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1.  SAMe Treatment Prevents the Ethanol-Induced Epigenetic Alterations of Genes in the Toll-Like Receptor Pathway 
Prior studies showed that Toll-like receptor (TLR) signaling pathway genes were up regulated in the liver of rats fed ethanol, but not in rats fed ethanol plus S-adenosylmethionine (SAMe). These results were obtained using a PCR microplate array analysis for TLRs and associated proteins such as proinflammatory cytokines and chemokine mRNA levels. A large number of genes were up regulated by the ethanol diet, but not the ethanol plus SAMe diet. In the present study, using the same experimental rat livers, DNA methylation analysis was done by using an Epitect Methyl DNA Restriction Kit (Qiagen, 335451) (24 genes). The results of all the genes combined shows a highly significant increase in methylation in the ethanol-fed group of rats, but not in the dextrose-fed, SAMe-fed or ethanol plus SAMe-fed groups of rats. There was also an increase in DNA methylation in rats with high blood alcohol levels compared to a rat with a low blood alcohol level. The individual genes that were up regulated as indicated by the increased mRNA measured by qPCR correlated positively with the increased methylation of the DNA of the corresponding gene as follows: Cd14, Hspa1a, Irf1, Irak1, Irak2, Map3k7, Myd88, Pparα, Ripk2, Tollip and Traf6.
doi:10.1016/j.yexmp.2012.09.024
PMCID: PMC3562371  PMID: 23047067
TLR (Toll-like receptor); SAMe (S-adenosyl methionine); BAL/blood alcohol levels; 5-methylcytosine
2.  The Heat Shock Protein Inhibitor Quercetin Attenuates Hepatitis C Virus Production 
Hepatology (Baltimore, Md.)  2009;50(6):10.1002/hep.23232.
The hepatitis C viral (HCV) genome is translated through an internal ribosome entry site (IRES) as a single polyprotein precursor that is subsequently cleaved into individual mature viral proteins. Non-structural protein 5A (NS5A) is one of these proteins that has been implicated in regulation of viral genome replication, translation from the viral IRES and viral packaging. We sought to identify cellular proteins that interact with NS5A and determine whether these interactions may play a role in viral production. Mass spectrometric analysis of coimmunoprecipitated NS5A complexes from cell extracts identified heat shock proteins (HSPs) 40 and 70.Weconfirmed anNS5A/HSPinteraction by confocal microscopy demonstrating colocalization of NS5A with HSP40 and with HSP70. Western analysis of coimmunoprecipitated NS5A complexes further confirmed interaction of HSP40 and HSP70 with NS5A.Atransient transfection, luciferase-based, tissue culture IRES assay demonstrated NS5A augmentation of HCV IRES-mediated translation, and small interfering RNA (siRNA)-mediated knockdown of HSP70 reduced this augmentation. Treatment with an inhibitor of HSP synthesis, Quercetin, markedly reduced baseline IRES activity and its augmentation by NS5A. HSP70 knockdown also modestly reduced viral protein accumulation, whereas HSP40 and HSP70 knockdown both reduced infectious viral particle production in an HCV cell culture system using the J6/JFH virus fused to the Renilla luciferase reporter. Treatment with Quercetin reduced infectious particle production at nontoxic concentrations. The marked inhibition of virus production by Quercetin may partially be related to reduction of HSP40 and HSP70 and their potential involvement in IRES translation, as well as viral morphogenesis or secretion.
Conclusion
Quercetin may allow for dissection of the viral life cycle and has potential therapeutic use to reduce virus production with low associated toxicity.
doi:10.1002/hep.23232
PMCID: PMC3846025  PMID: 19839005
3.  Divergent antiviral effects of bioflavonoids on the hepatitis C virus life cycle 
Virology  2012;433(2):346-355.
We have previously demonstrated that quercetin, a bioflavonoid, blocks hepatitis C virus (HCV) proliferation by inhibiting NS5A-driven internal ribosomal entry site (IRES)-mediated translation of the viral genome. Here, we investigate the mechanisms of antiviral activity of quercetin and six additional bioflavonoids. We demonstrate that catechin, naringenin, and quercetin possess significant antiviral activity, with no associated cytotoxicity. Infectious virion secretion was not significantly altered by these bioflavonoids. Catechin and naringenin demonstrated stronger inhibition of infectious virion assembly compared to quercetin. Quercetin markedly blocked viral translation whereas catechin and naringenin demonstrated mild activity. Similarly quercetin completely blocked NS5A-augmented IRES-mediated translation in an IRES reporter assay, whereas catechin and naringenin had only a mild effect. Moreover, quercetin differentially inhibited HSP70 induction compared to catechin and naringenin. Thus, the antiviral activity of these bioflavonoids is mediated through different mechanisms. Therefore combination of these bioflavonoids may act synergistically against HCV.
doi:10.1016/j.virol.2012.08.029
PMCID: PMC3478964  PMID: 22975673
HSP70; NS5A; IRES; HCV; Bioflavonoid
4.  Characterization of Timed Changes in Hepatic Copper Concentrations, Methionine Metabolism, Gene Expression, and Global DNA Methylation in the Jackson Toxic Milk Mouse Model of Wilson Disease 
Background
Wilson disease (WD) is characterized by hepatic copper accumulation with progressive liver damage to cirrhosis. This study aimed to characterize the toxic milk mouse from The Jackson Laboratory (Bar Harbor, ME, USA) (tx-j) mouse model of WD according to changes over time in hepatic copper concentrations, methionine metabolism, global DNA methylation, and gene expression from gestational day 17 (fetal) to adulthood (28 weeks).
Methods
Included liver histology and relevant biochemical analyses including hepatic copper quantification, S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) liver levels, qPCR for transcript levels of genes relevant to methionine metabolism and liver damage, and DNA dot blot for global DNA methylation.
Results
Hepatic copper was lower in tx-j fetuses but higher in weanling (three weeks) and adult tx-j mice compared to controls. S-adenosylhomocysteinase transcript levels were significantly lower at all time points, except at three weeks, correlating negatively with copper levels and with consequent changes in the SAM:SAH methylation ratio and global DNA methylation.
Conclusion
Compared to controls, methionine metabolism including S-adenosylhomocysteinase gene expression is persistently different in the tx-j mice with consequent alterations in global DNA methylation in more advanced stages of liver disease. The inhibitory effect of copper accumulation on S-adenosylhomocysteinase expression is associated with progressively abnormal methionine metabolism and decreased methylation capacity and DNA global methylation.
doi:10.3390/ijms15058004
PMCID: PMC4057715  PMID: 24810691
Wilson disease; copper; DNA; methylation; gene expression
5.  A Cell-Permeable Hairpin Peptide Inhibits Hepatitis C Viral Nonstructural Protein 5A Mediated Translation and Virus Production 
Hepatology (Baltimore, Md.)  2012;55(6):1662-1672.
NS5A is a key regulator of hepatitis C virus (HCV) life cycle including RNA replication, assembly, and translation. We and others have shown NS5A to augment HCV IRES-mediated translation. Further, Quercetin treatment and heat shock protein (HSP) 70 knockdown inhibit NS5A-driven augmentation of IRES-mediated translation and infectious virus production. We have also co-immunoprecipitated HSP70 with NS5A and demonstrated cellular colocalization leading to the hypothesis that the NS5A/HSP70 complex formation is important for IRES-mediated translation. Here, we have identified the NS5A region responsible for complex formation through in vitro deletion analyses. Deletion of NS5A domains II and III failed to reduce HSP70 binding, whereas domain I deletion eliminated complex formation. NS5A domain I alone also bound HSP70. Deletion mapping of domain I identified the C-terminal 34 amino acids (C34) to be the interaction site. Further, addition of C34 to domains II and III restored complex formation. C34 expression significantly reduced intracellular viral protein levels, in contrast to same size control peptides from other NS5A domains. C34 also competitively inhibited NS5A-augmented IRES-mediated translation, while controls did not. Triple-alanine scan mutagenesis identified an exposed beta-sheet hairpin in C34 to be primarily responsible for NS5A-augmented IRES-mediated translation. Moreover, treatment with a 10 amino acid peptide derivative of C34 suppressed NS5A-augmented IRES-mediated translation and significantly inhibited intracellular viral protein synthesis, with no associated cytotoxicity. Conclusion: These results support the hypothesis that the NS5A/HSP70 complex augments viral IRES-mediated translation, identify a sequence-specific hairpin element in NS5A responsible for complex formation, and demonstrate the functional significance of C34 hairpin-mediated NS5A/HSP70 interaction. Identification of this element may allow for further interrogation of NS5A-mediated IRES activity, sequence specific HSP recognition, and rational drug design.
doi:10.1002/hep.25533
PMCID: PMC3345309  PMID: 22183951
HSP70; NS5A; IRES; HCV; Protein binding
6.  The liver is populated by a broad spectrum of markers for macrophages. In alcoholic hepatitis the macrophages are M1 and M2☆ 
Background
Liver cell injury in alcoholic hepatitis (AH) is in part, due to macrophage generated proinflammatory cytokines i.e., M1, M2a, M2b, and M2c might be involved in ALD. The T cell response to chemokines and cytokines differs not only when M1 and M2 macrophages are compared but even when individual M2 subtypes are profiled.
Purpose
In AH, M1 monocytes in the blood show increased sensitivity in the TNF-α response to LPS. Immunohistochemistry (IHC) studies showed that the liver sinusoids in ALD are abundantly populated by CD163 expressing type 2 macrophages. In this report, we profile many of the molecules associated with M1 and M2 macrophages in livers with AH using IHC.
Methods
Using immunofluorescent antibody-labeling, we profiled the receptors, cytokines and chemokines observed in M1, M2a, M2b, and M2c macrophages in liver biopsies from patients with AH.
Results
The increased CD 163 expression found in previous studies was confirmed as well an additional macrophage phenotypic marker CD206, suggesting that AH pathogenesis at least partially involves M2a and M2c macrophages. TGF-β was found to be robustly over expressed by liver sinusoidal macrophages. Macrophage expression of the phenotypic markers TLR-2, TLR-4 and TLR-8 – found in both M1 and M2 macrophages – as well as the chemokines CCL-1 and CCL-18 was found. However, IRF-4, which is related to IL-4 production and M2a polarization as well as the cytokines CCL-1 and Il-1β and the chemokine CXCL-1 were also observed, suggesting that M2a and M2b also play a role in AH pathogenesis.
Conclusion
Livers with AH show robust macrophage over expression of TGF-β, a growth factor more commonly associated with M2 type macrophages and mostly known for its fibrogenetic properties. However, our immunoprofiling of macrophage over expression also shows that AH is driven by receptors, interferons, and cytokines that are commonly associated not just with M2 macrophages, but with M1 as well. Thus, a complex interplay between different types of macrophages expressing a diverse array of molecules and receptors is involved in AH.
doi:10.1016/j.yexmp.2013.09.004
PMCID: PMC3944995  PMID: 24145004
Alcoholic hepatitis; Macrophages; CD163; TLR-4
7.  FAT10 IS AN EPIGENETIC MARKER FOR LIVER PRENEOPLASIA IN A DRUG-PRIMED MOUSE MODEL OF TUMORIGENESIS 
There is clinical evidence that chronic liver diseases in which MDBs (Mallory Denk Bodies) form progress to hepatocellular carcinoma. The present study provides evidence that links MDB formation induced by chronic drug injury, with preneoplasia and later to the formation of tumors, which develop long after drug withdrawal. Evidence indicated that this link was due to an epigenetic cellular memory induced by chronic drug ingestion. Microarray analysis showed that the expressions of many markers of preneoplasia (UBD, Alpha Fetoprotein, KLF6 and Glutathione-S-Transferase mu2) were increased together when the drug DDC was refed. These changes were suppressed by S-adenosylmethionine feeding, indicating that the drug was affecting DNA and histones methylation in an epigenetic manner. The link between MDB formation and neoplasia formation was likely due to the over expression of UBD (also called FAT10), which is up regulated in 90% of human hepatocellular carcinomas. Immunohistochemical staining of drug primed mouse livers showed that FAT10 positive liver cells persisted up to 4 months after drug withdrawal and they were still found in the livers of mice, 14 months after drug withdrawal. The refeeding of DDC increased the percent of FAT10 hepatocytes.
doi:10.1016/j.yexmp.2007.12.003
PMCID: PMC2874461  PMID: 18280469
8.  Alcohol, nutrition and liver cancer: Role of Toll-like receptor signaling 
This article reviews the evidence that ties the development of hepatocellular carcinoma (HCC) to the natural immune pro-inflammatory response to chronic liver disease, with a focus on the role of Toll-like receptor (TLR) signaling as the mechanism of liver stem cell/progenitor transformation to HCC. Two exemplary models of this phenomenon are reviewed in detail. One model applies chronic ethanol/lipopolysaccharide feeding to the activated TLR4 signaling pathway. The other applies chronic feeding of a carcinogenic drug, in which TLR2 and 4 signaling pathways are activated. In the drug-induced model, two major methyl donors, S-adenosylmethionine and betaine, prevent the upregulation of the TLR signaling pathways and abrogate the stem cell/progenitor proliferation response when fed with the carcinogenic drug. This observation supports a nutritional approach to liver cancer prevention and treatment. The observation that upregulation of the TLR signaling pathways leads to liver tumor formation gives evidence to the popular concept that the chronic pro-inflammatory response is an important mechanism of liver oncogenesis. It provides a nutritional approach, which could prevent HCC from developing in many chronic liver diseases.
doi:10.3748/wjg.v16.i11.1344
PMCID: PMC2842526  PMID: 20238401
Toll-like receptor; Hepatocellular carcinoma; Methyl donors, Epigenetic processes; Inflammation; Alcohol; Drug toxicity; Lipopolysaccharides
9.  Epigenetic Events in Liver Cancer Resulting From Alcoholic Liver Disease 
Epigenetic mechanisms play an extensive role in the development of liver cancer (i.e., hepatocellular carcinoma [HCC]) associated with alcoholic liver disease (ALD) as well as in liver disease associated with other conditions. For example, epigenetic mechanisms, such as changes in the methylation and/or acetylation pattern of certain DNA regions or of the histone proteins around which the DNA is wrapped, contribute to the reversion of normal liver cells into progenitor and stem cells that can develop into HCC. Chronic exposure to beverage alcohol (i.e., ethanol) can induce all of these epigenetic changes. Thus, ethanol metabolism results in the formation of compounds that can cause changes in DNA methylation and interfere with other components of the normal processes regulating DNA methylation. Alcohol exposure also can alter histone acetylation/deacetylation and methylation patterns through a variety of mechanisms and signaling pathways. Alcohol also acts indirectly on another molecule called toll-like receptor 4 (TLR4) that is a key component in a crucial regulatory pathway in the cells and whose dysregulation is involved in the development of HCC. Finally, alcohol use regulates an epigenetic mechanism involving small molecules called miRNAs that control transcriptional events and the expression of genes important to ALD.
PMCID: PMC3860418  PMID: 24313165
Alcohol consumption; alcohol abuse; chronic alcohol use; alcoholic liver disease; ethanol metabolism; alcoholic liver disease; liver cancer; hepatocellular carcinoma; epigenetics; epigenetic mechanisms; DNA methylation; histone methylation; stem cells; micro RNAs
10.  Efficacy of Liposomal Amphotericin B and Posaconazole in Intratracheal Models of Murine Mucormycosis 
Mucormycosis is a life-threatening fungal infection almost uniformly affecting diabetics in ketoacidosis or other forms of acidosis and/or immunocompromised patients. Inhalation of Mucorales spores provides the most common natural route of entry into the host. In this study, we developed an intratracheal instillation model of pulmonary mucormycosis that hematogenously disseminates into other organs using diabetic ketoacidotic (DKA) or cyclophosphamide-cortisone acetate-treated mice. Various degrees of lethality were achieved for the DKA or cyclophosphamide-cortisone acetate-treated mice when infected with different clinical isolates of Mucorales. In both DKA and cyclophosphamide-cortisone acetate models, liposomal amphotericin B (LAmB) or posaconazole (POS) treatments were effective in improving survival, reducing lungs and brain fungal burdens, and histologically resolving the infection compared with placebo. These models can be used to study mechanisms of infection, develop immunotherapeutic strategies, and evaluate drug efficacies against life-threatening Mucorales infections.
doi:10.1128/AAC.00313-13
PMCID: PMC3697351  PMID: 23650163
11.  HOXB7 Promotes Invasion and Predicts Survival in Pancreatic Adenocarcinoma 
Cancer  2012;119(3):10.1002/cncr.27725.
BACKGROUND
The homeobox gene HOXB7 is overexpressed across a range of cancers and promotes tumorigenesis through varying effects on proliferation, survival, invasion, and angiogenesis. Although published microarray data suggest HOXB7 is overexpressed in pancreatic ductal adenocarcinoma (PDAC), its function in pancreatic cancer has not been studied.
METHODS
HOXB7 message and protein levels were examined in PDAC cell lines and patient samples, as well as in normal pancreas. HOXB7 protein expression in patient tumors was determined by immunohistochemistry and correlated with clinicopathologic factors and survival. The impact of HOXB7 on cell proliferation, growth, and invasion was assessed by knockdown and overexpression in PDAC cell lines. Candidate genes whose expression levels were altered following HOXB7 knockdown were determined by microarray analysis.
RESULTS
HOXB7 message and protein levels were significantly elevated in PDAC cell lines and patient tumor samples relative to normal pancreas. Evaluation of a tissue microarray of 145 resected PDACs found high HOXB7 protein expression was correlated with lymph node metastasis (P = .034) and an independent predictor of worse overall survival in multivariate analysis (hazard ratio = 1.56, 95% confidence interval = 1.02–2.39). HOXB7 knockdown or overexpression in PDAC cell lines resulted in decreased or increased invasion, respectively, without influencing proliferation or cell viability.
CONCLUSIONS
HOXB7 is frequently overexpressed in PDAC, specifically promotes invasive phenotype, and is associated with lymph node metastasis and worse survival outcome. HOXB7 and its downstream targets may represent novel clinical biomarkers or targets of therapy for inhibiting the invasive and metastatic capacity of PDAC.
doi:10.1002/cncr.27725
PMCID: PMC3867310  PMID: 22914903
HOXB7; invasion; metastasis; pancreatic adenocarcinoma; CCBP2; homeobox genes
13.  S-ADENOSYLMETHIONINE PREVENTS THE UP REGULATION OF TOLL-LIKE RECEPTOR (TLR) SIGNALING CAUSED BY CHRONIC ETHANOL FEEDING IN RATS 
Toll-like receptors (TLR) play a role in mediating the proinflammatory response, fibrogenesis and carcinogenesis in chronic liver diseases such as alcoholic liver disease, non-alcoholic liver disease, hepatitis C and hepatocellular carcinoma. This is true in experimental models of these diseases. For this reason, we investigated the TLR proinflammatory response in the chronic intragastric tube feeding rat model of alcohol liver disease. The methyl donor S-adenosylmethionine was also fed to prevent the gene expression changes induced by ethanol. Ethanol feeding tended to increase the up regulation of the gene expression of TLR2 and TLR4. SAMe feeding prevented this. TLR4 and MyD88 protein levels were significantly increased by ethanol and this was prevented by SAMe. This is the first report where ethanol feeding induced TLR2 and SAMe prevented the induction by ethanol. CD34, FOS, interferon responsive factor 1 (IRF-1), Jun, TLR 1,2,3,4,6 and 7 and Traf-6 were found to be up regulated as seen by microarray analysis where rats were sacrified at high blood alcohol levels compared to pair fed controls. Il-6, IL-10 and IFNγ were also up regulated by high blood levels of ethanol. The gene expression of CD14, MyD88 and TNFR1SF1 were not up regulated by ethanol but were down regulated by SAMe. The gene expression of IL-1R1 and IRF1 tended to be up regulated by ethanol and this was prevented by feeding SAMe. The results suggest that SAMe, fed chronically prevents activation of TLR pathways caused by ethanol. In this way the proinflammatory response, fibrogenesis, cirrhosis and hepatocellular carcinoma formation due to alcohol liver disease could be prevented by SAMe.
doi:10.1016/j.yexmp.2011.01.005
PMCID: PMC3092000  PMID: 21276439
Toll-like receptor (TLR); S-adenosylmethionine (SAMe); alcoholic liver disease (ASH)
14.  PROTECTIVE EFFECT OF QUERCETIN, EGCG, CATECHIN AND BETAINE AGAINST OXIDATIVE STRESS INDUCED BY ETHANOL IN VITRO 
There is a need for a nontoxic antioxidant agent to be identified which will prevent alcoholic liver disease (ALD) in alcoholic patients. We tested 4 candidate agents: quercetin, EGCG, catechin and betaine, all of which occur naturally in food. HepG2 cells over expressing CYP2E1 were subjected to arachidonic acid, iron and 100 mM ethanol with or without the antioxidant agent. All the agents prevented oxidative stress and MDA/4HNE formation induced by ethanol, except for EGCG. Catechin prevented CYP2E1 induction by ethanol. All the agents tended to down regulate the ethanol-induced increased expression of glutathionine peroxidase 4 (GPX4). All the agents, except catechin, tended to reduce the expression of SOD2 induced by ethanol. Heat shock protein 70 was up regulated by ethanol alone and betaine tended to prevent this. All 4 agents down regulated the expression of Gadd45b in the presence of ethanol, which could explain the mechanism of DNA demethylation associated with the up regulation of the gene expression observed in experimental ALD. In conclusion, the in vitro model of oxidative stress induced by ethanol provided evidence that all 4 agents tested prevented some aspect of liver cell injury caused by ethanol.
doi:10.1016/j.yexmp.2011.02.006
PMCID: PMC3113678  PMID: 21352821
Catechin; EGCG; Quercetin; Betaine; Oxidative Stress
15.  Systematic Analysis of Enhancer and Critical cis-Acting RNA Elements in the Protein-Encoding Region of the Hepatitis C Virus Genome 
Journal of Virology  2013;87(10):5678-5696.
Hepatitis C virus (HCV) causes chronic hepatitis, cirrhosis, and liver cancer. cis-acting RNA elements of the HCV genome are critical for translation initiation and replication of the viral genome. We hypothesized that the coding regions of nonstructural proteins harbor enhancer and essential cis-acting replication elements (CRE). In order to experimentally identify new cis RNA elements, we utilized an unbiased approach to introduce synonymous substitutions. The HCV genome coding for nonstructural proteins (nucleotide positions 3872 to 9097) was divided into 17 contiguous segments. The wobble nucleotide positions of each codon were replaced, resulting in 33% to 41% nucleotide changes. The HCV genome containing one of each of 17 mutant segments (S1 to S17) was tested for genome replication and infectivity. We observed that silent mutations in segment 13 (S13) (nucleotides [nt] 7457 to 7786), S14 (nt 7787 to 8113), S15 (nt 8114 to 8440), S16 (nt 8441 to 8767), and S17 (nt 8768 to 9097) resulted in impaired genome replication, suggesting CRE structures are enriched in the NS5B region. Subsequent high-resolution mutational analysis of NS5B (nt 7787 to 9289) using approximately 51-nucleotide contiguous subsegment mutant viruses having synonymous mutations revealed that subsegments SS8195-8245, SS8654-8704, and SS9011-9061 were required for efficient viral growth, suggesting that these regions act as enhancer elements. Covariant nucleotide substitution analysis of a stem-loop, JFH-SL9098, revealed the formation of an extended stem structure, which we designated JFH-SL9074. We have identified new enhancer RNA elements and an extended stem-loop in the NS5B coding region. Genetic modification of enhancer RNA elements can be utilized for designing attenuated HCV vaccine candidates.
doi:10.1128/JVI.00840-12
PMCID: PMC3648135  PMID: 23487449
16.  S-ADENOSYLMETHIONINE DECREASES THE PEAK BLOOD ALCOHOL LEVELS 3 HOURS AFTER AN ACUTE BOLUS OF ETHANOL BY INDUCING ALCOHOL METABOLIZING ENZYMES IN THE LIVER 
Introduction
An alcohol bolus causes the blood alcohol level (BAL) to peak at 1-2 hours post ingestion. The ethanol elimination rate is regulated by alcohol metabolizing enzymes, primarily alcohol dehydrogenase (ADH1), acetaldehyde dehydrogenase (ALDH), and cytochrome P450 (CYP2E1). Recently, S-adenosylmethionine (SAMe) was found to reduce acute BALs 3h after an alcohol bolus. The question, then, was: what is the mechanism involved in this reduction of BAL by feeding SAMe? To answer this question, we investigated the changes in ethanol metabolizing enzymes and the epigenetic changes that regulate the expression of these enzymes during acute binge drinking and chronic drinking.
Methods
Rats were fed a bolus of ethanol with or without SAMe, and were sacrificed at 3h or 12 h after the bolus.
Results
RT-PCR and Western blot analyses showed that SAMe significantly induced ADH1 levels in the 3h liver samples. However, SAMe did not affect the changes in ADH1 protein levels 12h post bolus. Since SAMe is a methyl donor, it was postulated that the ADH1 gene expression up regulation at 3h was due to a histone modification induced by methylation from methyl transferases. Dimethylated histone 3 lysine 4 (H3K4me2), a modification responsible for gene expression activation, was found to be significantly increased by SAMe at 3h post bolus.
Conclusion
These results correlated with the low BAL found at 3h post bolus, and support the concept that SAMe increased the gene expression to increase the elimination rate of ethanol in binge drinking by increasing H3K4me2.
doi:10.1016/j.yexmp.2010.08.010
PMCID: PMC3319153  PMID: 20828554
17.  BETAINE PREVENTS MALLORY-DENK BODY FORMATION IN DRUG-PRIMED MICE BY EPIGENETIC MECHANISMS 
Previous studies showed that S-Adenosylmethionine (SAMe) prevented MDB formation and the hypomethylation of histones induced by DDC feeding. These results suggest that formation of MDBs is an epigenetic phenomenon. To further test this theory, drug-primed mice were fed the methyl donor, betaine, together with DDC, which was refed for 7 days. Betaine significantly reduced MDB formation, decreased the liver/body weight ratio and decreased the number of FAT10 positive liver cells when they proliferate in response to DDC refeeding. Betaine also significantly prevented the decreased expression of BHMT, AHCY, MAT1a and GNMT and the increased expression of MTHFR, caused by DDC refeeding. S-Adenosylhomocysteine (SAH) levels were reduced by DDC refeeding and this was prevented by betaine. The results support the concept that betaine donates methyl groups, increasing methionine available in the cell. SAMe metabolism was reduced by the decrease in GNMT expression, which prevented the conversion of SAMe to SAH. As a consequence, betaine prevented MDB formation and FAT10 positive cell proliferation by blocking the epigenetic memory expressed by hepatocytes. The results further support the concept that MDB formation is the result of an epigenetic phenomenon, where a change in methionine metabolism causes global gene expression changes in hepatocytes.
doi:10.1016/j.yexmp.2008.11.002
PMCID: PMC3319154  PMID: 19073172
18.  INCREASED EXPRESSION OF FAT10 IN COLON BENIGN, PREMALIGNANT AND MALIGNANT EPITHELIAL NEOPLASMS 
Over expression of FAT10 is characteristic of numerous types of carinoma including liver, gastric and colon carcinomas. In the case of colon carcinoma it is possible to determine at the point in the progression from the benign to the malignant process of colon cancer development by determining which stage in the neoplastic process FAT10 overexpression occurs. This stage was determined by measuring the intensity of fluoresence of immunohistochemically stained normal mucosa, tubular adenomas, hyperplastic polyps, serrated adenomas, villotubular, villous adenomas and invasive adenocarcinoma stages. Using this approach it was found the overexpression of FAT10 began at the serrated adenoma stage and continued to include the villous and villotubular stages and the invasive adenocaricnoma stage. The FAT10 overexpression by invasive adenocarcinoma was accompanied by the expression of the catalytic subunits of the immunoproteasome which is functionally tied to overexpression of FAT10, Toll-like receptor activation and the proinflammatory response.
doi:10.1016/j.yexmp.2010.09.005
PMCID: PMC3319157  PMID: 20888811
FAT10; immunoproteasome; Toll-like receptors; interferon; proinflammatory response
19.  SAME PREVENTS THE INDUCTION OF THE IMMUNOPROTEASOME AND PRESERVES THE 26S PROTEASOME IN THE DDC-INDUCED MDB MOUSE MODEL 
Mallory-Denk Bodies (MDBs) form in the liver of alcoholic patients. This occurs because of the accumulation and aggregation of ubiquitinated cytokeratins, which hypothetically is due to the ubiquitin-proteasome pathway’s (UPP) failure to degrade the cytokeratins. The experimental model of MDB formation was used in which MDBs were induced by refeeding DDC to drug-primed mice. The gene expression and protein levels of LMP2, LMP7 and MECL-1, the catalytic subunits in the immunoproteasome, as well as FAT10, were increased in the liver cells forming MDBs but not in the intervening normal hepatocytes. Chymotrypsin-like activity of the UPP was decreased by DDC refeeding, indicating that a switch from the UPP to the immunoproteasome had occurred at the expense of the 26S proteasome. The failure of the UPP to digest cytokeratins would explain MDB aggregate formation. SAMe prevented the decrease in UPP activity, the increase in LMP2, LMP7, and MECL-1 protein levels and MDB formation induced by DDC. DDC refeeding also induced the TNFα and IFNγ receptors. SAMe prevented the increase in the TNFα and IFNγ receptors, supporting the idea that TNFα and IFNγ were responsible for the up regulation of LMP2, LPM7, and FAT10. These results support the conclusion that MDBs form in FAT10 over-expressing hepatocytes where the up regulation of the immunoproteasome occurs at the expense of the 26S proteasome.
doi:10.1016/j.yexmp.2010.03.001
PMCID: PMC3315394  PMID: 20223233
26S proteasome; Immunoproteasome; TNF alpha; Interferon gamma; inflammatory response; Mallory-Denk Body
20.  MALLORY BODY FORMATION IS ASSOCIATED WITH EPIGENETIC PHENOTYPIC CHANGE IN HEPATOCYTES IN VIVO 
Microarrays were done on the livers of mice fed DDC for 10 weeks, withdrawn 1 month (DDC primed livers) and refed 6 days,and compared with mice fed the control diet. The expression of a large number of genes changed when DDC was fed or refed. A Venn diagram analysis identified 649 genes where gene expression was changed in the same direction. The epigenetic memory of the DDC primed liver involved an increase in the expression of ubiquitin D, alpha fetoprotein, connective tissue growth factor, integrin beta 2, DNA methyl transferase 3a and DNA damage –inducible 45 gamma. DNA methyl transferase 3b was down regulated as was Cbp/p300. When DDC was refed, DNA methyltransferase and histone deacetylase were up regulated as shown by microarray analysis. Histone3 lysine9 acetylation was increased by DDC and DDC refeeding and DNA methyltransferases were not changed as shown by Western blot analysis. The data suggests the concept that the epigenetic memory that explains why DDC primed hepatocytes form MBs in 7 days of DDC refeeding is primarily the result of epigenetic modifications of gene expression through changes in histone acetylation and methylation, as well as DNA methylation.
doi:10.1016/j.yexmp.2007.03.003
PMCID: PMC3315395  PMID: 17531972
epigenetics; Mallory bodies; phenotypic change; genetic memory
21.  Reciprocal regulation by TLR4 and TGF-β in tumor-initiating stem-like cells 
The Journal of Clinical Investigation  2013;123(7):2832-2849.
Tumor-initiating stem-like cells (TICs) are resistant to chemotherapy and associated with hepatocellular carcinoma (HCC) caused by HCV and/or alcohol-related chronic liver injury. Using HCV Tg mouse models and patients with HCC, we isolated CD133+ TICs and identified the pluripotency marker NANOG as a direct target of TLR4, which drives the tumor-initiating activity of TICs. These TLR4/NANOG–dependent TICs were defective in the TGF-β tumor suppressor pathway. Functional oncogene screening of a TIC cDNA library identified Yap1 and Igf2bp3 as NANOG-dependent genes that inactivate TGF-β signaling. Mechanistically, we determined that YAP1 mediates cytoplasmic retention of phosphorylated SMAD3 and suppresses SMAD3 phosphorylation/activation by the IGF2BP3/AKT/mTOR pathway. Silencing of both YAP1 and IGF2BP3 restored TGF-β signaling, inhibited pluripotency genes and tumorigenesis, and abrogated chemoresistance of TICs. Mice with defective TGF-β signaling (Spnb2+/– mice) exhibited enhanced liver TLR4 expression and developed HCC in a TLR4-dependent manner. Taken together, these results suggest that the activated TLR4/NANOG oncogenic pathway is linked to suppression of cytostatic TGF-β signaling and could potentially serve as a therapeutic target for HCV-related HCC.
doi:10.1172/JCI65859
PMCID: PMC3696549  PMID: 23921128
22.  Diabetic murine models for Acinetobacter baumannii infection 
Objectives
Extremely drug-resistant (XDR; i.e. resistant to all antibiotics except colistin or tigecycline) Acinetobacter baumannii has emerged as one of the most common and highly antibiotic-resistant causes of infection. Diabetes is a risk factor for acquisition of and worse outcomes from A. baumannii infection. We sought to develop diabetic mouse models of A. baumannii bacteraemia and pneumonia and validate these models by comparing the efficacy of antibiotic treatment in these models with the established neutropenic mouse models.
Methods
Diabetic or neutropenic mice were infected via intravenous inoculation or inhalation in an aerosol chamber with an XDR A. baumannii. Treatment with colistin started 24 h after infection and continued daily for 7 days. Survival served as the primary endpoint while tissue bacterial burden and histopathological examination served as secondary endpoints.
Results
Lethal infection was achieved for the neutropenic and diabetic mice when infected intravenously or via inhalation. Neutropenic mice were more susceptible to infection than diabetic mice in the pneumonia model and equally susceptible in the bacteraemia model. Both models of bacteraemia were sensitive enough to detect virulence differences among different clinical strains of A. baumannii. In the pneumonia model, colistin treatment was effective in improving survival, reducing lung bacterial burden and histologically resolving the infection compared with placebo only in diabetic mice.
Conclusions
We developed novel models of A. baumannii bacteraemia and pneumonia in diabetic mice. These models can be used to study mechanisms of infection, develop immunotherapeutic strategies and evaluate drug efficacies against highly lethal A. baumannii infections.
doi:10.1093/jac/dks050
PMCID: PMC3584961  PMID: 22389456
bacteraemia; pneumonia; colistin; mice
23.  Epigenetic Mechanisms Regulate Mallory Denk Body Formation in the Livers of Drug-primed Mice 
The mechanism of Mallory Denk body formation is still not fully understood, but growing evidence implicates epigenetic mechanisms in MDB formation. In a previous study the epigenetic memory of MDB formation remained intact for at least four months after withdrawal from the DDC diet. In the present study, mice were fed a diet containing DDC or a diet containing DDC and S-adenosylmethionine (SAMe) to investigate the epigenetic memory of MDB formation. DDC feeding caused an increase in histone 3 acetylation, a decrease in histone 3 trimethylation, and an increase in histone ubiquitination. The addition of SAMe to the DDC diet prevented the DDC induced decrease of H3K4 and H3K9 trimethylation and the increase in histone ubiquitinylation. Changes in histone modifying enzymes, (HATs and HDACs) were also found in the liver nuclear extracts of the DDC/SAMe fed mice. Data mining of microarray analysis confirmed that gene expression changed with DDC refeeding, particularly the SAMe-metabolizing enzymes, Mat2a, AMD, AHCY and Mthfr. SAMe supplementation prevented the decrease of AHCY and GNMT, and prevented the increase in Mthfr, which provide a mechanism to explain how DDC inhibits methylation of histones. The results indicate that SAMe prevented the epigenetic cellular memory involved in the MDB formation
doi:10.1016/j.yexmp.2007.12.004
PMCID: PMC2874464  PMID: 18281034
24.  SIRT1 IS INVOLVED IN ENERGY METABOLISM: THE ROLE OF CHRONIC ETHANOL FEEDING AND RESVERATROL 
Sirt1, a deacetylase involved in regulating energy metabolism in response to calorie restriction, is up regulated after chronic ethanol feeding using the intragastric feeding model of alcohol liver disease. PGC1α is also up regulated in response to ethanol. These changes are consistent with activation of the Sirt1/PGC1α pathway of metabolism and aging, involved in alcohol liver disease including steatosis, necrosis and fibrosis of the liver. To test this hypothesis, male rats fed ethanol intragastrically for 1 month were compared with rats fed ethanol plus resveratrol or naringin. Liver histology showed macrovesicular steatosis caused by ethanol and this change was unchanged by resveratrol or naringin treatment. Necrosis occurred with ethanol alone but was accentuated by resveratrol treatment, as was fibrosis. The expression of Sirt1 and PGC1α was increased by ethanol but not when naringin or resveratrol was fed with ethanol. Sirt3 was also up regulated by ethanol but not when resveratrol was fed with ethanol. These results support the concept that ethanol induces the Sirt1/PGC1α pathway of gene regulation and both naringin and resveratrol prevent the activation of this pathway by ethanol. However, resveratrol did not reduce the liver pathology caused by chronic ethanol feeding.
doi:10.1016/j.yexmp.2008.08.002
PMCID: PMC2874466  PMID: 18793633

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