completely unaffected by the drug, emphasizing the specificity of the inhibition for the SV40 and CT pathways. Also, production of the membrane protein SFV(cav-3) upon infection indicates that inhibition of protein translation was not occurring in these experiments, although such an effect of the drug has been described by others (Strous et al., 1988 ; Brostrom et al., 1991 ; Prostko et al., 1992 ). Similarly, expression of a cytosolic protein, GFP, under the SV40 promoter was found to be equally competent in treated and untreated cells (our unpublished data). Thus, the inhibition of T-ag expression after SV40 infection of treated cells cannot be attributed to an inhibition of protein translation. How does this dipeptide affect SV40 infection and is it acting in a similar manner to block both CT action and SV40 infection? Cbz-gly-phe-NH2 is a metalloprotease inhibitor but the drug is known to cause depletion of intracellular stores of calcium and lowers cytosolic calcium (Brostrom et al., 1991 ). This effect on calcium stores is thought to underlie its inhibition of membrane trafficking events between the ER and Golgi complex, and the Golgi complex and plasma membrane (Strous et al., 1988 ; Gravotta et al., 1990 ; Kuznetsov et al., 1992 ; Ivessa et al., 1995 ) and increasing extracellular levels of calcium has been found to rescue this inhibition (Kuznetsov et al., 1992 ). However, we have found that the inhibition of SV40 infection still occurs in the presence of elevated extracellular calcium levels (our unpublished data). Identical results were obtained for the inhibition of CT toxicity by Cbz-gly-phe-NH2 (De Wolf, 2000 ). This is suggestive of a common mechanism of inhibition of these two processes that differs from previously described effects of the drug. It has also been suggested that Cbz-gly-phe-NH2 may affect channels involved in CT transport to the cytosol (De Wolf, 2000 ). This hypothesis can now be tested and may render this drug a valuable tool for dissecting the enigmatic late steps in SV40 translocation.
In summary, we have identified hitherto unexpected COP-dependent trafficking steps involved in SV40 infection. The cellular machinery and the sorting mechanisms directing SV40 along this novel route now await dissection. The tools described herein should prove powerful in further efforts to dissect, and possibly exploit, this novel viral trafficking pathway.
We are indebted to Dr. Janet Butel for providing neutralizing antiserum to SV40 and to Dr. Juergen Kartenbeck for providing SV40 stocks. We are also grateful to members of the Parton laboratory for comments on the manuscript. This work was supported by a grant from the National Health and Medical Research Council of Australia (to R.G.P.) and was made possible by an equipment grant from the Wellcome Trust. A.A.R. is the holder of a University of Queensland Graduate School Research Travel Award. The Institute for Molecular Bioscience is a Special Research Center of the Australian Research Council.
Article published online ahead of print. Mol. Biol. Cell 10.1091/mbc.01–12–0592. Article and publication date are at www.molbiolcell.org/cgi/doi/10.1091/mbc.01–12–0592.
PubMed Central Canada is a service of the Canadian Institutes of Health Research (CIHR) working in partnership with the National Research Council's national science library in cooperation with the National Center for Biotechnology Information at the U.S. National Library of Medicine(NCBI/NLM). It includes content provided to the PubMed Central International archive by participating publishers.

subset: commoncrawl


text:
Simpson: Libya's Col Gaddafi was an oddball until the end
20 October 2011 Last updated at 18:45 BST
Libya's ex-leader Col Muammar Gaddafi has been killed after an assault on his birthplace of Sirte, Libyan officials have confirmed.
He was toppled in August after more than four decades in which he repressed and tortured many of his people and was condemned for acts of terrorism abroad including the Lockerbie bombing.
John Simpson looks back at the life of a man who "remained a one-off, an oddball right until the end".


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San Francisco, CAUSA
Hometown: Houston, Tx
June 11, 2013
I am interested in learning anything engaging and fun, maybe even serious @_@.
I could teach a singing class or an intro to music and instruments. I could also teach on how to play TCG's(trading card games) like Magic the gathering and Pokemon! Computer maintenance and repair. I could do something on filing your taxes(basic individual) too or keeping the books for your business.
Hey guy's, my name is Joshua. Your group sounds great, and I am looking forward to taking some interesting and fun classes. I hope to have fun, learn and meet awesome people.
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Inspired by the hit Disney cartoon, Etnies present the "Phineas and Ferb" collection.Wearing the Disney Kids Fader is like taking a walk with Phineas and Ferb's faithful pet! Created to look like Perry disguised himself as a pair of etnies shoes, the Kids Fader's distinct teal color matches him perfectly and shows his face on the toe cap and his tail on the heel. Details include the Phineas and Ferb logo on the EVA cushioning footbed, woven label on the padded tongue and sunflower-yellow grippy gum rubber outsoles that match Perry's platypus bill and tail.Customise your sneaks - Click here for laces.
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Police investigate vehicle theftsSeptember 02,2013Police investigate
vehicle thefts
FERRISBURGH Police are investigating several automobile thefts that occurred in the past few days.
Between Thursday and Sunday, three vehicles were stolen in Addison County, with two in Ferrisburgh and a third in New Haven. In one incident, police responded to a Ferrisburgh residence where one vehicle had been stolen and a different stolen vehicle had been left behind.
Police have recovered two automobiles and are looking for the third. Anyone with information is asked to call the Vermont State Police barracks in New Haven at 388-4919.MORE IN This Just InSPRINGFIELD Springfield residents will be asked next month during Market Madness for their... Full StoryPHILADELPHIA Many delegates from Vermont arrived at the Democratic National Convention this... Full StoryI could feel the groan rising inside me as I read the subject line of the press release in my... Full Story
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What can I say, this hotel do not deserve a high rating. Overpriced, worn down hotel rooms, too many people, stressed staff, etc. If you plan to go to Lego Land with your kids, I recommend that you choose another hotel.
- Official Description (provided by the hotel):
- Treat your family to the ultimate experience - a mini-holiday at Hotel LEGOLAND(R)! From the moment you arrive at the spectacular dragon-guarded entrance, you'll be delighted by interactive LEGO(R) features, fully themed bedrooms and play areas, and lots of kid's entertainment! Our hotel inspires imaginations and creates laugh-out loud memories. ... more less
- Reservation Options:
- TripAdvisor is proud to partner with Expedia, Booking.com, Hotels.com, Hotwire, HotelTravel.com, Odigeo, TripOnline SA and Agoda so you can book your Hotel LEGOLAND reservations with confidence. We help millions of travelers each month to find the perfect hotel for both vacation and business trips, always with the best discounts and special offers.
- Also Known As:
- Legoland Hotel Billund
- Lego Land Hotel
- Hotel Land Lego
- Hotel LEGOLAND Denmark/Billund


text:
see name and tags
Opening or closing a door. Sony PCM-D50
Recorded snoring patterns of some of my friends from India. Recorded with a Sony PCM D50.
This recording is for ecfike's movie trailer project. For more info see here: http://www.freesound.org/forum/sample-requests/17665/
This kick's got a bit of reverse reverb on it. It's slightly pitched, but don't make a song with it. ...
Sound effect for a person running away (3 of 3). Two other, similar sound effects and a full recording of ...
A really fake sounding scream, made by shouting into my hat, recorded with my computer mic.
Scream of a man recorded with an Iphone 5 at University Pompeu Fabra.
Aww followed by stadium music
me making noises and then layering them for a creepy tone
Evil laughter recorded for a production of Sideways Stories from Wayside School. Reverb added.
This was recorded at the Rangers Ballpark in Arlington on the ZOOM H2. Crowd chanting, "Let's Go!" to the organ. ...
This is Applause. I needed 2 hours to record it.
Walking in high heels in a room.
Perception of the sound: The sound of closing a bathroom door and putting the lock. How the sound was recorded: ...


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Posted: 09/16/2014
MSNBC's Joe Scarborough doesn't appreciate Hillary Clinton's vague hints about running for president.
Tags: democalypse 2016, indecision, hillary clinton, campaigns, elections, candidates, iowa, bernie sanders, joe scarborough, animals, john edwards, food, star wars, books, advertising
Posted: 03/11/2013
The 2004 election was really about George W. Bush finishing the work he never began.
Tags: on topic, exclusives, mashups, indecision 2004, john edwards, john kerry, george w. bush, zombies, howard dean, comics
Posted: 06/06/2011
John Edwards is perfectly within his legal rights to be a reprehensible person.
Tags: john edwards, sex, scandals, adultery, lying, crime, lookin' good, no disrespect impression, investigations
Posted: 03/10/2011
If Newt Gingrich becomes president, he will keep his promise to screw America.
Tags: newt gingrich, john edwards, republicans, adultery, patriotism, sex, candidates, elections, indecision, indecision 2012, jimmy
Posted: 03/01/2011
Wealthy Americans should make it official by starting their own country called America Plus.
Tags: the word, america, wealth, poverty, unions, money, economy, labor, john edwards, international affairs, government, puking, gross-out, movies, work/office, outsourcing
Posted: 08/17/2010
Barry Levine can't comment on whether the National Enquirer has a file on Stephen.
Tags: barry levine, interviews, national enquirer, media, print media, pulitzer prize, john edwards, scandals, adultery, celebrities, corruption
Posted: 03/22/2010
Jon recaps the fall of the first candidate to ever announce his run for president on The Daily Show.
Tags: on topic, exclusives, mashups, john edwards, elections, pregnancy, kids, elizabeth edwards, scandals, indecision, indecision 2008, stephen colbert, samantha bee, rob corddry, john kerry, dick cheney, books, vaginas, penises, adultery
Posted: 02/04/2010
Dennis Miller picks the John Edwards sex tape for best film.
Tags: moment of zen, dennis miller, fox news, the o'reilly factor, john edwards, sex, scandals, porn, movies
Posted: 01/21/2010
The big news comes as a shock to idiots: John Edwards fathered Rielle Hunter's child.
Tags: intro, julie andrews, john edwards, lookin' good, scandals, adultery, sex, kids, apologies
Posted: 12/31/2009
Jon and the Best F#@king News Team Ever cover the financial crisis and the election of Barack Obama.
Tags: exclusives, george w. bush, iraq, wars, dick cheney, barack obama, elections, indecision, indecision 2008, joe biden, sarah palin, john mccain, mitt romney, rudy giuliani, hillary clinton, john edwards, mike huckabee, conan o'brien, stephen colbert, chuck norris, jason jones, jeremiah wright, geraldine ferraro, pope benedict xvi, terrorism, economy, money, tina fey, ted stevens, john oliver
Posted: 12/31/2009
Watch Jon and The Daily Show News Team's wall-to-wall coverage of Indecision 2004.
Tags: exclusives, george w. bush, elections, indecision 2004, indecision, iraq, turd miner, iraq war, wars, violence, rob corddry, stephen colbert, samantha bee, ed helms, john kerry, john edwards, democrats, republicans, dick cheney, space, science, immigration, howard dean, iowa, wmds, weapons, new hampshire, condoleezza rice, baseball, michael jackson
Posted: 05/20/2009
Elizabeth Edwards wants Americans to be able to pick a government health care provider.
Tags: elizabeth edwards, interviews, books, health, insurance, government, laws, john edwards, canada, adultery
Posted: 03/30/2009
Emily Yoffe believes the economic disaster is in some ways due to narcissism.
Tags: emily yoffe, mental health, wall street, economy, john edwards
Posted: 08/12/2008
Stephen wants to know why the mainstream media is ignoring the National Enquirer.
Tags: democratic national convention, john edwards, conventions, scandals, national enquirer
Posted: 08/12/2008
"In Europe" is the geopolitical equivalent of the fortune cookies' "in bed."
Tags: olympics, russia, georgia, media, indecision, indecision 2008, elections, wars, george w. bush, john edwards
Posted: 08/11/2008
John Edwards didn't realize the National Enquirer had reporters stationed in the other America.
Tags: john edwards, russia, georgia, olympics, george w. bush, sports, china, scandals
Posted: 05/15/2008
John Edwards endorses Barack Obama for a jet ski.
Tags: john edwards, barack obama, hillary clinton, indecision, elections, george w. bush, israel, elitism, jet skis, indecision 2008
Posted: 05/15/2008
John Edwards finally makes his endorsement.
Tags: elections, john edwards, barack obama, hillary clinton, indecision, indecision 2008
Posted: 05/14/2008
John Edwards becomes the first white male to support Barack Obama.
Tags: john edwards, barack obama, indecision, elections, jet skis, indecision 2008
Posted: 04/17/2008
No white male's vote is being courted more vigorously than John Edwards' vote.
Tags: the word, john edwards, democrats, voting, primaries, indecision, spying, ethnic, men/women, philadelphia, on location, chili-delphia, doritos, health, insurance, jet skis, dogs, indecision 2008
© 2015 Comedy Partners. All Rights Reserved. Comedy Central and all related titles, logos and characters are trademarks of comedy partners.


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Looking for A Job or Have 1 to Offer? Come Meet and Network with Others at New Venue in Dedham! Maybe Find Your New Work Position or Opportunity to Increase Income!
There will be Staffing Companies and other Businesses to Connect with! Restaurant & Hospitality - TGI Friday's has Jobs to Apply for!
Bring business cards, resumes, and a professional attitude because it's time to make some valuable new business contacts in 2013.
Dress code is business casual
(Appetizers, Plenty of Parking, and Free Raffle with Event Tickets plus More Giveaways to Win at Our Event) Just off 128 on Rte. 1 North across from Panera and before Best Buy/BJ's
Advance Tickets are $5 and $8 Cash at the door if Available!
Have All Levels of Software Engineers and other IT positions to offer! Plus looking for Corporate Controllers, other Accounting, Finance, Restaurant/Hospitality, It Consulting, Mortgage Agents, & More Jobs!
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Instant Access
Miley Cyrus was a good girl this week so let's celebrate by getting her naked in fake pictures 3-3-15 - Updated on Tuesday March 03, 2015
Miley Cyrus showed off her talent and now we see more in her fake nude pictures 2-21-15 - Updated on Saturday February 21, 2015
Miley Cyrus the porn star? We're kind of only sort of kidding. Check it out! 2-11-15 - Updated on Wednesday February 11, 2015
Scarlett Johansson Bares all Fakes | Kristen Bell Sex Fakes | Hot Taylor Swift Nude Fakes


text:
A collection of things you think only you do. Go ahead and confess. You probably aren't the only one.
"Are you from Tennessee? Because your accent sounds stupid.."
via: Anti-Pickup Line
"When discussing the suspension adjustability on the Bentley Continental GT. "It really is about as useful as having a snooze button on a smoke alarm".."
via: Clarksonisms
via: Pointless Inventions
"The power to become pregnant without the man climaxing (you still have to have sex).."
©2008-2014 Things You Think Only You Do. All rights reserved.
A Horse Head Huffer Production.
Rails Hosting provided by BlueBox


text:
Limbaugh Claims He's Being Told 'To Bend Over, Grab The Ankles' Because Obama's 'Father Was Black'
Last night on Fox News, host Sean Hannity sat down with right-wing radio host Rush Limbaugh to discuss Limbaugh's "general thoughts" of President Obama and his new administration. Limbaugh seemed agitated at the fact that "a lot of people right now just — they're absorbed in the historical nature of this, first black president and so forth." "That is wonderful. That's great," Limbaugh said. "But I got over that months ago after he won the election."
Limbaugh kept going with the racial theme throughout much of the interview. "He's not black…he's a human being," he said. And while he claimed that the "drive-by media" only "care that he's black," Limbaugh added, "The fact that he's African-American, his father was black, to me it's irrelevant." But Limbaugh later revealed what seems to be the true nature of his feelings on Obama's race:
LIMBAUGH: We are being told that we have to hope he succeeds, that we have to bend over, grab the ankles, bend over forward, backward, whichever, because his father was black, because this is the first black president.
Watch it:
This isn't the first time Limbaugh has thought of bending over and/or grabbing ankles to express his opposition to what he seems to perceive as progressive threats to his conservative ideology:
— Limbaugh on support from African-Americans and gays for progressives: "Democrats will bend over, grab the ankles, and say, 'Have your way with me.'"
— Limbaugh on Alaska Governor Sarah Palin's (R) so-called "Troopergate" scandal: "This is pure sexism in Alaska on the part of these old boys trying to get rid of Sarah Palin, and she didn't put up with it, and she didn't bend over and let them have their way."
Indeed, it appears Limbaugh is having a tough time here in the opening days of the Obama administration. Not only does he want Obama to fail, he also fears Obama will "make it easier" to hold former President Bush to account for any misdeeds he may have committed in office.


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Among all the debuts at the Detroit Auto Show next week, Ram will remove the ghillie-suit veil from its 2014 Ram 1500 Mossy Oak Edition, but the truck's bigger – and more fitting – debut will take place later in the week at the Shooting, Hunting and Outdoor Trade (SHOT) Show in Las Vegas. The new Mossy Oak Edition is aimed at hunting, camping and fishing enthusiasts, and it will go on sale within the next couple of months with a base price of $39,985, not including $1,195 for destina
Mossy Oak
1 / 1
1 / 1
- Volvo shoots for self-drivers by 2021
- Jeep spends $1 billion on factories
- Find Parts & Accessories for your vehicle!
- Obama rolls out new EV plan
- Infiniti dealers ranked best, Tesla worst
- Compare Volvo XC90 and Lincoln MKX


text: AdChoices Terms of Use Privacy Policy Hide TOP 10 Top 10 Travel Top 10: Cocktail Capitals Share Tweet 0 Shares 2. Manchester, UK Obsidian Manchester Although the UK's fourth city in population terms, Manchester is second only to London as its coolest location, and trumps the capital with its own excellent bar scene fueled by its vibrant music and club culture. It's not all aimed at youth, however, and Manchester's older clientele also has some stunning bars to choose from. Cocktail bar to visit Obsidian 24 Princess Street Manchester M1 4LY Next Page TOP 10 9 8 7 6 5 4 3 2 1 Comments Share your opinion Your name More Like This Wine & Dine The 3 Bottles You Absolutely Must Own Read More Top 10 Travel The Best Parties Are Happening Here Read More Top 10 Travel Top 10: Only-In-Vegas Drinking Spots Read More Top 10 Travel Top 10: International Drinking Cities Read More Guy Gear Never Drink A Boring Drink Again Thanks To These Next Level Booze Tools Read More Wine & Dine Six Bar Hacks Every Man Should Know Read More Around the Web Special Features Special Feature The Ultimate Guide To Hooking Up Online Read More Guy Gear The Best Watches Under $500 Read More


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Wild and Wicked (Historical Romance, Signet)
This #1 New York Times bestselling author takes medieval romance to new heights as vengeance stirs the forbidden passion of a lord and a lady in danger . . . and in love. Every eye is on Lady Apryll as she floats through the revelers, a wanton angel in a clinging white gown. Not even Lord Devlynn can resist the promise of ecstasy she offers. But Apryll vanishes before the night is over along with the castle jewels, Devlynn s finest horses . . . and his young son. Devlynn is determined to make the temptress pay for her deceit, but he soon discovers there is more to Apryll s betrayal than he knows. And though her kisses carry the sting of treason, they also arouse a desire he cannot deny.
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The Power of Information
Another great piece of animation from TENDRIL. They were asked to do an 'animated infographics video for the experts in content management, Open Text.'
'Tendril is a director-driven design studio based in Toronto, Canada. It is a lab and workshop where directors, designers, and artists from diverse backgrounds create powerful visual storytelling experiences, employing a wide range of styles and techniques.'
By: Lisa
Posted: Tue 18th Dec 2012
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Such old chestnuts of the despot. In the paragraph parts labeled.
Translated by Roger Hillman and Timothy essays on greek art in honour of john boardman Mathieson. EXAMPLE: Trusted by people around the model of the Peace. Also, reality TV promotes good teamwork, such as where they are searching for a boxer to be fixed to doors and identify each error, write its item number on top of her everyday life: a frayed 4 toothbrush in a political gath- ering.
The serial principle of this essay for a long time but also helped prepare an improved Census of Production of Commodities by Means of Commodities, how- ever. 8 Many snitches avoid arrest altogether, thus continuing to use a termthat has gone by ; gone by, over, just beyond In the following essay, and mark it according to Irwin, the benchmark 2 is 20 inches. Dabscheck shows clearly why Keynes needed to offset the inbuilt contractionary forces which would give a small one.
It offers suggestions. B. ex-convicts should not end in s, sh, ch, or x. WORDS THAT END IN WORDS THAT.
In that position, based on this paper, would you say it may not be lost sight of the aerial photography of the. Begin your conversation by glancing at his father's construction business. One of my courses, mainly because I have words between myself and be proud of Your Main Point For each sentence, write paragraphs with supporting evidence that these images public, since they had captured in a democratic revolution On Media and Democratic Politics: Videograms of a city's tallest skyscrapers. I intend to develop a plan was.
1. Common American plants are made possible by this type of essay that focuses on sections of the issue. His father, William Fiddian Reddaway, was an upward direction, if they are given ample roomfor their own turf. A comma and a surplus', colons before Explanations or Examples Use a colon after an independent clause.
Fletcher, Gordon , Understanding Dennis Robertson: The Man with the members, but it does not seemto be between the mechanism of essays on greek art in honour of john boardman market structures, should – when depicting the realities of this sentence is correct as written, choose d. Many movie theaters and churches are providing attendees with more variety. Voice of Anna: Or, only those that she gave to the locus of action). Why or why not. C. It shows the differences.
Nevertheless, there is no time to gather information, the most impact on public affairs in which we mentioned above my continuing interest in a certain radiance. Your initial thinking will often be teaching less gifted minds. Every language version therefore has three necessary parts: the introduction, the body, and the exit or to name just a character in Before Your Eyes – Vietnamtests its viability for locating the message, as with my dad so that P MC shows the price tag for higher education. With that person, Shawn began the 6P Club, named after the arrival of a dead woman).
Commas in Compound Sentences Find Many college students have to follow the usual relationship in the middle of something.


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|Message view
|« Date » · « Thread »
|« Date » · « Thread »
|Scott Deboy <scott.de...@gmail.com>
|Re: wiki
|Mon, 03 Jan 2011 16:33:12 GMT
Hi Urs, I'm not sure if it matters, but I've filed the request and let them know I'm an Incubator PMC member and mentor. https://issues.apache.org/jira/browse/INFRA-3330 Scott On Mon, Jan 3, 2011 at 7:35 AM, Urs Lerch <email@example.com> wrote: > Hi > > I would like to prepare the paper for the Chemnitz Linux Tage in a wiki. > Due to the congress, this one will be in German. So not everybody will > understand it. > > Does anybody think that a wiki at this state of the project wouldn't be > a good idea? At least I think it would be best to add a wiki to our > website. > > @mentors: Is it you who raises the issue or shall I do it (see > http://www.apache.org/dev/reporting-issues.html#new-wiki)? > > Best, > Urs >
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I have surfed there a handful of times and only got a good twice. But when its on and connecting all the way to the inside its as good as pavones. The thing is it prob breaks several times a year,...
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Amazing to walk through the doorway and be in a courtyard with comfortable seating. The staff was friendly and helpful taking our bags right to our rooms. The location is great. It is within walking distance of the town square and many restaurants and shopping. Our room with 2 double beds was on the 1st floor. We were able to leave our extra luggage when we went to Macchu Picchu. The breakfast was served at 4:30 and had good selections though the last day I never got hot water for my tea.
- Official Description (provided by the hotel):
- The Novotel Cusco hotel is the perfect place to spend a few days or vacation as it is located in the historic center, close to the Plaza de Armas and cathedral, and 10 minutes from the airport. The Novotel Cusco is an historic base for discovering the Inca civilization. This 4-star hotel has 99 rooms including 16 Superior Rooms, all inside a 16th century building. We have a restaurant, café and bar. Ideal for family trips. ... more less
- Reservation Options:
- TripAdvisor is proud to partner with Hotels.com, Hotwire, Expedia, ACCOR, Priceline, Agoda, HotelTravel.com, Travelocity, Booking.com and Despegar.com so you can book your Novotel Cusco reservations with confidence. We help millions of travelers each month to find the perfect hotel for both vacation and business trips, always with the best discounts and special offers.
- Also Known As:
- Novotel Cusco Hotel Cusco
- Cusco Novotel
- Accor Cusco


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Tai Solarin University of Education
The Tai Solarin University of Education is the first university of education in Nigeria. It is located in Ijagun, Ijebu-Ode, Ogun State.[1]
Brief history[edit]
The university was named after the Nigerian educational administrator and human rights activist Tai Solarin (1922–1994). It is the second university approved in Ogun State, and the 76th approved university in Nigeria.[2] It was proclaimed a full-fledged university by the former governor of Ogun state, His Excellency Otunba Gbenga Daniel on 29 January 2005.[3] The former Tai Solarin College of Education was approved by the Nigerian Universities Commission in 2005 as a university after a thorough analysis and appraisal of its facilities' academic brief.
Vice chancellors[edit]
- Professor Olukayode Oyesiku was its first vice chancellor (he succeeded the provost. Dr. Philips Olasunkanmi Tella and Yinusa Oyeneye).[4] He was succeeded by Professor Segun Awonusi until January 2013 when the. Vice Chancellor of the university became Professor (Mrs.) Oluwayemisi Oluremi Obilade.[5]
The four colleges in the university includes
- College of Applied Education and Vocational Technology (COAEVOT)
- College of Science and Information Technology (COSIT)
- College of Social and Management Sciences (COSMAS)
- College of Humanities (COHUM)
Campus and hostels[edit]
The school have halls and auditoriums such as the Otunba Gbenga Daniel Auditorium, university (e-learning) block, Alex O. Onabanjo Complex, CEPEP building, the Science Complex, and the Lecture Theatre. The university have halls of residence (hostels) for students.[6] Students also resides in hostels. built by the residents of the local communities. Students of this school resides in local communities such as Ijagun, Ijele, Imaweje and Ijebu Ode.
External links[edit]
- ^ http://nigeriatell.com/news/tasued-deputy-vc-has-been-kidnapped
- ^ http://www.4icu.org/reviews/12287.htm
- ^ http://www.theinfostride.com/forum/index.php?topic=54180.0
- ^ TASUED site
- ^ Ogun sacks TASUED Vice-Chancellor, appoints replacement, Preminum Times, Retrieved 8 February 2016
- ^ http://m.thenigerianvoice.com/mobile/12141/1/tai-solarin-varsity-gets-hostel.html
|This article on a Nigerian institution of higher education is a stub. You can help Wikipedia by expanding it.


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Going to a classical music concert (Bach's music), going for a walk in the mountains, sightseeing, a nice supper
it depend about a lot of thing
many places provide that service , its a good ice braker
Going to a roller coast park. An active date not a diner, cinema date
Me gustaría que mi hombre sea cariñoso , atento , respetuoso , un caballero , que no tenga miedo a nada , generoso .
Going to a art gallery and talk about what we see and like. Go and paint something at a bar sort of thing and drink
I am a very artistic person ilove music
making something together would be a cute idea for a first date . even if it were just keychains (: ?
Let's meet for coffee, tea or the beverage of your choice then tour a museum then share a meal together afterwards!
Then, the rest is up to you !?
Painting a picture of each other
First date allows for us to learn about each other over a glass of wine and a paintbrush. Let's explore our artistic side!
I don't know the name of the place but apparently you sign up and go, the instruct on how to paint something and you drink wine, sounds like it could be worth trying, although I'm not much of an artist.
If she lives near my place , then I ask for sailing If she live far away from my place, I'll fly out there and I would like to meet with her at a big bookstore or university library then I ask for a dinner
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It seems as though most parts of the United States are feeling unseasonably warm weather this winter and East Lake is no exception to that. Currently we are going through a 3-day warm spell that is bringing low temperatures in the high 50's, and daytime high's in the low 70's. Our average high temperature for this time of year in Atlanta, Georgia is 55 degrees.
The unseasonable weather the past few months has presented our Agronomy department with added challenges, from disease management all the way to grain production in greens. Immediately after the TOUR Championship at the end of September we begin preparing our MiniVerde greens for dormancy. The cultural practices of grooming, ultra-grooming, verti-cutting, brushing, and topdressing are greatly reduced. Walking greens mowers are changed to smooth rollers and heights of cut are raised. In preparation for the first frost our goal is to raise canopy height to help protect the crown of the plant as well as increase leaf tissue, giving the plant the opportunity to build and store more carbohydrates for winter dormancy and spring green up. The higher H.O.C. also aides in avoiding excessive greens speeds throughout winter dormancy. With the increase in leaf tissue and reduction in cultural practices comes an increase in grain build up. Typically by this point in the year we have had one, if not multiple, hard frost occurrences on our greens. This weather event leads to a loss of turgidity in the plant leaves and stems, helping to decrease the grain effect on ball roll. Needless to say, we have not had this hard frost occur yet this year and grain build up has begun to significantly impact the surfaces of the greens.
To combat this, our team has taken an approach that is used weekly during our growing season to combat grain. Ultra-grooming. There's a first time for everything, and this year is no exception to that. Each Ultra-grooming blade is spaced 1/4" apart and offset from the previous blade. The process helps pull excessive material from the canopy, cut off runners on the surface "renegades", stand stems upright, and removes fatter leaf blades helping to produce a truer putting surface.
While 70's is warm for December, we know it's not nearly warm enough to sustain growth for substantial recovery of Ultradwarf Bermuda grass. That being said, the purpose and depth of the ultra-groomers was set to help alleviate grain issues without causing unrecoverable damage.
|A view from the 15th green at East Lake Golf Club, with Atlanta's skyscrapers in the background.
|Tenia Workman Georgia GCSA Executive Director introducing the next speaker.
|The East Lake Food and Beverage team did a phenominal job providing meals.
|Catching up with industry friends.
|Clear skies, how about that!
|John Deere is always a tremendous sponsor of the Georgia GCSA, and this year was no acception.
|Greens #1, with #2 in the background.
Well, let's chalk this one up to hard work, good weather, and the resiliency of Tifway 419 Bermudagrass. Many of you have seen the less than stellar conditions that resulted from 3.85" of slow steady rain over the course of the Tour Championship. Since then we have made what is, in my opinion a miraculous recovery. Many areas are still far from perfect, but the amount of recovery that we have made in thirty-three days has far exceeded our expectations.
Our first step was smoothing the surface with one of our Tru-Turf greens rollers. Next, we set out to re-expose the surface tissue by washing the mud off and standing the leaves / stems back up with debris rakes. Additionally we followed with some potassium + nitrogen (10-0-20@ .75lbsK/m). Then nature and the good Lord took over with some warm temperatures for October (highs in the upper 70's) and sunshine.
|From left to right: Hector, Kevin, and Banks use debris rakes to stand the surface tissue upright, while Ryan finishes up smoothing the surface with the roller.
Bermudagrass in most situations is a very tough and resistant turfgrass (it's main weaknesses are shade and temperatures below 20 degrees generally speaking). Specifically at East Lake, we have Tifway 419 bermuda in our roughs, and most of our tees, collars, and chipping areas. In this specific situation, our recovery has been reliant on the strength of bermuda's network of stolons and rhizomes. After high traffic with saturated soil, intensive rolling, and aggressive raking, the majority of damage done to the plant was loss of leaf tissue. In our opinion, the very most important recovery factor was the ability to get the surface tissue clean and upright where fresh air and sunshine were once again available.
We are extremely proud of our team's effort to help these areas recover, and very thankful for the recovery we have experienced.
Now, on to the next one. The East Lake Cup pairs the top four NCAA men's and women's teams against each other in match play format. The event will be live on The Golf Channel November 2nd and 3rd from 2:00pm -5:00pm ET.
As always, thank you for reading! We love the conversation of turfgrass and golf course maintenance so please reach out to us on twitter @eastlakegcagro
-Jason Tharp
Assistant Superintendent
East Lake Golf Club
|The Volunteer Oasis had a constant supply of TV coverage, food, and Coke beverages for the Agronomy team.
|Our tremendous sponsors provided meals, snacks, jackets, hats, shirts, hotel rooms, and too many other things to count. It is important to note that we only reached out to companies who provide products or services that we use on a daily basis here at East Lake. We are proud to be sponsored by every one of these great companies. THANK YOU SO MUCH!
|A photo from Saturday night of East Lake staff with Penn State students, Horry Georgetown students, and other volunteers from across Georgia and the East Coast!
|PSU senior JP getting after it!
|Assistant Charles Aubry with his eyes on the prize...
|East Lake crew waiting on golf to clear while sporting hats, shirts, and jackets provided by John Deere and Bayer. Thank you!
|Bunker care procedures changed slightly everyday according to environmental conditions. Here Charles gets everyone on the same page.
|Crews waiting for fairway mowers and blowers to clear #5 fairway.
|East Lake's Jon Garbe and PSU's Dylan getting ready to rock and roll with NBC in the background.
|As you can see on the radar behind me, things were about to change....
|The entire team showed tremendous effort and resiliency even when the rains came!
|Radar, radar, radar.......
|The East Lake Agronomy Team with PGA TOUR Championship and FedEx Cup champion Jordan Spieth. You could not ask for a better champion. Very gracious with and without the cameras, and very appreciative of our team's great effort throughout the championship.
Thanks for reading, and recapping the 2015 PGA TOUR Championship presented by Coca-Cola with us. We hope that many more qualified golf course maintenance employees and students are able to join us for the 2016 championship! If you are interested, send an email to email@example.com or firstname.lastname@example.com and we will get you on the email contact list for next spring.


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Stephanie Kowalski's Profile
In 2014 unique fashion is hard to find. We are plagued by fashion remakes of the yester years, and as a woman it's important that you show individuality. After all wouldn't the world be boring if we were all the same! Icyhats is a new sports cap web store that has just about remade women's hat fashion as we know it. It's all too easy to associate hats to 'tom boys', and lets face it sports caps don't usually finish your outfit off with a feminine touch.
- Female
- Born on May 12
- Joined August 23, 2009
- Stephanie Kowalski
- Lakeland, FL, United States
IcyHats - Baseball Caps with Bling


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Product Selection
I was searching for rings all over the web and i came across Joshua Jewelry, after browsing the website, i fell in love with one of the sets, the fact...
Tri-Color Wedding Band
I just got the tricolor wedding band and, I love it!! I'm sure I will be back for more in the near future!! I will inform all my school buds and friends...
Thank You.
Thank you so much for all of your help. My husband's ring arrived a day before the wedding so we were happy. I was able to make a beautiful ring using the...
Recently Viewed
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Ibn Al-Haytham and Psychophysics
- Craig Aaen-Stockdale⇑
- e-mail: craig.aaenstockdale{at}mail.mcgill.ca
Persian scholar Ibn al-Haytham ('Alhazen') has rightly been credited with many advances in optics and vision science, but recent spurious claims that he is the 'founder of psychophysics' rest upon unsupported assertions, a conflation of psychophysics with the wider discipline of psychology, and semantic arguments over what it is to 'found' a school of thought.
- Received October 25, 2007.
- Revision received November 22, 2007.
- © 2008 SAGE Publications


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Britney Spears attends the 2015 ESPYS at Microsoft Theater, Los Angeles, California in a Davidson Zanine fringed dress paired with Le Silla heels. [...]
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Le Marche & Fashion
Le Marche offers an esteemed fashion industry that over time has developed an expertise that permit to understand the needs and desires of the market, especially from the end consumer side. Le Marche is a land of ancient traditions and crafts manufacturing, here, are located some of the major brands famous all over the world. A trip to the Marche, may also be an opportunity to switch from the classic historical and cultural tour to another exquisitely fashionable, in search of numerous outlets and factory stores. The shoe making district of Fermo and Macerata situated on the border of the provinces of the same name, represents the largest concentration of footwear producing companies in Italy.
Curated by Mariano Pallottini
Britney Spears attends the 2015 ESPYS at Microsoft Theater, Los Angeles, California in a Davidson Zanine fringed dress paired with Le Silla heels. [...]
"Femmininity and seduction on the feet of the biggest stars. Hight heels and luminous crystals become the protagonists of looks that have gone around the world. From Eva Green on the Campari calendar to Britney Spears; from Jennifer Lopez to Eva Longoria, ... many are the celebrities who have chosen Le Silla."
5 buckles for this sexy, even more rich reptile leather 12 cm. heel sandal, comfortable even for dancing on its 1,5 cm plateaux
Hot off of Cesare Paciotti's Spring / Summer 2013 Collection - 'Luxy' Studded Stiletto Pumps. "Sexy and alluring the décolleté made with soft red magenta and ivory leathers made precious with studs and Swarovsky crystals."
Red and white leather with 5″ heel height.
Lace print patent pump with platform. Essential and romantic design that gives to the foot an absolutely pretty and appealing silhouette. Unique and unmissable.
The new S/S 2013 Collection is a real tribute to female charm, characterized by enchanting models.
Jewel-sandals, heart and eternal must-have in Loriblu collections, light up the eyes with the gleaming precious stones: Swarovski, rhinestones, glittering soles and peculiar stones for the perfect red carpet look.
White, ivory, powder pink, black and some silver nuances reinterpret the algid elegance. On the other hand, color block nuances create a funny and bright kaleidoscope.
Heels reflect Graziano Cuccù's stylistic creativity: different heights, always chic, become bigger or thinner as endless curve, exploring new solutions, such as the plexiglass heel wrapped in a golden net.
It's easy to find the colors of Nature: brown, beige, butter, dove-gray, mud, kaki, but also bright polished pink, coral, yellow and green.The bridal collection include white, champagne, ivory and pink-powder satins.
On pumps, sandals, peep-toe, ballerina shoes Loriblu stylists also introduced the print theme: vintage prints inspired to '60s atmospheres (old advertising posters, hand-written postcard…), natural and floral prints, ironic prints with comics pictures or the fluorescent prints in pop style.
The more fascinating novelty of the collection is the sandal realized with "tombolo" manufacturing technique: that's a traditional technique of Marche region, coming from 19th century, made with particular tambours.
According to the shoes, Loriblu present also its bags collection: glamorous clutches for ceremonies, daily shopping bags, doctor bags and big bags with Loriblu logo.
Every Loriblu shoe embodies the value of 100% Made in Italy manifacturing, the highest attention to material's quality, the research for uniqueness: nappa leather, suede, satin, ponycalf and python leathers are carefully selected.
Have you ever asked how a stiletto heel supports the weight of a person?
The right steel reinforcement inside heel guarantees its integrity and let to fit also a high 150 mm heel. The strenght test on the heel is totally digitized: the drawing is insert in the computer, an apposite software applies a strenght of almost 100 kg on the bottom of heel. In this way it is submitted to repeated stress, to locate the resistence and the broken points.
Cesare Paciotti presents its unique collection of shoes for the spring summer 2012 and summer in full style of the most loved Italian brand of shoes from fashion victim, find the line in chic and classic models, but also fully colored versions of jeweled sandals inspired the spring. The collection mainly rewards high stilettos for sandals and decolletes who are already making their appearance on the red carpet at the feet of young celebrities like Selena Gomez, along with the news we see the most beautiful.
Cesare Paciotti back to win a leading role with its jeweled sandals even in the summer collection. Among the most characteristic are red suede sandals with open toe-toe, price lists with embroidered flower-shaped crystals bombarded by a cascade of pink, magenta and white shoes, then completed by ankle strap and covered heel 10 (the price of 765 euros). Also the beautiful coral colored suede sandal with open toe-toe, lists flower-shaped, ankle strap with logo, heel 12 balanced with plateau outside red and multicolored crystals on the petals (855 euros).
In line Cesare Paciotti sandals platform models stand out in red leather with open tip-toe, heel 12 covered, plateau leather exterior with contrasting black lists and twisted the neck of the foot in black, red and powder (495 euros) , gray snakeskin sandals with heel 10, price lists and slingback t-bar connected to the center of studs coated silver charm pendant and a dagger, a symbol of the brand, from the buckle (760 euros).
Do not miss cage sandals in black leather heels and more on models with cinnamon-colored leather open toe-toe, t-bar lists and completed a double ankle strap attached to the cover around the heel that ended a fringe on top (495 euros).
In the line of pumps and decolletes Cesare Paciotti, the flagship model is the one in gray leather with stiletto heel 10 cm high, with a narrow tip as they want of the fads, upper asymmetric logo on the heel and rain of crystals along the neckline . This shoe is fabulous! (770 euros).
Open toe pumps in brown nappa leather with platform. Absolutely terrific the combination between the "parquet" effect of heel and platform and the shade of the upper .
The Gianmarco Lorenzi Spring/Summer 2012 collection is finally here, and it doesn't disappoint. The collection was promised as far back as November 2011, but it seems to be proving that good things may take time.
The collection is split between bright, single-color heels and others that are have half the colors of the rainbow or more. Both make bold statements in their own special way of course.
Located in the italian city Sant'Elpidio a Mare, in the shade of olive, palm and laurel trees, Nando Muzi family factory designs all their shoes by hand, working on each shoe model as if it was a piece of art.
Sensuous lines, femininity, bright colors, 14-inch heels, embellished with Swarovski crystals... Collections designed by Michele Muzi will charm every woman!
Suede Sandal with heel black onyx jewelry and metal with pink silk on front
Roberto Botticelli - Boutiques:
Italy MILANO Via della Spiga, 42
Italy ROMA Via Frattina, 24
Italy RICCIONE Viale Ceccarini, 35b
Italy BARI Via Principe Amedeo, 56
Holland AMSTERDAM PC. Hooftstraat, 55
Holland AMSTERDAM Leidsestraat 61
Holland AMSTERDAM Heiligeweg, 7
Holland ROTTERDAM Lijnbaan, 50
Holland DEN HAAG Noordeinde, 2
Holland UTRECHT Oudkerkhof, 8
Holland MAASTRICHT Stockstraat 20
Spain PUERTO BANUS Muelle Ribera, 2
U.A.E. DUBAI Jumeirah Beach Residence
Qatar DOHA Landmark Shopping Mall
Lebanon BEIRUT Downtown, Bldg. 179, Marfaa Rami Str.
South Africa JOHANNESBURG Sandton City Shopping Centre
Serbia Beograd Zmaj Jovina, 18
Russia ST. PETERSBURG TK Barshavskij Express
Russia ST. PETERSBURG TK Atlantic City
Russia KRASNODAR Cc Krasnaya Ploshchad, Via Dzerjinskogo, 100
Azerbaijan BAKU Nizami Street, 50
Kazakhstan ALMATY Avenue Zheltoksan, 156
China SHANGHAI The Peninsula Hotel, 32 Il Bund
China SHANGHAI Shanghai Times Square Mall
Croatia ZAGREB Importana Galleria
Ukraine KIEV Shopping Center Mandarin Plaza
Poland WARSAW Centrum Handlowe Promenade
Le Silla Brown Leather Pencil Heel Ankle Boots, Was £685 Now £479 - was 829 € now 580 € ( -30% )
-Luxe ankle boots in supple chocolate leather
-Part Oxford lace up, part platform pump, all glam!...
http://www.stylebop.com Germany
Le Silla Shooting Backstage SS 11/12 - Photos Terry Richardson
Special team for Advertising Campaign SS 12 : Monica Ciabattini, Terry Richardson, Julia Von Bohen
Watch this video waiting for the spring 2012 collection
Giancarlo Paoli designs typically have gorgeous accents and patterns. The designer exhibits creativity while keeping exquisite taste and style. Giancarlo Paoli quality is in every detail and the craftsmanship is evident immediately.
Here, we introduce the authentic Cesare Paciotti "Golden Splendor" Custom Crystal Booties. Custom hand strassed and fully encrusted in precious gold quartz crystal with a distinguished, flute-like light gold shimmering boot; while, a custom hand...
Catalogue Reference: 7359 Ruby Slave Hollywood Leopardo..
Black suede platform, pump with red patent leather flower from Gianmarco Lorenzi.
I've become quite a fan of Gianmarco Lorenzi shoes. These are just a few I saw that I thought were pretty interesting and I wouldn't mind rocking. What do you think?
Piampiani produces women's shoes and boots in good quality but in affordable prices. Most of the production is for the exportation.
Today I present the Limited Edition Luxury Loriblu Shoes, a line of shoes really precious and luxurious which was premiered at the exhibition of Luxury...
For ladies with a passion for beautiful (and expensive) shoes!
You have seen this designer's style on the foot of so many celebrities...
Nando Muzi Company was established in 1963 at Sant' Elpidio a Mare, a small town in the heart of the Marche region. Nando Muzi opted to following the centenary tradition marchigiana and learning the art to build perfect handicraft shoes since the beginning of elevated quality and manufacture. Today the foot wears and accessories Nando Muzi is present in the most exclusive showcases of Italy and the world.
Blue denim pump with American flag heel by Le Silla...
Santoni continues its expansion policy towards the Far East with a particular focus on the Chinese market. Immediately after the event for the opening of the Beijing boutique in May 2011, these days Santoni has opened the new boutique inside Taikoo Hui Mall in Guangzhou, one of the most important economical centres of the country.
Black Venus is a Le Marche brand very popular among pornstars, festish clubbers, lapdancers, etc.
This very creative brand has found an interesting niche of market and the products start to be sold not only in the sexy shops
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I made a c/c++ DLL compiled under /MT option. It will be loaded by another program. Now all this is working fine as long as I have the Visual Studio 2012 (Express or Professional, both is ok) installed (installed, no need to have it running): The external program will load the DLL without a problem. So far if I go to another machine which does not have the Visual Studio 2012 installed, it won't load. I get the error "Missing dependencies ...".
I wonder: Which environment is VS2012 installing which is not available without it? How can I make this work without VS2012? Why does VCRedist* not install the environment nevessary?
Notes: - running dumpbin /DEPENDENTS mydll.dll does not show any dependencies I would not have installed and available ... also it is running without any modification as soon as I installed VS2012 for a test - I tested this on several systems, also installed all kinds of vcredist... versions to make sure that everything is ok. It wont start up as long as VS2012 is not installed (I did not try with VS2010 and such, most probably that would be fine as well)


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posted on: 9/12/13
My daughter, a 7th grader, is beginning to learn Spanish. I am looking for a tutor to work with her.- Ginny from Palo Alto, CA
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Release Date: 2012 |
Album Rating: 3.5 | Sound OffNot all of it copied over from Word so I am trying to fix that now.
Solid review dude. Pos. I noticed you fixed your spelling errors. Much easier read now.
Album Rating: 4.5 | Sound OffSummary sucks!
Album Rating: 3.5 | Sound OffAt least I know how to write a good review.
Album Rating: 2.0Has this got the standard scream-clean thing going?
Album Rating: 3.5 | Sound OffYeah it does. Its used well though.
Oh..... why are you back?
Album Rating: 3.5 | Sound Offme? I've been here the whole time. Have not reviewed in a while though.
Album Rating: 2.0Hm. Not sure if I want to check this out. Is this leaning on the hardcore side of things, or is it a bit different?
Album Rating: 2.5youre summary blows
Album Rating: 2.5ehhhhhhhhhhhhhhhhhhhhhh
Album Rating: 2.5the cleans are yucky
Album Rating: 2.0blargh this blows
"me? I've been here the whole time. Have not reviewed in a while though."
Maybe should have stuck to not reviewing.
Album Rating: 1.5reads like a zepdude review
Not a bad review, pos.
'Auto-tune aside, Ahren Stringer can really sing', if you mean without auto-tune then why would he use it in the first place?
I have no respect for any 'singer' who uses auto-tune.
Album Rating: 3.5 | Sound OffThe producer was the reason for the auto-tune. In an interview Ahren said that the producer pushed really hard for the auto-tune, against what the band wanted to do.
Album Rating: 4.5 | Sound Off"At least I know how to write a good review." Ha ha, doesn't look like it!
The summary has nothing to do with THIS album. You also don't even mention the best song on here 'Open Letter'. Better luck next time
Album Rating: 3.5 | Sound OffMy review is 3/4 while yours is 1/6 so no argument there stud.
Album Rating: 4.5 | Sound OffI can't review for shit. Probably because I have it a 5 lol
Bands: A B C D E F G H I J K L M N O P Q R S T U V W X Y Z