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We have previously shown that treatment of mice with pyrazole or acute ethanol potentiated Fas agonistic Jo2 antibody-induced liver injury by a mechanism involving induction of CYP2E1 and elevated oxidative stress. The current study evaluated whether chronic alcohol feeding potentiates Fas-induced liver injury and whether CYP2E1 plays a role in any enhanced hepatotoxicity. Wild type and CYP2E1 knockout mice were fed ethanol or isocaloric dextrose for four weeks followed by a single treatment with either saline or Jo2. Mice were killed 8 h after the Jo2 challenge. There were 3–5 fold increases of transaminases and more extensive eosinophilic necrosis, hemorrhage and infiltration of inflammatory cells in the central zone of the hepatic lobule in the ethanol-fed mice treated with Jo2 compared to the dextrose/Jo2 or ethanol/saline treated mice. Liver injury was blunted in ethanol-fed CYP2E1 knockout mice treated with Jo2. The chronic ethanol feeding produced steatosis, elevation of CYP2E1 and oxidative stress in wild type but not CYP2E1 knockout mice. These changes in wild type mice fed ethanol were similar after saline or Jo2 treatment. The Jo2 treatment produced activation of JNK and p38 MAP kinase, increased activity of caspases 8 and 3, and lowered hepatic GSH levels in both the dextrose- and alcohol-fed mice. JNK was activated at early times after Jo2 treatment in the ethanol-fed mice. Serum TNF-α levels were strikingly elevated in the wild type ethanol/Jo2 group which showed liver injury compared to all the other groups which did not show liver injury. Inhibition of JNK or p38 MAPK partially, but not completely, prevented the elevated liver injury in the wild type ethanol/Jo2 mice. These results show that chronic ethanol feeding enhances Fas-induced liver injury by a mechanism associated with induction of CYP2E1, elevated serum TNF-α levels and activation of MAPK.
Alcoholic liver disease (ALD) is a common clinical complication of long-term alcohol abuse. Its morphological features include alcoholic fatty liver (steatosis), alcoholic hepatitis, and alcoholic cirrhosis. The pathogenesis of ALD involves multifactorial processes such as genetic, nutritional and environmental factors, and a number of injurious factors such as oxidative/nitrosative stress, bacterial lipopolysaccharide and cytokines [1,2]. Accumulating evidence suggests that oxidative stress and lipid peroxidation play a key role in mechanisms of alcohol-induced liver injury. Many pathways participate in mechanisms as to how ethanol induces oxidative stress, including induction of CYP2E1, mitochondrial dysfunction, activation of MAP kinases, elevated cytokine formation, elevated expression of the inducible isoform of nitric oxide synthase (iNOS), and decreases in hepatic antioxidant defense [3–7]. Induction of CYP2E1 contributes to the alcohol-mediated hepatotoxicity in cultured cell models such as ethanol-sensitive E47 HepG2 human hepatoma cells and RALA hepatocytes transduced with ethanol-inducible CYP2E1, and in the intragastric ethanol infusion model of ALD [8–10]. However, there are reports that CYP2E1 may not play a role in alcohol liver injury based on studies with gadolinium chloride or CYP2E1 knock-out mice [11,12]. Therefore, there is a need to further understand the role and significance of CYP2E1 induction via ethanol exposure so as to explore the mechanisms of ALD and provide a basis for preventing ALD complications and severe liver damage.
The Fas/Fas ligand system may play a central role in ethanol-induced hepatic apoptosis [13–15]. Galle, et al [16] showed up-regulation of Fas ligand mRNA expression in hepatocytes of alcohol-damaged liver. Apoptosis induced by low concentrations of ethanol in HepG2 cells was associated with Fas-receptor activation and subsequent caspase-8 and caspase-3 activation [17]. Minana, et al [14] showed that ethanol and acetaldehyde caused apoptosis in hepatocytes after chronic ethanol feeding and that acetaldehyde elevated Fas ligand levels; they suggested that Fas may play a role in ethanol-induced apoptosis. However, Nakayama, et al [18] reported that in HepG2 cells, ethanol-induced apoptosis was not mediated via Fas or TNF-α receptors, and that ethanol did not up-regulate Fas expression. Hepatocytes were recently shown to express both Fas and Fas ligand which can induce death of other co-cultured cells, and it is interesting to speculate that apoptosis by autocrine or paracrine mechanisms involving Fas may play a role in alcohol-induced liver injury [19]. Pyrazole treatment to induce CYP2E1 potentiated Fas-mediated liver injury via elevated oxidative and nitrosative stress [20].
Our previous data showed that acute ethanol pretreatment potentiated Fas agonistic antibody Jo2-induced hepatic toxicity by elevating CYP2E1-dependent oxidative/nitrosative stress, suggesting that acute ethanol treatment increases sensitivity to a low-toxic Jo2 challenge resulting in severe synergistic liver damage [21]. Other studies reported that fatty liver or obesity or chronic ethanol consumption cause increased sensitivity to hepatotoxins such as LPS [22,23], suggesting the multifactorial nature and complex interactions among primary mechanistic factors and between primary and secondary factors as the basis for elucidation of mechanisms of ALD [24]. In this study we evaluated whether chronic ethanol consumption potentiates the hepatotoxicity of Jo2 Fas agonistic antibody. We fed wild type mice containing CYP2E1 and CYP2E1 knockout mice with ethanol chronically, followed by subliminal Jo2 antibody treatment to evaluate whether an increased hepatotoxicity involves CYP2E1.
Animal experiments were approved by the Laboratory of Animal Care and Use Committee of the Mount Sinai School of Medicine and carried out under the guidelines of the National Institutes of Health for the humane use of animal subjects. CYP2E1 knockout (−/−) mice, Cyp2e1 tm1Gonz, weighing 17.8±2.7 g at 6–8 weeks of age, were kindly provided by Dr. Frank Gonzalez, National Institutes of Health and were bred and maintained in the animal center at Mount Sinai. The wild-type (CYP2E1+/+) 129/SV mice, weighing 16.9±1.6 g at 6–8 weeks of age, were purchased from Charles River Breeding Laboratory (Boston, MA). The CYP2E1 −/− (KO, n=16) and +/+ (WT, n=16) mice were pair-fed a liquid ethanol diet (ETOH) or control dextrose diet (Dex) (Bio-Serv, Frenchtown, NJ) at ethanol calorie of 10%, 15%, 20%, 25% each for 5 days, respectively and 35% for 10 days. After the 4 weeks feeding period, body weights of the dextrose-fed wild type and CYP2E1 knockout mice were 18–20 g (gains of 2–3 g body wt) while body weights of the ethanol-fed wild type and CYP2E1 knockout mice were 14–15 g (loss of body wt of about 2–3 g). Mice were withdrawn from ethanol and food and received intraperitoneally one dose of 0.1 µg of Jo2 anti-Fas antibody (Jo2) (BD Pharmingen, San Diego, CA) or saline control (Sal). At 8 hours after administration of Jo2 or saline, the mice were sacrificed and serum and liver samples were collected. Blood ethanol levels were similar in the wild type and the CYP2E1 knockout mice fed ethanol. In some experiments assaying the time course of injury, ethanol or dextrose-fed wild-type (CYP2E1+/+) 129/SV mice were sacrificed at 2 (n=4), 4 (n=4) and 8 (n=4) hours after administration of Jo2 or saline.
Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were assayed using a diagnostic kit (ThermoDMA, Louisville, CO). The serum TNF-a level was assayed using a mouse TNF-α ELISA kit (Pierce Biotichnology, Rockford, IL). A total of 50 µl of Standards (standard curve concentration: 0, 50, 350 and 2450 pg/ml) or serum were added into each well in duplicate. The absorbance was measured at 450 minus 550 nm and the results were calculated using the standard curve.
Small pieces of liver tissue were fixed in 10% neutral buffered formalin and processed into paraffin sections for hematoxylin-eosin (HE) or immunohistochemical staining. Morphological changes were observed by two pathologists who were blinded from the experimental information. All pathological changes of fatty degeneration, apoptosis and necrosis by HE staining and light microscope were graded as none (0), mild (<25%), moderate (25–50%), and severe (>75%). Immunohistochemical staining was performed by using the IHC Select Detection System (Chemicon, Temecula, CA) for MDA with rabbit-anti-MDA antibody (1:100). Slides were visualized with 3,3-diaminobenzidine (DAB) and positive staining was reflected by the brownish yellow color. In each case, a negative control (non-immune serum) was used. The evaluation of a specific positive reaction was marked as negative (−), weakly positive (+), moderately positive(++), and strongly positive (+++).
Liver tissue homogenates were freshly prepared in 5–10 volume of ice-cold 150 mM KCl. Microsomes, mitochondria and the cytosol fractions were prepared using differential centrifugation. The protein concentration of the different fractions was determined using a protein assay kit based on the Lowry assay (BioRad, Hercules, CA). The levels of CYP2E1, FAS, cFLIP, JNK and P38 MAPK protein in 50 µg of protein samples from freshly prepared microsome, cytosol or homogenate fractions were determined by Western blot analysis with anti-CYP2E1 (1:10000) (a gift from Dr. Jerome Lasker, Hackensack Biomedical Research Institute, Hackensack, NJ), anti-FAS (1:2000) (Santa Cruz Biotechnology, Santa Cruz, CA), anti-cFLIP (1:1000) (Stressgen Biotechnology, British Columbia, Canada), anti-p-JNK and anti-JNK (1:1000), anti-p-P38 and anti-P38 antibody (1:1000) (Santa Cruz Biotechnology), respectively. All specific bands of proteins detected by Western blot were quantitated with the Automated Digitizing System (ImageJ gel programs, version 1.34S, National Institute of Health).
CYP2E1 activity was measured in liver microsome fractions by the spectrophotometric analysis at 510 nm of the oxidation of p-nitrophenol to p-nitrocatechol in the presence of NADPH and oxygen [25]. Protein carbonyl adducts were assayed in liver homogenates using 20 µg of protein samples and the OxyBlot Protein Oxidation Detection Kit (Chemicon, Temecula, CA). The DNP-derivatized protein samples were separated by SDS-PAGE followed by Western blot as described above. Reduced non-protein thiols, mainly glutathione (GSH) was analyzed in liver tissue homogenates by a fluorescence assay with the proluminescent substrate o-phthalaldehyde.
Caspase-8, caspase-3, caspase-6 and caspase-9 activities were determined in liver tissue homogenates by measuring proteolytic cleavage of the proluminescent substrates Z-IETD-AFC, AC-DEVD-AMC, AC-VEID-AFC and AC-LEHD-AFC (Calbiochem, La Jolla, CA). The fluorescence was determined based on the amount of released AFC (caspase-8, −6, −9, λex=400, λem=505) or AMC (caspase-3, λex=380, λem=460). The results were expressed as arbitrary units of fluorescence (AUF) per milligram of protein.
In order to assess the possible protection against Jo2 hepatoxicity by JNK or P38 MAPK inhibitors--- SP600125 (Sigma, St Louis, MO) or SB203580 (Tocris, Ellisville, MO), wild type 129/SV mice were fed the liquid ethanol diet or control dextrose diet at total ethanol calorie of 10% and 20% for 10 days, respectively and 35% for 10 days. Mice received intraperitoneally one dose of 0.1 µg of Jo2 anti-Fas antibody plus SP (15 mg/kg, body wt) (n=6) , or Jo2 plus SB (15 mg/kg, body wt) (n=6) or Jo2 plus saline control (n=6). At 8 hours after administration of Jo2 plus SP or plus SB or saline control, the mice were sacrificed and serum and liver samples were collected for detection of serum transaminase, liver morphology or p-JNK/JNK and p-P38/P38 MAPK protein levels as described above.
Values reflect means±SD. One-way ANOVA (post-hoc test Tukey HSD comparisons) analysis was performed by SPSS 10.0 statistic software. Statistical data were corrected for multiple group comparison. P values of less than 0.05 were considered statistically significant.
Eight groups of mice were studied in this report. Wild type mice were fed dextrose or ethanol and after 4 weeks treated with either saline or Jo2; these are referred to as WT Dex/Sal, WT Dex/ Jo2, WT ETOH/Sal and WT ETOH/Jo2. Similarly, CYP2E1 knockout mice were fed dextrose or ethanol and after 4 weeks treated with either saline or Jo2; these are referred to as CYP2E1 KO Dex/Sal, CYP2E1 KO Dex/Jo2, CYP2E1 KO ETOH/Sal and CYP2E1 KO ETOH/Jo2. Treatment with Jo2 elevated ALT and AST levels in dextrose-fed WT mice compared to saline treated dextrose-fed mice. A similar increase by Jo2 was found in CYP2E1 KO mice fed dextrose (Fig.1A,1B). Thus Jo2 causes some liver injury in dextrose-fed mice by a CYP2E1-independent pathway. In ethanol-fed mice, Jo2 administration produced a high increase in serum ALT and AST levels compared to saline treated ethanol-fed mice. This large increase by Jo2 was blunted in the CYP2E1 KO mice (Fig.1A,1B). Elevated steatosis and macrovesicular fat were observed in the WT ETOH mice treated with either saline or Jo2 (Fig. 1C3, C4) compared to the CYP2E1 KO ETOH mice treated with either saline or Jo2 (Fig.1 C7, C8). More severe pathological changes were observed in the WT ETOH/Jo2 (Fig.1 C4) than that in the CYP2E1 KO ETOH/Jo2 (Fig.1 C8) group; in the WT ETOH/Jo2 group, many hepatocytes displayed extensive eosinophilic necrosis, hemorrhage and infiltration of inflammatory cells in the central zone of the hepatic lobule. Jo2 treatment produced some hepatocyte degeneration or focal necrosis in both the dextrose-treated wild type and CYP2E1 knockout groups compared to the saline-treated WT dextrose and KO dextrose groups (Fig.1 panels, C2 and C6 compared to C1 and C5); however, the injury by Jo2 in the WT dextrose-fed mice was much less than that in the WT ethanol-fed mice (C2 compared to C4). Thus, chronic ethanol feeding potentiated Jo2-induced liver injury in WT mice but not in CYP2E1 KO mice.
Experiments were carried out to evaluate whether the enhanced hepatotoxicity found in the WT ETOH/Jo2 group was associated with an elevated formation of reactive oxygen species and whether CYP2E1 contributes to such an increase. The level of protein carbonyl adducts was higher in the WT ETOH/Jo2 or WT ETOH/Sal group compared to WT Dex/Jo2 or WT Dex/Sal group (Fig.2A). The ethanol feeding also elevated protein carbonyls in the CYP2E1 knockout mice compared to the dextrose fed KO mice (Fig.2A). This suggests that ethanol elevates carbonyl adducts by a CYP2E1-independent mechanism. Nevertheless, greatest levels of protein carbonyls were found in the ethanol-fed wild type mice, suggesting that CYP2E1 contributes to the increase in protein carbonyl adducts by chronic ethanol feeding with or without Jo2 administration. The Jo2 treatment did not elevate protein carbonyl levels in any group; indeed, carbonyls were less in Jo2-treated than saline-treated groups e.g. WT Dex and WT ETOH. The reason for this decrease by Jo2 is unknown. MDA was assayed as one index of lipid peroxidation; MDA expression in situ was mainly found in areas of steatosis or in macrovesicular fat in the centrilobular zone or portal area of the liver. The MDA levels were highest in the WT ETOH/Jo2 or WT ETOH/Sal group (+++) (Fig.2B3, B4); smaller increases were also found in all the other groups (+) (Fig.2B2,2B6,2B7,2B8) except for the lack of obvious positive staining in the WT Dex/Sal (Fig.2B1) or KO Dex/Sal group (Fig.2B5). These results, similar to the protein carbonyl adduct data, show that ethanol treatment elevates MDA levels, CYP2E1 contributes to this increase, but Jo2 treatment does not further elevate MDA levels over the chronic ethanol increase.
After 4 weeks of chronic ethanol feeding, the oxidation of PNP, a reflection of CYP2E1 catalytic activity, was significantly higher in the WT ethanol-fed mice treated with either saline or Jo2 compared to the WT dextrose-fed mice treated with either saline or Jo2 (Fig.3A). In the CYP2E1 KO ETOH/Sal or KO ETOH/Jo2 group, the PNP activity was very low, validating the absence of CYP2E1 (Fig.3A). The slight increase in PNP oxidation in the ethanol-fed CYP2E1 KO mice compared to dextrose-fed mice might reflect ethanol induction of other CYPs capable of oxidizing PNP e.g. CYP3A. Increases of 1.7-fold of CYP2E1 protein expression were detected by western blot analysis in the WT ETOH/Sal group compared to the WT Dex/Sal group, while the CYP2E1 level in the WT ETOH/Jo2 group was 3.8-fold higher than the CYP2E1 level in the WT Dex/Jo2 group. This appears to be due to a decrease in CYP2E1 by Jo2 in the WT Dex (Fig.3D). Thus, Jo2 administration does not alter the increase in CYP2E1 activity or even increases CYP2E1 protein levels about 2-fold after ethanol feeding. In the CYP2E1 KO groups, PNP activity or CYP2E1 protein expression could barely be detected compared to the WT groups (Fig.3A, 3D). Administration of Jo2 lowered the GSH level in the WT dextrose-fed and ethanol-fed mice compared to the WT saline-treated dextrose or ethanol-fed mice, respectively (Fig.3C). However, Jo2 did not lower hepatic GSH levels in the CYP2E1 knockout mice (Fig.3C), analogous to the low extent of increase in protein carbonyls and MDA by ethanol in CYP2E1 KO mice.
The content of TNF-α in serum was markedly higher in the WT ETOH/Jo2 group compared to the WT Dex/Jo2 group or WT ETOH/Sal group (Fig.3B). Importantly, it was found that the serum TNF-a level was significantly lower in the CYP2E1 KO ETOH/Jo2 group compared to the WT ETOH/Jo2 group (Fig.3B). Jo2 treatment lowered the level of cFLIP in both the WT and the KO group compared to the Sal-treated mice in both groups (Fig.3D). In the WT ETOH/Jo2 and the KO ETOH/Jo2 groups, the cFLIP level was significantly lower than that in the WT Dex/ Jo2 groups (Fig.3D). The decline in cFLIP level was similar in the WT ETOH/Jo2 group and the KO ETOH/Jo2 group (Fig.3D), thus appears to be independent of CYP2E1. There was no difference in Fas levels in all groups either in the WT mice or in the KO mice (Fig.3E). Thus, the enhanced hepatotoxicity found in the WT ETOH/Jo2 group is not due to altered levels of Fas.
The activity of caspase-8 was significantly higher in the Jo2-treated group compared to the Sal-treated group either fed the dextrose diet or the ETOH diet. There was no significant difference in caspase-8 activity in the Jo2-treated WT dextrose or ethanol-fed mice compared to the Jo2-treated dextrose or ethanol-fed CYP2E1 KO mice (Fig.4A). Similarly, the activity of caspase-3 was significantly higher in the Jo2-treated WT dextrose or ethanol-fed mice compared to the Sal-treated groups. There was also no significant difference in caspase-3 activity in the WT Dex/Jo2 or WT ETOH/Jo2 group compared to the KO Dex/Jo2 or KO ETOH/Jo2 group (Fig.4B). Activity of caspase-6 or caspase-9 was not significantly different between all the groups or between WT versus KO groups (Fig.4C, 4D).
Immunoblot assay showed that the ratio of phosphorylated p-JNK to total JNK was elevated 1.9-fold in the Dex/Jo2 groups but only 1.2-fold in the ETOH/Jo2 compared to the ETOH/Sal group (Fig.5A). Note that JNK was activated by the chronic ethanol feeding even in the absence of Jo2: 1.7-fold ratio of p-JNK/JNK in the ETOH/Sal group compared to a ratio of 1.0 in the Dex/Sal group (Fig.5A). There were increases of 1.5-fold and 1.3-fold in the ratio of phosphorylated p-P38 to total P-38 MAPK in the Dex/Jo2 or ETOH/Jo2 group compared to the Dex/Sal or ETOH/Sal group, respectively (Fig.5A). Thus, Jo2 activated JNK and p38 MAPK in both the dextrose-fed mice and alcohol-fed mice. Time course experiments indicated that JNK was activated at 2 h in the ethanol-fed mice compared to the dextrose-fed mice (Fig.5B). The JNK activation began to decline at 8 h after Jo2 treatment in ethanol-fed mice and began to increase in the dextrose-fed mice (Fig.5B). P38 MAPK was activated only at 8 h after administration of Jo2 in the ethanol-fed mice, in contrast to the early activation of JNK by Jo2.
A time course for Jo2 hepatotoxicity was carried out. Levels of serum ALT or AST were significantly elevated in the ethanol/Jo2 (and to a small extent in the dextrose/Jo2) group at 8 h but not at 2 or 4 h after Jo2 administration (Fig.6A,6B). Thus, the time point where elevated hepatotoxicity is observed in the ethanol-fed mice treated with Jo2 occurs at times where JNK and a lesser extent p38 MAPK are activated.
To initiate studies on the significance of the elevation of JNK or P38 MAPK in the enhanced toxicity of chronic ethanol feeding plus Jo2 treatment, the ability of SP600125 or SB203580 to protect against this toxicity were carried out. After administration of SP, the ratio of phosphorylated p-JNK to total JNK was significantly lower in the ETOH or Dex group at 8 h after Jo2 treatment (Fig.5B). Similarly, the ratio of phosphorylated p-P38 to total P38 MAPK was also significantly lower in the ETOH group 8 h after administration of SB (Fig.5B). Levels of serum AST were significantly lowered in the ETOH/Jo2/SP or ETOH/Jo2/SB group compared to the ETOH/Jo2 group (Fig.6D). The level of serum ALT was also lowered in the ETOH/Jo2/SP or ETOH/Jo2/SB group compared to the ETOH/Jo2 group but because of variability, p values were 0.05<p<0.1 (Fig.6C). Histopathology showed that severe injury was observed in the ETOH/ Jo2 group at the 8 h time point (Fig.6E3) including fatty accumulation, extensive eosinophilic necrosis and some infiltration of inflammatory cells as compared to 2 or 4 h after Jo2 administation (Fig.6E1, E2). There was a partial amelioration of liver injury, including limited necrotic focus, in the ETOH/Jo2/SP (Fig.6E4) or ETOH/Jo2/SB (Fig.6E5) group compared to the ETOH/Jo2 group (Fig.6E3).
CYP2E1 is induced under a variety of pathophysiological conditions such as fasting, diabetes, obesity and high-fat diet [26–28]. CYP2E1 protein expression and activity are elevated after ethanol exposure [29,30]. CYP2E1 is responsible for metabolizing and activating a large number of chemical solvents, industrial monomers and precarcinogens [31] and is active in catalyzing the production of ROS and lipid peroxidation in microsomal membranes [32–34]. In vitro, ethanol-mediated production of ROS, cell death and GSH depletion were observed in both the cytosolic and mitochondrial fraction of CYP2E1 transfected cell lines [8,35]. The in vivo role of CYP2E1 in alcohol-induced liver injury is controversial, especially in the early stage of experimental ALD. In this study, we used a chronic ethanol feeding plus Jo2 treatment model in wild type mice and in CYP2E1 knockout mice to evaluate whether chronic ethanol treatment increases Jo2 hepatotoxicity, whether CYP2E1 or CYP2E1-mediated ROS contributes to an increased liver injury formed after chronic ethanol consumption, and what key factors participate in this potentiated hepatotoxicity. Hepatotoxicity occurs in the wild type mice fed the alcohol diet chronically and treated with Jo2 compared to the wild type mice fed dextrose and treated with Jo2 or CYP2E1 knockout mice fed the dextrose or alcohol diet followed by treatment with Jo2. Steatosis was more severe in the CYP2E1 wild-type mice than that in the CYP2E1 knockout mice after alcohol feeding and treatment with either saline or Jo2. We have previously shown that CYP2E1 plays a role in ethanol-induced fatty liver [36]. Interestingly, no significant toxicity was found in the saline treated ethanol-fed groups, suggesting that steatotitic histopathological changes alone did not promote further liver injury. However, the fatty liver may provide a basis for a second toxic challenge e.g. with Jo2. CYP2E1 protein and catalytic activity were highly induced in wild type mice compared to the CYP2E1 knockout mice fed alcohol and treated with either saline or Jo2 treatment. These results show that a) chronic ethanol feeding produced more steatosis in wild type mice than in CYP2E1 knockout mice; b) the ethanol feeding elevated CYP2E1 in wild type mice; c) the increase in fatty liver and in CYP2E1 are not sufficient by themselves to result in significant liver injury i.e. necrosis; d) however, the increases in fatty liver and CYP2E1 may set the stage for the liver to become sensitive to a second challenge as provided by Jo2 administration at concentrations which do not cause liver injury to dextrose-fed mice without fatty liver or elevated CYP2E1.
Experiments were subsequently carried out to evaluate possible mechanisms which play a role in the elevated Jo2 toxicity in the ethanol-fed WT mice. Since CYP2E1 contributes to this enhanced toxicity, our initial hypothesis was that oxidative stress was elevated in the ethanol plus Jo2-treated wild type mice. Indeed, protein carbonyl, MDA or lipid peroxidation and GSH depletion were highly elevated in the wild type mice fed ethanol and treated with Jo2, as compared to the CYP2E1 knockout mice fed ethanol and treated with Jo2. However, similar increases in these markers of oxidative stress also occur in the saline-treated ethanol-fed mice (without Jo2 treatment) as compared to saline-treated ethanol-fed CYP2E1 knockout mice. These results show that CYP2E1 plays a major role in the elevation of oxidative stress produced by chronic ethanol feeding. However, since this elevation was not different between the saline-ethanol-fed mice which do not show liver injury, and the Jo2-ethanol-fed mice which do show liver injury, differences in the extent of oxidative stress alone can not explain the liver injury formed in the ethanol/Jo2 mice compared to ethanol/saline mice (although differences in oxidative stress can contribute to the liver injury in the ethanol/Jo2 mice compared to the dextrose/Jo2 mice).
Abnormal cytokine metabolism is a major feature of ALD, especially LPS mediated-hepatitis [8,37,38], and increased serum TNF-α levels are associated with the disease severity and mortality of alcoholic hepatitis [39]. Rats after chronic alcohol feeding are more sensitive to the hepatotoxic effects of administration of LPS and have higher plasma levels of TNF-α than control rats [23,40,41]. Gadolinium chloride was found to protect against alcoholic liver injury via down-regulation of TNF-α production in Kupffer cells [11]. Anti-TNF-α antibody prevented alcohol liver injury in rats [42], and mice lacking the TNF-R1 receptor did not develop alcohol liver injury [43]. These studies clearly indicate TNF-α as a major risk factor for the development of alcohol liver injury. Could differences in TNF-α levels be important for the liver injury found in the ethanol/Jo2 group compared to the other groups ? There was a small increase in serum TNF-α levels in wild type mice and CYP2E1 knockout mice fed the dextrose diet followed by Jo2 treatment as compared to the wild type or knockout mice fed the dextrose diet and treated with saline. Thus, Jo2 elevates TNF-α comparably in the absence or presence of CYP2E1 in dextrose fed mice. Chronic ethanol feeding alone did not elevate serum TNF-α levels in wild type or knockout mice compared to the dextrose controls, showing that fatty liver or elevated CYP2E1 are not sufficient to increase TNF-α in this model. However, Jo2 elevated serum TNF-α levels to high levels in wild type mice fed ethanol; this increase was not observed in the ethanol/Jo2-treated knockout mice indicating a role for CYP2E1 in the ethanol/Jo2-mediated elevation of TNF-α. CYP2E1 is present in hepatic Kupffer cells and is inducible by ethanol [44]. How CYP2E1 and Jo2 may interact to elevate production of TNF-α will require further study. Interestingly, the extent of TNF-α elevation is associated with the severity of hepatotoxicity being highest in CYP2E1 wild-type mice fed alcohol which display liver injury, and low in the 3 other groups which do not show liver injury. These results suggest that the elevation of TNF-α may contribute to the increased hepatotoxicity in the wild type ethanol/Jo2 mice.
The Fas/Fas-L system may play a key role in ethanol-induced hepatic apoptosis [13–15]. Fas recognizes Fas ligand (FasL) or Fas antibody to initiate the receptor cross-linking and cell apoptosis via receptor oligomerization and recruitment of the Fas-associated protein with death domain, which eventually leads to the activation of caspase-8 and downstream caspases such as caspase-3 [45–47]. Liver is particularly sensitive to Fas-mediated toxicity and the injection of Fas antibody in mice results in extensive hepatocyte necrosis and even fulminant liver failure [48]. The current study shows that Jo2 Fas antibody-mediated toxicity was potentiated by chronic alcohol feeding with development of extensive acidophilic necrosis, hemorrhage and infiltration of inflammatory cells in damaged areas. Levels of Fas were similar in all groups. While there were increases of caspase-8, and caspase-3 activities in the alcohol/Jo2 mice, there were similar increases in the dextrose/Jo2 mice, thus elevated caspase activities cannot explain the differences in liver injury between the alcohol/Jo2 versus the dextrose/Jo2. There was no significant difference in the Jo2 elevation of caspase 3 or 8 activities between wild type mice and CYP2E1 knockout mice, thus the Jo2 elevation of caspase 3 or 8 activities is independent of CYP2E1 status. c-FLIP has been identified as a regulator of death ligand-induced apoptosis downstream of death receptors and FADD [49,50]. c-FLIP was decreased after alcohol feeding plus Jo2 treatment compared to the dextrose feeding plus Jo2 treatment. However, there was no significant difference in downregulation of c-FLIP between wild type alcohol/Jo2 mice with liver injury and CYP2E1 knockout alcohol/Jo2 mice without liver injury. These results suggest that the enhanced hepatotoxicity found in the CYP2E1 wild type mice fed alcohol followed by Jo2 treatment is not due to the altered levels of Fas, the upregulation of caspase-8, −3 and the decreased levels of cFLIP. These results do not rule out an important role for any of the above in the overall injury, but appear to rule out an important role for any of the above in explaining the differential liver injury produced by Jo2 in the ethanol-fed mice.
Mitogen activated protein kinases (MAPKs), JNK, p38 and ERK, are important cellular signaling molecules that convert various extracellular signals into intracellular responses via serial phosphorylation [51–54]. Basically, prolonged activation of either JNK or p38 MAPK promotes cell death with an associated decrease in mitochondial membrane protential, whereas ERK activation may serve as a cell survival factor [55–57]. But the exact role of MAPK in the cellular response in different hepatotoxicity models remains controversial. As to the role of MAPK in CYP2E1–dependent toxicity, Hoek and colleagues found that ethanol increased TNF-α toxicity in CYP2E1 expressing liver cells via a p38 MAPK-dependent pathway [58]. Czaja and colleagues showed that increased CYP2E1 expression sensitized hepatocytes to TNF-α toxicity through prolonged activation of JNK [10]. CYP2E1 plus arachidonic acid toxicity was mediated either by ERK or p38 MAPK [59, 60]. Koteish, et al [23] reported that chronic ethanol exposure potentiated LPS liver injury in mice despite inhibiting JNK activity whereas Chung, et al [61] reported that six months of ethanol treatment in rats increased JNK activation. With respect to the role of MAPK on Fas-induced liver damage, Brunner, et al reported that TRAIL-induced amplification of Fas-induced liver damage required JNK activation and phosphorylation of its downstream target and proapoptotic Bcl-2 homolog Bim [62]. We evaluated the possible role of MAPK on the ethanol/Jo2 enhanced liver injury. Jo2 alone was able to promote the activation of JNK and p38 MAPK in both the ethanol fed and dextrose fed mice; there was no further increase in activation of JNK or p38 MAPK in the ethanol/Jo2 mice which display elevated hepatotoxicity. In order to explore the exact role and dynamic changes of JNK or p38 MAPK activation on the enhanced hepatotoxicity, time course experiments and inhibitors experiments were carried out. The results showed that chronic ethanol feeding was able to significantly promote the activation of JNK at an early stage after Jo2 treatment, with activation reaching a peak value at 2 h followed by gradual decline at 4 and 8 h. A time course of liver injury in the ethanol-fed mice showed injury occurring at 8 h but not at 2 or 4 h after addition of Jo2. Thus, the liver injury occurs after the activation of JNK and it is possible that the early activation of JNK in the ethanol/Jo2 mice may play a role in the developing liver injury. Treatment with inhibitors of JNK or p38 MAPK partially but not completely prevented the liver injury in the ethanol/Jo2 mice. These results suggest that JNK or p38 MAPK activation, at least partially, contribute to the enhanced Jo2-mediated hepatotoxicity following chronic alcohol feeding, however, detail mechanisms of the enhanced hepatotoxicity remain to be further defined.
In conclusion, chronic alcohol feeding leading to induction of CYP2E1 and to liver steatosis potentiates Fas Jo2-mediated hepatotoxicity in mice. Chronic alcohol feeding produces extensive steatosis and over-expression of CYP2E1 in liver, events which promote the early stage of ALD. CYP2E1 plays an important role in the steatosis, mediating oxidative stress and lipid peroxidation, events independent of Jo2 administration. Jo2 activates Fas internalization and increases activation of caspases 8 and 3, activation of JNK and P38 MAPK, and decreases GSH, events independent of CYP2E1. The combination of alcohol induction of CYP2E1 plus Jo2 administration leads to an increase in TNF-α levels. A scheme to summarize the enhanced hepatotoxicity and events envisioned to occur in the chronic alcohol diet feeding plus Jo2 treatment model is shown in Fig.7. Neither of the factors associated with the induction of CYP2E1 (fatty liver, oxidative stress) or addition of Jo2 (activation of MAPK, caspase activation, decline in GSH) are sufficient to promote significant liver injury. However, the combination of alcohol, CYP2E1 and Jo2 is sufficient to promote liver injury. Thus, the enhanced hepatotoxicity is associated with the over-expression of CYP2E1, increase of TNF-α production and upregulation of MAPK activation especially JNK.
This work was supported by USPHS Grants AA 017425 and AA 03312 from the National Institute on Alcohol Abuse and Alcoholism. We thank Dr. Frank J. Gonzalez (Laboratory of Metabolism, National Cancer Institute, Bethesda, MD) for providing breeder CYP2E1 knockout mice used for this study and Dr. Syllvan Wallenstein (Biostatistician, Department of Community and Preventive Medicine, Mount Sinai School of Medicine, New York, NY) for help with the statistics in the manuscript preparation.
Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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.


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The AuthorsChris Rowan
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The case of the disappearing lake
High in the Oregon Cascades, there is a lake attracting quite a bit of attention this summer. That's because it is disappearing. The disappearing lake, appropriately called Lost Lake, isn't just disappearing because of the drought that Oregon has been … Continue reading
One year ago yesterday: volcanoes and fossils and elephant seals, oh my!
Our last day in Antarctica was filled with a few of my favorite things, and I wished at our last two stops, as I had at many, that we had lots more time to explore and soak in the scenic … Continue reading
One year ago today: Into the icy Weddell Sea and Antarctic Sound
One year ago today was a day of icy splendor, with plenty of penguins too. We've already shown you some of our photos from this portion of the trip, in our explainer on ice in a multitude of forms. Here's … Continue reading
Antarctica field log: Penguin Island? Surely you mean Volcano Island!
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A land of basalt and chalk
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Currently I have actually been to Miami on a regular basis, It's type of a 2nd residence for me. From my item of experience right here I would certainly such as to share a short quick recap of traveler destinations in Miami.
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A location constantly set upon with individuals, the South Coastline- South situated in the Southern part of Miami complied with by a beachfront roadway is constantly hectic with vacationers throughout the year. Managing the gold sands and also moving down sea drive is a life time experience. Simply wake up and also begin your early morning with fresh croissants as well as head on to the coastline to delight in amongst those swimsuit-clad groups.
It is one of the most historic locations that talks regarding just how Miami has actually been all these years. On the contrary, I have actually discovered that some of the Miami's deco structures typically aren't initial yet were repainted with white and also refined pastel trim for getting focus.
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Photo sharing instagram and video sharing apps Instagram is very familiar. About 30 corer users uploaded their happy moment's pictures or video at private photo sharing apps Instagram. But the size of the video or picture had been fixed by private photo sharing apps Instagram authority. The rules and regulations are that the uploading video and pictures must be squared. Now this rule has been smooth by the Instagram authority.
New feature has been added with new update of Instagram. As a result the portrait or landscape size photo can be uploaded by anyone. The specialist thinks that to attract various firms' Instagram authority brings this change so that the firms can easily upload their advertisement. The private photo sharing apps Instagram authority says about this update at their blog site. The main objective is that the user can easily upload their video or pictures at Instagram. It knows that the picture every five in one is not squared and we know that the size is not proper for the private photo sharing apps Instagram. It may happen that either delete the friend from picture or repel the video. To remove this problem all types of size Instagram accept. The blog post also says that for video graph it is the good news. Widescreen video gives more cinematic look that user upload at private photo sharing apps Instagram. Instagram was established in 2010 and from that time user can upload only square size file. They break this regulation in 2015. On the other hand in 2013 they started the option of video uploading. More than 15 seconds length video was not accepted by private photo sharing apps Instagram. But this facility is very helpful for various brand and firms. Now from this site they want to great feedback.


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As well as its enriching properties, Pureology Essential Repair Conditioner rebuilds and repairs hair and defends against damage with protein-rich soy milk and pure biotin.
Pureology Essential Repair Condition is formulated with AntiFadeComplex, maximising colour retention while protecting hair from thermal damage. The smell of jasmine, ylang ylang and rosewood relaxes and rejuvenates.
How to use Pureology Essential Repair Conditioner:
- Massage the conditioner tenderly into hair and scalp.
- Wait 1-2 minutes before rinsing.
- For best results use after shampooing with Essential Repair Shampoo.
FREE Standard UK delivery available on orders over £20.
Rush Shop ships internationally, as well as to the UK and Ireland. Services include:
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Royal Mail Standard Delivery service: 2 - 10 working days
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Royal Mail International Delivery Network: 5 - 21 working days
Please read our Delivery Policy for more information on International and UK delivery times before completing purchase.


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View from SquidLips, 8x10 Oil on Canvas Panel, Florida Scene
painting by artist Carmen Beecher
10 in X 8 in (25.4 cm X 20.3 cm)
Oil on Canvas Panel, Florida Scene
This painting is already sold. However, I may have other paintings available.
More Paintings by Carmen Beecher
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The Daily Painters Art Gallery - www.dailypainters.com
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Images may not be reproduced without the permission of the artist.


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My family of 5 stayed at the Embassy Suites Paloma Village as part of our spring break trip. We often stay at Embassy Suites and this was the best! While the room was not the biggest for an Embassy Suites, everything else was superb, from the breakfast to the staff to the reception. Our only regret is that we couldn't stay longer. It's location is convenient to many attractions in central or north Tucson (Sonora Desert Museum and Sabino Canyon are two we visited), be warned that south Tucson locations will require quite a drive. There are many upscale restaurants within walking distance of the hotel.
- Official Description (provided by the hotel):
- The new Embassy Suites Tucson's Paloma Village is nestled in the breathtaking foothills of the Catalina Mountains overlooking Tucson. Our upscale hotel features 119 suites. All are equipped with two 32-inch flat screen televisions, work desk, microwave, refrigerator and coffee maker. We offer complimentary breakfast and a nightly reception. We have a heated-pool, hot tub, state-of-the-art fitness and business center. Walk to a great variety of dining, spa facilities, art galleries and trendy shopping. ... more less
- Reservation Options:
- TripAdvisor is proud to partner with Hilton Hotels Direct, Expedia, Booking.com, Orbitz, Hotels.com, Priceline, TripOnline SA, Travelocity, Hotwire, Cheap Tickets, HotelQuickly, Cancelon and getaroom.com so you can book your Embassy Suites by Hilton Tucson Paloma Village 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:
- Embassy Suites Tucson Paloma Village Hotel Tucson
- Tucson Embassy Suites


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Very Easy Vogue 8136; ca. 1982; Misses' Skirt and Culottes. A line skirt one inch (2.5cm) below mid-knee or culottes, mid-calf length has front and back gathered into lined hip yokes with high waistline with or without belt carriers and side seam pockets. Purchased belts and tops.
Links to reviews/blog postsEdit
Add a photo to the gallery by clicking the "modify" button below.
[insert your username, and make sure your preferences allow for people to contact you via e-mail. Do not list your email address here!]


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It's nearly Halloween (Yikes! better get those sweets in), but strangely, this type of 'scary' has nothing to do with this blog.
In the summer I decided to try something new for my business. Having always focussed on designing training materials for sale, or delivering bite-size training sessions to specific businesses, I decided to run an 'open' course. I priced it well, gave lots of detail and promoted it heavily on facebook, LinkedIn, Twitter and via email.
It was a resounding failure.
Well, I say it was a failure, but actually if I look at it another way, it was quite successful. True, the workshop didn't run due to lack of participants, but I did get quite a lot of enquiries from people who were interested but found either the date or the location inconvenient.
This has resulted in organising more 'informal' events for smaller groups on dates to suit them, and I have picked up a new client as a result. I also sold the licence for the materials to a company in China who will translate them and run the workshop with a local trainer (it was completely impractical and not cost-effective for me to go over there to do it).
So my 'failed' attempt has not only resulted in the same amount of income as if it had succeeded, but it has also opened new doors for me. So if you run a small business, I would advise you to step out of your comfort zone and do something new and untested. It may succeed, it may fail. It may result in unexpected benefits, but either way, you'll be wiser for it.
Louise Gelsthorpe


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The Loft
Buy the book now – CLICK HERE
Annie Doyle believes the answer is yes, but will she find it in time?
Fifty years of memories are hidden in the walls of the loft. Now Ophelia Browne is leaving the house and she's leaving some very powerful memories behind. Annie needs to find just one… the one that will save Oliver's life.
On the day of their wedding, Annie sees only happiness ahead, but when an accident calls her and Oliver back to Memory House, her world is changed forever.
After only three nights in the loft, Annie must now find the single most meaningful memory in Oliver's mind. If she finds it in time, she can save his life, if she doesn't…well that's something she can't afford to think about.
Readers will welcome back the much-loved characters from Memory House and enjoy a few new friends!
ADVANCE PRAISE for THE LOFT, Book Two…
Crosby is one of those authors that does more than leave you with memories of her books, she leaves footprints on a reader's soul. The Loft is literature at it's finest. – Suzie, Bunny's Review
I love all of Bette's book – they are a great combination of sweet & happy and a little sorry/conflict. there are so many things to like about this book… – Leslie, StoreyBook Reviews
This book was amazing. Bette has a way of getting her characters to come to life in her books. – Mommysmoose
If you like a nice heartwarming book with a little bit of suspense, then read this book. – Rachel
Very lovely and heart warming read. I always find myself wanting to be friends with the characters in Bette's books. – Sandy
Literary Awards for The Loft –
Social Media Links:
Email: firstname.lastname@example.com
Find Author Bio, Author Interview, Author Pictures, Book Covers, Press Releases CLICK HERE
The Loft is in the hands of my Advance Reviewers
and I trust them implicitly to let me know what they think of my upcoming release, The Loft.
The Loft will be live September 15th! Check out all the reviews that will be posted by my advance reviewers wherever books are sold.
Also follow the hashtag #theloft on Twitter and Facebook for more updates.


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Solid Wood Crafts and Toys Collection
This multicolored marble tulip tree is provides six feet of fun for people of all ages. Put this marble tree in your play room as a decoration which people are able to have fun with! Watch as the marbles bounce from petal to petal all the way down to the base. The marbles are included in this fun toy so it is ready the minute you receive it.
Please call with questions on our American made wooden toys or order them online through our safe and secure checkout. This is a great educational toy for your child or grandchild.
© Copyrights 2003 - 2017 DutchCrafters All rights reserved.


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Decision makers
- Jack In the Box
Bring back the Bacon Cheddar Potato Wedges!!!
I don't think there is much debate as to why people are upset that the Bacon Cheddar Potato Wedges have mysteriously disappeared. What's important is getting them back! Sign the petition and hopefully Jack will realize that some decisions are just a mistake...
- Jack In the Box
A few weeks ago, the patrons of Jack in the Box were taken by surprise to discover the Bacon Cheddar Potato Wedges were yanked from the menu, only to be replaced with a poor (and I'm sure cheaper) substitute – French Fries!
This will not do. Many of us have been enjoying the cheesy, bacon-y goodness of the wedges for years and are appalled by the decision. Listen to the people, Jack…
Kim Harrison started this petition with a single signature, and now has 91 supporters. Start a petition today to change something you care about.
Report abuse
Please report any content that may violate Change.org's Terms of Service or Community Guidelines.
Thank you for taking the time to report content. Our team will review your claim and contact you if we need more information.


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This Forum has been archived
Forums: Admin Central Index → General Questions → How do I promote someone on my wiki to admin?
Central's forums are a place for the community to help other members.
To contact staff directly or to report bugs, please use Special:Contact.
To contact staff directly or to report bugs, please use Special:Contact.
Note: This topic has been unedited for 1596 days. It is considered archived - the discussion is over. Do not add to unless it really needs a response.
—This unsigned comment is by Joshualeverburg1 (talk • contribs) . Please sign your posts with ~~~~!
- Go to Special:UserRights on your wiki.--GodPray 03:45,1/17/2012
- You do want to make sure you only promote them to admin, and not to bureaucrat. Admin you can fire; bureaucrats can only be de-opped by Wikia staff. A common mistake new bureaucrats/founders make is to simply copy the user rights that they themselves have. When you give someone bureaucrat status, though, you're essentially making them your irrevocable equal. Though it doesn't happen often, this can then lead to your new bureaucrats promoting people of whom you don't approve, and before you know it, you've completely lost control of the wiki you've founded. So take care to give only those rights that you really need to give in order to maintain your wiki efficiently. See also Help:User access levels. czechout ☎ ✍ fly tardis <span style="">17:03: Tue 17 Jan 2012


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July 17th, 2017
Tyree Guyton benefit auction is now live on paddle8
Now is your chance to purchase a Tyree Guyton original! All proceeds from this auction will support The Heidelberg Project in Detroit, MI.
CLICK HERE to view the collection, this special benefit auction closes on Jul 31st at 5 PM ET.
February 1st, 2017
Tyree Guyton to take part in 2017 Monument Lab exhibition
Mural Arts Philadelphia announces the roster of 22 artists, hailing from Philadelphia and from locations around the world, who will create temporary public art in 10 locations across the city for Monument Lab this fall. This year's exhibition asks participating artists to answer the question "What makes a monument in the 21st Century?"
September 7th, 2016
Gold Street special edition print drops today via 1xrun!
Get yours now -- there are only 25 available of this special edition print by Tyree Guyton! Visit the Murals in the Market 2016 Print Suite via 1XRUN today at 12:00 p.m. EDT.
August 14th, 2016
An end, new beginning of the heidelberg project
After 30-years, Tyree Guyton announced that he will be dismantling the Heidelberg Project to make way for a new beginning. Read more via Detroit Free Press
July 25th, 2016
face-ology to open at inner state gallery
Opening August 13th at Inner State Gallery, Detroit's Tyree Guyton will celebrate the 30th anniversary of The Heidelberg Project with his latest solo exhibition Face-ology a career spanning showcase, featuring new works by the founder and creative force of the city's most iconic public art installation.
July 6th, 2016
Murals in the Market festival announces 50 artists
Tyree Guyton is to take part in the second annual Murals in the Market this September.
March 7th, 2016
The Heidelberg Project to celebrate 30 years in April
Read more via Black Magazine
January 28th, 2016
What Time Is It? Tyree Guyton, New Work Opens at DCDT February 5th
Join us at the opening reception for What Time Is It? Tyree Guyton, New Work on Friday, Feburary 5th from 5 p.m. - 7 p.m. at the Detroit Center for Design + Technology (DCDT) in Midtown. This exhibit, hosted by Lawrence Technological University College of Architecture and Design will be open February 5th - 26th at DCDT from 10 a.m. - 4 p.m. daily.
November 16th, 2015
Tyree Guyton to represent U.S. at Shenzhen Biennale of Architecture / Urbanism in China
August 1st, 2015


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Connecting Families to Senior Living
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You know your family, A Place For Mom knows Senior Living. Our dedicated local Bethesda, MD advisors have helped 3,366 families make the right choice for their needs. Get full details, pricing and read 334 reviews of our hand-picked communities. Typically prices range from $1500.00 to $792900.00 per month.
Bethesda in MD offers so many senior living options, how will you know what is the right fit for you? Our compassionate Senior Living Advisors are local experts who offer personalized assistance, financial resources and time-saving advice. We have helped families in the Bethesda Maryland area find the right senior living community options to match their unique needs and budget.
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Options For Senior America Corp. started providing home care services in late 1989 to the residents of the Washington, DC metropolitan area. What started as a handful of customers grew substantially due to the personal customer service that the company's management has always been keen to provide....
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Marshall used a combination of freshman Modupe Ishola and senior Catie Knable in limiting KSU's 6-6 center Andrea Csaszar to only 5 points in the game.
"It was just her (Ishola) presence," added Chadwick. "She played hard on both ends of the floor." Ishola finished the game with 7 points, 6 rebounds, and two blocks in 24 minutes of action.
Modupe Ishola looked for room around Kent State's Lindsay Shearer in MU's 56-51 win.
Kent State had a 31-27 lead at the half, but could never pull away from Marshall by any more than five points. In the second half, The Herd shot out to a six point lead at 41-35, but KSU went on an 8-0 run to go ahead at 43-41.
Kent State tied the game once again with 2:56 left in the game, but Sikeetha Shepard-Hall's three pointer with 1:23 proved to be the knockout punch that KSU never saw coming.
"That was two heavyweights going to-to-toe," quipped Chadwick of the slow-paced physical game. "We got the ball in the paint and our players did the right things with it."
Behind Ishola and Knable, Marshall out-rebounded Kent State by a whopping 49-31 margin, including 18 offense rebounds by Marshall. Sikeetha Shepard-Hall led Marshall in scoring with 13 points, with Ishola, KaShawna Curry, and Teyonka Hodge each hitting for 7 points each. Mary Pat Statler hit for 8 points, while Knable was off her normal pace with just 4 points, but did grab 9 rebounds.
Kent State All-MAC selection Andrea Csaszar was held to only 5 points, well off her 14.2 ppg scoring pace this season.
"Our responsibility was to beat her (Csaszar) up," said Marshall's Modupe Ishola, "and keep her out of the paint."
Marshall is now 9-3 overall and 1-1 in the MAC, while Kent State drops to 7-5 overall and 0-1 in the conference. Marshall next plays on Saturday afternoon at 3pm, facing the Ohio Bobcats at the Convocation Center in Athens, Ohio.


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Clarion Hotel and Conference Center near Busch Gardens
Grid of 40 images
Get an overview of this hotelHotel with 2 outdoor swimming pools, near University of South Florida
Popular property highlights
A stay at Clarion Hotel and Conference Center near Busch Gardens places you in the heart of Tampa, convenient to University Mall and University of South Florida.
This hotel is within close proximity of University of South Florida Botanical Gardens and Moffitt Cancer Center.
Make yourself at home in one of the 255 air-conditioned rooms featuring refrigerators and microwaves. Complimentary wireless Internet access keeps you connected, and cable programming is available for your entertainment. Private bathrooms with shower/tub combinations feature complimentary toiletries and hair dryers. Conveniences include safes and desks, as well as phones with free local calls.
Dip into one of the 2 outdoor swimming pools or enjoy other recreational amenities including a waterslide and a fitness center. Additional amenities at this hotel include complimentary wireless Internet access, wedding services, and a television in a common area. Getting to nearby attractions is a breeze with the complimentary area shuttle that operates within 3 mi.
Enjoy a satisfying meal at a restaurant serving guests of Clarion Hotel and Conference Center near Busch Gardens. Quench your thirst with your favorite drink at a bar/lounge. A complimentary hot/cold buffet breakfast is served daily.
Featured amenities include a 24-hour business center, complimentary newspapers in the lobby, and a 24-hour front desk. Planning an event in Tampa? This hotel has 10000 square feet (929 square meters) of space consisting of conference space and meeting rooms. Free self parking is available onsite.Information missing or incorrect? Tell us!Opens in a new window
Clarion Hotel and Conference Center near Busch Gardens
Hotel Amenities
Hotel Amenities
Clarion Hotel and Conference Center near Busch Gardens features 2 outdoor swimming pools, a waterslide, a fitness center, and a children's pool. The hotel offers a restaurant. A bar/lounge is on site where guests can unwind with a drink. Guests can enjoy a complimentary breakfast each morning. Public areas are equipped with complimentary wireless Internet access.
This hotel offers access to a 24-hour business center and meeting rooms. Event facilities measuring 10000 square feet (929 square meters) include conference space. This business-friendly hotel also offers multilingual staff, a picnic area, and coffee/tea in a common area. A complimentary area shuttle within 3 mi is available for guests. Complimentary self parking is available on site.
Clarion Hotel and Conference Center near Busch Gardens has designated areas for smoking.
- Total number of rooms - 255
- Number of floors - 2
- Elevator/lift
- Fitness facilities
- Designated smoking areas (fines apply)
- 24-hour front desk
- Luggage storage
- ATM/banking
- Multilingual staff
- Picnic area
- Restaurant
- Safe-deposit box at front desk
- Conference space size (meters) - 929
- Free RV, bus, truck parking
- Free self parking
- Bar/lounge
- Coffee/tea in common areas
- Free newspapers in lobby
- Free breakfast
- Conference space
- Waterslide
- Number of outdoor pools - 2
- Designated smoking areas
- Free WiFi
- Laundry facilities
- Meeting rooms
- Free area shuttle
- 24-hour business center
- Conference space size (feet) - 10000
- Children's pool
- Television in common areas
- Wedding services
Family Friendly Amenities
- Family Hotel
- Outdoor pools
- Children's pool
- Waterslide
- Free Wi-Fi
- Area shuttle (complimentary)
- Laundry facilities
- Refrigerator
- Microwave
- Cribs/infant beds (complimentary)
Available in all rooms: Free WiFi
Available in some public areas: Free WiFi
Free self parking, Free RV, bus, truck parking
Room Amenities
- Air conditioning
- Phone
- Free local calls
- Microwave
- Refrigerator
- Coffee/tea maker
- Private bathroom
- Hair dryer
- Shower/tub combination
- Iron/ironing board
- Desk
- Cable TV service
- Free WiFi
- In-room safe
- In-room climate control (air conditioning)
- Free toiletries
- Free cribs/infant beds
- Rollaway/extra beds (surcharge)
- Connecting/adjoining rooms available
- Television
Where to Eat
A complimentary hot/cold buffet breakfast is served each morning between 7 AM and 10 AM.
Clarion Hotel and Conference Center near Busch Gardens has a restaurant serving breakfast and dinner.
Nearby Things to Do
2 outdoor swimming pools are on site along with a children's pool. Other recreational amenities include a waterslide and a fitness center.
If you have requests for specific accessibility needs, please note them at check-out when you book your room.
- Accessible bathroom
- Roll-in shower
Hotel Policies
Check-in time starts at 4 PM
Check-in time ends at 2 AM
Special check-in instructions:
Check-out time is 11 AM
Payment types
Children and extra beds
- Children are welcome.
- Kids stay free! Children 18 years old and younger stay free when using existing bedding.
- Rollaway/extra beds are available.
- Free cribs (infant beds)!
- Pets allowed for an extra charge of USD 30 per night
- 1 per room (up to 35 lb)
You need to know
Extra-person charges may apply and vary depending on property policy.
Government-issued photo identification and a credit card or cash deposit are required at check-in for incidental charges.
Special requests are subject to availability upon check-in and may incur additional charges. Special requests cannot be guaranteed.
Service animals exempt from fees/restrictions
No rollaway/extra beds available
This hotel will place a credit card or debit card authorization for $100.00 USD for incidentals upon guest arrival. For more details, please contact the office using the information on the reservation confirmation received after booking.
- Deposit: USD 100.00 per stay
Optional extras
- Pet fee: USD 30 per night
- Rollaway beds are available for an additional fee
Hotel Name
- Clarion Busch Gardens
- Clarion Hotel Busch Gardens
We should mention
Children 18 years old and younger stay free when occupying the parent or guardian's room, using existing bedding. The property has connecting/adjoining rooms, which are subject to availability and can be requested by contacting the property using the number on the booking confirmation.


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Tech survivors rally for gun control
By Katherine Coates, Capital News Service 01/19/11
Armed with duct tape, Colin Goddard balanced on a chair to hang a screen so people could watch a film at the Richmond YWCA gymnasium.
Goddard appears to be fairly athletic. But four years ago, he was barely able to do one spin on a bicycle because of gunshot injuries he suffered during the shooting rampage at Virginia Tech on April 16, 2007.
Goddard and others gathered in Richmond this week to share their thoughts about gun violence as "Living for 32" – a documentary about the Tech massacre – was screened at the YWCA.
Goddard was one of the people who survived when a fellow Virginia Tech student, Seung-Hui Cho, entered their classroom and began shooting. Cho, who had a history of mental health problems, killed 32 people before killing himself.
Since then, Goddard and other survivors have become activists for gun control.
The film screening in Richmond was organized Monday by the Virginia Center for Public Safety. Colin Goddard's father, Andrew Goddard, is president of the nonprofit group.
Gun control is the subject of debate in the General Assembly as the 2011 legislative session gets under way. It's also a national issue after a gunman killed six people and wounded a dozen, including U.S. Rep. Gabrielle Giffords, in Arizona on Jan. 8.
Several bills before the assembly would relax existing firearms laws. Some would make it easier to get a permit to carry a concealed weapon, prohibit the state from restricting firearms or exempt guns made in Virginia from federal regulation. The Virginia Center for Public Safety opposes such measures.
The center supports bills that would provide more gun control. One measure, for example, would require criminal background checks before people can buy firearms at gun shows. Other would ban firearms from libraries, the Capitol and the General Assembly Building.
"They have to keep guns out of the State Capitol," Andrew Goddard said. "We don't want consequences."
To underscore how lax existing laws are, Colin Goddard goes to gun shows and demonstrates that he can buy weapons with cash – and without an ID or background check.
The film screening at the YWCA drew an audience of concerned citizens and victims and survivors of gun violence.
Omar Samaha, a Virginia Tech graduate, joined Colin Goddard in presenting the film and answering questions from the audience.
Samaha's sister, Reema Samaha, was killed during the Virginia Tech shooting. Samaha now works with Students for Gun Free Schools, a grass-roots campaign to ban concealed weapons from college campuses. The campaign was started in honor of Samaha's sister.
"Students feel they need to carry because they don't feel safe ... it is a variety of social issues," Samaha said.
The film recalls events that happened nearly four years ago. For many in attendance, the emotions were as raw as if the ordeal happened yesterday.
Alex Evans was the chaplain for the Blacksburg police and pastor at Blacksburg Presbyterian Church. He was present the day of the shootings and said the film carried him back to the day.
"I felt a very emotional response," Evans said. "But also very logical and practical. [Colin Goddard] is calling on us to improve our law and society."
Goddard says he is not anti-gun; however, he believes society needs more control over violent weapons.
"Living for 32" will premiere at the Sundance Film Festival in Utah at the end of January and at Virginia Tech in February. Students can request the film to be shown at their university.
To learn more about "Living for 32," visit the documentary's website, http://livingfor32.com/
'It's such a blessing'
By Ashlee Korlach, Special to the Citizen 07/24/2017 FeaturesGeneral News
On Independence Day, a Brazilian-born Henrico resident took the Naturalization Oath of Allegiance on the steps of the Virginia Historical Society and felt peace finally settle around her.
Jeanette Hamlett's journey to U.S. citizenship is a story of faith and love, which she told with her husband, John Hamlett, by her side. > Read more.
Preschoolers give pillows to families in need
By Ashley Luck, Special to the Citizen 07/24/2017 FeaturesGeneral News
A group of preschoolers from Chesterbrook Academy Preschool donated 56 pillows to local families in need at the Housing Families First shelter. The shelter is always in need of pillows and linens as, since families take the donated ones with them to their new homes.
Housing Families First provides shelter and support for homeless families and assist them in finding a permanent housing solution. The shelter serves people year-round, said Terri Iguina, operations and volunteer manager at Housing Families First. > Read more.
Dairy Queen's Blizzard sales July 27 to benefit Children's Hospital of Richmond
Citizen Staff Reports 07/24/2017 BusinessGeneral News
Dairy Queen's 13th Annual Miracle Treat Day – Thursday, July 27 – will raise fund to benefit sick and injured children being treated at Children's Miracle Network hospitals throughout the United States. Locally, $1 or more from every Blizzard Treat sold at participating locations will be donated to the Children's Hospital of Richmond at VCU. Last year, the event raised more than $14,400 for the hospital. > Read more.
Crime Stoppers' Crime of the Week: July 24, 2017
Citizen Staff Reports 07/24/2017 General NewsPublic SafetyCrime of the Week
Crime Stoppers needs your help to find the person who struck a pedestrian in the City of Richmond.
On July 21 at 12:52 a.m., a woman was crossing the street at Forest Hill and Sheila Lane when she was struck by a dark colored four-door sedan that was traveling eastbound on Forest Hill. She was transported by ambulance to Chippenham Hospital for minor injuries and released. > Read more.
Henrico Police locate missing man
Citizen Staff Reports 07/24/2017 General NewsPublic Safety
Henrico Police have located a missing 46-year-old Henrico man.
Police had reported Kevin William Cannelli missing this weekend, after he was last seen July 18. He was located safe in the Richmond area July 24. > Read more.
Police had reported Kevin William Cannelli missing this weekend, after he was last seen July 18. He was located safe in the Richmond area July 24. > Read more.
Henrico Police locate missing man
Business in brief
Mother, son work to raise funds for Make-A-Wish
Eyes in the sky
Rock on!
- More News
Print Edition
Jul. 20, 2017Current edition | Archive
Restaurant Watch
Weekend Top 10
'Five Flights' opens July 20 at CAT Theatre
Henrico actress lands role in Christian TV series
All's fair in food and fun
- More Entertainment
Obituary – Hoyle S. "Chick" Walters
Obituary – Janet Sue Vest
Obituary – Barbara Jean (Chilton) Crider
- More Obituaries
Glen Allen woman raising companion dog
Guided tour of Malvern Hill planned as part of 'History at Sunset' series
Anthem LemonAid registration now open
- More Community
Growing a grown-up: Part II
How to grow an adult
Make 'em take the pledge: no more snuff boxes!
- More Opinions
Code Lacrosse joins Richmond Strikers
5 Henrico student-athletes win scholarships at awards dinner
RIR to host playoff race in 2018
- More Sports
|July 2017
Place an Ad | More Classifieds
CalendarSPARC's teen summer musical "Oliver!" will be at the Henrico Theatre at 7:30 p.m. July 28-29 and at 2 p.m. July 29-30. The award-winning musical adaptation of the classic Dickens' novel springs to life with some of the most memorable characters and songs ever to hit the stage. Book, music and lyrics by Lionel Bart. Tickets are $14. The July 20 performance will be sign-interpreted. For details and to purchase tickets, visit http://www.sparconline.org/tickets. Full text


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I ate at Kelly's Roast Beef in Revere several years ago, and remember liking it (I had fried clams), but haven't been back since. I'm curious about how the food at the original location compares to other branches (Medford, Allston, etc). Are there noticeable differences in terms of the food quality between Revere and elsewhere?
I just read this thread, and people seem to indicate that the food is pretty comparable at all locations....
Is this still the case?
Dave MP


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Employment Opportunities in the College of Engineering and Applied Sciences
The College of Engineering and Applied Sciences is one of nine schools and colleges within the University at Albany SUNY (UAlbany). Currently, UAlbany is making a major investment in building engineering degree programs, and the College of Engineering and Applied Sciences is in an exciting period of rapid expansion. The college is also the home of the recently established Institute for Advanced Data Analytics.
Strategically located in the state capital of New York, UAlbany is an internationally recognized public research institution that brings "The World Within Reach" to more than 17,300 students at the graduate and undergraduate levels. UAlbany is close to the metropolitan centers of New York City, Boston, and Montreal as well as centrally located between the Catskills and the Adirondacks; the region provides abundant opportunities for outdoor recreation.
The College of Engineering and Applied Sciences is in an exciting period of rapid expansion as the new department in environmental and sustainable engineering will join our long-standing programs in computer science and information science, with programs in electrical and computer engineering just launched. This is just the beginning; programs in several other engineering disciplines are currently in development. The College has also launched a dual degree program in Computer Science with Chongqing University of Posts and Telecommunications in China. Please read more about current employment opportunities in Computer Science, Electrical and Computer Engineering, Environmental and Sustainable Engineering, and in the Institute for Advanced Data Analytics below.
For additional information about the College and its departments, please visit: http://www.albany.edu/ceas/.
Positions available
Computer Science, Full-time Professors of Practice and LecturersBeginning Fall 2017. Positions are three-year, renewable, 10 month appointments. Successful applicants will teach in the program core both at UAlbany and, every second or third semester, at Chongqing – with additional compensation and all expenses covered. A variety of courses must be covered including, but not limited to, programming and data structures, software engineering, programming languages, compiler design, operating systems, databases, data analytics, discrete math, and related topics.
For the title of Professor of Practice (Lecturer), a minimum Ph.D. (MS) degree in Computer Science or a related discipline from a college or university accredited by the U.S. Department of Education or an internationally recognized accrediting organization is required. For a complete job description and application procedures, visit: https://albany.interviewexchange.com/jobofferdetails.jsp?JOBID=80792&CNTRNO=2&TSTMP=1487264140647
Questions regarding the position may be addressed to email@example.com.
Electrical and Computer Engineering, Open Rank FacultyBeginning Fall 2017. Areas of particular interest include, but are not limited to, hardware and circuit design, control systems, communications, electromagnetics, RF systems, or energy sources and systems.
Applicants must have a PhD in Computer Engineering, Electrical Engineering, or a closely related discipline. For a complete job description and application procedures, visit: https://albany.interviewexchange.com/jobofferdetails.jsp?JOBID=80892&CNTRNO=3&TSTMP=1487264140647
Questions regarding the position may be addressed to firstname.lastname@example.com.
Environmental and Sustainable Engineering, Professor and ChairBeginning July 2017. The successful candidate will have an established record of scholarship and research with demonstrated potential to guide the growth and development of the department faculty and its programs, BS through PhD, in environmental and sustainable engineering at the University at Albany.
Applicants must have a PhD in Environmental Engineering, Civil Engineering, or a closely related discipline. For a complete job description and application procedures, visit: https://albany.interviewexchange.com/jobofferdetails.jsp?JOBID=81280&CNTRNO=0&TSTMP=1487347234292
Questions regarding the position may be addressed to firstname.lastname@example.com.
Founding Director: The Institute for Advanced Data AnalyticsBeginning Fall 2017. The Director will be responsible for leveraging the University's deep assets in advanced data analytics into a nationally and internationally recognized Institute. The successful candidate will have an international reputation as both a data scientist and a leader in building novel multi-institutional, multidisciplinary, funded research collaborations, will be able to demonstrate strong visionary and inspirational leadership skills, will be an effective and creative communicator able to convey the Institute's mission to multiple diverse audiences, and will have demonstrated success in fundraising and/or entrepreneurial revenue generation. The position will include a faculty appointment in the College of Engineering and Applied Sciences, possibly joint with another discipline closely related to the scientific mission of the Institute. The successful candidate will have a track record commensurate with the academic rank of full Professor in his or her primary field.
Required qualifications for the Director of the Institute of Advanced Data Analytics are; a doctoral degree in computer science, engineering, or another discipline closely related to the core scientific mission of the Institute; demonstrated history of creating and organizing interdisciplinary teams with a computational component; advanced oral and written communication skills to reach diverse audiences; successful experience in connecting and engaging government agencies in large funding opportunities including fundraising and other entrepreneurial revenue generation; evidence of scholarly accomplishments such as refereed publications, technical reports, books and grantsmanship appropriate for appointment at the rank of full Professor at a research-intensive university. For a complete job description and application procedures, visit: https://albany.interviewexchange.com/jobofferdetails.jsp;jsessionid=B75532E406C1EBEEA4CCD96DDB27485F?JOBID=81492
Questions regarding the position may be addressed to email@example.com.


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Genre : Drama, Comedy, Music
Release Date : May 15, 2015
Duration : 115 minutes
Country : United States
Language : English
- Anna Kendrick
- Rebel Wilson
- Hailee Steinfeld
- Brittany Snow
- Skylar Astin
- Adam DeVine
- Katey Sagal
- Anna Camp
- Alexis Knapp
- Hana Mae Lee
- John Michael Higgins
- Elizabeth Banks
After the Barden Bellas' performance on President Obama's birthday disastrously ends with Fat Amy's (Rebel Wilson) awry airlifting solo, they are suspended from performing in any a cappella showcase. The Bellas' leader, Beca Mitchell (Anna Kendrick), makes a deal to allow the Bellas be reinstated if they win the Worlds a cappella tournament. Meanwhile, Beca secretly starts a recording studio internship that only her boyfriend, Jesse Swanson (Skylar Astin), knows about. Beca impresses her boss with her mashup skills for Snoop Dogg's Christmas album and is given a chance to become a producer. Fat Amy soon finds out and advises Beca to tell the other Bellas. Freshman Emily Junk (Hailee Steinfeld) stops by the Bellas' house for an audition and succeeds when the Bellas learn that Emily's mother, Katherine Junk, was once a Bella. That night, the Bellas have a party with the Treblemakers, where Benji (Ben Platt) awkwardly develops a crush towards Emily. The Bellas later attend a car show which they could have performed at, to meet their replacers "Das Sound Machine" (DSM) – led by intimidating duo Pieter Krämer (Flula Borg) and Kommissar (Birgitte Hjort Sørensen). They then attend an exclusive riff off, where they lose against DSM in the final round when Emily tries to sing her own song. (From : Wikipedia.org)
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Listing information
- Beds: 3
- Stories: 2
- Foreclosure: No
- Lot Area: 6.40
- County: Warren
- Body of Water: None
- Style: Contemporary
- Baths: 3 (Full: 2 3/4: 0 1/2: 1 Other: 0)
- Parking Type Description: None 0 cars
- Total Square Footage: 2,624 ($152.06 per sq. foot)
- Lot Size: 353.95x1021.85x240.07x1281.82
- High School: Hadley/Luzerne
- Subdivision: None
- Year Built: 2010
- Status: Active with Contingency
- New Construction: No
- Above Ground Sq. Feet: 2,624
- Estimated Annual Taxes: $6,143
- Elementary School: Hadley/Luzerne
Appliances Included Dishwasher, Microwave, Refrigerator, Stove - Gas
Attic Floored
Attic Access Crawl Opening
Basement Exterior Entry, Full, Heated, Interior Entry, Walk Out
Cooling Type Window Units
Custom Features Generator - Automatic
Dining Features Great Room
Documents Deed, Deed Restrictions, Survey
Exterior Feature Deck, Porch, Stone Drive, Underground Utilities
Exterior Finish Vinyl Siding
Flooring Carpet, Ceramic Tile, Hardwood, Tile
Garage No Garage
Heat Distribution H/W Baseboard
Heat System Fuel Propane
Interior Features Ceiling Vaulted, Dry Wall, Dryer Connection, Fireplace - Wood, Granite Countertop, Internet - Cable, Internet - DSL, Other Interior Features, Smoke Alarm, Walk-in Closets, Washer Connection
Kitchen Features Custom, Island, Modern, Pantry
Land Features Garden Space, Landscaped, Road Frontage, Secluded Lot, Stream on Property
Other Amps - 200, Main Road Frontage - 353.95, Water Type: None - Stream/Brook
Possession At Closing
Roof Composition Shingle
Sewer Septic Tank/Leach Field
Style Contemporary
To Be Built No
Water Well Drilled
Water Heater Propane
Waterfront Lake/River View, Pond/Stream
Zoning Residential
Bedroom 2 2
Bedroom 3 2
Dining Room 1
Kitchen 1
Laundry Room 1
Living Room 1
Master Bedroom 1
Listing Courtesy of Davies-Davies & Associates Real Estate, Llc