The large blocks of contiguous forest in and around KSNP have bee

The large blocks of contiguous forest in and around KSNP have been designated as a ‘Level 1 Tiger Conservation Landscape’, because they are considered to provide one of the best chances for the long-term survival of tigers (Dinerstein et al., 2007). Nevertheless, tigers living in the KS region are threatened,

principally by loss of habitat, and then by poaching of tiger and prey. Deforestation (i.e. complete forest conversion to farmland) has fragmented KSNP into two parts and deforestation rates from 1995 to 2001 were 34.6 km2/year (0.28%/year) inside KSNP and 213.1 km2/year (0.96%/year) across the KS region (Linkie et al., 2008b). Camera-trap field data for tiger and their presumed prey were collected from four study areas from 2004 to 2007: (1) Renah Kayu Embun – 26 camera traps set from 3 September to 30 November 2004 in 112 km2 ranging from 947 to 1941 m a.s.l. located in Jambi province; (2) Sipurak – 28 camera traps set from 3 January to 29 March 2005 in 88 km2 ranging from 694 to 1254 m a.s.l. located in Jambi province; (3) Bungo

– 32 camera traps set from 16 April to 23 November 2006 in 237 km2 ranging from 363 to 1745 m a.s.l. located in Jambi province; (4) Ipuh – 40 camera traps set from 24 August 2006 to 2 May 2007 in 569 km2 ranging SB203580 ic50 from 194 to 1064 m a.s.l. located in Bengkulu province. These four study areas were selected 5-FU because of their presumed importance for tiger, for which the KSNP management authority had requested detailed information. A combination of TrailMaster and Photoscout passive infrared camera traps, activated by a heat-motion sensor, was used. Cameras were set along ridge trails and medium-large bodied animal trails, as identified through the presence of tiger sign. Cameras were checked every 2 weeks and their films replaced. To investigate the temporal tiger–prey activity patterns, photographs that were recorded within 30 min of a previous photograph of the same species and at the same camera placement were not used, because they were not

considered to be independent. The remaining data were regarded as a random sample from the underlying distribution that describes the probability of a photograph being taken within any particular interval of the day. The probability density function of this distribution was then referred as the activity pattern, which presupposes that the animal is equally likely to be photographed at all times when it is active (Ridout & Linkie, 2009). A two-step procedure for quantifying the extent of overlap between two activity patterns, based on a sample from each species, was performed. For the first step, each activity pattern was estimated separately, either non-parametrically, using kernel density estimation or by fitting a distribution from the flexible class of non-negative trigonometric sum distributions (Fernández-Durán, 2004).

01, and 6 4 ± 1 in Pkd2cKO mice treated with 60 mg/kg/daily, P <

01, and 6.4 ± 1 in Pkd2cKO mice treated with 60 mg/kg/daily, P < 0.01) (Fig. 1C). Consistent with the increase in liver cysts, the liver/body weight ratio of Pkd2cKO mice was also significantly higher in sorafenib-treated animals (Pkd2cKO vehicles: 0.058 versus 0.0762 in mice treated with 20 mg/kg/day, P < 0.01, and 0.079 in mice treated with 60 mg/kg/day, P < 0.01) (Supporting Fig. 1). Previous studies have shown that the growth of liver cysts is dependent upon an increased

proliferation and a decreased apoptosis of cystic cholangiocytes.7, 8, 21 Consistent with the increased volume of liver cysts, the immunohistochemical expression of Ki67, a nuclear antigen present JQ1 supplier only in the nuclei of proliferating cells,22 was significantly selleck products increased in mice treated with sorafenib (Pkd2cKO vehicles: 6.8 ± 1% versus 11 ± 2% in Pkd2cKO mice treated with 20 mg/kg/day, P < 0.01, and 10.5 ± 2.1 in Pkd2cKO mice treated with 60 mg/kg/day, P < 0.01) (Fig. 2A). Apoptosis was assessed by measuring the immunohistochemical expression of CC3.7, 8 The number of CC3-positive cells in the liver cyst epithelium was significantly decreased in mice treated with sorafenib (Supporting Fig. 2) (Pkd2cKO vehicles: 11.0 ± 0.8% versus 8.2 ± 0.8% in Pkd2cKO mice treated with 20 mg/kg/day, P < 0.01, and 7.9 ± 0.7 in Pkd2cKO mice treated with 60 mg/kg/day; P <

0.01). These data suggest that sorafenib increases liver cyst growth through increased cell proliferation and decreased apoptosis in the liver cystic epithelium. Cyst proliferation in Pkd2cKO mice is sustained by a PKA-dependent Raf/MEK/ERK1/2 pathway.7 ERK1/2 is downstream of Raf and therefore should be inhibited by sorafenib. On the contrary,

the expression of phosphorylated ERK1/2 (pERK1/2) was significantly increased in cholangiocytes lining the cysts in mice treated with sorafenib, with respect to untreated Pkd2cKO mice (Pkd2cKO vehicles: 3 ± 0.7% versus 4.9 ± 1.1% in Pkd2cKO mice treated with 20 mg/kg/day, P < 0.01, and 5.2 ± 1 in Pkd2cKO mice treated with 60 mg/kg/day; P < 0.01) (Fig. 2B). No differences in the percentage of pERK1/2 positive hepatocytes were observed (Pkd2cKO vehicles: 2.2 ± 0.8% versus 2.8 ± 0.97% in Pkd2cKO mice treated with 20 mg/kg/day, P value not significant). These data suggest that increased proliferation in cystic Rutecarpine cells in sorafenib-treated Pkd2cKO mice is a consequence of increased ERK1/2 signaling. In apparent contrast to our in vivo data, Yamaguchi et al.23 reported that sorafenib inhibits ERK1/2 activation and cell proliferation in kidney cells isolated from cysts of ADPKD patients. To clarify whether sorafenib has inhibitory effects on isolated PC2-defective cholangiocytes, we measured cell proliferation (by MTS and BrdU assays) and the levels of phosphorylated ERK1/2 in cholangiocytes isolated from normal controls and from liver cyst epithelial cells of Pkd2cKO mice, as described.

This was suggested by the known interaction with the KRTK motif o

This was suggested by the known interaction with the KRTK motif of thrombospondin, the major activator of TGF-β in vivo,40 leading us Pirfenidone ic50 to propose that the KTFR sequence could play a similar

role in ADAMTS1 (Fig. 3). The inhibition of ADAMTS1-mediated activation of TGF-β by KTFR peptides indicates that the mechanism of interaction is similar—if not identical—to that reported for thrombospondin-mediated activation.24 In this case, a “conformational” mechanism is in full agreement with the results of our experiments using proteolysis-deficient ADAMTS1 mutants and protease inhibitors, which show that TGF-β activation by ADAMTS1 is independent of the proteolytic activity of the latter. Although it might occur via similar interactions, activation of TGF-β by ADAMTS1 and thrombospondin are unlikely to overlap in vivo. Thrombospondin is expressed in freshly isolated human HSCs,22 but we show that their activation induces a decrease in thrombospondin expression MG-132 research buy counterbalanced by a dramatic increase in ADAMTS1 expression. The physiological relevance of this activation process is fully supported by our finding that depleting ADAMTS1 in HSCs strongly diminishes the release of TGF-β-dependent transcriptional activity. A conformational model of TGF-β

activation by ADAMTS1 predicts that interfering with KTFR/SLKL interactions should lead to a decrease Terminal deoxynucleotidyl transferase in available active TGF-β. We demonstrate that such a mechanism is, indeed, at play in vivo, using a murine model of induced liver fibrosis. In full agreement with the activation pathway described above, injection of the KTFR peptide in CCl4-treated mice that develop liver fibrosis reduces the levels of biological markers associated with hepatic damage (Fig. 7). This conclusion is further borne out by the demonstration, using highly sensitive SHG analysis,41 that concomitant injection of KTFR dramatically reduces collagen deposition

associated with the early onset of fibrosis (Fig. 8). The full conservation of KTFR and LSKL motifs between humans and mice suggests that this important proof of concept can be extrapolated to humans, identifying ADAMTS1 as a new therapeutic target during chronic liver injury. Many factors have been implicated in TGF-β activation, including thrombospondin, proteases (e.g., plasmin, thrombin, and MMP), and integrins, but also heat, acid, reactive oxygen species, and mechanical force. All these components build up an environmental network, in which the role of each one is obviously part of the sum and depends on dynamic tissue changes, especially as the liver proceeds from a healthy to a fibrotic state.

We demonstrated a decrease of TSLC1 protein in ≈80% of HCCs, sugg

We demonstrated a decrease of TSLC1 protein in ≈80% of HCCs, suggesting its involvement in the tumorigenic process of human HCCs. One miRNA usually affects several target genes, thus other genes besides TSLC1 could also be affected by miR-216a and contributing to the increase of cell proliferation and migration activities. For example, the AMOT, MTSS1,

INTS7, and IL2RG genes (as revealed by the microarray analysis of Table 2S) could also be the putative targets for miR-216a and worthy to be investigated. The mechanisms for the decrease of TSLC1 have been extensively investigated in many Poziotinib in vitro tumors that were caused either by allelic loss or by promoter methylation.23-25 Because our current study demonstrated that the elevation of miR-216a, through its putative target sites at the 3′ UTR of the TSLC1 gene, could decrease its protein expression, it thus provided a novel mechanism for the decrease of this important tumor suppressor gene in early hepatocarcinogenesis. No significant change in the mRNA levels of TSLC1 in most HCCs has been supported further by the microarray analysis released by the oncoHCC database.26 Thus, it argued against the promoter methylation. Intriguingly, we noted that a similar situation for the decrease of TSLC1 protein levels, independent of promoter methylation, was also reported in neuroblastoma

and bladder cancers.27, 28 The contribution of a miR-216a-mediated PI3K Inhibitor Library clinical trial decrease of TSLC1 in these tumors warrants further investigation. Although it is well documented that the androgen Anacetrapib pathway is involved in hepatocarcinogenesis,13, 20, 21 the target genes affected by

this pathway remain largely unidentified. Recently, Feng et al.29 identified the cell-cycle-related kinase as one putative target gene activated by the androgen pathway and contributing to male HCC. In addition to the protein coding gene, our current study suggested that AR is able to regulate the transcription of specific miRNAs, indirectly affecting the genes with the potential for tumorigenesis. As we noted, the androgen pathway was reported to regulate the expression of several miRNAs in the prostate cancer cells, including miR-141,30 miR-125b,31 and miR-21,32 which all showed a functional effect on the carcinogenic process. Whether the precancerous liver tissues of male HBV patients also affect these miRNAs awaits further clarification. The results from the current study warranted initiating nonbias genome-wide approaches for identifying the whole spectrum of miRNAs regulated by the androgen pathway in hepatocarcinogenesis. We thank the Taiwan Liver Cancer Network (TLCN) for providing the hepatocellular carcinoma tissue samples and related clinical data (all are anonymous) for our research work. This network currently includes five major medical centers (National Taiwan University Hospital, Chang-Gung Memorial Hospital-Linko, Veteran General Hospital-Taichung, Chang-Gung Memorial Hospital, Kaohsiung, and Veteran General Hospital, Kaohsiung).

7 versus WM: 2,775 ± 492 5, n = 5, P < 0 05; and Fig 4C, liver h

7 versus WM: 2,775 ± 492.5, n = 5, P < 0.05; and Fig. 4C, liver histology). To confirm that Gsk3β inactivation functioned downstream of PI3 kinase activation, SB216763 and wortmannin were administered in concert prior to the ischemia insult. Gsk3 inhibition remained hepatocytoprotective against IRI in the presence of PI3 kinase inhibition (Fig. 4D, sALT: Ctl, 7,825 ± 583.9 versus SB, 3,511 ± 809.0; P < 0.01; WM, 8,863 ± 826.9 versus SB/WM, 3,069 ± 741.7; P < 0.01). Thus, PI3 kinase-dependent Gsk3β phosphorylation serves as a self-regulatory mechanism of liver homeostasis

to limit the excessive IR-triggered tissue damage. It has been well established that TLR4 activation is the key step in liver inflammatory immune response against IR.5, 9 To investigate the cellular mechanism of our in vivo findings, we analyzed the effects of Gsk3 inhibition in macrophage Apoptosis inhibitor response to TLR4 stimulation in vitro. Bone marrow-derived macrophages were stimulated with LPS in the absence or presence of SB216763. As shown in Fig. 5A, Gsk3 inhibition significantly reduced IL-12p40 and IL-1β, but increased IL-10 gene induction

at 1 hour of culture. In contrast, the induction of TNF-α, IL-6, and CXCL10 were unaffected at this early timepoint. By 6 hours, whereas the IL-12p40 expression remained lower, levels of TNF-α, IL-6, IL-1β, and CXCL10 all became significantly reduced. IL-10 levels were comparable between the two groups. Gsk3 inhibition did not alter LPS-induced MAP kinase activation, as the phosphorylation kinetics of JNK, Erk, and p38 were similar in control and SB-treated macrophage cultures (Fig. 5B). The disparities between IL-12/IL-10 and TNF-α/CXCL10 genes at the timepoints

regulated by the Gsk3 inhibition indicated a possible difference in their regulatory mechanisms, i.e., the early regulated genes were the primary targets of Gsk3, whereas the later ones were regulated by the primary gene products. To test whether IL-10 may represent such a primary gene regulating the late inhibition of TNF-α/CXCL10 induction, we added anti-IL-10 Ab in SB216763-treated macrophage cultures. RAS p21 protein activator 1 Indeed, LPS-induced TNF-α/CXCL10 levels at 6 hours, which remained diminished by Gsk3 inhibitor alone, became restored (or even enhanced) after adjunctive anti-IL-10 Ab and SB216763 (Fig. 5C). Interestingly, anti-IL-10 Ab restored otherwise suppressed IL-12p40 gene induction by SB216763. Thus, Gsk3 inhibition regulates macrophage TLR4 response by directly down-regulating the pro-inflammatory IL-12 gene, yet up-regulating the induction of immune regulatory IL-10, which, in turn, further suppresses the pro-inflammatory gene expression programs. Although Gsk3β has been shown to regulate macrophage cytokine production and hepatocyte apoptosis,12, 21 its role in liver IRI cascade, an inflammation-mediated hepatocellular injury process, has not been explored.

An ice pack can be applied to the injection area for 5 min before

An ice pack can be applied to the injection area for 5 min before injection. The smallest gauge needle available (usually 25–27 gauge) should be used. Pressure should

be applied to the injection site for at least 5 min [18]. Live virus vaccines (such as oral polio vaccine, MMR) may be contraindicated in those with HIV infection. People with hemophilia who have HIV should be given pneumococcal and annual influenza vaccines. Immunization to hepatitis A and hepatitis B is important for all persons with hemophilia. These immunizations may not be as effective in those with HIV infection. (Level 4) [ [19, 20] ] Patients and their families should be provided with psychological and social support [21, 22]. Hemophilia is also a financial burden that places restrictions on several aspects of normal living [23]. The PLX3397 social worker and/or

other members of the comprehensive care team should: provide as much information as possible about the physical, psychological, emotional, and economic dimensions of hemophilia, in terms the patient/parents can understand. be open and honest about all aspects of care. allow the patient/parents to work through their emotions and ask questions. Provide care and support patiently. talk to affected children, not just their parents. Children can often understand a good deal about their illness and can work with the physician if properly informed and educated. buy Dabrafenib remind parents Glutamate dehydrogenase not to ignore siblings that are healthy. be able to recognize warning signs of burnout and depression, which are common with chronic illness, and provide suggestions for coping. recognize that cultural background may affect patients’ views

of illness. encourage patients to engage in productive and leisure activities at home and in the workplace. work in partnership with the patient organization to advocate for hemophilia care and to provide education to families and members of the community. enlist the assistance of local groups and organizations where social workers are unavailable. Patients with hemophilia can have normal sexual intercourse [24]. Muscle bleedings (e.g., iliopsoas) may sometimes be the result of sexual activity. Complications of hemophilia can be accompanied by sexual dysfunction, which may include lack of libido or impotence. Pain or fear of pain may affect sexual desire, and hemophilic arthropathy may place limitations on sexual intercourse. Sexuality is also affected by chronic HCV and HIV infection, age-related diseases like hypertension and diabetes mellitus, and certain medications. In some cases, oral phosphodiesterase-5 inhibitors (sildenafil, tadalafil) may be helpful. These medications mildly inhibit platelet aggregation in vitro, and may cause epistaxis due to nasal congestion. Aging patients with hemophilia will inevitably suffer from age-related diseases [24, 25].

62–64 RAGE’s interaction with DAMPs also results in activation of

62–64 RAGE’s interaction with DAMPs also results in activation of p38 SAPK, the transcription factors STAT-3 and AP-1.62,63 Intriguingly, animals treated with extracellular ligand binding domain of RAGE (sRAGE) displayed increased survival after total hepatic IR.62 Moreover, the remnants of sRAGE treated livers revealed diminished activation of JNK, STAT3 and NF-κB.62 Since the author’s 2003

review in the Journal, many advances have been made in elucidating the mechanisms underlying hepatic IR injury.23 These include clarification of interactions between different cell types, a variety of signalling pathways between inflammatory cells, response to oxidative stress, new molecules promoting the release of selleck chemical cytokines, expression of chemokines, and

neutrophil recruitment. However, little progress has been made in pinning down the ultra-early events, or critical mediators post-IR that initiate the plethora of late phase responses. Because so many events complicate the later stages of IRI, efforts in the next decade should be focused on designing optimal interventions that will inhibit these very early events, or intercepting the critical mediators that trigger the signalling cascades leading to end-organ damage. “
“Recent data suggest that the chemokine receptor CXCR3 is functionally involved in fibroproliferative disorders, including liver fibrosis. Neoangiogenesis is an important pathophysiological feature of liver scarring, but a functional role of angiostatic CXCR3 chemokines Alpelisib manufacturer in this process is unclear. We therefore investigated neoangiogenesis in carbon tetrachloride (CCl4)-induced liver fibrosis in Cxcr3−/− and wildtype mice by histological, molecular, and functional imaging methods. Furthermore, we assessed the direct role of vascular endothelial growth factor (VEGF) overexpression

on liver angiogenesis and the fibroproliferative response using a Tet-inducible bitransgenic mouse model. The feasibility of attenuation of angiogenesis and associated liver fibrosis by therapeutic treatment with the angiostatic chemokine Cxcl9 was systematically analyzed in vitro and in vivo. The results demonstrate that fibrosis progression in Cxcr3−/− mice was strongly linked to enhanced neoangiogenesis and VEGF/VEGFR2 expression compared with wildtype littermates. Systemic VEGF overexpression Fossariinae led to a fibrogenic response within the liver and was associated with a significantly increased Cxcl9 expression. In vitro, Cxcl9 displayed strong antiproliferative and antimigratory effects on VEGF-stimulated endothelial cells and stellate cells by way of reduced VEGFR2 (KDR), phospholipase Cγ (PLCγ), and extracellular signal-regulated kinase (ERK) phosphorylation, identifying this chemokine as a direct counter-regulatory molecule of VEGF signaling within the liver. Accordingly, systemic administration of Cxcl9 led to a strong attenuation of neoangiogenesis and experimental liver fibrosis in vivo.

Indeed, we also observed a 5-fold basal up-regulation of PDGF-Rβ

Indeed, we also observed a 5-fold basal up-regulation of PDGF-Rβ in CcnE2−/− HSC, which indicates that these cells are already primed for activation and proliferation. We thus conclude that CcnE2 does not share

overlapping functions with CcnE1 in HSCs, but acts as an antifibrotic. Based on our experiments, we suggest an essential role of CcnE1 for HSC activation and fibrosis induction (as illustrated in Fig. 7D): In WT cells, the peak of CcnE1 expression occurs before the maximum expression of α-SMA and PDGF-Rβ. We conclude that CcnE1 drives profibrogenic CHIR-99021 mw mechanisms, predominantly through the targeting and activation of hitherto quiescent HSCs. Previous work demonstrated that E-type cyclins are dispensable for the continuous proliferation of embryonic fibroblasts—sharing some similarities with hepatic myofibroblasts—whereas they are essential for the exit from quiescence.9 In our present study, the same

phenomenon seems to operate in HSCs, except that they rely only on CcnE1 for cell-cycle reentry, because CcnE2 is not induced during liver fibrogenesis. Accordingly, CcnE1-deficient HSCs are unable to normally reenter the cell cycle from G0. Our results raise the question of whether our findings are model specific and may only apply for the CCl4-fibrosis buy Mitomycin C model. Although we cannot exclude that some of our results (e.g., cell-cycle arrest of CcnE1−/− hepatocytes) are CCl4 specific, our data from ex vivo analyzed primary HSCs describe a general, model-independent biological function showing that CcnE1 is an essential cell cycle and survival factor for HSCs. In summary, we demonstrate that CcnE1

is a novel key mediator of hepatic fibrosis in mice because it provides essential functions for the proliferation and survival of HSCs. Future work will evaluate whether the targeted inhibition of CcnE1 might be a therapeutic option to prevent liver fibrosis. The excellent 5-Fluoracil research buy assistance of Sibille Sauer-Lehnen, Carmen C. Tag, and the Core Unit “Q3-Cell Isolation” of the SFB/TRR57 with the isolation of primary HSCs is gratefully acknowledged. We are also grateful to Kanishka Hiththetiya and Christiane Esch for their technical support with the histological analysis of liver samples. Confocal microscopy was performed in the Interdisciplinary Center for Clinical Research (IZKF) Aachen within the Faculty of Medicine at RWTH Aachen University with the kind support of Gerhard Müller-Newen. Additional Supporting Information may be found in the online version of this article. “
“Medicine is expected to benefit from combining usual cellular and molecular studies with high-throughput methods (genomics, transcriptomics, proteomics, and metabolomics). These methods, collectively known as omics, permit the determination of thousands of molecules (variations within genes, RNAs, proteins, metabolites) within a tissue, cell, or biological fluid.

01), but transcriptome data showed no significant changes in gene

01), but transcriptome data showed no significant changes in genes related to cell cycle. Conclusions: Baseline

histology and hepatic gene expression differ according to clinical outcome in patients with AH. Higher liver macrophage expansion, increased proliferative hepatocyte and LPC number as well as up-regulation of cell proliferation related genes are associated see more with a favourable outcome (decrease in MELD score at 3 months). Stem cell transplantation has no effect on hepato-cyte proliferation. Disclosures: The following people have nothing to disclose: Nicolas Lanthier, Laura Rub-bia-Brandt, Nathalie Lin-Marq, Sophie Clement, Jean-Louis Frossard, Nicolas Goossens, Laurent Spahr “
“Metabolic syndrome (MS) is likely to be associated with non-alcoholic fatty liver disease (NAFLD). The prevalence of NAFLD in visceral fat type MS (V-type MS) is known to be higher than in subcutaneous fat type MS (S-type MS) in men with MS, and a larger subcutaneous fat area is reported to be not associated with NAFLD in women. We elucidated differences between V-type S-type MS in Japanese women with MS. The subjects were 276 women with MS who

underwent a medical checkup including abdominal ultrasonography. We examined for the prevalence of fatty liver and investigated biochemical parameters, and we also made a distinction between V-type and S-type MS. Triglyceride, uric acid, alanine PF-562271 in vivo aminotransferase (ALT), aspartate aminotransferase (AST), γ-glutamyltransferase, the frequency of fatty liver and impaired glucose tolerance (IGT) were significantly higher in V-type learn more MS than in S-type MS. On logistic regression analysis with NAFLD (in our study,

fatty liver with ALT ≥31 IU/L was defined as NAFLD) as a dependent variable, body mass index, dyslipidemia, AST and V-type MS were significant predictors of an increased prevalence of NAFLD (odds ratios [OR] = 18.85, 3.119, 59.77 and 3.205; 95% confidence intervals [CI] = 3.585–99.15, 1.195–8.142, 18.03–198.2 and 1.198–8.573; P < 0.001, <0.05, <0.001 and <0.05, respectively). Women with V-type MS are more likely to have fatty liver, IGT and liver dysfunction than those with S-type MS. V-type MS is one of the significant predictors for NAFLD in Japanese women with MS. "
“Aim:  Previous studies have shown significantly elevated levels of interleukin (IL)-6 in cirrhotic patients with minimal hepatic encephalopathy (MHE), but the relationship between circulating levels of IL-6 and ammonia is unclear. The aim of this study is to investigate the relationship between both variables in cirrhotic patients with MHE. Methods:  Psychometric tests including number connection test part A (NCT-A) and digit symbol test (DST) were performed to diagnose MHE in 85 cirrhotic patients. Simultaneously, circulating levels of IL-6 and ammonia were measured. Results:  Thirty-two (37.6%) cirrhotic patients were diagnosed with MHE.


Fifty SB431542 microliters of tissue extract was used for GSH measurement. GSH was measured according to the manufacturer’s instructions with a GSH assay kit (Promega, Madison, WI). Luminescence was detected with a Synergy 2 multimode microplate reader with Gen5 data analysis software (BioTek, Winooski, VT). DNA was extracted from 25 mg of mouse liver. Total DNA purification was performed with a DNeasy blood and tissue kit (Qiagen, Germantown, MA) according to the manufacturer’s instructions. RNA was eliminated by incubation in 5 μg/mL RNase (Roche, Indianapolis, IN). A 1.5-μg DNA aliquot was loaded onto 1.5% agarose gel for separation to assess for DNA fragmentation.

TUNEL staining was conducted with an in situ apoptosis detection kit from R&D Systems according to the manufacturer’s instructions. Six to seven animals were selleck screening library used per time point per treatment group. At least 1000 cells (TUNEL-positive cells and TUNEL-negative cells) were counted in each of eight separate low-power fields for each animal, and the percentage of TUNEL-positive cells was calculated. Two hours prior to sacrifice, animals received 30 μg of BrdU/g of body weight intraperitoneally. Liver

specimens were obtained, fixed in 4% paraformaldehyde, processed for histological analysis, and stained with the Amersham cell proliferation kit (Amersham Pharmacia Biotech, Ltd., United Kingdom). BrdU incorporation was measured at 24, 36, 48, and 72 hours. There were four to seven mice per treatment group per time point. At least 1000 cells (BrdU-positive cells Benzatropine and BrdU-negative cells) were counted

in each of eight separate low-power fields for each animal, and the percentage of BrdU-positive cells was calculated. Hepatic cytoplasmic and nuclear proteins were extracted with the NE-PER nuclear and cytoplasmic extraction reagent kit (Pierce Biotechnology, Rockford, IL) according to the manufacturer’s instructions. Liver samples were homogenized in a radioimmunoprecipitation assay buffer (50 mM trishydroxymethylaminomethane–hydrochloric acid, pH 7.4, 150 mM sodium chloride, 1% Nonidet P40, 0.1% sodium dodecyl sulfate, 0.25% sodium deoxycholate, 1 mM ethylene diamine tetraacetic acid, and protease and phosphatase inhibitors). Four hundred micrograms of protein was used for immunoprecipitation. The lysate was precleared with 1 μg of isotype immunoglobulin G (IgG) and 50 μL of protein A/G agarose at 4°C for 1 hour. Five micrograms of an immunoprecipitating antibody or isotype IgG was added to the supernatant, and it was rocked overnight at 4°C. Next, 50 μL of protein agarose was added, and the mixture was rocked for 2 hours at 4°C. The protein bound to the agarose conjugate was centrifuged, and the pellet was washed three times with a radioimmunoprecipitation assay buffer.