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HIV protease inhibitors are important pharmacological agents used in the treatment of HIV-infected patients. One of the major disadvantages of HIV protease inhibitors is that they increase several cardiovascular risk factors, including the expression of CD36 in macrophages. The expression of CD36 in macrophages promotes the accumulation of cholesterol, the development of foam cells, and ultimately atherosclerosis. Recent studies have suggested that α-tocopherol can prevent HIV protease inhibitor-induced increases in macrophage CD36 levels. Because of the potential clinical utility of using α-tocopherol to limit some of the side effects of HIV protease inhibitors, we tested the ability of α-tocopherol to prevent ritonavir, a common HIV protease inhibitor, from inducing atherosclerosis in the LDL receptor (LDLR) null mouse model. Surprisingly, α-tocopherol did not prevent ritonavir-induced atherosclerosis. However, cotreatment with the nucleoside reverse transcriptase inhibitors (NRTIs), didanosine or D4T, did prevent ritonavir-induced atherosclerosis. Using macrophages isolated from LDLR null mice, we demonstrated that the NRTIs prevented the upregulation of CD36 and cholesterol accumulation in macrophages. Treatment of LDLR null mice with NRTIs promoted the ubiquitination and downregulation of protein kinase Cα (PKC). Previous studies demonstrated that HIV protease inhibitor activation of PKC was necessary for the upregulation of CD36. Importantly, the in vivo inhibition of PKC with chelerythrine prevented ritonavir-induced upregulation of CD36, accumulation of cholesterol, and the formation of atherosclerotic lesions. These novel mechanistic studies suggest that NRTIs may provide protection from one of the negative side effects associated with HIV protease inhibitors, namely the increase in CD36 levels and subsequent cholesterol accumulation and atherogenesis.
- macrophage
- proteasome
- low-density lipoprotein receptor
highly active antiretroviral therapy (HAART) is essential to decreasing the morbidity and mortality of HIV-infected individuals (10, 22, 27). However, prolonged use of HAART is associated with hyperlipidemia, lipodystrophy, insulin resistance, and accelerated atherosclerosis (7, 10, 11, 17, 26). Of the various classes of compounds used in HAART, the HIV protease inhibitors appear to be associated with negative cardiovascular risk factors (7, 10, 11, 17, 26). Because of the importance of HIV protease inhibitors in HAART, it is crucial to understand the mechanisms whereby HIV protease inhibitors induce cardiovascular side effects.
HIV protease inhibitors affect multiple cellular mechanisms, including inhibiting the degradation of the nuclear form of sterol regulatory element binding proteins (nSREBP) in the liver and adipose tissue (2, 15, 24). Accumulation of nSREBP in the liver increases fatty acid and cholesterol biosynthesis whereas in adipose tissue accumulation of nSREBP induces lipodystrophy, insulin resistance, and decreases leptin levels (2, 15, 24). HIV protease inhibitors also decrease the degradation of nascent apolipoprotein B by suppressing proteasome activity (16). Insulin resistance and diabetes are promoted by the ability of HIV protease inhibitors to attenuate glucose transporter GLUT4 activity in muscle and adipose tissues (12).
In addition to the above effects, Dressman et al. (5) demonstrated that HIV protease inhibitors contribute to the formation of atherosclerosis by promoting the upregulation of CD36 and the subsequent accumulation of sterol in macrophages. CD36 is a class B scavenger receptor that facilitates the uptake of modified lipoproteins into macrophages, which promotes foam cell formation and the development of atherosclerotic lesions. The use of CD36 blocking antibodies, CD36 antisense morpholino oligonucleotides, and CD36 null mice, clearly demonstrated that CD36 upregulation was necessary for HIV protease inhibitor-induced sterol accumulation in macrophages and atherosclerotic lesion formation (5). The fundamental finding that HIV protease inhibitors increase the amount of CD36 in macrophages has been confirmed by Munteanu et al. (19).
The reported mechanism for HIV protease inhibitor upregulation of CD36 involved the upregulation of peroxisome proliferator-activated receptor-γ (PPARγ) (5). PPARγ has been previously reported to be upregulated by molecules found in modified lipoproteins such as oxidized low-density lipoprotein (6, 20). Importantly, the upregulation of PPARγ by HIV protease inhibitors and modified lipoproteins leads to increased CD36 expression, which then promotes further uptake of modified lipoproteins, foam cell formation, and subsequently atherosclerotic lesions (6, 20). HIV protease inhibitors stimulated the expression of PPARγ via protein kinase C, but the entire molecular pathway has not been elucidated (5, 6). In contrast, Munteanu et al. (19), using THP-1 monocytes, suggested that the primary mechanism for increasing the amount of CD36 in macrophages is due to the inhibition of the proteasome. This conclusion was based on the use of the proteasome inhibitor N-acetyl-Leu-Leu-norleucinal (ALLN), which significantly increased CD36, whereas only a modest increase in CD36 mRNA was observed in the presence of the HIV protease inhibitor ritonavir (19). Presently it is unclear how many mechanisms contribute to HIV protease inhibitor-induced increases in the level of CD36 and the relative importance of each mechanism.
Importantly, the study by Munteanu et al. (19) detailed a finding of potentially great clinical significance, namely that α-tocopherol, in THP-1 monocytes, prevented ritonavir-induced increases in CD36 levels by preventing the inhibition of proteasome activity. If α-tocopherol prevents HIV protease inhibitor-induced increases in CD36 levels in humans, a simple and safe treatment would be available to counteract at least one of the negative side effects of HIV protease inhibitors. While the cell culture studies done by Munteanu et al. (19) are intriguing, the in vivo effects of α-tocopherol have not been determined. In the present study we tested the ability of α-tocopherol to prevent HIV protease inhibitors from increasing CD36 levels in an animal model. As a control, we used nucleoside analog reverse transcriptase inhibitors (NRTIs), which are commonly used in combination therapy with HIV protease inhibitors. To our surprise, α-tocopherol did not prevent HIV protease inhibitor-induced increases in CD36 or the development of atherosclerotic lesions. However, NRTIs completely prevented the upregulation of CD36 and the development of atherosclerosis.
RPMI medium 1640, DMEM high-glucose medium, fetal calf serum, l-glutamine, trypsin-EDTA, and penicillin-streptomycin were purchased from Life Technologies (Grand Island, NY). Percoll, polyvinylidene difluoride (PVDF) membranes, Tween 20, α-tocopherol, and chelerythrine chloride were purchased from Sigma (St. Louis, MO). Bradford reagent was purchased from Bio-Rad (Hercules, CA). The anti-PKCα IgG was from BD Biosciences (San Diego, CA), the anti-actin IgG was from Sigma, the anti-mouse CD36 (IgM) was from BioDesign International (Kennebunk, ME), the anti-SRA (scavenger receptor, type A) was from Serotec, anti-ubiquitin was from Zymed Laboratories (San Francisco, CA), and Quality Control Biochemicals (St. Louis, MO) generated the SR-BI (scavenger receptor, type B, class I) antibody as a fee for service. Horseradish peroxidase-conjugated IgGs were supplied by Cappel (West Chester, PA). Super Signal chemiluminescent substrate was purchased from Pierce (Rockford, IL). Bristol-Myers Squibb provided the didanosine and Abbott Laboratories provided the ritonavir. The mouse feed was obtained from Harlan Tekland (Madison, WI). The protein kinase C assay kit was from Calbiochem (San Diego, CA). [γ-32P]adenosine 5′-triphosphate (109 TBq/mmol) was from Perkin Elmer (Boston, MA). The ubiquitin enrichment kit was from Pierce.
Sample buffer (5×) consisted of 0.31 M Tris·HCl, pH 6.8, 2.5% (wt/vol) SDS, 50% (vol/vol) glycerol, and 0.125% (wt/vol) bromophenol blue. Tris-buffered saline (TBS) consisted of 20 mM Tris·HCl, pH 7.6, and 137 mM NaCl.
All animals were housed in the University of Kentucky animal facilities. Animals were maintained in constant temperature conditions on a 14:10-h light/dark cycle (lights on at 0400 h), and they were provided food and water ad libitum. The LDLR null mice were obtained from The Jackson Laboratory (Bar Harbor, ME). At 6 wk of age mice were placed on a chow diet containing 0, 400, or 800 mg/kg of α-tocopherol and given either vehicle control (0.01% ethanol), ritonavir (50 μg/day), didanosine (75 μg/day), or D4T (75 μg/day) in the drinking water. Where indicated, chelerythrine chloride (5 mg/kg) was injected intraperitoneally every 48 h for the duration of the study (3). This regimen of ritonavir has previously been described as producing atherosclerotic lesions in male LDLR null mice without altering plasma cholesterol levels (5). Mouse peritoneal macrophages were obtained by lavage (29).
Cholesterol and cholesteryl ester mass quantification.
Cholesterol and cholesteryl ester mass was quantified using a commercially available kit from Wako Chemicals (Richmond, VA) per manufacturer's instructions.
SDS-PAGE and immunoblotting.
Samples were concentrated by trichloroacetic acid precipitation and washed in acetone. Pellets were suspended in sample buffer that contained 1.2% (vol/vol) β-mercaptoethanol (Laemmli sample buffer) and heated at 95°C for 3 min before being loaded into gels. Proteins were separated in 12.5% SDS-polyacrylamide gels by using the method of Laemmli (13). The separated proteins were then transferred to PVDF membranes. Membranes were blocked in TBS + 5% dry milk for 1 h at room temperature. Primary antibodies were diluted in TBS + 1% dry milk and incubated with membranes for 1 h at room temperature. Membranes were then washed four times, 10 min per wash, in TBS + 1% dry milk. The secondary antibodies (all conjugated to horseradish peroxidase) were diluted 1/20,000 in TBS + 1% dry milk and incubated with membranes for 1 h at room temperature. The membranes were then washed in TBS, and the bands were visualized by chemiluminescence.
Northern blot hybridization.
Total RNA was extracted from cells using TRIzol (Invitrogen, Carlsbad, CA). RNA was quantified by spectrometry, and 20 μg of total RNA was loaded into each lane of a 5% denaturing agarose gel. Following electrophoretic separation, RNA was transferred to nylon membranes and probed for PPARγ, PKCα, and GAPDH mRNAs, as previously described (5).
Protein A-Sepharose beads were first blocked by incubation for 4 h at 4°C with a peritoneal macrophage cell lysate (200 μg/ml), plus 30 mg/ml of BSA in immunoprecipitation buffer (150 mM NaCl, 0.5% Triton X-100, 50 mM Tris·HCl, pH 8.0). Blocked beads were then used to preclear the experimental fractions that had been adjusted to 0.5% (vol/vol) Triton X-100. Precleared fractions were incubated for 2 h at 4°C with the appropriate antibody before adding blocked Protein A-Sepharose beads and incubating an additional 1 h at 4°C. The beads were collected by centrifugation, washed four times in high-salt (500 mM NaCl) immunoprecipitation buffer, and finally treated with Laemmli sample buffer. Immunoprecipitated proteins were detected by Western blotting.
Quantification of atherosclerotic lesions.
After 8 wk of treatment, plasma was collected for cholesterol, cholesteryl ester, and triglyceride determinations. The mice were then processed to quantify the surface area of atherosclerotic lesions. Atherosclerotic lesions were quantified as we have done previously (4, 5). Briefly, the aorta from the arch to the ileal bifurcation was collected, the extraneous tissue was dissected away, and the intimal surfaces were exposed by a longitudinal cut. The aortas were placed under a dissecting microscope equipped with a charge-coupled device camera attachment that captures the image directly to a computer file. Atherosclerotic lesions on the intimal aortic surface appear as bright white areas, compared with the thin and translucent aorta. Areas of intima covered by atherosclerosis were quantified with ImagePro software 6.0 (Media Cybernetics, Silver Spring, MD).
Least squares analysis of variance was used to evaluate the data with respect to sample, treatment, time, and their interactions, using the ANOVA procedure of Statistica (StatSoft, Tulsa, OK). When appropriate, samples were compared using the Tukey HSD test. Means were considered different at P < 0.01.
Effect of α-tocopherol on ritonavir-induced atherosclerosis.
Munteanu et al. (19) have reported that α-tocopherol can prevent HIV protease inhibitor-induced accumulation of cholesterol in THP-1 macrophages. These in vitro data suggested that α-tocopherol may be effective at preventing or reducing HIV protease inhibitor-induced atherosclerosis. To test whether α-tocopherol can prevent the atherogenic effect of ritonavir, 6-wk-old male LDLR null mice fed a standard chow diet were treated with ritonavir (50 μg/day), vehicle control (0.01% ethanol), α-tocopherol (400 mg/kg), or ritonavir (50 μg/day) and α-tocopherol (400 mg/kg or 800 mg/kg) in their drinking water for 8 wk, as we described previously (5). LDLR null mice were used for these studies because this mouse model develops few atherosclerotic lesions in the absence of a high-fat diet or HIV protease inhibitors, as we previously demonstrated (5). Thus the effects of ritonavir and α-tocopherol on atherosclerotic lesions can be studied independently of other factors that affect lesion formation (i.e., serum cholesterol). At the conclusion of the study, the ascending and descending aortas were removed and opened, and the areas covered by atherosclerotic lesions were quantified by image analysis (4, 5) (Fig. 1). Animals treated with vehicle or α-tocopherol did not have significant lesions, whereas animals treated with ritonavir developed significant lesions, as previously demonstrated by Dressman et al. (5). Animals treated with ritonavir and α-tocopherol developed atherosclerotic lesions to the same extent as animals treated with ritonavir only. Importantly, two doses of α-tocopherol (400 and 800 mg/kg) did not protect animals from ritonavir-induced atherosclerotic lesion formation.
Effect of NRTIs on ritonavir-induced atherosclerosis.
In HAART, patients are often treated with protease inhibitors, in combination with other anti-retroviral drugs, such as nucleoside reverse transcriptase inhibitors, to increase the efficacy of anti-retroviral activity. Since we predicted that α-tocopherol would decrease protease inhibitor-induced atherosclerotic lesion size, we also used two different NRTIs, didanosine and D4T, presumably used as negative controls that would not affect ritonavir-induced atherosclerosis. Six-week-old male LDLR null mice fed a standard chow diet were treated with didanosine (75 μg/day), D4T (75 μg/day), ritonavir (50 μg/day) and didanosine (75 μg/day), or ritonavir (50 μg/day) and D4T (75 μg/day) in the drinking water for 8 wk. Animals treated with didanosine or D4T alone did not develop significant atherosclerotic lesions (Fig. 1). Surprisingly, animals treated with ritonavir and didanosine, or ritonavir and D4T did not develop atherosclerotic lesions. To our knowledge these are the first data that indicate that NRTIs can limit the atherogenic effects of ritonavir.
NRTIs prevent an increase in macrophage cholesterol and CD36 levels.
Figure 1 clearly demonstrates that didanosine and D4T can protect mice from ritonavir-induced atherosclerosis. Thus we next investigated the mechanism whereby protection is afforded by NRTIs. We previously demonstrated that ritonavir induces the accumulation of cholesterol in macrophages and that cholesterol accumulation and atherosclerotic lesion formation were dependent on the upregulation of CD36 (5). To determine whether didanosine and D4T affected macrophage cholesterol accumulation and CD36 protein levels, peritoneal macrophages were isolated from mice treated as described in Fig. 1. Consistent with previous studies (5), ritonavir increased the cholesterol associated with macrophages (Fig. 2A) by two- to threefold. Didanosine and D4T alone did not alter cholesterol levels compared with the vehicle control. However, didanosine and D4T both inhibited ritonavir-induced increases in macrophage cholesterol. Consistent with Fig. 1, α-tocopherol did not affect macrophage cholesterol levels or prevent ritonavir-induced increases in macrophage cholesterol. Figure 2B illustrates that didanosine and D4T prevented ritonavir upregulation of CD36 in macrophages. Again, α-tocopherol was ineffective at preventing ritonavir-induced upregulation of CD36. Although it has been established that ritonavir induces the generation of atherosclerotic lesions in LDLR null mice by the upregulation of CD36 (5), other proteins may affect lesion development. To determine whether didanosine or D4T was altering other atherogenic proteins, we examined the levels of SR-BI and SRA (Fig. 2B). Importantly, none of the treatments altered the levels of SR-BI and SRA, suggesting that these proteins were not involved in the protective effects of didanosine and D4T. Figure 2B shows the actin loads from the described samples, indicating that equal protein was loaded into each well. These data suggest that the mechanism whereby didanosine and D4T prevented HIV protease inhibitor-induced atherosclerosis was by preventing the upregulation of CD36 in macrophages.
NRTIs promoted the loss of PKCα protein in macrophages.
We previously used molecular and genetic models to demonstrate that ritonavir-induced CD36 upregulation was mediated by a PKC-dependent increase in PPARγ, which subsequently increased the expression of CD36 (5). To determine whether didanosine and D4T prevented ritonavir-induced CD36 upregulation, and atherosclerotic lesion formation by a PPARγ mechanism, we isolated RNA from peritoneal macrophages obtained from LDLR null mice, treated as described for Fig. 1. Ritonavir increased PPARγ mRNA levels without altering PKCα mRNA levels (Fig. 3A). Treatment with α-tocopherol did not inhibit ritonavir-induced upregulation of PPARγ, in agreement with Figs. 1 and 2. Importantly, didanosine and D4T prevented ritonavir-induced upregulation of PPARγ mRNA. Since PKCα mRNA levels were not changed, we next examined the levels of PKCα protein. Figure 3B demonstrates that didanosine and D4T in the absence or presence of ritonavir promoted the loss of PKCα protein, whereas animals treated with α-tocopherol had normal levels of PKCα. These data demonstrated that didanosine and D4T caused the loss of PKCα, which may be responsible for the inhibition of PPARγ upregulation.
NRTIs promoted the ubiquitination of PKCα.
The data shown in Fig. 3 demonstrated that didanosine and D4T promoted the loss of PKCα protein, without affecting the level of PKCα mRNA, which suggested that a post-translational mechanism is responsible for the loss of PKCα protein. A common mechanism for promoting cytosolic protein degradation is the ubiquitination of proteins, so that they are targeted to the proteasome. To determine whether ubiquitination was involved in the loss of PKCα, we immunoprecipitated PKCα from macrophages isolated from animals treated as described in Fig. 1. Each immunoprecipitation was done with 500 μg of cell protein, whereas only 20 μg of protein was analyzed in Fig. 3, which likely accounts for the apparent lack of PKCα in Fig. 3, and the presence of a small amount of PKCα in Fig. 4. The precipitated material was then resolved by SDS-PAGE and immunoblotted by using an anti-ubiquitin antibody. The amount of cross-reactive material was quantified by image analysis, normalized to the amount of actin in the original immunoprecipitation sample and plotted in Fig. 4A. The data demonstrate that didanosine and D4T dramatically increased the amount of PKCα that has undergone ubiquitination. Importantly, the vehicle, ritonavir, and α-tocopherol samples did not affect PKCα ubiquitination. The non-NRTI-treated samples contained ∼90-fold more PKCα than the NRTI-treated samples but had 5–8-fold less ubiquitinated PKCα (Fig. 4B). These data suggest that didanosine and D4T prevented CD36 upregulation by promoting the degradation of PKCα.
In vivo inhibition of PKC prevents HIV protease inhibitor-induced increases in macrophage CD36.
Collectively, the data presented thus far suggest that didanosine and D4T protected against ritonavir-induced atherosclerosis by downregulating PKCα and consequently preventing the upregulation of CD36. To determine whether the lack of functional PKCα can prevent ritonavir-induced atherosclerosis, we treated 6-wk-old LDLR null mice with 2.5 mg/kg chelerythrine, a PKC inhibitor, along with ritonavir (50 μg/day) for 8 wk. Peritoneal macrophages were isolated from the mice at the conclusion of the study, and the extent of PKC activity was determined with a commercially available enzymatic assay system. Figure 5A demonstrates that ritonavir increased PKC activity sixfold above the vehicle control. Importantly, chelerythrine decreased basal PKC activity and ritonavir-induced PKC activity, thereby demonstrating the effectiveness of chelerythrine at inhibiting macrophage PKC. Consistent with a decrease in PKC activity, chelerythrine prevented ritonavir-induced upregulation of PPARγ (Fig. 5B) and an increase in the amount of CD36 protein (Fig. 5C). These data support the concept that PKC is necessary for ritonavir-induced increases in CD36 protein levels.
In vivo inhibition of PKCα prevents HIV protease inhibitor-induced atherosclerotic lesions.
The data suggested that didanosine and D4T inhibit ritonavir-induced atherosclerosis by causing the ubiquitination and downregulation of PKCα. The PKC inhibitor chelerythrine prevented the upregulation of PPARγ and CD36 in macrophages. To determine whether the inhibition of PKC activity protected against ritonavir-induced atherosclerosis, LDLR null mice were treated with chelerythrine as described in Fig. 5. Figure 6A demonstrates that chelerythrine prevented the accumulation of cholesterol in macrophages from mice treated with both ritonavir and chelerythrine. Dramatically, chelerythrine, like didanosine and D4T, completely inhibited ritonavir-induced atherosclerosis (Fig. 6B). These data demonstrate that NRTI-induced depletion of PKC can account for the ability of NRTIs to prevent HIV protease inhibitor-induced atherosclerosis.
The development of aspartyl endopeptidase protease inhibitors, which catalyze the cleavage of the HIV gag and gag-pol polyproteins, has been one of the most significant advances in HIV therapy (10, 22, 27). The usefulness of HIV protease inhibitors is tempered by significant side effects, including lipodystrophy, hyperlipidemia, visceral adiposity, and insulin resistance (7, 10, 11, 17, 26). The ability of HIV protease inhibitors to interfere with the function of the proteasome results in an increase in sterol regulatory element binding proteins (SREBPs) and apoB, both of which may contribute to lipodystrophy, hyperlipidemia, and cardiovascular diseases, such as atherosclerosis (7, 10, 11, 17, 26). However, a study by Dressman et al. (5) demonstrated that in mouse models, HIV protease inhibitors can promote atherosclerosis independent of changes in plasma lipid levels. The increase in atherosclerosis was shown to be dependent on the increase in macrophage CD36 protein levels (5). Studies by Munteanu et al. (18, 19) confirmed that HIV protease inhibitors increase macrophage CD36 protein levels. This observation is potentially of clinical significance because reducing the dose of protease inhibitor to lessen the increase in plasma lipids may not correspond to a decrease in risk for cardiovascular disease.
The mechanism(s) whereby HIV protease inhibitors increase CD36 is not completely clear. Dressman et al. (5) reported that HIV protease inhibitors increased PPARγ via a PKC intermediate signaling step. Treatment of macrophages with oxidized LDL has been shown to result in an increase in PPARγ activity and subsequently the expression of CD36; thus the involvement of PPARγ is a plausible mechanism (5, 6). In vitro studies with human peripheral blood monocytes demonstrated that PKC inhibitors prevented ritonavir-mediated upregulation of CD36 (5). In addition, the addition of a PPARγ agonist could overcome the PKC block, suggesting that PKC is upstream of PPARγ (5). In contrast, Munteanu et al. (18, 19) reported that the level of PPARγ mRNA in the monocyte cell line THP-1 was not affected by therapeutic doses of ritonavir. Furthermore, they provided data (18, 19) that inhibition of the proteasome with ALLN increased CD36 levels, and they suggested that ritonavir-induced increases in CD36 are caused by decreased proteasome degradation of CD36. Since different cells and different concentrations of ritonavir were used in the two studies, it is difficult to directly compare the data. However, at the present time both mechanisms are possible, and in fact it is conceivable that both mechanisms may be in part responsible for the increase in macrophage CD36.
Studies by Munteanu et al. (19) also indicated that relatively high levels of α-tocopherol (50 μM) could prevent ritonavir-induced increases in CD36 in THP-1 cells. The authors provided data demonstrating that α-tocopherol could partially relieve the ritonavir-mediated inhibition of the proteasome. Although α-tocopherol has been shown to be ineffective in preventing cardiovascular disease (28), it is entirely possible that α-tocopherol could prevent HIV protease inhibitor-induced atherosclerosis. Consequently, we tested this exciting possibility in our mouse model. To our surprise and disappointment, α-tocopherol, even at a very high concentration, did not prevent ritonavir-mediated atherosclerosis or increases in macrophage CD36 levels. Despite convincing data with THP-1 cells (19), in vivo studies with a mouse model could not reproduce the effect of α-tocopherol on macrophage CD36 protein levels. However, we made the unexpected observation that the NRTIs didanosine and D4T did prevent ritonavir-induced atherosclerosis.
NRTIs are incorporated into the viral genome during replication but are not efficiently removed because the viral reverse transcriptase does not have a proofreading exonuclease activity. NRTIs are a critical component of HAART; however, toxicity has been observed, related to inhibition of mitochondrial DNA replication (14) and fat redistribution syndrome (21). In the current study, treating cells with ritonavir and didanosine, or ritonavir and D4T, presumably was to serve as a negative control for the putative effects of α-tocopherol. Stunningly, the NRTIs prevented ritonavir-induced increases in macrophage CD36 levels and ritonavir-induced atherosclerosis formation. Consistent with Dressman et al. (5), ritonavir induced an increase in PPARγ expression and the NRTIs prevented this ritonavir-induced increase. The NRTIs did not affect the level of PKCα mRNA; however, the NRTIs promoted the loss of PKCα protein in the presence or absence of ritonavir. By using anti-ubiquitin antibodies and immunoprecipitation, we demonstrated that the NRTIs promoted the ubiquitination of PKCα, which led to degradation of the protein by the proteasome. The mechanism by which NRTIs play a role in the ubiquitination of PKCα is not known.
Previous studies by Gogu et al. (8, 9) demonstrated that zidovudine, the first drug in the 2′,3′-dideoxynucleoside class approved by the FDA for treatment of AIDS, prevented erythroid progenitor cell differentiation by inhibiting PKC. Although these investigators did not demonstrate the mechanism of PKC inhibition, their findings are consistent with our didanosine data demonstrating the degradation of PKC. Interestingly, Gogu et al. (9) also demonstrated that α-tocopherol could overcome the inhibitory effect of zidovudine on erythroid progenitor cell differentiation. Although it was not demonstrated, it is possible that α-tocopherol prevented the degradation of PKC. If α-tocopherol prevented the degradation of PKC, based upon our current data, one would predict that animals receiving ritonavir, didanosine, and α-tocopherol would develop atherosclerosis, because didanosine no longer caused the degradation of PKC. The interactions between HIV protease inhibitors, NRTIs, and α-tocopherol are complex and still incompletely understood.
The current study along with the previous study by Dressman et al. (5) strongly points to PKC as being a major regulatory point in mediating the effects of protease inhibitors and NRTIs on the expression of macrophage CD36. To further explore the role of PKC we treated mice with chelerythrine, a PKC inhibitor, in the presence or absence of ritonavir. Since chelerythrine was only given to intact mice, and the peritoneal macrophages were isolated, and were immediately assayed for PKC activity, this indicates that chelerythrine was effective at inhibiting PKC activity in vivo. Chelerythrine was effective at inhibiting PKC phosphorylation activity, inhibiting PPARγ upregulation, and inhibiting increases in CD36 protein levels. Importantly, chelerythrine prevented ritonavir-induced macrophage cholesterol accumulation and ritonavir-induced atherosclerotic lesion formation. It is important to point out that the ability of chelerythrine to prevent atherosclerosis is most likely limited to ritonavir-induced atherosclerosis and not atherosclerosis caused by the other multitude of risk factors.
Although we were unable to confirm the protective effect of α-tocopherol, with regards to ritonavir-induced increases in macrophage CD36, with our in vivo studies, we did discover that NRTIs can prevent an increase in macrophage CD36 levels. Interestingly, NRTIs increased the ubiquitination of PKC, which resulted in the degradation of PKC, and consequently prevented the upregulation of PPARγ, and the increase in macrophage CD36. This mechanism was surprising because it has been suggested that HIV protease inhibitor-related increases in CD36 are due to inhibition of the proteasome (18, 19). However, HIV protease inhibitor effects on the proteasome are complicated. For instance, Schmidtke et al. (25) has demonstrated that ritonavir inhibited the chymotrypsin-like activity of the proteasome but increased the tryptic activity of the proteasome. This finding offers one explanation as to why NRTIs could induce the degradation of PKC, in the presence of HIV protease inhibitors. Alternatively, Piccinini et al. (23) demonstrated that the use of a single protease inhibitor (as we did in the current studies) only had a minimal impact on proteasome activity. In fact, Piccinini et al. (23) showed that the use of three HIV protease inhibitors in combination only caused ∼43% inhibition of the proteasome. Therefore, another possible explanation for our results is that the concentration of ritonavir used in our studies did not sufficiently inhibit the proteasome to affect the degradation of PKC. Finally, Andre et al. (1) have demonstrated that concentrations of ritonavir that do not affect proteasome function can inhibit the presentation of antigen to cytotoxic T lymphocytes; thus it is possible that HIV protease inhibitors and NRTIs impact PKC independent of the proteasome.
The mechanism whereby NRTIs promote the ubiquitination of PKC is unknown. Our current working hypothesis is that HIV protease inhibitors and NRTIs both impact on PKC, and since PKC is upstream of PPARγ, the ability of PPARγ to cause an increase in macrophage CD36 protein levels is increased by HIV protease inhibitors and decreased by NRTIs. This hypothesis does not exclude the contribution of other possible mechanisms nor should it be extended to other non-HIV drug mechanisms for affecting macrophage CD36 protein levels and atherosclerosis.
This work was supported, in part, by National Institutes of Health Grants HL-68509 (to E. J. Smart), DK-63025 (to E. J. Smart), HL-073693 (to M. E. Wilson), and RR-O15592.
We thank the Kentucky Pediatric Research Institute work group for providing invaluable advice and assistance.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
- Copyright © 2006 the American Physiological Society


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Darkest Hour
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subset: commoncrawl


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FRANKFURT, Sept 22 (Reuters) - Profit and sales at German tyremaker Continental AG in 2005 will not be below their level this year, chief executive Manfred Wennemer said at a trade fair in the German city of Hanover. "2005 is still far away. But you can assume that 2005 will certainly not be worse than 2004," he said, adding that this applied to both profit and sales. Earlier the company said it would beat last year's operating profit in 2004.
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Dog Park Alliance Holding Shreveport's First-Ever Doggie Speed-Dating Event and Pet Wedding
A new and unusual fundraiser is coming up this weekend for the Shreveport Dog Park Alliance. The group's leader Cynthia Keith says it's a huge pet wedding. She says the group is always trying to think outside the box on these fundraising ideas.


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Property listings have increased sharply throughout the spring selling season, in what is good news for buyers.
According to RP Data's latest property pulse, the current volume of stock on the market is now higher than during the depths of the global financial crisis.
Over the past five years, spring has been the season to record the greatest number of listings while autumn tends to be the busiest season for property sales.
RP Data's research analyst Cameron Kusher said during spring 2010, the number of properties available for sale rose sharply at a time when housing finance volumes trended lower since September of last year, and when property value growth has been flat since June of this year.
For the week ending August 2010, the total number of listings nationally has increased by 16.5 per cent. Across the capital city markets, listings have climbed 21.9 per cent higher.
"Currently 75,655 properties are advertised for sale nationally which is the highest volume since RP Data began tracking listing volumes in 2007," Mr Kusher said.


text: Dr. Otto Scharmer is a Senior Lecturer at MIT, Boston, a Thousand Talents Program Professor at Tsinghua University, Beijing, and co-founder of the Presencing Institute. He chairs the MIT IDEAS program for cross-sector innovation that helps leaders from business, government, civil society to innovate at the level of the whole system. He is author of Theory U and co-author of Presence and Leading from the Emerging Future. In 2015 he co-founded the MITx u.lab, a massive open online course for leading profound change that has since activated a global platform and eco-system of societal and personal renewal involving more than 100,000 users from 185 countries. With his colleagues he delivered award winning programs for companies including Alibaba, Daimler, Eileen Fisher and Google and co-facilitated innovation labs on reinventing education, health, business, government, and well-being. Otto received the Jamieson Prize for Excellence in Teaching at MIT (2015) and the EU Leonardo Corporate Learning Award for the contributions of Theory U to the future of management and learning (2016). For more detail: ottoscharmer.com


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EAPA News
IARC issues Health Classification for Occupational Exposures during Road Paving
The International Agency for Research on Cancer (IARC), based in Lyon, France, issued a statement on 20 October assigning occupational exposures to straight-run bitumens and their emissions during road paving a Group 2B ("possibly carcinogenic to humans") classification. This Group 2B classification puts road paving in the same category as numerous exposures, including cell phones and coffee.
EAPA, with its colleagues in North America, the National Asphalt Paving Association (NAPA), and in the bitumen industries on both sides of the Atlantic, underwrote a series of studies that were designed and led by IARC over the last ten years or so. None of these studies reported any direct link between bitumens used in road paving and the onset of cancer in the workforce. However, with health and safety being the highest priority for the industry and its employees, EAPA will continue to work with all its stakeholders towards reducing emissions of all sorts, so as to minimise any possible irritation or other impacts from them.
IARC, which brings together international panels of scientists, is based in Lyon, France. Its assessments go to national health agencies for possible guidance. IARC has reviewed more than 900 chemicals and other agents since its founding in 1971. IARC categorizes chemicals, agents, mixtures and exposures.
To put this classification into proper context, it is worth quoting Dr. Jim Melius, PHD, MD, the top occupational physician at the Laborers International Union of North America (LIUNA): "People working in the asphalt paving industry should not be concerned about this new IARC classification. The two key animal studies on paving asphalt did not show any evidence of cancer risk, and the major IARC cancer study of people working in the paving industry in Europe did not show any increased risk for cancer."
The Asphalt Paving Industry: A Global Perspective
NAPA and EAPA have published the Second Edition of The Asphalt Paving Industry: A Global Perspective.
This joint publication gives the reader an overview of the world's asphalt pavement industry. It provides a description of the industry, its scope, and its economic impact. The document includes chemical and physical data about asphalt pavement material, describes asphalt mixing plants and explains how transport and placement of asphalt mixes is accomplished. In addition, it discusses bitumen fume exposure and measures that have been taken to reduce exposure including engineering controls and warm-mix asphalt.
Manifesto of the European Construction Forum launched in the European Parliament
The press release of the event can be found here
If you want to get further information on the manifesto, the ECF in general and EAPA's involvement please go to www.ecf.be
EAPA's Gulay Malkoc spoke at the NorBit Seminar 2010
In October, a yearly binder seminar was held in Oslo, Norway, organized by the Norwegian Bitumen Association (NorBit). NorBit is a non-profit association, which was established in August 2009 and continues the work of the NaBin, which is no longer active in Norway.
The purpose of NorBit is to spread information and knowledge on bituminous binders through an annual one-day seminar by inviting international lecturers from Europe and USA in combination with other Nordic people.
The NorBit seminar in 2009 focused on moisture susceptibility of asphalt mixes and safety in bitumen storage and handling. However, this year the main topic was Polymer Modified binders (PmB's).
The seminar was moderated by Roar Telle, who is a member of the Technical Committee of the Norwegian Asphalt and Road Contracting Association (FAV), which is a member of EAPA .
Gulay Malkoc from EAPA was invited to give a presentation on the HSE aspects of PMB's and CRM binders. Gulay also took the opportunity to give a short introduction to EAPA's activities and presented the upcoming E&E Congress 2012 in Istanbul and invited the participants to attend the congress.
MPA publishes Asphalt Application Documents
The UK EAPA Member MPA published several documents on different asphalt applications.
You can find them under Publications > Member Publication.


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The Message New Testament with Psalms and Proverbs : The New Testament in Contemporary Language
This New Testament is different from any other New Testament on the market. Written in the rhythms of today's language, The Message New Testament with Psalms and Proverbs brings out the Scripture's expressive flavor that will bring a unique freshness whether you are reading it for the first time or have been studying it for years.
Publisher: The Navigators
608Pages / Publication Date: 20160701
Subject: BIB020030-BIBLES / The Message / New Testament & Portions,BIB020060-BIBLES / The Message / Text
$9.97 $13.99
For those of you have been familiar with Crossroads for some time, you'll know that Crossroads' Founder David Mainse is an epic storyteller. This Far By Faith, features over 70...
Prodigals come in many forms. We often think of the defiant ones as prodigals, but there are other types that break our hearts: embarrassing, intellectual, lukewarm, and religious prodigals. The...
Why is forgiveness so hard? People who refuse to forgive often sabotage their future and create an emotional cancer that spreads into every other aspect of their lives. Even those...
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Journey into Joy-Now you can enjoy reading these practical and inspiring insights from Jesus' famous Sermon on the Mount as recorded in Matthew 5-7. Based on the original transcripts of the Journey Into Joy...


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Photo Upload Print - Heart Frames, 5 Photos
A thoughtful gift with a lot of heart...
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Product Description
We all have photographs we love, don't we? From the snapshot of you walking down the aisle to your honeymoon in the Caribbean - it's no wonder that you want to display them with pride around your love nest. Yet when you can't decide on which snap to frame, your mantlepiece soon fills up and starts looking like a hall of frame, sorry, fame!
Our photo upload print is here to solve any future cluttered mantlepiece problems! With a charming white decking-effect background, the print features five photos in a variety of square, rectangular and heart-shaped frames, finished with a red love heart and image of a wax letter seal with a heart. You can add five of your favourite photos to be professionally printed and choose your frame colour, size and mount options to make it extra special.
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Read full description
Customer Reviews (1)
100% would recommend this to a friend
Customer Reviews (1)
100% would recommend this to a friend
Photo Upload Print - Heart Frames, 5 Photos
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The Purple One will always be full of magic. Prince has two new LPs on the way, and the posthumous releases promise unreleased material. If you want something you can listen to now, stick around. Trent Reznor said he's working on Nine Inch Nails music, Cher did a duet with James Corden, Red Bull Music Academy has a new video game series with musicians, and Kim Gordon returns with Body/Head for a new live album.
The first posthumous release by Prince is on the way, and according to Prince's estate, it includes never before heard music. Two albums are on their way, and both have new songs. The first is a 40-song greatest hits compilation called Prince 4Ever. The LP comes out on November 22. Lesser-known favorites like "Why You Wanna Treat Me So Bad" will be next to official releases of "Moonbeam Levels" and other songs that never got an official release. The second, a deluxe reissue of Purple Rain, comes out in early 2017. The plans for this one were approved by Prince before he passed away, including a second album of previously unreleased material. What that is has yet to be disclosed. Stay tuned here though for more information regarding The Purple One. [Rolling Stone]
Trent Reznor and Atticus Ross just finished a score for Leonardo DiCaprio's climate change documentary, Before the Flood. Turns out that wasn't the only thing they were working on. The musicians went on Zane Lowe Beats 1 radio for an interview and, when there, revealed they've been working on Nine Inch Nails material, too. "Things are going good and when we have something that we think is excellent, we will unleash it on the world," said Reznor. To that, we say it's worth the wait. [Rolling Stone]
James Corden's TV show gets celebrities to indulge in fun activities, and it makes you wish you were friends with them in the process. His newest episode didn't let up on that. Corden spent his October 20 show singing a duet with the one and only Cher. The song? A spin on her classic 1965 hit "I Got You Babe" (remembered fondly in Groundhog's Day), only now reworked to be "I Got You Bae." It's goofy, touching on everything from Netflix and chill to Facebook-official relationships. If you need a laugh, this video is for you. [Rolling Stone]
Red Bull Music Academy has a new series called Diggin' In the Carts. The show explores video game music past and present with host Nick Dwyer, a writer and co-director of the Red Bull Music Academy documentary video series. The first season zones in on Japanese video game music to be exact. Expect 8-bit goodness, 16-bit revolutions, and arcade songs, all of which include a rich history about tones, melodies, and forms of appreciation. To expand on that, modern musicians like Thundercat come by to talk about what these soundtracks mean to them. It's a big series that we're already heart-eyed over. Episodes air every Thursday at 7pm EST. Head here to learn more. [Red Bull Music Academy]
Sonic Youth may be dead, but that band's spirit lives on through Kim Gordon. She and Bill Nace create music together under the moniker Body/Head. They announced a new live album is on the way, and the record, called No Waves, sounds like the gut-busting rock we want to keep living in now that Sonic Youth is no more. They recorded it at Big Ears Festival in 2014. Surprisingly, the LP only has three tracks, but it clocks in around 40 minutes, drawing on experimental improvisation and drone-like work to offer fans a taste of their live shows. The whole thing comes out November 11 via Matador. Check out the album art above. [Consequence of Sound]
See you back here on Monday for another Music Dispatch!
Image: NPG Records/Warner Bros


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Today is Memorial Day.
A day to remember and honor those who have fallen for our sake. To protect our freedoms. To protect our kids' freedoms.
Our freedoms to say what we want when we want in whatever way we want. Our freedoms to bear arms if we so choose. Our freedoms to print what we want. Our freedoms to live the American Dream. To work hard and choose our own destiny - not being beholden to what someone else chooses for us.
My husband's grandpas both fought in WWII. He is a WWII historian who is writing several books on the subject. One of them is a compilation of interviews that he did with over 500 WWII vets, telling their stories. Let me tell you: the hell that they went through - for our sake - is unbelievable. And that's just WWII. I can only imagine what soldiers in Korea, Vietnam, Iraq, Afghanistan, and others have gone through too.
I encourage you to take some time today and remember and honor those who have fallen for our sake. Also? Thank a soldier/veteran. Not just today, but any time you see one. They put everything on the line for us.
If you are a soldier or military veteran, or a family who stayed behind while your loved ones went away to fight for me, I thank you from the bottom of my heart for your service. My family honors you today and every day.


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Hatch Projects are 5-year research plans with measurable goals and impacts designed to contribute to the broad research mission of the USDA-NIFA. These peer-reviewed projects allow the Nebraska Agricultural Experiment Station (ARD-Agricultural Research Division) to support faculty salaries with federal funds, which in turn free up state resources to be used for operating, travel, personnel, and graduate research assistantship support across the Institute of Agricultural and Natural Resources. Prior to developing a Hatch project, PIs/Co-PIs should engage with their unit administrator(s) and colleagues in research planning. We strongly recommend that faculty develop collaborative team-based Hatch Projects that have achievable goals; units must develop policies that allow funds provided by ARD (GRAs, staff-lines, operating and travel) to support approved Hatch projects.
Note: There is also a competitive internal-grant program associated with Hatch-Multistate projects. Hatch-Regular and Hatch-Multistate projects serve different purposes and are programmatically managed differently.
Who needs a Hatch Project?
ARD Tenure-Track faculty MUST have Hatch project
• If faculty research aligns with mission of USDA
• Not for faculty with biomedical research interests
Non-Tenure Track faculty with an ARD appointment are not required to have a Hatch project but are not prevented from leading or collaborating on a Hatch project.
ARD Hatch Resources
The resources below provide general guidance for the development and review of Hatch projects.
- Individual Hatch Project Review Process (Process/Timeline for Creating a Hatch Project)
- Team Hatch Project Review Process (Process/Timeline for Creating Team Hatch Project)
- Hatch Peer Review Form (Reviewer Form)
- Hatch Project Outline Format (Proposal Outline)
- Hatch Project Resources Form (Financial Support Form)
- Hatch Project Approvals Form (Departmental Signatures)
- Team Hatch Participation Agreement (Team Accountability Form - Optional/Internal Use Only)


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GraphicRiver Yachtsman 4220976
Category: Stock Vector | Comment: ( 0 )
GraphicRiver Yachtsman 4220976
Stock Vector \ Characters \ People
Palm \ Race \ Ocean \ Movement \ Navigation \ Recreation \ Regatta \ Sailor \ Sailing
Stock Vector - Yachtsman 4220976 by Ellf @ GraphicRiver
Stock Vector \ Characters \ People
Graphics Files Included: JPG Image, Vector EPS; Layered: No; Minimum Adobe CS Version: CS.
Illustration with happy people. Handwritten text in the works
Office Worker. Illustration.; Tennis Player; Toothache. Illustration.; Skier. Illustration.; Trumpeter; Toothache. Illustration.; Smoker. Illustration..
Keywords: active, activity, adventure, and, background, boat, captain, colors, expression, fish, fun, gulls, hobbies, holidays, island, leisure, movement, navigation, ocean, of, palm, race, recreation, regatta, sail, sailboat, sailcloth, sailing, sailor, sea.
Download Link


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Scuba and I Are Awesome
Posted: Sun, Jul 10, 2011
Location: Savanna Bay, Virgin Gorda
After waking up on the blue lagoon, we ate breakfast. It was cereal and it got everywhere so Lulu and Chloe had a lot of work to do. After breakfast Nick, Matt, and I were jumping off the boat whilst everyone else either relaxed or was waterskiing. We took many photos. After this a dinghy came and got us and we went windsurfing. It was ok and we ended up mucking around in the water with our instructor Nick. We had an ordinary lunch, after which we went to drive dinghies. We also learnt how to tie a clove hitch. At about 3 we went scuba diving. In my group was Reyes, Fede, Pietro, and Matt. This was my first day of scuba diving. Tor (my other instructor) waited on the beach for us. Our chefs Blake and Allan cooked rice and chilli for dinner. There was a little bit of singing. We went to bed on the blue lagoon.


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Selecting the characteristics of a powered support in an inclined deposit and in a longwall with a large opening
Supports have developed according to the aperture and slope of the longwall concerned. Increases in load-bearing have generally been in response to problems resulting from the lie of the land. The study was carried out in two ways: an analytical approach which was as advanced as possible allowing for the available means and conditions at the bottom and an overall study using appropriate statistical methods. The latter confirmed the previous results but did not provide any new ones. On the other hand the analytical approach, applied at high performance working points, made it possible to specify the properties of the face by reference to the mechanics of the stratified deposits. Old results can be re-interpreted in the light of the new, and rules can be put forward for selecting the support according to the nature of the hanging roof. Two situations may be encountered: a) It is technically possible to envisage a load-bearing capacity which would prevent the roof from caving in at the face and would deposit it behind the support; under these conditions the required support can be calculated exactly. b) It is impossible to avoid crevasses in the face, and the support must prop up the blocks formed. Conventional estimating methods are used in this case.
Additional information
Bibliographic Reference: EUR 12314 FR (1989) MF, 99 pp., ECU 8, blow-up copy ECU 12.50
Availability: (2)


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Doll Talking Spanish Teacher Barbie back to school toy
Sold for:
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- Sold Date:
- Source: eBay
Barbie Talking Spanish Teacher is a Toys R Us exclusive! Press the button on the back of Barbie doll to hear over 60 words and phrases in Spanish and English!Oprime el boton en el dorso de la muneca Barbie par oir mas de 60 palabras y frases en ingles y espanol. When you speak two languages, you can make twice as many friends! That's why Barbie loves to speak in English and Spanish. Barbie is new and never removed from her box. Winning bidder to pay $12.00 shipping in U.S., international to be determined. Good Luck. Check out my other items ! After purchasing,


text: I work in a restaurant in the east village and we always get out around 130 and are normally wanting to grab some food. We normally grab a slice of pizza or an occasional stop at San Loco, but those options are starating to wear on us. Any suggestions where we can get some good food a little bit later on.


text: Had the Droid a week, love it so far, although I am still getting used to trying to hold it without touching things accidentally. I really like the fact that my employer insisted I get one and is paying for it. I am already missing the Memo and ToDo features from the Palm. I used them heavily and had them all categorized. I am sure there is something similar out there for Android but I have not found it yet. It would be nice if the ToDo's synched with the Task list on my Gmail account, so I could access them when I am out of service. I played with the Car navigation feature and found it worked well. Google Sky is way cool. Are there any apps out there that can measure you car's 0 to 60 time for you, using the onboard GPS? James


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Models For Intermediate Ring
Intermediate Ring by Indesit
£13.43 Discontinued
Normally £13.74
Email me when back in stock - we'll try to re-stock quickly!
The product Intermediate Ring is suitable for the models listed below.
View full product details for Intermediate Ring.
Search our range of models by entering your model number to perform a search.


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Stock Graphic >> Illustration - Vector Hand Drawn Floral Brushes 326802 by Alps View Art @ CreativeMarket
File Size: 2.29 MB; Vector: Yes.
Hi there,
this product was created to help designers & illustrator fans create wonderful design in a second. It's perfect for creating all kinds of wedding design (invitations, save the date cards, thank you cards, etc), scrapbook elements, postcards, personal cards or company logo. You get some wreaths and laurels, decorative elements, flowers, flower bundles, ribbons and floral brushes. All of these brushes can be applied to any stroke in Adobe Illustator. Please note - corner segments are not included, so this brushes would better fit curved paths, circles or straight lines.
All the elements were hand drawn, scanned and carefully re-drawn into vectors.
You get.
- 34 ribbons, floral and decorative elements in two .eps10 files
- 1 seamless pattern in one .eps10 file
- 10 Handdrawn Illustrator Pattern Brushes in one .ai file
Not sure about how to install and use illustrator brushes? It's easy!
1 Unzip Brushes
2 Go to Window - Brush Libraries - Other Library and locate brushes
3 That's all! Enjoy :)
Please Note: in order to use this brushes you need Adobe Illustrator
These brushes work on all versions of Illustrator. If you experience any problems with loading & using brushes, please contact me.
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Vector Hand Drawn Floral Brushes
by Alps View Art
Keywords: floral, flower, brush, vector, design, marriage, wedding, wreath, element, ribbon, laurel, pastel, card, template, logotype, logo, incitation, save the date, heart, love.


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EXCELLENT Form Plugin
This form plugin is GREAT! I have used other paid and free form plugins and have primarily used CF7 for most of my form needs but I'm thinking that Ninja Forms might be taking over that a little. I just starting using this form plugin but from initial results it is a GREAT plugin. It works very well and doesn't have many problems, like some of the other form plugins do.
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Ninja Forms was just added to my top ten plugins for every site.
- The topic 'EXCELLENT Form Plugin' is closed to new replies.


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Papagaio (Djambi) Trailer (2014)
01 January 2014 43 mins
A major new 16mm film work Papagaio (Djambi) 2014, shot in São Tomé and Príncipe (a Portuguese speaking Island nation off the western coast of Central Africa), bears witness to a West African voodoo ritual, known locally as D'Jambi. Whilst intoxicated, the participants dance and enter a state of trance in which they channel the spirits of the dead. At times the footage is shot by the artists, and at other moments the camera becomes an alibi, held and manoeuvred by one of the participants.
Watch the official Papagaio (Djambi) 2014 trailer in HD below or find more Papagaio (Djambi) videos on Vidimovie.


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Hi! My name is Ryan Eidson.
Friends and clients have told me that I make complex ideas easy to understand, that I'm a patient teacher, and that I get things done fast.
My portfolio of experience includes:
- Author of fiction and nonfiction works such as the book, A Couple with Common Cents, and short stories
- Co-manager of a farm in East Asia
- Public speaker to audiences of 20 to 2,000
- Teacher to children, teens, and adults in summer camp and classroom settings
- Founder of a newspaper, Macon The News
I have written on a diverse range of topics, from educational issues in a Midwestern school, to daily living in rural Asia. A few of my past adventures include direct sales, pig farmer, and alto saxophone player in high school jazz band.
The following media have featured me and my work:
- Beyond Evangelical blog by Frank Viola
- Horizons magazine
- The 48 Days podcast by Dan Miller
- "This Week in the Valley" TV program
- and more (click to view the full list…)
I sum it up this way: I'm a creative writer, systems thinker, and flexible planner.
Originally from a small family farm in Illinois, I learned responsibility at an early age as I took care of cattle, pigs, chickens, and other animals.
I currently live with my wife, Lori, in rural Missouri. My last name is pronounced ED⋅son. (I'm not related to Thomas Alva Edison, as far as I know.)
Need to contact me? Click here.


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FREE SHIPPING on orders of $85 or more
An excerpt from www.HouseOfNames.com archives copyright © 2000 - 2017
Origins Available: Dutch, German
Vorderstrasse Early Origins
Vorderstrasse Spelling Variations
Vorderstrasse Spelling Variations
Spelling variations of this family name include: Vorden, Forden, van Vorden, Voorduin, Van Doorn, Van Forden, Van Doorne, Van Foorden, Doorn, Doorne and many more.
Vorderstrasse Early History
Vorderstrasse Early History
This web page shows only a small excerpt of our Vorderstrasse research. Another 319 words (23 lines of text) covering the years 1829 and 1880 are included under the topic Early Vorderstrasse History in all our PDF Extended History products and printed products wherever possible.
Vorderstrasse Early Notables (pre 1700)
Vorderstrasse Early Notables (pre 1700)
More information is included under the topic Early Vorderstrasse Notables in all our PDF Extended History products and printed products wherever possible.
The Great Migration
The Great Migration
Some of the first settlers of this family name were:
Vorderstrasse Settlers in United States in the 19th Century
Vorderstrasse Family Crest Products
Vorderstrasse Family Crest Products
See Also
See Also
Other References
The Vorderstrasse Family Crest was acquired from the Houseofnames.com archives. The Vorderstrasse Family Crest was drawn according to heraldic standards based on published blazons. We generally include the oldest published family crest once associated with each surname.
This page was last modified on 16 March 2010 at 08:17.
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November 3rd, 2005
Thanksgiving Poem, which later became the song Over the River and Through the Woods, plus how the writer of the poem was a real-life heroine When November comes, life becomes a whirlwind of activity. In the middle of it all, we start thinking about what we have to be thankful for. I'm thankful we have […]
Please contribute a traditional song or rhyme from your country.
receive a free copy of Mama Lisa's Christmas Around The World when you purchase our best selling collection Kid Songs Around The World.
People everywhere love Christmas - but we don't all celebrate it the same way. Mama Lisa's Christmas Around The World is a celebration of the diversity and love with which many different cultures mark this joyful time of year.You'll read about traditions and songs from many different nations. The songs are given in their original languages and with English translations.What makes the book really special is the many comments from Mama Lisa's correspondents who have shared stories and memories from their own lives. The memories of childhood touch us forever!
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In Kid Songs Around The World we've gathered 100 of our favorite songs and rhymes from all the continents of the globe.
Each song includes the full text in the original language, with an English translation, and most include sheet music. All include links to web pages where you can listen to recordings, hear the tune or watch a video performance. Each includes a beautiful illustration.
Many have commentary sent to us by our correspondents who write about the history of the songs and what they meant in their lives.
Christmas Around The World and Kid Songs Around The World are downloadable e-books, which you will gain access to immediately. (They are not physical books.)
We hope these books will help foster a love of international children's songs!
$3.99 for Both Downloadable E-Books Order Here!


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Karen Soroca
Marketing Consultant
Karen has more than fifteen years of experience in developing marketing and public relations strategies and implementing campaigns for technology and healthcare companies.
Before joining nToggle, Karen led the marketing rollout of the community hospital program for UpToDate, a leading global provider of information for healthcare professionals and students. Prior to UpToDate, Karen was a senior account executive at Collaborative Insight, where she was responsible for implementing marketing and public relations programs for a broad range of B2B and B2C entrepreneurs in the technology, education and energy sectors.
Karen graduated with a MS in Public Relations from Boston University and a BA in Journalism and Political Science from Temple University.


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Saturday, Nov. 7, 2010
By George M. Wilcox
Palatine's last scoring play Saturday is called "midget." The trick play was designed specially for 5-foot-6, 154-pound running back Terry Halloran.
"I'm the smallest guy on the team," said Halloran, who did not seem to mind the lack of political correctness behind the play's name.
But for Halloran, the trick play resembling a fumblerooski turned into an unlikely trip to the end zone. It also placed the final dagger into a Glenbrook South defense, which gave up 28 second-half points in a 42-14 loss Saturday in the second round of the Class 8A playoffs in Glenview. The matchup featured the state playoffs' only game between four-loss teams.
On fourth-and-1 at the GBS 31, 14th-seeded Palatine packed all of its lineman at the line of scrimmage. Halloran took a handoff to his left and pulled out of the grasp of linebacker John Heles to rumble 31 yards for a touchdown on just his second carry of the game.
Glenbrook South (6-5), a No. 11 seed, scored twice in the second quarter to tie the game at 14 at halftime, but Palatine quarterback Cody Bobbit (28 carries, 199 yards) scored two of his three rushing touchdowns in the third quarter to lead the Pirates (7-4). Bobbit suffered a leg cramp on the first play of the fourth quarter and did not return.
"We went back to running the ball," said Bobbit, who completed his only second-half pass attempt of seven yards. "That's our bread and butter."
Running back Jimmy Smearman (19 carries, 91 yards) played quarterback the rest of the game and scored on an 18-yard run early in the fourth quarter. Halloran's trick play ended an eight-play scoring drive with 3:51 remaining in the fourth.
"I was jumping for joy (before the play)," Halloran said. "I had to settle down for us to make sure where the defense was lined up. I had a lot of energy."
Palatine will host 24th-seeded and crosstown rival Fremd (6-4) or second-seeded Loyola (9-1) in the quarterfinals. Fremd defeated Palatine 17-16 on Oct. 8. The Pirates advanced to the quarterfinals for the first time since 1997 and for the first time under coach Tyler Donnelly, who is in his sixth season.
"We said, 'Do you want to play next week or not?'" Donnelly said at halftime. "'Do you want to let this team beat you?' It was the seniors and we let No. 11 (Bobbit) take over."
GBS, which was hoping to make back-to-back quarterfinal appearances, had only 28 total yards and three first downs in the second half. Senior Matt Jenkins (4 of 15, 27 yards) threw two interceptions. The first turned into a 38-yard interception return for a touchdown by Palatine linebacker Lucas Rago to put the Pirates ahead 14-0. Bobbit ended the game's first possession with a 2-yard TD run to cap an 11-play, 78-yard drive.
"It was a tough act to follow after (quarterback) Mike Pullano last year," Jenkins said. "We had a hell of a year. I'm proud of us. We had a chance to go to the quarterfinals. Palatine played better than us."
Senior John Strickland (17 carries, 114 yards) scored both of the Titans' touchdowns on runs of 7 and 42 yards. He juked past three or four tacklers and then bowled over safety Dan Haze into the end zone to score his final TD with four seconds remaining before the half.
"He loves to play football," GBS coach Mike Noll said of Strickland. "He'll be a good college football player. I think he'll play defense. For us, he did a lot for us. Jeepers."


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Jaber Albakr
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We respect this country and its people, its government and its laws. We don't want something like this to happen here.feedback
Oct 12 2016 Refugees
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Almost exactly a year ago we were confronted with the realization that the way we defined ourselves as a church was outdated and failing. Using every relevant measure we were an institution in decline. Membership, worship attendance, finances, all in a kind of free fall. We knew that if we held to the status quo, we were not going to make it.
And so, step by step, we have begun to abandon old categories that used to define what it means to be a church. Even the word "church" may not be adequate to what is happening to us. Step by step we are letting go of twentieth century notions of what a church is and does. Whatever those images are for you, let them go, because God's is moving in our midst, challenging customs and expectations.
The first thing you did (for better or worse) was to let me and Laurie step in as volunteer coordinating pastors. Do you know that there is no such thing in the United Methodist Church? Together, we made that up. You may not realize it, but every week we are subverting the very notion of a twentieth century paid, professional, pastoral ministry. Nothing in the recent history of Methodism, at least in America, prepares us for this. This is just all new.
Next you did away with the formal committees and structures outlined by the Methodist rule books. We just made up new ways to organize ourselves. The Bishop looked over at what we are doing and said, well … go for it. See what you can accomplish as the people of God.
And so we are. But we are not and cannot be done.
In the days and weeks ahead your coordinating committee will present new ways to bring ministry into our neighborhood. For example, imagine a small group of recent college graduates, from across the region, living in our parsonage, funded by us to bring new kinds of worship and ministry to our neighborhood. Is that possible? We don't know. How would it work? We don't know. But then a year ago we did not know how any of this was going to work, and yet here we are.
On March 3rd we will get together as a whole church to talk together about these kinds of ideas for our future. Exciting ideas, because God is not done with us yet. The Holy Spirit reminds us over and over that we are not here to preserve an institutional heritage, or to maintain institutional inertia. We are of course grateful for the faithfulness of all those who have gone before us and who worked faithfully in their day. However, we are not here to do exactly what they did just because it worked then, or because others expect us to do it now.
What we are here to do is to join together as the Body of Christ. We are here to figure out what that means now in our little community. We are here to proclaim without embarrassment that the kingdom of God is at work here. That God is real, that the needs of the world (right here) are great, and that we have something to do and say about that.
Come and join us.


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Today was the first day of work for the new year, I'm glad I'd finished work early and was the first one to arrive at home. I was surprised when hubby arrives home, wake me up from an afternoon nap in the lounge chair, give me a lovely greeting and gave me a card. I wonder why? I thought it was a "sorry card" for making me upset the other day but to remind me it's January 3, the memorable date when we had our first chat and the day we've met on lavalife. I was impressed by his memory, sometimes he can be the worst forgetful man I knew but he has some special thoughtfulness hidden in some ways, well done my love for remembering this day.
Its 7 years now that we are facing the life's challenge, excitement,sharing happiness and sadness, and learning from each other as we live our lives everyday together as a couple. This coming March 8 was the day we exchange our vows and so proud to wear our wedding bands, is our 4th year anniversary.
I was sad because the day before he upsets me for some reason but now happy to remember this unforgettable day of our lives. Sometimes it doesn't mean if one is upset or there is an argument or an exchange of hurtful words because of anger sometimes you can think of silly thought that it would be the end of everything but nooo.. it's just a matter of dealing the situation and it will bind the two of us to be more mature and responsible individual to live in sure foundation of marriage.
Now we are just sitting in the backyard gazing at each other and reminiscing all the happy memories that we've shared since then and now.


text: Marco Perrin Daily updated news, gossip, rumors, pictures, videos and more about Marco Perrin.TranslationsFrançaisEspañolDeutschItalianoPortuguês中文 Tweeter ABCDEFGHIJKLMNOPQRSTUVWXYZ Marco Perrin1927 - 2014Marco Perrin was a French actor of Serbian descent , best known for his roles in Going Places or Army of Shadows. He died on February 17, 2014 in Issy-les-Moulineaux, aged 86.Quick FactsName: Marco PerrinOccupation: actorBorn: May, 16 1927 in Aix-en-ProvenceDied: February, 17 2014 in Issy-les-Moulineaux (aged 86)Citizenship: FranceOrigin: SerbiaSelected Filmography1974: Going Places (Bertrand Blier): Supermarket inspector1973: The Dominici affair (Claude Bernard-Aubert): the cook1969: Army of Shadows (Jean-Pierre Melville): Octave BonnafousThe latest Marco Perrin newsRumorsIs Marco Perrin Still Alive?AMP™, 22-11-2017 | [November 2017 update] News of Marco Perrin's death on February 17, 2014 stunned the world. Ever since, millions of fans worldwide have come to believe the actor's death was a hoax and that he was still alive and well.Read It »Marco Perrin'The Dominici affair' named Best Film of All TimeAMP™, 22-11-2017 | According to the latest Watch and Listen magazine poll just out yesterday (Tuesday, November 21), 'The Dominici affair' is now considered to be the Greatest Film in the History of Cinema.Read It »


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I'm all for merchandise if it comes in the form of a tasteful t-shirt or an official poster, but some people always have to take things a step too far!
In China [The Vampire Diaries](series:200853) enthusiasts have been getting their kicks from chugging down drinks designed to look like blood from blood bags. Tasteful, eh?
Thankfully, the Chinese authorities are putting an end to this gruesome fad , but it isn't just because it's a bit macabre... The English-only labeling and a lack of official permits for sale in China are also to blame.
But, of course, there are also undertones of moral panic about how this marketing will affect China's youth. The China Food and Drugs administration said that;
This kind of sales strategy, which sells no-name products that pander to peoples' desire for things exciting and new, violates social integrity and moral principles and fails to conform to related national laws. It misleads customers and damages the health of youth
I don't know about you guys, but I think China might actually be right about this one. I don't think drinking fake raspberry flavor blood will cause moral degeneration or damage social integrity, but I do think there are other people to think about here.
Young children and the chronically squeamish do not need to have to see people slurping out of pretty realistic looking blood bags for a cheap kick. Can't you just enjoy the show, guys?!
Would you drink out of a blood bag?
(Source: The Hollywood Reporter)


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My Nonprofit Reviews
Review for California Shakespeare Theater, Berkeley, CA, USA
Our daughter attended CalShakes' 5 week conservatory this past summer and the 2 week conservatory the summer before. We have been attending special performances for students for years. CalShakes has wonderful outreach to the youth community and always has interactive presentations before plus Q&A with the cast after performances. Our daughter (& indeed hundreds of youth) has broadened her acting abilities & knowledge greatly through the in-depth programs provided by this organization.
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I've personally experienced the results of this organization in...
the expansion of my daughter's acting ability.
The kinds of staff and volunteers that I met were...
helpful and very involved with the students they are teaching.
How frequently have you been involved with the organization?
About every month
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How to Make "Welcome to Night Vale" Perfecter
It became #1 most downloaded podcast on iTunes for a time in 2013. It's been praised to the by everyone from the A.V. Club to Nerdist. It's been so successful that it's performing live shows nation wide, something podcasts don't typically pull off. Mara Wilson of Mara Wilson Writes and Jackson Publick of The Venture Brothers have both played parts on it. Yes, it does seem that Welcome to Night Vale is close to as good as it gets when it comes to audio drama being released today
But still, there's something that seems essentially wrong about this show about a friendly desert community where mysterious lights pass overhead while we all pretend to sleep. It hardly ruins the show or, as clearly indicated by the large and loyal following it has accumulated in the past two years, even hurts the show to a significant degree. Yet it's an error that could, if corrected, could move Welcome to Night Vale away from near the top of audio projects to the upper echelons of fiction. And I would know: I've listened to, like, a whole radio show.
For the uninitiated, Welcome to Night Vale is about a town where basically every paranormal thing that can be thought up has happened, every tin foil hat conspiracy of a non-prejudiced nature is true (i.e., they're aware there's a World government, but it's not related to a supposed international Jewish conspiracy or anything of the kind) and where the laws of science are often just ignored. This is all presented from almost entirely one host reading off local news, public service announcements,
Here's a fan animation that gives a general sense of what Welcome to Night Vale is about which includes an audio sample:
Welcome to Night Vale is full of surprisingly funny elements and rich characters for how creepy of an atmosphere it creates. There's flustered Mayor Pamela Winchell, Hiram McDaniels the five-headed dragon running for Mayor (Jackson Publick) , the faceless old woman who lives in your home, the Sheriff's Secret Police, Night Vale's pen pals from the obliterated Soviet city of Nulogorsk that arrive at the desert town in a submarine, the hooded figures, the cat which floats in the bathroom named Khoshekh, the little girl who was born as a fully grown adult's hand,
the angels we're not supposed to know about the complete lack of angels, the yellow helicopters, the Glow Cloud which became School Board President, Eternal Scouts whom adorn the town forever, the NRA who insist we're all immune to bullets, the faceless old woman in your home (Mara Wilson) and of course Carlos, perfect Carlos the scientist who becomes the boyfriend of our humble narrator Cecil Palmer. And that really just scratches the surface.
As you can see from all the above, Welcome to Night Vale is kinda crammed full of weirdness and incidents. It's full of conflicting parties with conflicting interests… that don't conflict. Herein lies the problem this article is about: while Night Vale is full of good jokes and interesting ideas, there are so many of them that they start to get in each other's way and make it impossible to get invested in any of them.
Simply put, there's no way to define or make sense of Night Vale, and beyond a jokey context, that can be detrimental to properly enjoying it. How are we supposed to be creeped out by anything when it's all a bunch of one-offs? How are we supposed to develop empathy for any of the characters when their interactions don't make sense? There are roughly nineteen shadowy forces that seem to rule Night Vale in close competition with each other. How are we supposed to invest in any of them when their overabundance keeps calling into question how so many can possibly coexist in the same small town? I'm not saying that it's impossible, but there should be some sense of the interplay between these various groups. That would be much more compelling to hear than Cecil read a non-sequiter public service announcement
Add to that, it's hard to understand why a listener should be too worried about anything that happens to Night Vale anymore. How can anyone get too worried about the approaching army in episode #31 or the arriving submarine in episode #40 when we've already seen threats to Night Vale like the otherworldly children that came with the Eternal Scouts in episode #23 go away after during the weather segment? Anyway, Night Vale has been said to be a place where dying is a "privilege," so it's impossible to properly get a sense on how much danger to get a gauge on when the town's actually in danger. And for a horror comedy of this type to work, you need that sort of underlying tension and suspense for the comedy to work against.
This might not seem like a significant problem so long as Cecil Baldwin continues to say funny things, but this is the sort of thing is exactly how a show becomes stagnant or spins its wheels. Eventually the formula of "something threatens Night Vale, but it's essentially a non-issue by the end of the weather report" will make the show seem more like comfort food than an interesting and humorous way to bring yourself painlessly twenty-something minutes closer to death.
The writers are taking steps in that direction. For example, there's the character Intern Dana. She's had a relatively long-running story starting in episode 20 of entering the Dog Park and seeming to walk into another plane of existence in her attempt to escape. At one point, she walks out of a house that's not really there. This has the very satisfying effect of tying into an incident in the pilot, where one of the first things Perfect Carlos is shown doing is monitoring that house.
Welcome to Night Vale has more than laid an exceptional groundwork for a very entertaining podcast. Now if its writers concentrate on doing more to build on the existing points and connect them in relevant ways that deepen our understanding of Night Vale without overexplaining it, it might have a truly Twin Peaks-type enduing legacy. Here's crossing my fingers, all eighteen of them, that it delivers.


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Train Orleans → AmboiseFrom HK$0
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Overview The average travel time between Orleans and Amboise is 1h22 minutes. The quickest route is 39 minutes. The first train leaving Orleans is at 00:07, the last at 21:42. There is an average of 22 trains a day between Orleans and Amboise, leaving approximately every 50 minutes.
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JODPIE Bar
JODPIE Bar is a remotely hosted script that dynamically provides live Open Directory data through an elegant programming solution based on the combination of server-side scripting (PHP) and client-side scripting (JavaScript). JODPIE Bar is pure javascript you can embed it in any web page, even static HTML pages. JODPIE Bar is customizable and is designed to fit in a little column of web page display; the script provides a partial and modified presentation of ODP data using an on the fly randomizer. Links are displayed using thumbshots provided by Thumbshots.org. With this script it�s possible to embed live Open Directory data in a web page just in seconds; after inserting few lines in a section of a web page and live Open Directory data will be displayed! JODPIE Bar has been developed having in mind blogging platforms; remotely provided html is as simple as possible to integrate with all html designs. The script works fine with most blogging platforms, mainly on sidebar and run transparently with any theme.Visit publisher site: JODPIE Bar
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The vegetarian diet is generally beneficial to health, provided of course to practice correctly.
By launching its call to say "no thank you to the turkey," the former Beatles singer was not intended that this poultry. Convinced vegetarian, Paul McCartney counted encourage Americans to adopt the vegetarian diet as him. That is to say to banish from their plate red and white meats, poultry, fish, shellfish – in short, all animal flesh. Vegetarians eat plant indeed but unlike vegans, they do not exclude foods derived from animals such as milk and dairy products, eggs or honey.
Generally advocated on behalf of religious or ideological belief, vegetarian diet, properly practiced, is generally beneficial to health. "Especially in Western countries where people eat a lot of sugars, animal fats and foods whose quality is often questionable," says Dimitrios Samaras, physician associated with the nutrition unit of the University Hospitals of Geneva (HUG ). In addition, the followers of this diet are more successful at the famous consume five fruits and vegetables daily. These plants, associated with legumes and cereals complete, accurate Esther Guex, HES dietician at Lausanne University Hospital (CHUV), provide "a diet rich in fiber and antioxidants" that protect our body against various diseases.
Good for the heart and arteries
Vegetarianism is so deemed to be good for the heart and arteries. Animal fats, "especially when they are fried," said Dimitrios Samaras, indeed contain saturated fatty acids that increase the levels of "bad cholesterol", which promotes the development of cardiovascular disease.
The vegetarian diet also decreases the risk of colorectal cancer. That makes sense, since studies have established a link between the development of these tumors and the consumption of red meat. "The mortality from this cancer is 10% higher in people who eat one serving per day, says nutritionist HUG. It is even 16% higher in those who daily consume a portion of sausage. "
Eliminate red meat menus could also have beneficial effects on inflammation of the body that contributes to the development of diabetes, atherosclerosis and even some dementias like Alzheimer's disease. "These inflammatory syndromes are influenced by food, resumes Dimitrios Samaras, vegetarians and seem slight advantage compared to the rest of the population."
About the benefits of the vegetarian diet in obesity is a derivative gain, according to the nutritionist who admits he "rarely seen in obese vegetarians." This is probably due mainly to lifestyle, for those who adopt this type of diet are generally more concerned about their diet, take great care of their bodies and doing more exercise than others.
Possible deficiencies
The vegetarian diet is rather positive for health, provided, insist our two experts, "to document and learn." Otherwise, it may have some risks. Starting with a lack of protein because "those plants are of less value than those from animals," says Dimitrios Samaras. We must compensate by consuming eggs and dairy products. But also recommends Guex Esther, "in properly combining cereals and legumes, in order to achieve a good protein complementarity".
One could also fear a lack of omega-3 highly in fish. But this is actually not a problem, since according to the dietitian, "as we find it in canola oil."
We must also be careful not to suffer from deficiencies in minerals such as calcium, iron and zinc. The latter "is less well absorbed by the body due to the presence of substances (phytates) in plants," warns nutritionist Geneva. Furthermore, vegetarians ingest little vitamin B12, "almost only present in animal products' and vitamin D. All of which leads to Dimitrios Samaras that" you should discuss with their physician the need to take dietary supplements ".
For an adult and warned vegetarian, there is no great danger. The risk is greater, according to Esther Guex for "pregnant and lactating women, who have greater needs in proteins and minerals, as well as for children and adolescents." For them, says the nutritionist HUG, "this kind of restrictive diet should be under medical supervision."
Finally, "I would not put anyone vegetarianism" said Dimitrios Samaras. It is quite different, conclude our two experts, the vegetarian diet. By also removing eggs and dairy products, it can cause serious nutritional deficiencies, especially in children.