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BHMT (betaine–homocysteine methyltransferase) remethylates homocysteine to form methionine. SAM (S-adenosylmethionine) inhibits BHMT activity, but whether SAM modulates BHMT gene expression is unknown. Transcriptional regulation of the human BHMT is also unknown. The present study examined regulation of the human BHMT gene by SAM and its metabolite, MTA (5′-methylthioadenosine). To facilitate these studies, we cloned the 2.7 kb 5′-flanking region of the human BHMT gene (GenBank® accession number AY325901). Both SAM and MTA treatment of HepG2 cells resulted in a dose- and time-dependent decrease in BHMT mRNA levels, which paralleled their effects on the BHMT promoter activity. Maximal suppression was observed with the BHMT promoter construct −347/+33, which contains a number of NF-κB (nuclear factor κB) binding sites. SAM and MTA treatment increased NF-κB nuclear binding and NF-κB-driven luciferase activities, and increased nuclear binding activity of multiple histone deacetylase co-repressors to the NF-κB sites. Overexpression of p50 and p65 decreased BHMT promoter activity, while blocking NF-κB activation increased BHMT expression and promoter activity, and prevented SAM but not MTA's ability to inhibit BHMT expression. The NF-κB binding site at −301 is responsible, at least in part, for this effect. Lower BHMT expression can impair homocysteine metabolism, which can induce ER (endoplasmic reticulum) stress. Indeed, MTA treatment resulted in increased expression ER stress markers. In conclusion, SAM and MTA down-regulate BHMT expression in HepG2 cells in part by inducing NF-κB, which acts as a repressor for the human BHMT gene. While SAM's mechanism is NF-κB-dependent, MTA has both NF-κB-dependent and -independent mechanisms.
- betaine–homocysteine methyltransferase (BHMT)
- endoplasmic reticulum stress
- HepG2 cell
- 5′-methylthioadenosine
- nuclear factor κB (NF-κB)
- S-adenosylmethionine
BHMT (betaine–homocysteine methyltransferase) is a zinc metalloenzyme that catalyses the transfer of a methyl group from betaine to homocysteine, forming dimethylglycine and methionine respectively [1]. BHMT is primarily expressed in the liver and kidney and is responsible, along with methionine synthase, for the remethylation of homocysteine to regenerate methionine [1]. In the rat, hepatic BHMT gene expression is induced by a methionine-deficient diet. This is further induced if the methionine-deficient diet contains dietary methyl groups such as betaine or choline [1]. In humans, betaine supplementation also increases homocysteine remethylation [2]. Despite these well-known effects of diet on BHMT gene expression, the molecular mechanism remains unknown.
Previously, we reported that hepatic BHMT mRNA levels are significantly increased in the MAT1A knockout mouse where hepatic SAM (S-adenosylmethionine; also known as AdoMet and SAMe) levels are reduced by 75% [3]. Another well-known condition where hepatic BHMT activity is induced is ethanol feeding in rodents [4]. We reported that ethanol feeding depleted both hepatic methionine and SAM levels [5]. In liver the bulk of methionine is metabolized to SAM so that methionine deficiency results in SAM deficiency [6]. This raises the possibility that the effects observed with a methionine-deficient diet may be exerted at least in part via SAM. Indeed, Finkelstein and Martin [7] showed that SAM inactivates BHMT. However, whether SAM can also influence BHMT at the mRNA level is unknown. Previously, Bose et al. [8] showed that the activity of recombinant human BHMT is not regulated by SAM, which challenges previous observations [7].
BHMT mRNA levels and activity are decreased in rat liver cirrhosis [9], which may contribute to the well-known hyperhomocysteinaemia that occurs in cirrhosis [10]. We have also shown that most of the patients with liver cirrhosis have either undetectable or reduced BHMT mRNA levels [11]. Given the importance of homocysteine in atherosclerosis and as an inducer of ER (endoplasmic reticulum) stress [12,13], it is important to better understand the regulation of the BHMT gene. The purpose of the current study is to examine whether SAM and its metabolite, MTA (5′-methylthioadenosine), can influence the expression of BHMT and, if so, define the molecular mechanisms.
Cell culture medium and FBS (fetal bovine serum) were obtained from Gibco BRL Life Technologies (Grand Island, NY, U.S.A.). The Luciferase Assay System was obtained from Promega (Madison, WI, U.S.A.). All restriction endonucleases were obtained from either Promega or Gibco. [32P]dCTP (3000 Ci/mmol) was purchased from New England Nuclear (DuPont, Boston, MA, U.S.A.). SAM, in the stable form of sulfate-p-toluenesulfonate salt was from Knoll Farmaceutici (Milan, Italy). MTA and cycloleucine were from Sigma (St. Louis, MO, U.S.A.). All other reagents were of analytical grade and were obtained from commercial sources.
Cell culture
HepG2 cells were obtained from the Cell Culture Core of the USC Liver Disease Research Center and grown according to instructions provided by the American Type Culture Collection (Rockville, MD, U.S.A.).
Recombinant plasmid and expression vectors
NF-κB (nuclear factor κB)-luciferase construct and empty vector pluc-MCS were obtained from Stratagene (La Jolla, CA, U.S.A.). pCMV-p50 and pCMV-p65 expression plasmids were kindly provided by Dr Richard Rippe (University of North Carolina at Chapel Hill) as described in [14]. Recombinant, replication-defective adenoviruses expressing IκBSR [IκB (inhibitory κB) super-repressor], which expresses a mutant IκB that cannot be phosphorylated and therefore irreversibly binds NF-κB and prevents its activation, have been previously described [14].
Cloning of the 5′-flanking region of the human BHMT gene and construction of 5′-deletion constructs
Overlapping clones containing the BHMT gene were isolated from a human genomic library (Genome Walker, Clontech). The library was screened using a random 32P-labelled probe of the human BHMT cDNA [15]. Five positive plaques were selected and DNA was isolated and digested with EcoRI. The insert fragment was subcloned into a TA vector (Invitrogen) and sequenced in both directions using the automated ABI Prism dRhodamine Terminator Cycle Sequencer performed by the Sequencing and Genetic Analysis Core Facility, Norris Cancer Institute, Keck School of Medicine USC. The initial primers were universal primers for the TA vector, all subsequent primers were nested primers designed using the available sequence information and the MacVector software program. The nucleotide sequence was verified by multiple bi-directional sequencing reactions. Sequences were aligned and a consensus sequence was generated using the ASSEMBLIGN software program. A 2.7 kb 5′-flanking region of the human BHMT gene was cloned between MluI and SmaI sites of a promoterless pGL-3-basic vector (Promega) creating the recombinant plasmid −2698/+33 BHMT-LUC. The desired 5′-deletion constructs (−2386/+33, −1920/+33, −1471/+33, −1176/+33, −839/+33 and −347/+33) were generated using the upstream primers: GTGGATATTATCATCTATTATG, GTTTTGCCACATTTTGAGG, GTATTTCAAACACATGTCACTTTAG, GTTCAAGCGATTCTCCT, GTAACTACTGTTAACATCTC and GTGCAAGAGGCAAAAAGGGAC respectively. The universal downstream PCR primer is GGTGGCATCTTTGTGGTGTCCAG.
Transcription start sites of the human BHMT
Transcription start sites were determined using the GeneRacer™ kit (Invitrogen). The 5′ ends of cDNA using the reverse primer 5′-TGTCGGTGCCCATGGCAGCTGCC-3′, which is complementary to −10 to +13 of the human BHMT [15], were obtained. RACE (rapid amplification of cDNA ends) PCR products were cloned using TOPO TA cloning for sequencing and sequencing analysis was performed by the Sequencing Facility, Norris Cancer Centre, Keck School of Medicine USC.
Analysis of promoter constructs in cell culture
To study the relative transcriptional activities of the BHMT promoter fragments, HepG2 cells (0.8×106 cells in 2 ml of medium) were transiently transfected with 2.5 μg of BHMT promoter luciferase gene construct or the promoterless pGL3-basic vector (as negative control) using the Superfect transfection reagent (Qiagen, Valencia, CA, U.S.A.) as we described [14]. To control for transfection efficiency, cells were co-transfected with the Renilla pRL-SV40 vector (Promega, Madison, WI, U.S.A.), which is a plasmid containing the Renilla luciferase gene driven by a SV40 (simian virus 40) promoter. After 24 h, cells were harvested and lysed in 200 μl of reporter lysis buffer (Luciferase Assay System, Promega). Aliquots of the cell lysates were sequentially assessed for firefly and Renilla luciferase activities using a TD-20/20 luminometer (Promega). The luciferase activity driven by the BHMT promoter constructs was normalized to the Renilla luciferase activity. Each experiment was done with triplicate samples.
Effect of SAM and MTA on BHMT expression assessed by Northern-blot analysis and real-time RT (reverse transcriptase)–PCR
HepG2 cells were treated with SAM (0.25–5 mM) or MTA (0.25–1 mM) for up to 18 h. We had previously shown that these agents could induce apoptosis in HepG2 cells after 18 h of treatment [16]. Indeed, both 5 mM SAM and MTA did not cause apoptosis after 12 h of treatment but apoptosis was induced by 18 h (results not shown). Following treatment, total RNA was extracted and Northern hybridization analysis was performed using specific BHMT cDNA probes that correspond to 879–1090 of the human BHMT sequence [15] as described in [17]. To ensure equal loading of RNA samples and transfer in each of the lanes, prior to hybridization, membranes were rinsed with ethidium bromide and photographed and the same membranes were also rehybridized with a 32P-labelled β-actin cDNA probe as described in [17]. Auto-radiography and densitometry (Gel Documentation System, Scientific Technologies, Carlsbad, CA, U.S.A. and NIH Image 1.60 software program) were used to quantify relative RNA. Results of Northern-blot analysis were normalized to β-actin.
Real-time quantitative PCR was also carried out on the above RNA samples from HepG2 cells treated with SAM (0.25–5 mM) or MTA (0.25–1 mM) for 12 h. Total RNA (1 μg) was used in a 20 μl reverse transcription reaction volume. Following reverse transcription (M-MLV RT; Life Technologies, Gaithersburg, MD, U.S.A.), the real-time PCR was run in the Mx3005PTM thermo-cycler (Stratagene, La Jolla, CA, U.S.A.) in triplicates with the thermo-cycle profile of stage 1: 95 °C for 10 min, stage 2: 95 °C for 15 s, 60 °C for 1 min, 40 cycles. The primer and TaqMan probe [BHMT 00156084, HPRT1 (hypoxanthine–guanine phosphoribosyltransferase 1) Hs99999909, UBC (ubiquitin C) Hs00824723, ready-in-use mixture] and Universal PCR Master mix were purchased from ABI (Foster City, CA, U.S.A.). HPRT1 and UBC were used as housekeeping genes as described in [18].
The expression of BHMT RNA was checked by normalizing the Ct (threshold cycle value) of BHMT to that of the control housekeeping gene (HPRT1 or UBC) [19]. The ΔCt obtained was used to find the relative expression of BHMT in treated cells compared with untreated or empty vector-treated cells according to the formula: where ΔΔCt=(ΔCt of BHMT in treated cells)−(ΔCt of BHMT in control cells).
Effect of SAM and MTA on recombinant BHMT promoter and NF-κB-driven reporter activity
To assess the effect of SAM or MTA on BHMT promoter activity, HepG2 cells were transfected with recombinant human BHMT promoter constructs and treated with SAM (5 mM) or MTA (1 mM) during the last 12 h of the transfection. In some experiments, cells were pretreated with cycloleucine (20 mM) for 2 h in order to block the conversion of MTA back to SAM [16]. This was followed by MTA (1 mM) or SAM (5 mM) treatment for another 12 h. Luciferase activity driven by these promoter luciferase gene constructs was measured as described above.
To assess the effect of SAM or MTA on NF-κB-driven luciferase activity, HepG2 cells were transfected with recombinant NF-κB-LUC (contains five κB sequences linked to the reporter) and treated with SAM (5 mM) or MTA (1 mM) for 12 h. Luciferase activity driven by this NF-κB promoter was measured as described above.
Effect of p50 and p65 expression vectors on BHMT promoter activity
To see if overexpression of p50 or p65 can influence the BHMT promoter activity, HepG2 cells were first transfected with either a p50 or p65 expression vector (1.5 μg per well for 12 h) and then transfected with the recombinant BHMT promoter luciferase construct −347/+33-LUC or the pGL-3-basic vector for 24 h. Luciferase activity was measured as described above.
Effect of blocking NF-κB on BHMT expression, promoter activity and effects of SAM and MTA
To study the effect of blocking NF-κB activation, HepG2 cells were infected with recombinant adenoviruses carrying IκBSR or empty vector for 12 h as described previously [14]. After 12 h of infection, the viruses were removed and replaced with fresh medium for SAM (5 mM) or MTA treatment (1 mM) for 12 h and RNA was isolated for real-time PCR. In other experiments, following infection with adenoviruses carrying IκBSR or empty vector for 12 h, HepG2 cells were transfected with the BHMT promoter construct −347/+33-LUC, the pGL-3-basic vector, or the NF-κB-LUC for 12 h. Luciferase activity was measured as described above.
Effect of an NF-κB site mutation on BHMT promoter activity
Site-directed mutagenesis was performed to examine the functional roles of two putative NF-κB binding sites located at −301 and −135 of the BHMT gene (relative to the translation start site, see Figure 1). Each site was mutated (−301 site, from 5′-GTGGCAACCT-3′ to 5′-CTCGCAACCT; −135 site, from 5′-GGGAGGCCCT-3′ to 5′-AGTAGGCCCT-3′), where the mutated sequence is underlined. The mutation was performed by PCR and confirmed by restriction enzyme digestion and sequencing. HepG2 cells were transfected with −347/+33-LUC that contained the wild-type, a mutation at the −301 site (Mut−301), a mutation at the −135 site (Mut –135), or a double mutant (both NF-κB sites are mutated) and the effect of the mutation was assessed by measuring luciferase activity. HepG2 cells transfected with these constructs were also treated with SAM (5 mM) or MTA (1 mM) for 12 h and luciferase activity was measured as described above.
EMSA (electrophoretic mobility-shift assay) and supershift assay
EMSAs for consensus (obtained from Santa Cruz Biotechnology, Santa Cruz, CA, U.S.A.) and putative NF-κB binding sites were performed as described in [14]. The probes are 32P-end labelled double-stranded DNA fragments (−142 to −114, −271 to −246 and −310 to −285 of BHMT) or containing a consensus NF-κB binding site (5′-AGTTGAGGGGACTTTCCCAGGC-3′). The effect of SAM (0–5 mM) and MTA (0–2 mM) on nuclear binding activity to the consensus NF-κB probe was first determined following treatment of HepG2 cells with these agents for 12 h. The effect of SAM (5 mM for 12 h), MTA (1 mM for 12 h), or p50 overexpression for 24 h on binding to the putative NF-κB binding sites of the BHMT promoter was next determined. Supershift assays were performed with anti-p50 and anti-p65 antibodies (Santa Cruz Biotechnology) as described in [14].
Western-blot analysis
A time course of NF-κB activation was determined using Western-blot analysis to assess the level of nuclear p65 and p50 as described in [20].
ChIP (chromatin immunoprecipitation) assay
To see if SAM or MTA treatment of HepG2 cells induces NF-κB and HDAC (histone deacetylase) binding to the BHMT promoter in an endogenous chromatin configuration, a ChIP assay was carried out following the ChIP assay kit protocol provided by Upstate (Waltham, MA, U.S.A.). HepG2 cells were treated with SAM (5 mM), MTA (1 mM) or vehicle control and processed for the ChIP assay as per the protocol except for minor modifications. Briefly, proteins were cross-linked to DNA by treating cells with 1% formaldehyde at 37 °C for 10 min. After fixation, cells were washed with ice-cold PBS, resuspended and lysed in SDS lysis buffer (Upstate) and centrifuged for 5 min at 110 g. Cell lysates were sonicated at 25–30% power, 5×10 s using a Sonic Dimembrator Model F60 (Fisher Scientific, Pittsburgh, PA, U.S.A.) to fragment the chromatin to approx. 1 kb or less. The sonicated cell lysates were spun in a microcentrifuge at 13000 g for 10 min at 4 °C. The supernatant containing the soluble chromatin sample was treated with 1 μl of 5 M NaCl and heated at 65 °C for 4 h to reverse the protein–DNA cross-links. The reversed soluble chromatin sample (20 μl) was removed and used as the input control (total chromatin fraction) for the final PCR reaction. The remaining chromatin solution was split into equal fractions and subjected to immunoprecipitation in the presence or absence of anti-p50, anti-p65, anti-HDAC1, anti-HDAC2 and anti-HDAC3 antibodies (Santa Cruz Biotechnology). PCR of the BHMT promoter region across the three NF-κB sites within −301 to −135 of the BHMT promoter was performed using the forward primer 5′-AAATCTGAGCGAGTGGCAACCTCGC-3′ (bp −313 to −289 relative to the ATG start codon) and the reverse primer 5′-ATCTTCGTGGTGTCCAGACAGGTGG-3′ (bp −23 to +2 relative to the ATG start codon). All PCR products were run on 8% (w/v) acrylamide gels and stained with ethidium bromide for 15–30 min.
SAM levels
SAM levels were measured in HepG2 cells treated with cycloleucine (20 mM, 2 h pretreatment) or vehicle followed by SAM (5 mM) or MTA (1 mM) with or without cycloleucine (20 mM) for another 12 h as we described previously [16].
Markers of ER stress
To study the effect of SAM and MTA on the ER stress response, the expression of the ER stress markers, GRP78 (glucose-regulated protein 78) and GADD153 (growth-arrest and DNA-damage-inducible protein 153) [13], was analysed in HepG2 cells treated with these agents for 12 h. Real-time PCR quantification of GRP78 and GADD153 RNA from SAM- or MTA-treated HepG2 cells was performed using the following custom made primers and probes (GenScript Corporation): GRP78 forward primer: TCCTGCGTCGGCGTGT; GRP78 reverse primer: GTTGCCCTGATCGTTGGC; and GRP78 probe labelled with FAM (6-carboxy fluorescein) and TAMRA (6-carboxytetramethylrhodamine): AAGAACGGCCGCGTGGAGATCAT. GADD153 forward primer: GTGAATCTGCACCAAGCATGA; GADD153 reverse primer: AAGGTGGGTAGTGTGGCCC; and GADD153 probe labelled with FAM and TAMRA: CAATTGGGAGCATCAGT-CCCCCACT. HPRT1 was used as a control housekeeping gene for these experiments. The real-time PCR was run in triplicate with the thermo-cycle profile of stage 1: 95 °C for 10 min; stage 2: 95 °C for 15 s, 60 °C for 1 min for 40 cycles.
Statistical analysis
Data are given as means±S.E.M. Statistical analysis was performed using ANOVA followed by Fisher's test for multiple comparisons. For changes in mRNA levels, ratios of BHMT to β-actin densitometric values were compared with ANOVA. Significance was defined by P<0.05.
Cloning and sequencing of the 5′-flanking region of the human BHMT
The sequence of the 2.7 kb product is shown in Figure 1. Analysis of the transcription factor binding site was performed using Transcription Factor Search (http://www.cbrc.jp/research/db/TFSEARCH.html) and MatInspector (http://www.genomatix.de/cgi-bin/eldorado/main.pl). The 5′-flanking region of the human BHMT contains multiple consensus binding sites for NF-κB, Sp1 (stimulating protein-1), SREBP (sterol-regulatory-element-binding protein), C/EBP (CCAAT/enhancer-binding protein) and HNF (hepatocyte nuclear factor). In addition, consensus binding sites for GRE (glucocorticoid receptor), CREB (cAMP-response-element-binding protein), STAT (signal transducer and activator of transcription), ISRE (interferon-stimulated response element), c-Myc, c-Myb and transcription factor E2F are also present.
Transcriptional start site
The transcriptional start site was determined by cloning products of 5′-RACE using cDNA from HepG2 cells, followed by sequencing. The primer corresponding to −10 to +13 of the human BHMT cDNA was used and yielded a product beginning 80 nt upstream of the translational start site (Figure 1, indicated by forward arrow).
Effect of SAM and MTA on BHMT expression
We next examined the effect of SAM and MTA on BHMT expression in HepG2 cells using Northern-blot analysis (Figure 2A) and real-time RT–PCR (Figures 2B and 2C). Both agents exerted a dose-dependent down-regulation of BHMT mRNA levels, with the maximum inhibitory dose of SAM at 5 mM (50% fall) and MTA at 1 mM (84% fall) at the 12 h time point (Figure 2B). At these doses, no apoptosis was evident (results not shown). However, treatment with 5 mM MTA for 12 h induced apoptosis (results not shown). Up to 12 h, the effect of MTA and SAM (results not shown) treatment on BHMT mRNA levels was time-dependent (Figure 2C). All subsequent studies were done using non-toxic regimens, namely SAM (5 mM) or MTA (1 mM) for 12 h.
Effect of SAM and MTA on BHMT promoter activity
To delineate sequences that drive the expression of the human BHMT gene, deletion mutants ranging from −2698 to +33 were cloned into the promoterless luciferase reporter gene vector pGL3-basic. The promoterless construct pGL3-basic served as the background control. Luciferase activity was measured after transient transfection of HepG2 cells with these constructs. Figure 3 shows that the human BHMT promoter was able to efficiently drive luciferase expression in HepG2 cells. Construct −347/+33 has maximal promoter activity. Treatment of transfected HepG2 cells with SAM (5 mM) or MTA (1 mM) during the last 12 h of the transfection led to a significant inhibition in promoter activity, which was most pronounced with the construct −347/+33 and closely paralleled the effect of these agents on endogenous BHMT gene expression (Figure 2B).
To see if the effect of MTA may be mediated by its conversion into SAM, cells were pretreated with cycloleucine (20 mM for 2 h) prior to treatment with MTA or SAM. Indeed, cycloleucine treatment increased the reporter activity driven by the BHMT promoter construct −347/+33 but did not block the ability of MTA to inhibit the BHMT promoter activity (see supplementary data at http://www.BiochemJ.org/bj/401/bj4010087add.htm.). The effectiveness of cycloleucine treatment is demonstrated by measuring cellular SAM levels. Cycloleucine lowered SAM levels since it is an inhibitor of methionine adenosyltransferase; while MTA treatment raised SAM levels since it can be converted back to SAM [16]. Cycloleucine treatment blocked the MTA-mediated increase in cell SAM levels, which is consistent with its ability to block the conversion of MTA into SAM (see supplementary data).
SAM and MTA treatment induces NF-κB nuclear binding in HepG2 cells
The −347/+33 region of the human BHMT gene contains three NF-κB binding sites (Figure 1). To see if SAM and MTA treatment affects NF-κB nuclear binding, we first examined this using a consensus NF-κB probe. Indeed, treatment with these agents led to a significant increase in NF-κB nuclear binding (Figure 4A). SAM and MTA activated NF-κB in a time-dependent fashion, with appearance of p65 and p50 in the nuclear compartment by 2 h after treatment (Figure 4B). We next examined whether binding to each of the three NF-κB sites (at −301, −271 and −135) of the BHMT gene is affected by SAM or MTA treatment. Figure 4(C) shows that SAM and MTA treatment led to an increase in NF-κB binding to −135 and −301 sites, while binding to −271 site is minimally affected (results not shown). Overexpression with p50 led to increased p65/p50 and p50/p50 nuclear binding (Figure 4C), and overexpression with p65 also led to increased p65/p50 nuclear binding (results not shown). Increased NF-κB nuclear binding occurred following MTA or SAM treatment not only with recombinant probes for NF-κB (EMSA), but also in the endogenous chromatin configuration as demonstrated using the ChIP assay (Figure 5). Furthermore, both SAM and MTA also induced increased nuclear binding of HDAC1, HDAC2 and HDAC3 to the region spanning the NF-κB sites (Figures 5A and 5B).
Role of NF-κB in SAM- and MTA-mediated inhibition of BHMT expression in HepG2 cells
To see if increased NF-κB nuclear binding translated to a functional change, we determined the effect of SAM and MTA on NF-κB-driven reporter activity. Figure 6(A) shows that treatment of HepG2 cells with either SAM or MTA nearly doubled the NF-κB-driven reporter activity. To demonstrate that increased NF-κB nuclear binding can impact on BHMT promoter activity, we determined the effect of overexpression of p50 or p65 on BHMT promoter activity. Figure 6(B) shows that overexpression of either p50 or p65 lowered the BHMT promoter activity by nearly 50%.
To further examine the role of NF-κB in regulating human BHMT gene expression, HepG2 cells were infected with adenoviruses carrying IκBSR to block NF-κB activity. Infection with IκBSR reduced the NF-κB-driven luciferase activity to 4±0.4% (mean±S.E.M. from two experiments performed in duplicate) and led to an increase in BHMT promoter activity by 215±51% (mean±S.E.M. from four experiments, P<0.05 versus control BHMT promoter activity). Blocking NF-κB activation led to a slight increase in endogenous BHMT mRNA levels, prevented the ability of SAM to lower BHMT expression and blunted the inhibitory effect of MTA (Table 1).
To conclusively demonstrate the importance of the two NF-κB sites present in the −347/+33 region of the human BHMT (at −301 and −135), we performed site-directed mutagenesis of these sites and determined the effect of these mutations and the effect of SAM and MTA treatment on BHMT promoter activity. Table 2 shows that, when the NF-κB site at −301 was mutated, the BHMT promoter activity increased, SAM no longer inhibited but MTA was still able to inhibit promoter activity. However, the effect of MTA is slightly blunted, from 63% inhibition with wild-type or the Mut–135 construct to 50% inhibition with the Mut–301 construct.
Effect of SAM and MTA on ER stress markers
Homocysteine has been linked to increased ER stress [21]. To see if inhibition of BHMT expression by SAM and MTA can lead to increased ER stress, the expression of two ER stress markers GRP78 and GADD153 was examined. MTA treatment increased GRP78 and GADD153 mRNA levels to more than 350 and 500% of control levels respectively (GRP78 was increased to 362±63% and GADD153 was increased to 524±30% of control values; results represent means±S.E.M. from three to four experiments, P<0.005). However, SAM treatment did not increase these ER stress markers (results not shown).
Homocysteine is a risk factor for atherosclerosis [12] and an inducer of ER stress that may participate in the pathogenesis of alcoholic liver disease [13]. In the liver, homocysteine lies at the junction of two intersecting pathways, the transsulfuration pathway (the pathway that converts the sulfur atom of methionine into cysteine and glutathione), and the remethylation pathway (the pathway that conserves homocysteine as methionine), which is coupled with cobalamin, folate and betaine metabolism [22]. In the liver and kidney, BHMT is responsible, along with methionine synthase, for remethylation of homocysteine [1]. Hyperhomocysteinaemia commonly occurs in liver cirrhosis due to impaired homocysteine metabolism [10]. Betaine, a cofactor for BHMT, has been shown to prevent the hyperhomocysteinaemia and liver injury in rodent models of alcoholic liver disease [13,23].
Previous studies in the rat have shown that hepatic BHMT expression is induced by a methionine-deficient diet [1]. However, the molecular mechanism(s) are unknown. SAM has been shown to inactivate BHMT [7]; however, whether it influences BHMT at the mRNA level has not been studied. In our current study, we examined the influence of SAM and its metabolite, MTA, on BHMT gene expression. The rationale for studying MTA is that MTA is a product of SAM metabolism in the polyamine pathway [7] as well as non-enzymatic hydrolysis [24]. We have shown that SAM's effect on cell growth, apoptosis and TNFα (tumour necrosis factor α) expression can be mimicked by MTA [16,25,26]. In contrast with SAM, MTA does not contribute to GSH synthesis, is not a methyl donor and inhibits methyltransferases [27]. Thus additional insights can be gained by comparing the effect of these two agents.
We began these studies by cloning the 2.7 kb 5′-flanking region of the human BHMT. One transcriptional start site was identified 26 nt downstream from a putative TATA box and 80 nt upstream of the translational start site. These results are in agreement with the results published by Park and Garrow [1]. In the work of Park and Garrow [1], the human BHMT promoter was not functionally characterized. The sequence of the BHMT 5′-flanking region contains numerous NF-κB binding sites as well as HNF binding sites, which may play a role in conferring high expression in the liver. Transfection studies showed that maximal promoter activity was obtained with the BHMT promoter construct −347/+33-LUC, denoting the presence of enhancer element(s) in this region. Additional enhancer element(s) are likely present between −1920 and −1471. In contrast, the presence of repressor element(s) is suggested between −1471 and −347, as well as −2698 and −2386 as the promoter activity fell significantly.
Treatment of HepG2 cells with SAM and MTA led to a dose- and time-dependent decrease in BHMT mRNA levels. MTA is a more potent inhibitor than SAM, which is reflected by both dose–response curves as well as the magnitude of inhibition. We had also observed this in our other studies [16,25], and it raises the possibility that the effect of SAM may be mediated at least in part by MTA. The effect of these agents on the endogenous BHMT gene expression is comparable with their effects on the recombinant BHMT promoter activity. The maximum inhibitory effect occurred with the shortest promoter construct −347/+33.
Since MTA can be converted back to SAM via the methionine salvage pathway [16], we examined whether blocking this conversion by using cycloleucine (an inhibitor of methionine adenosyltransferase) would have any effect. Cycloleucine treatment lowered cellular SAM levels and increased BHMT promoter activity. SAM and MTA treatment raised cellular SAM levels by 268 and 160% respectively. Cycloleucine pretreatment completely blocked the effect of MTA on cellular SAM levels but had no influence on MTA's ability to inhibit BHMT promoter activity. These results are consistent with the following: (i) MTA's effect is not mediated via SAM, and (ii) there is no direct correlation between cellular SAM levels and BHMT expression. While we cannot exclude that cellular SAM levels may influence BHMT expression, our results suggest that the inhibitory effects of SAM and MTA occurred by some other mechanism(s). Furthermore, BHMT expression falls in human and rat liver cirrhosis [9,11], where SAM biosynthesis is inhibited [6]. Thus it is unlikely that the intracellular SAM level is the main regulatory control for BHMT expression.
Struck by the presence of multiple NF-κB binding sites in the −347/+33 region, we examined whether SAM and MTA treatment may influence NF-κB nuclear binding activity. Indeed, treatment with either of these agents led to an increase in NF-κB nuclear binding to two of the three NF-κB sites present within this region of the BHMT gene. Increased NF-κB nuclear binding to this region of the BHMT gene in its endogenous chromatin configuration was also demonstrated using the ChIP assay. Increased nuclear binding translated to a functional change as SAM and MTA treatment increased the NF-κB-driven reporter assay in HepG2 cells.
Overexpression of NF-κB inhibited BHMT promoter activity, whereas blocking NF-κB activation increased BHMT expression and promoter activity, supporting the notion that NF-κB acts as a repressor for this gene. Blocking NF-κB activation completely prevented the ability of SAM to inhibit BHMT expression, but it only blunted MTA's inhibitory effect. These results suggest that SAM exerts its effect on BHMT expression via NF-κB but MTA exerts its effect on BHMT expression by both NF-κB-dependent and -independent mechanisms. Finally, mutagenesis experiments suggest that SAM exerts its inhibitory effect via transactivation of the NF-κB site at −301, which is functioning as a repressor. However, we cannot rule out participation of other NF-κB sites in the regulation of BHMT. Much more work will be required to elucidate the NF-κB-independent mechanisms of BHMT transcriptional regulation.
While NF-κB typically acts as an enhancer to transactivate promoters, it has also been shown to act as a repressor [28,29]. In this regard, several co-repressor complexes for NF-κB have been described, including SMRT (silencing mediator for retinoic acid receptor and thyroid hormone receptor), NcoR (nuclear receptor co-repressor), HDAC1, HDAC2, HDAC3 and HDAC6 [28,30]. Interestingly, both SAM and MTA also induced recruitment of HDAC1, HDAC2 and HDAC3 to the BHMT promoter region containing the NF-κB binding sites. HDAC1 has been shown to physically interact with NF-κB and HDAC2 is known to associate with HDAC1 [29]. Presumably these HDAC proteins regulate the activity of NF-κB through their deacetylase activity since their effect can be blocked by HDAC inhibitors [28]. The mechanisms regulating the interaction between NF-κB and HDAC proteins are largely unknown and results from the present study suggest that SAM and MTA may enhance this interaction. This possibility will require further examination. Even though SAM and MTA may inhibit BHMT expression by recruiting HDACs to the BHMT promoter, the fact that overexpression of p50 and p65 can inhibit BHMT promoter activity would suggest that participation of these co-repressors may not be necessary to inhibit BHMT expression. The ability of NF-κB to repress BHMT gene expression may help to explain the marked down-regulation of BHMT expression in human hepatocellular carcinoma [11], where we have described increased NF-κB nuclear binding activity [31]. It may also help to explain decreased BHMT expression in liver cirrhosis [11], where inducers of NF-κB are often up-regulated (i.e. oxidative stress).
Since impaired homocysteine metabolism can lead to ER stress and apoptosis [13], we also examined whether treatment with SAM or MTA can induce ER stress in HepG2 cells. Consistent with this notion, MTA treatment more than tripled two ER stress markers. However, SAM treatment did not induce the SAM markers. This may be due to the fact that SAM's inhibitory effect on BHMT gene expression is only 40–50% at best, while MTA is much more potent in its inhibitory effect. This also suggests that blocking homocysteine metabolism via BHMT and inducing ER stress may be another mechanism for these agents (at least in the case of MTA) to exert a pro-apoptotic effect on liver cancer cells [16].
Castro et al. [32] have published in a meeting proceeding characterization of the human BHMT promoter and demonstration of SAM's ability to inhibit BHMT promoter activity. Our results of the basal human BHMT promoter activity and SAM's ability to down-regulate BHMT promoter are in full agreement with this work. These investigators found that methionine and ethionine did not affect BHMT promoter activity and had no influence on SAM's inhibitory effect, suggesting a direct effect of SAM on BHMT promoter. However, the molecular mechanism of SAM's inhibitory effect was not pursued in their work. Our current study extends these observations, shows an even more pronounced inhibition of BHMT expression by a SAM metabolite, MTA, and elucidates the molecular mechanism of SAM's inhibitory effect.
It should be stressed that our work used pharmacological concentrations of SAM. The plasma concentration of SAM is extremely low (∼0.05 μM) [33] and can increase up to 1 mg/l or 2.5 μM following oral administration of 1000 mg of SAM [34]. However, liver SAM levels can reach 0.5 mM following a methionine load [35]. Thus our data are likely relevant for the liver, particularly after a methionine load or for patients taking large doses of SAM. Nevertheless, the key finding of the present paper is that using SAM and MTA at pharmacological doses can inhibit BHMT expression by activating NF-κB, which acts as a repressor for this gene. While SAM's inhibitory effect is NF-κB-dependent, MTA exerts its inhibitory effect by both NF-κB-dependent and -independent mechanisms. Inhibiting BHMT expression can lead to increased ER stress and contribute to the pro-apoptotic effects of these agents on liver cancer cells.
This work was supported by NIH (National Institute of Health) grants DK51719 (S.C.L.), AA12677, AA13847 and AT1576 (S.C.L. and J.M.M.) and Plan Nacional of I+D SAF2002-00168 of the Ministerio de Educación y Ciencia (to J.M.M.). HepG2 cells were provided by the Cell Culture Core of the USC Research Center for Liver Diseases (DK48522). K.R. is a recipient of the Postdoctoral Fellowship of the Training Program in Alcoholic Liver and Pancreatic Diseases (T32 AA07578), and A.I.A. is a recipient of the Postdoctoral Fellowship supported by the CIC bioGUNE, Centro de Investigación Cooperativa en Biosciencías.
Abbreviations: BHMT, betaine–homocysteine methyltransferase; ChIP, chromatin immunoprecipitation; Ct, threshold cycle value; EMSA, electrophoretic mobility-shift assay; ER, endoplasmic reticulum; FAM, 6-carboxy fluorescein; GADD153, growth-arrest and DNA-damage-inducible protein 153; GRP78, glucose-regulated protein 78; HDAC, histone deacetylase; HNF, hepatocyte nuclear factor; HPRT1, hypoxanthine–guanine phosphoribosyltransferase 1; MTA, 5′-methylthioadenosine; IκB, inhibitory κB; IκBSR, IκB super-repressor; NF-κB, nuclear factor κ B; RACE, rapid amplification of cDNA ends; RT, reverse transcriptase; SAM, S-adenosylmethionine; TAMRA, 6-carboxytetramethylrhodamine; UBC, ubiquitin C
- The Biochemical Society, London


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Comment Re:Still depends on gcc? Still needs root? (Score 2) 29
You don't need to be root to add the challenge file, just have permission to write to the website folder.
You can also use DNS verification, although that method is not present in Certbot yet. There are a few third-party clients that do support that method, however.

subset: commoncrawl


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Following a brief foreword and introduction the reader will find presentations on the problems of crime and punishment, the relation of war and medicine, individual and collective aggressiveness, behavior influences of babies, and the effect of society and war on each other. Also included is an address by Sir Norman Angell on plans for peace. While much of these offerings are interesting they tend to be mostly psychologic and philosophic considerations and in this sense are closely related to human behavior. Perhaps such material may not be interesting to all readers, but the present lectures fill a useful place in the series of lectures for the public which has become so well known in New York.
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Old boring dirty, cancer causing, acid rain causing coal. The biggest threat to global health next to nuclear war.
Sentiment: Strong Sell
Don't you have a tree that needs a hug or something?
Caol - you have to give Queen4aday a break. It's stressful getting free welfare, food stamps ,cell phones and not having to work for them. It's got to be a lonely boring life never having to leave the house to work. for a living........imo
When NG heads back to 8 bucks in about 5 years, you better hope your Utility Voucher has an inflation clause. You'll be begging for a coal fired supplier. Buy American Coal now.
Sentiment: Buy
I guess you want to power the United States on hamsters then? Or let's turn your house into an Algae farm to power your microwave? Until infrastructure is built... Wake up coal is needed. Not to mention your EPA is about to kill natural gas fracking.


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We have quite a few, but the TOP Jamaican newspapers are probably the ones below.
That is, of course, is topped by the prominent and prestigious: Jamaica Gleaner and The Jamaica Observer, both of which are now online.
And they don't occupy the top spots by chance, they are genuinely the more reputable and established newspapers.
They deliver responsible, high quality impartial reporting- on news and current happenings in Jamaica.
You get the usual; news, sports, politics, commentary, lifestyle, business, editorial, letters, classifieds and all!
By the way, both now have mobile applications. The blackberry ones can be found at Blackberry World. I have both on my blackberry phone, although, admittedly, I use the Observer app more then the Gleaner's. The Observer application is a lot more nifty and clean - to me.
That said, I never miss my Sunday Gleaner, the hard copy! And I can safely say it is also the most sought after Sunday paper here- primarily for it's 'Careers' section, which shows vacancies and job openings across the country.
Other notable local newspapers include:
Another notable resource, although not a newspaper, is the government owned Jamaica Information Service (JIS).
It provides loads of information on current news and happenings in Jamaica. If you can't get around to all of them, don't miss the JIS.
Make sure you bookmark my current events in Jamaica page as well. It keeps you up with the very latest from Jamaica.
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yesterday + target raised today = downsomething wrong possold half and will watch itcan buy them
CRDN is also featured in the stocks in the news on page B8 in IBD. Mentions that CRDN appears to be finding support at the 10 week moving average. Analysts expect Q4 EPS growth of 44% and sales gains of 29%.
Well dont let word get out but this stock has been put on cabots model portfolio. I would expect a rise in volume tomorrow.
Maybe he is tshih, I don't know. Don't care either. Still bullish on this stock. Got out entirely on Dec ist when it hit a high and have not gotten back in. The late december order from the Marines is a fine indication that revenue keeps on coming in for this company from a variety of sources. I didn't expect that order so late in the year. Management has done exactly what they said they would do and I have no reason to doubt their estimates. We're still undervalued.
Volume is very low, so I don't think something is wrong. It does seem like CRDN has passed the baton to AH.


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DeMarco's hat trick helps Villa Maria lacrosse team prevail
Grace DeMarco's hat trick lifted the Villa Maria girls lacrosse team to a 7-6 win over McDowell on a rainy Monday night at Gus Anderson Field.
A 3-2 Trojans (2-3) lead at halftime quickly evaporated at the hands of DeMarco, who scored two of her three goals in the second half. Diana Oduho had two goals for the Victors (2-1).
Leading 7-4 with 5 minutes left in the game, Villa was able to keep the ball away from McDowell and run the clock down to the final 2 minutes. Once the Trojans were able to get back on the offensive, they netted two goals, but it wasn't enough as time ran out.
Sophia Garck and Alex Zielinski had two goals each for the Trojans.


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"Proud Cub Scout Mom" Ladies T-shirt
Starting at:
$14.99You're raising a super-cool Cub—show your pride in style! Bright-Red tee features "Proud Cub Scout Mom" lettering with Cub Scouts® logo in background. Short-sleeve T-shirt is made of pre-shrunk, ringspun 100% cotton. Imported. Learn More
Washable Wool Jac-Shirt
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$69.99Introducing a convenient update to the still-classic red uniform jac-shirt! A new Adult blend of 85% wool and 15% nylon creates a comfortable, washable fabric that's not only durable but delivers all the warmth you expect from wool. It's a versatile layering piece that's made to last. Look for added details enhancing quality and style: back yoke for easier fit, double-needle stitching, elbow patches, and a BSA® emblem that can be sewn to the front pocket. Imported. Learn More
Waterproof Breathable Adult Rain Jacket with Reflective Detail
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$29.99This one's sealed tight for premium protection and accented for added safety. Full-zip-front design features a BSA® reflective, heat-seal logo at left chest and reflective taping on back for enhanced visibility in bad weather or at night. Offering seam-sealed waterproofing and breathability in a lightweight, diamond-patterned ripstop nylon, this hooded jacket is comfort-rich and weather-ready. Includes hood, two front pockets, and elasticized cuffs. Lime Green. Imported. Learn More
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AT&T Inc (T) Earnings: After-Hours News
AT&T Inc. (NYSE: T) reported fiscal third-quarter earnings excluding items of 66 cents per share on revenue of $32.16 billion, versus Wall Street estimates for earnings of 65 cents per share on revenue of $32.19. During the same year-ago period, the telecom giant issued earnings of 62 cents on revenue of $31.46 billion.
The company's earnings came in-line with forecasts as AT&T saw strong wireless gains and record U-verse results. In the quarter, AT&T added nearly 1 million subscribers and the company sold a third-quarter record of over 6.7 million smartphones. Smartphones accounted for a record 89 percent of postpaid phone sales in the period.
"We're setting the standard for 4G LTE speeds and network reliability. Our fiber and U-verse expansion projects are ahead of schedule bringing high-speed broadband to millions more customers," said Randall Stephenson, AT&T chairman and CEO. "With these initiatives, we're seeing excellent growth across our major platforms — mobility, U-verse and strategic business services."
The communications firm said U-verse had its first billion-dollar revenue month, up 28.1 percent, and total subscribers topped 10 million.
Shares of AT&T edged up 0.14 percent to $35.33 in extended-hours trading after the news.
The Dow Jones industrial average fell 54.33 points, or 0.35 percent, to close at 15,413.33. The S&P 500 Index was down 8.29 points, or 0.47 percent, to end at 1,746.36. The Nasdaq Composite Index was down 22.49 points, or 0.57 percent, to finish at 3,907.07.
Video Credit: Reuters
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Lemon and Olive Oil Dressing
Total Time
Prep 10 mins
Cook 0 mins
A classic Mediterranean salad dressing that's simple and so good – makes a great marinade for grilled chicken, too.
Ingredients Nutrition
- 1 tablespoon sea salt
- 3 garlic cloves, minced
- 1⁄2 cup fresh lemon juice
- 1⁄2 cup high-quality extra virgin olive oil
- 1⁄2 cup freshly grated parmesan cheese (optional)
- 1 grated lemon, zest of (optional)
- With a mortar & pestle, make a paste of the salt and minced garlic.
- Mix together lemon juice and olive oil. Add the salt and garlic mixture to the lemon juice and olive oil in a tightly closed container.
- Shake well. Sprinkle optional grated Parmesan or lemon zest on top of salad before tossing.
Most Helpful
The one-to-one ratio of EVOO to lemon juice makes it way too tart. Use the tried-and-true 3 parts EVOO to one part vinegar (in this case, lemon juice).
This was so salty it was inedible as salad dressing. I didn't use the cheese or zest but I don't think that would have made a difference. After tripling the oil and lemon juice it was passable. Is 1T salt a typo? I'm shocked anyone gives this 5 stars as written. Maybe it would be good as a marinade, but not for dressing.
This is a terrific, fresh


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Blog Entry# 467387
Posted: Jun 28 2012 (5:23PM)
243 Responses
Last Response: Jul 01 2012 (5:39PM)
Jun 28 2012 (5:23PM) 12259/Sealdah - New Delhi AC Duronto Express
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Please join hands and welcome the new king of indian railways. Now BPL shatabdi hand over crown to the new IR KING.....Sealdh Duranto...
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Jun 28 2012 (5:53PM)
Begins Falls Rises ™*~ 21448 blog posts 1207 correct pred (63% accurate)
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I never consider the paper remarks. Not even the TT or the AVG speed. U can also remember tat we saw Nagavali SF Express made a impressive run despite it had a slack time. 40mins of 61km isn't an easy job for any express train. Even here, Duronto gets clearance almost til its tech halts. even BPL Shatabdi follows the same rule. There are days, when Shatabdi used to arrive early too. what is wrong when 5-10mins are added. Nothing will change. It'll do its old job except we are just fighting for the crown prince.
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149 Old Solomons Island Rd
Directions Annapolis, MD 21401
Higher Standards
Click on one of the people below to find out more information.
Hometown: Red Bank, NJ
With over 40 years experience in the automotive industry, Frank learned the automotive business from the ground up. He broke into the industry as a sales representative in Baltimore and eventually became a sales manager and general manager at a Volvo Retailer. He is a graduate of the NADA General Dealership Management Program. Frank has been associated with Subaru since 1991 when he purchased the Annapolis franchise.
In 1999, Frank moved the Subaru franchise along with the Volvo franchise from its West Street location to a newly constructed home just off Rt. 50 on Busch's Frontage Road. Anticipating the growth of the brand, he created an exclusive, flagship home for Annapolis Subaru at its current Annapolis location. Year after year since its move in 2004, Annapolis Subaru sells more new and preowned Subaru models than any retailer in the region.
In addition to Annapolis Subaru, Frank is the President of Gateway Subaru, Annapolis Hyundai, Annapolis Volvo and Chesapeake Cycles.
Active in the community, he is a former member of the Maryland Auto Dealers Association Board of Directors, Subaru of America Dealer Advisory Board and the BB&T Advisory Board.
He is a former member of the Board of Directors of The Summit School and the Volvo Cars of North America Dealer Advisory Board. Most recently Frank was awarded the Commercial Stewards Award from the Chesapeake Bay Trust. The trust's Commercial Stewards Award annually is given to recognize an outstanding corporate or commercial entity that strives to make a difference in the community and for the Bay. Frank was the recipient of the award for his work to implement stormwater management projects and best practices at his dealerships.
Patrick began his career as a 15 year old lot attendant at a Volvo retailer working with our owner and CEO, Frank Ferrogine. While attending the university of Baltimore, he maintained the position of used car buyer for Annapolis Volvo/Subaru. He is a graduate of the NADA General Dealership Management Program. Upon completion of his education, he became the Pre-Owned Sales Manager. In January of 1999, Pat was made Sales Manager and in January of 2003, he became General Manager overseeing all operations of Annapolis Subaru. Pat is now the Vice President and part owner of the corporation.
Hometown: Redbank, N J
Chris Kelly has been in Sales and Sales Management in the automotive industry since 1995. In March of 2003 he became the Pre-Owned Manager at Annapolis Subaru. His experience, ability as a manager and strong communication skills made Chris the perfect candidate for advancement. In July of 2004 when he moved to our new state of art facility on Old Solomon's Island Road, Chris was made the General Sales Manager for Annapolis Subaru, and he's remained there ever since!
Hometown: Calvert County
Joe has worked in the automotive industry for 17 years. He began his career in automotive and internet sales and than moved to the F& I area of the industry, where he remains today.
When Joe is not working hard at Annapolis Subaru he loves spending as much time as possible with his Wife and 4yr old daughter. He especially enjoys that quality time when they all manage to get away to their favorite destination, Kiawah Island. Joe is also a Die Hard Redskins fan!!!!!
Hometown: Charlottesville, VA
Billy has worked in the sales industry for several years, but working at Annapolis Hyundai was his first venture into the automotive field of sales. He was encouraged by his father in-law to join the family business, as Sales & Leasing Consultant. Billy has since learned the Finance side of the industry and is now our Finance Manager. His friendly and comedic personality is a winner with customers and associates alike.
Billy grew up in Charlottesville, Virgina but his family roots are in Baltimore. Billy returned to his roots in 2004 when he was recruited by CCBC-Essex for Lacrosse. During his time here Billy became a two-time first team NJCAA All-America selection, was named as MVP in the 2004 NJCAA Championship Game after leading the Essex Knights to the title, and was named as Maryland Junior College Defensive Player of the Year in 2005. After playing with CCBC-Essex Billy transferred to Towson University where he played his final season of Lacrosse as a Towson Tiger and graduated. He now resides in Edgewater with his wife Lindsay, daughter Sloan and their golden retriever, Penny Lane.
Mike began his automotive career in Michigan in 1997. He recently relocated to Annapolis and joined the Annapolis Cars family. Mike initially began as Annapolis Volvo's Pre-Owned Lot Sales Manager and was elevated to Sales Manager at Annapolis Subaru.
Mike has two children: a son and daughter. Outside of work he enjoys activities with his family like fishing and playing sports.
Hometown: Annapolis, MD
A lifelong resident of Anne Arundel County, Chris has been involved in the automotive industry for nearly a decade. Chris and his team are knowledgeable about what makes your Subaru a Subaru. They are experts at helping you personalize your Subaru. That's why, with his leadership, Annapolis Subaru sells more genuine Subaru Parts and accessories than any other Subaru retailer in the market.
Hometown: Baltimore, MD
Ed began selling cars in 2005. Since joining Annapolis Subaru, he is the perennial Sales Leader. Before his career in the automotive industry, Ed retired from 19 years in law enforcement. Ed enjoys motorcycling, traveling, attending family events with his wife and 2 sons. Ed has excellent follow-up skills and great customer service.
Hometown: Baltimore, MD
Paul has been selling cars for 10 years. He has many awards & recognitions both for Annapolis Subaru & our sister store Annapolis Volvo, including Salesman of the Year. Paul enjoys cruising down the Chesapeake Bay, swimming, fishing and spending time with his 2 daughters. Paul has good communication skills and makes his customers feel welcomed and relaxed in a honest manner.
Annapolis resident Ottie Jones joined Annapolis Subaru in 2013. He was elevated to Leasing & Sales Associate in 2015. When not working you might run into him at the gym or at the beach with friends.
Kyle grew up in Sunderland, MD, the youngest of 5 children. He currently resides in Annapolis with his dog Happy.
Prior to joining Annapolis Subaru, Kyle served in the Army as a 19D Cavalry Scout stationed in Ft. Hood, Texas as well as a 9 month tour in Afghanistan serving as a MRAP Gunner
Kyle enjoys sports, visiting his local watering hole and taking Happy to the local dog park. Sometimes Happy stops by Annapolis Subaru too so you might get to meet this cool pup.
Roy is an "import" from Australia and began selling cars in November of 2015. Before starting his career in the automotive industry, Roy spent 27 years in law enforcement both here in the USA and in Australia. Upon retiring from public service Roy spent about 5 years in engineering and Federal Government Security. Having been the owner of 5 Subaru vehicles over the past 12 years he draws on his Love for Subaru vehicles to introduce customers to the brand and to it's advantages.
Sonja is a true Southern Girl raised and married into a Military family. Her two college-aged daughters currently attend Ole Miss and Mississippi State and her son is enrolled in Annapolis High School.
Sonja attended high school in Heidelberg Germany. She drove the Autobaun, which contributed to her respect for fast cars. Thanks to her father she learned about tools and how to rebuild cars as well as riding dirt bikes.
With a career that combines her interests with her life experience as a mom, Sonja has found a home at Annapolis Subaru. Her knowledge of our technologically advanced vehicles engineered for your family's safety when driving makes enables her to help you make a well-informed choice for your new or preowned car.
Since joining Annapolis
Subaru in Spring, 2010, Daz has been assisting Annapolis Subaru
customers in
the Parts Boutique. Prior to beginning his career at Annapolis Subaru, Daz was adopted from ARF (Animal Resource Foundation) in Chester, MD by Parts Manager, Chris Parks. His after work activities include wrestling, tug-of-war, and swimming. Since he loves attention and companionship, Daz is always ready to help Chris attend to his nephews when needed. Daz is happy to meet Annapolis Subaru customers and encourages you to visit the Parts Department so that he can meet you.
Whether they are searching for a specific automotive part or accessory or just stopping by to chat, Daz is ready to assist. Like all of our associates, nothing makes Daz happier than pleasing our customers.
Prior to beginning his career at Annapolis Subaru, Daz was adopted from ARF (Animal Resource Foundation) in Chester, MD by Parts Manager, Chris Parks.
His after work activities include wrestling, tug-of-war, and swimming. Since he loves attention and companionship, Daz is always ready to help Chris attend to his nephews when needed.
Daz is happy to meet Annapolis Subaru customers and encourages you to visit the Parts Department so that he can meet you.


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Rediscovered Flatware
36 USDassorted
Rated 3.3 out of 5 by 24 reviewers.
Rated 5 out of 5 by Anthrobsessed2 Question for Anthro I want to buy this and I want to buy it so it will be enough for 6 people. I am wondering about the description where it says: Set of five assorted pieces. Does that mean each time I purchase a "set" that it will most likely be different quantities of forks, spoons and knives? Thanks October 30, 2010
Rated 3 out of 5 by ericaruth just okay to the previous reviewer: a set of five is one place setting. so you'd order a set for each place setting that you want. as for the product itself. i got four place settings and this rang true for all of them. the salad forks and teaspoons were tiny, like baby silverware, in length. the size of the spoon/fork part was normal but they were all so short that it was weird looking. other than that, everything was cute. i just think i'm going to return them because they weren't cute ENOUGH for the money. every piece i got was different, which was good. most of them were lovely like the product pictures, with engraved flowers and monograms etc, but about two pieces were completely hideous and art-deco style. they just looked really out of place. November 13, 2010
Rated 5 out of 5 by ilieherecharmed Answer to previous question No, "assorted" doesn't mean you could possibly get 5 knives or 4 forks and 1 spoon, it means that the patterns on the handles will not match. The sets are consistently made up of the pieces shown (2 spoons, 2 forks, 1 knife). November 14, 2010
- Five-piece setting
- Silver plated metal
- Hand wash


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posted on: 4/17/14
Hi. I have a short assignment in ITM ( Information Technology MGMT ). I need ur help for doing this assignment. The due date on Tuesday April 22. Would u help me? Thanks.- Alaa from Hayward, CA
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Sept 24, 2015
The Association met the District at the bargaining
table Sept. 24 and we have four more meetings scheduled for this semester: 10/8, 10/15. 11/19 and 12/3. Articles sunshined by the Association are 8 (compensation), 10 (Health and Welfare Benefits), 12 (Hours of Service), and 13 (Stipends, Reassigned and Release Time). The District sunshined article 14 (Distance Ed.).
Memorandum of Understanding between
Desert Community College District
and the
College of the Desert Faculty Association
March 17, 2015The parties agree to extend the current collective bargaining agreement, including all ratified/agreed to tentative agreements, signed MOUs, grievance settlements, and side letters of understanding from June 30, 2015 to December 31, 2015.
Ratification of the tentative agreement arrived at between CODFA and DCCD means the amendment to the 2014-15 bargaining agreement between CODFA and College of the Desert which includes a 4.5% off-schedule settlement that was paid February 27th, 2015 and will be retroactive to 7/1/14. Going forward, new longevity stipends will be applied beginning with the February paycheck. The new annual health insurance cap of $15,600 will be applied retroactively to July 1st. The deduction on your regular paycheck will be adjusted to capture the correct amount through your May paycheck.If you have any questions, please contact John Ramont in the fiscal office at 760-773-2513.
The Fountain of Knowledge.
CODFA Officers:
President: Denise Diamond.Members at-Large: Heather Benes and Wendy Ansley.
Vice President/Sectry: Jose Simo
Treasurer: KarenTabor
Bargainaing Team:
Geoff Hagopian, Fred Sangiorgio, Dustin Culhan, Karen Tabor and the invaluable CTA staffer, Marianne Reynolds.
Newsletters:June, 2015
- January, 2015
- May, 2014
- Special Retirements Edition
March, 2014
October, 2012
February, 2012
Collective Bargaining Agreement for 7/1/13 - 6/30/15
Change logs for contract for 12/13
CODFA Constitution and Bylaws (draft)Academic Senate Bylaws
Plundering of Pension Funds
Complaint Ltr From CFT Lawyer ACCJC Gone Wild
(update by Hittelman)
- CFT Complaint against ACCJC
CCSF Protest against ACCJC
United Public Workers for Action Conference in SF
How America's Universities Became Hedge Funds - Bob Samuels
Selling Schools Out - Lee Fang
BlumGate: Why two Bay Area newsrooms dismissed my story about conflicts of interest in UC investment deals - Peter Byrne
The Lumina Foundation's idea of how to do better higher ed (indoctrination) on the cheap.
Key Ruling Finds ACCJC broke law
Read More(Tha'ts just a recommendation.)


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Internet Explorer
by Mat Snow
|February 23, 2016
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Together they were a phenomenon, but separated they became a fascination. The Beatles as a group released 12 studio albums in 8 years. As solo artists, however, John Lennon, Paul McCartney, George Harrison, and Ringo Starr have released a collective 70 LPs and…
|October 10, 2014
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They've frequently been described as the biggest band in the world. The Joshua Tree alone has sold more than fifteen million copies worldwide. They hold the record for the most Grammy wins by a rock act. And their 360 tour, which started in 2009, attracted more…
by Ledley King
|September 12, 2013
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Born in Bow in 1980, Ledley King joined Tottenham Hotspur as a trainee at the age of sixteen, and was a White Hart Lane talisman from his 1999 debut through to his retirement in 2012. Telling it how it was behind the scenes at Spurs during his years progressing…
|October 15, 2014
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Iconic drug-rock frontman, brutally poetic songwriter, cult novelist, and critically acclaimed screenwriter – Nick Cave is one of the most revered and singular artistic talents of the past three decades. This collection of interviews tells the story of Cave's…
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|October 1, 2013
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The Beatles as solo artists have collectively released over 900 songs since 1968. This beautifully illustrated four-book set examines their individual triumphs and tragedies in a post-Beatles world. They were a phenomenon together and a fascination apart. The…
|November 24, 2014
arg1-9782732466668.html Les Beatles, et après, dans un coffret cadeau richement illustré. Les Beatles ont sorti douze albums en huit ans. Mais depuis leur séparation à la fin des années 1960, John Lennon, Paul McCartney,…
|November 2, 2015
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Explore 50 years of The Who with stories, photos, memorabilia, and more. Famed music journalist Mat Snow celebrates fifty years of the Who's debut album, My Generation , with this complete illustrated history of the legendary rock band. Loaded with photos,…
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In the near future water falls from the sky whenever someone lies (either a mist or a torrential flood depending on the intensity of the lie). This makes life difficult for Matt as he maneuvers the marriage question with his lover and how best to "come out" to his traditional Chinese parents.
Publisher: Tom Doherty Associates (February 20, 2013)
Collection: Tor Books
Format: EPUB
File size: 580.1 KB
Protection: DRM free
Language: English


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Dam how long has it been?1year?
Dam how long has it been?1year?
Aug 04, 2005 5:25AM PST
Well as you may have guess it or noticed, i rearlly come to 1up.com . i been busy my self.My dad leaves this or next month to NC. Then from there we all head out to Okinawa, aniways still love this site and much love to all my buddies.
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A Room with a View
adapted from E M Forster's novel by Simon Reade, directed by Adrian Noble
28 September - 8 October 2016
Felicity Kendal stars in the stage adaptation of EM Forster's much-loved novel.
Half Life
by John Mighton, directed by Nancy Meckler
29 September - 5 November 2016
This award-winning comedy is an octogenarian love story told with humour and tenderness.
Mr Gandy's Grand Tour
Alan Titchmarsh talks about his new novel
30 September 2016
Britain's favourite gardener and presenter talks about his new best-selling novel.
6 November 2016
BalletBoyz are back with an all-male company of ten incredible dancers.


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All of the attention after the United States' 5-1 victory over El Salvador in the quarterfinals of the CONCACAF Gold Cup will go directly toward the attacking display put on by the Yanks, and rightfully so.
But if you look deeper into the match, the offensive firepower from the home side overshadowed a few major defensive lapses.
The offensive strategy for Jürgen Klinsmann's side worked to perfection against an El Salvador team that had a blatant advantage at M&T Bank Stadium with the crowd decked out in blue.
Klinsmann's master plan succeeded for the first time in the 22nd minute as a short corner played by José Torres to Landon Donovan resulted in a goal from defender Clarence Goodson.
WATCH: Clarence Goodson's opening goal for the #USMNT - http://t.co/1dR1XkI9Il— U.S. Soccer (@ussoccer) July 21, 2013
Donovan's cross from just outside the right edge of the penalty area found the crossing Goodson, and before El Salvador keeper Dagoberto Portillo could even react, the San Jose Earthquakes defender had his fifth international goal.
Just six minutes after Goodson's opening goal, Joe Corona finished off a piece of individual brilliance as he slotted home his second career international goal into the bottom left corner of the net.
With the Americans on an attacking high, they forgot to defend before the halftime whistle as they let Rodolfo Zelaya run across the penalty area before DaMarcus Beasley intervened by bumping the El Salvador winger.
As Zelaya fell to the ground, the referee signaled for a penalty, and the disgust from American fans about their back four reared its deserved ugly head yet again.
Concern about an equalizer continued for the first 10 minutes of the second half as Zelaya ran past Matt Besler on a few occasions.
With a spark needed for the Yanks, Klinsmann inserted the newly-blonde Eddie Johnson into the lineup, and within 30 seconds of the Seattle Sounders forward being on the pitch, he delivered the required energy boost.
Once Johnson entered the match and scored in the 60th minute, the Americans took control of the match and pummeled the team with the most fans in the stands.
WATCH: Eddie Johnson scores with his 1st touch in Baltimore - http://t.co/M38EWkAkwm #DemolitionMan #USAvSLV— U.S. Soccer (@ussoccer) July 21, 2013
Landon Donovan and Mix Diskerud added a goal apiece toward the end of the second half. Both of those goals mirrored the Klinsmann attitude of continuing to score instead of being content with scoring just one or two goals.
While a five-goal outburst against the 94th-best team in the world according to FIFA is a spectacular result, the Yanks still have to be concerned about their defending.
Right-back Michael Parkhurst looked vulnerable after moving forward in attack, and while Besler did have a great match at centre-back alongside Goodson, he was caught out a few times by Zelaya in the second half.
With either Honduras or Costa Rica looming in the semifinals at Cowboys Stadium on Wednesday night, the back four of the Yanks must be more securely knit than they were in the 15-minute span around halftime to ensure safe passage to next Sunday's final at Soldier Field in Chicago.
#USMNT extends its team-record win streak to nine straight games.— Paul Carr (@PCarrESPN) July 21, 2013
Follow me on Twitter, @JTansey90.


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William Pratt (MP for Southwark)
From Wikipedia, the free encyclopedia
William Pratt (fl. 1589) was an English politician.
He was a Member (MP) of the Parliament of England for Southwark in 1589.[1]
|This article about a Member of the Parliament of England (up to 1707) is a stub. You can help Wikipedia by expanding it.


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H.Res.104 - Supporting the goals and ideals of National Engineers Week, and for other purposes.112th Congress (2011-2012)
Subject — Policy Area:
One Policy Area term, which best describes an entire measure, is assigned to every bill or resolution. Find Bills by Subject and Policy Area provides guidance for using Policy Areas in your research.
Subjects: H.Res.104 — 112th Congress (2011-2012)
- Advanced technology and technological innovations
- Commemorative events and holidays
- Research and development
- Science and engineering education


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There are a lot of cool things happening this week at the San Diego Comic-Con, but by far one of the coolest just landed in our inbox: a chance to eat tacos served by Robert Rodriguez and several cast members from Machete, followed by an outdoor screening of exclusive SDCC footage from the film and a party that's open to the public on a first-come, first-served basis.
The details of the July 22nd event can be found in the below invite, which you can print out and redeem for a free taco, but that's not even all! Rodriguez has recorded a special greeting just for the Comic-Con fans.
Machete stars Jessica Alba (Sin City), Robert De Niro, Michelle Rodriguez (Fast & Furious), Lindsay Lohan (Mean Girls), Cheech Marin (Once Upon a Time in Mexico), Jeff Fahey ("Lost"), Don Johnson, Steven Seagal (Under Siege), and of course Danny Trejo as the lead character. Look for it in theatres September 3rd, 2010.
The feature finds Machete (Trejo), a renegade former Mexican Federale, roaming the streets of Texas after a shakedown from drug lord Torrez (Seagal). Reluctantly, Machete takes an offer from spin doctor Benz (Fahey) to assassinate McLaughlin (De Niro) a corrupt Senator. Double-crossed and on the run, Machete braves the odds with the help of Luz (Rodriguez), a saucy taco slinger; Padre (Marin), his "holy" brother; and April (Lohan), a socialite with a penchant for guns. All while being tracked by Sartana (Alba), a sexy ICE agent with a special interest in the blade slinger.
Machete – Robert Rodriguez's Comic-Con Greeting
Uploaded by dreadcentral. – Check out other Film & TV videos.
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Addison Place
5100 Abbotts Bridge Rd
Alpharetta, GA 30005
25 Google user reviews
Sebastian Murphy
9 months ago
Used to live here, decent facilities but the management would never take care of problems quickly. Trash compacter would break down often, and the pools would be opened weeks after any other neighborhoods in the area and they would open later than the management would announce.
Sarah Harper
a year ago
Windows leaked, it took two months to replace our A/C unit in the middle of summer, they broke our water heater and took weeks to fix it, washer and dryer didn't work well and they still haven't fixed the dryer (we had to fix it ourselves). Maintenance took forever and the quality of the apartment was lackluster. We had water damage when we moved in and stains on the wall. Lots of kids running around the tennis court and being extremely loud. Very inconsiderate neighbors (noise wise) that the front office won't do anything about. The gates are always broken and never closed. In the year I have lived there the gates have been closed for maybe three weeks total. Too expensive for what you get. They did get new management so maybe they can turn the place around.
rk s
a year ago
One of the worst apartments to live in Alpharetta. Constant problems with A/C and heating units. Most apts in this community have broken A/C units and the service is very very poor. We had to spend several nights without the heater working. Some of my neighbors faced the same problem. The service men were helpless and complained about the mgmt. refusing to upgrade the A/c heating units. There is always an additional 200$ bill to your rent irrespective of whether you will live in single, double or triple bedroom apt. You're forced to take an internet/cable of their choice whether you need or not. Trash compacter always overflows. Btw, be prepared to share your apt with roaches as they are very common here
zumma a
a year ago
If I could rate this as lower than one star I would I live here. Please be careful before you move in. My family is not very happy living here. This place is the worst ever in my life. 1 kids playground with trash compactor overflowing 2 utility fee is extremley high after new maintenance company.($300) 3 comcast is OBLIGATION ($100/mo) - monopoly? 4 Family Electiricy is separated (~$100/mo) 5 Roaches everywhere( a lot in kitchen) 6 Gates are completely open 24/7.
vidya sankar
2 years ago
I live here now..It is NOT a GATED community as it is open to Abbotts Bridge and Jones Bridge road on the sides.The trash compactor is a constant problem(atleast once a month the trash is overflowing).The Gates are completely open during the morning and afternoon school times. Atleast once a month the gates stop functioning and remain open all the time.. Inspite of all this the 3 stars is because of the people who live here are super friendly..lots of friends for my kids..soo convenient to Publix,Kroger,CVS and restaurants
Abby Potter
3 years ago
This place is a joke. At this very moment, there are two dumpsters outside of the trash compactor (broken for weeks). All three (total) are overflowing with trash. Trash is all along the street. It is so gross. How long exactly does it take to have a trash compactor repaired.....makes you wonder what they do with hundreds of residents monthly trash fees.
Jackie Robinson
2 years ago
Great Location, poorly designed. There is NO insulation what so ever. I can say this because the pipes froze over the winter and I saw first hand what was there when the wall was cut into. Very few with pets pick up after them. Parking is lacking. Phone calls are either not answered nor are they returned. There are no activities to promote getting to know your neighbors. Getting your payment posted in a timely manner is next to impossible. I just moved after a 1 1/2 and am so glad I am out of there.
Tori Smith
3 years ago
Looks are deceiving! Speaking from experience...These apartments are at best a farce!! Management changed 5 times while I was there, NO KIDDING. Maintenance, well if you can't say anything nice then say nothing at all, so I'll spare you the sordid details. Gated, yeah right, the gate NEVER worked consecutively yet you pay a mandatory included in the rent Gate Fee! People sit in cars and wait for a resident with "the precious" key to entry WHEN the gate does work and follow in behind you. Oh right, the gate allows one car per entry says a sign that's posted, NOT!!! The trash compactors on both sides were always overflowing with pungent odors and mounds of trash on weekends...embarrassing when guests visited, and you pay a fee for this lovely feature too! The rats come for FREE, yes they've moved in too! Teens ran around on late nights, as in the wee morning hours knocking on doors, yep happened to me. There is theft, package taken right from my front door! And don't waste your time calling The Connor Group, the owners, you have a snowball's chance in hell of getting someone, they NEVER answer the phone...just "show them the money". Reputable realtor shared they send those that can't get a home to Addison Place because they are really low-income based apartments, without a rigid credit check, it shows. Wish I'd known that prior to moving in! Things SOMETIMES may look good on the outside but inside the units are outdated and to bait you with a lease renewal that goes up by the hundred$ you're offered updated appliances. Luxury apartments, a resort styled community....Really? No Really?! More like gated ghetto. Would not recommend these apartments to anyone!!!!!!!!!!
17 more reviews


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Head OfficeEurope, Middle East & Africa
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Tel: +44 (0)1908 200022, #2
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In truth I've got tons, but only one that really matters. How much fun is the game for more casual players? Is it easy to play it for a little while here and there and have a good time with it? I hate to be this way, but with the number of games I have over all my systems (a LOT) I just won't be able to give this game the time that it deserves, however, I REALLY wanna get it, I've been playing Sim City games from the start and haven't had one for a long time, and watching the gameplay footage from IGN the game looks to be awesome.
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credit suisse group ag-reg
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