Check out our New Publishers' Select for Free Articles

Biochemical Pharmacology (v.76, #2)

Editorial Advisory Board (pp. iii).

IL-13 as a therapeutic target for respiratory disease by Marion T. Kasaian; Douglas K. Miller (pp. 147-155).

Interleukin-13 (IL-13) is a critical mediator of asthma pathology. On B cells, monocytes, epithelial cells, and smooth muscle cells, IL-13 acts through the IL-13Rα1/IL-4Rα complex to directly induce activation responses that contribute to atopic disease. In human populations, genetic polymorphisms in IL-13, its receptor components, or the essential signaling element STAT6, have all been associated with increased risk of atopy and asthma. Animal studies using IL-13 deficient mice, IL-13 transgenic animals, and IL-13 neutralization strategies have confirmed an essential role for this cytokine in driving major correlates of asthma pathology, including airway hyperresponsiveness (AHR), lung eosinophilia, mucus generation, and fibrosis. Ongoing studies continue to define both overlapping and distinct roles for IL-13 and the related cytokine, IL-4, in promoting asthmatic changes. Furthermore, new evidence concerning the role of the “decoy” receptor, IL-13Rα2, has prompted re-evaluation of the receptor forms that underlie the numerous activities of IL-13. In this review, we summarize the essential role of IL-13 in asthma, compare the relative contributions of IL-13 and IL-4 to key aspects of the asthmatic phenotype, and outline novel therapeutic strategies to target this critical cytokine.

Keywords: Abbreviations; AHR; airway hyperresponiveness; BAL; bronchoalveolar lavage; NHP; nonhuman primateAirway hyperresponsiveness (AHR); Bronchoalveolar lavage (BAL)

Selective targeting of the HIV-1 reverse transcriptase catalytic complex through interaction with the “primer grip” region by pyrrolobenzoxazepinone non-nucleoside inhibitors correlates with increased activity towards drug-resistant mutants by Samantha Zanoli; Sandra Gemma; Stefania Butini; Margherita Brindisi; Bhupendra P. Joshi; Giuseppe Campiani; Caterina Fattorusso; Marco Persico; Emmanuele Crespan; Reynel Cancio; Silvio Spadari; Ulrich Hübscher; Giovanni Maga (pp. 156-168).

PBO (pyrrolobenzoxazepinone) derivatives are non-nucleoside reverse transcriptase inhibitors (NNRTIs), which display a selective interaction with the catalytic ternary complex of HIV-1 reverse transcriptase (RT) and its substrates. In order to develop novel PBOs with improved resistance profiles, we synthesised additional PBO derivatives, specifically designed to target highly conserved residues in the β12–β13 hairpin, the so-called “primer grip” region of HIV-1 RT. Here, we investigated the biochemical and enzymological mechanism of inhibition of HIV-1 RT wild type and carrying NNRTIs-resistance mutations, by these derivatives. Our kinetic analysis indicates that the ability of PBOs to selectively target the catalytic ternary complex of RT with its substrates directly correlates with greatly reduced sensitivity to NNRTIs-resistance mutations, particularly the K103N substitution. Molecular modeling and docking studies provided an explanation for this correlation at the structural level.

Keywords: HIV-1; Reverse transcriptase; NNRTIs; Kinetics; Drug resistant; Molecular modeling

Validating the mitotic kinesin Eg5 as a therapeutic target in pancreatic cancer cells and tumor xenografts using a specific inhibitor by Min Liu; Haiyang Yu; Lihong Huo; Jianchao Liu; Minggang Li; Jun Zhou (pp. 169-178).

Pancreatic cancer is a devastating disease with a high mortality rate. Treatment of this malignancy remains a big challenge in oncology, and none of the currently available chemotherapeutic agents has a remarkable impact on improving patient survival. Consequently, it is important to explore new targets and find effective drugs for the management of this disease. Here we report that inhibition of the mitotic kinesin Eg5 by a pharmacological compound effectively prevents the proliferation of pancreatic cancer cells by halting mitotic progression, resulting in robust apoptosis. The mitotic arrest induced by this agent is attributed to its interference with spindle formation and activation of the spindle checkpoint. Impairment of the spindle checkpoint significantly compromises both mitotic arrest and apoptosis induced by the Eg5 inhibitor, suggesting the importance of the spindle checkpoint in monitoring Eg5 inhibitor sensitivity. Furthermore, treatment of nude mice bearing tumor xenografts of human pancreatic cancer results in pronounced tumor regression by triggering apoptosis. These data thus indicate Eg5 as a potential target for pancreatic cancer treatment.

Keywords: Pancreatic cancer; Eg5 inhibitor; Spindle checkpoint; Apoptosis; Tumor xenograft

The anti-proliferative potency of celecoxib is not a class effect of coxibs by Susanne Schiffmann; Thorsten Jürgen Maier; Ivonne Wobst; Astrid Janssen; Heike Corban-Wilhelm; Carlo Angioni; Gerd Geisslinger; Sabine Grösch (pp. 179-187).

Celecoxib, a COX-2 (cyclooxygenase-2)-selective inhibitor (coxib), is the only NSAID (non-steroidal anti-inflammatory drug) that has been approved for adjuvant treatment of patients with familial adenomatous polyposis. To investigate if the anti-proliferative effect of celecoxib extends to other coxibs, we compared the anti-proliferative potency of all coxibs currently available (celecoxib, rofecoxib, etoricoxib, valdecoxib, lumiracoxib). Additionally, we used methylcelecoxib (DMC), a close structural analogue of celecoxib lacking COX-2-inhibitory activity. Due to the fact that COX-2 inhibition is the main characteristic of these substances (with exception of methylcelecoxib), we conducted all experiments in COX-2-overexpressing (HCA-7) and COX-2-negative (HCT-116) human colon cancer cells, in order to elucidate whether the observed effects after coxib treatment depend on COX-2 inhibition. Cell survival was assessed using the WST proliferation assay. Apoptosis and cell cycle arrest were determined using flow cytometric and Western blot analysis. The in vitro results were confirmed in vivo using the nude mouse model. Among all coxibs tested, only celecoxib and methylcelecoxib decreased cell survival by induction of cell cycle arrest and apoptosis and reduced the growth of tumor xenografts in nude mice. None of the other coxibs (rofecoxib, etoricoxib, valdecoxib, lumiracoxib) produced anti-proliferative effects, indicating the lack of a class effect and of a role for COX-2. Our data emphasize again the outstanding anti-proliferative activity of celecoxib and its close structural analogue methylcelecoxib in colon carcinoma models in vitro and in vivo.

Keywords: Abbreviations; APC; adenomatous polyposis coli; cel; celecoxib; COX-2; cyclooxygenase-2; eto; etoricoxib; FAP; familial adenomatous polyposis; FCS; fetal calf serum; HPLC; high performance liquid chromatography; LC–MS/MS; liquid chromatography coupled with tandem mass spectrometry; lum; lumiracoxib; LPS; lipopolysaccharide; DMC; methylcelecoxib; NSAID; non-steroidal anti-inflammatory drug; PARP; poly (ADP)-ribose polymerase; PGE2; prostaglandin E; ₂; rof; rofecoxib; val; valdecoxibColon cancer; NSAIDs; Cyclooxygenase-2; Apoptosis; Celecoxib

The cytostatic activity of pyrimidine nucleosides is strongly modulated by Mycoplasma hyorhinis infection: Implications for cancer therapy by Annelies Bronckaers; Jan Balzarini; Sandra Liekens (pp. 188-197).

Nucleoside analogues are widely used as chemotherapeutic agents in the treatment of cancer. Several cancers are reported to be associated with mycoplasmas (i.e. Mycoplasma hyorhinis), which contain a number of nucleoside-metabolizing enzymes. Pyrimidine nucleoside analogues, such as 5-fluoro-2′-deoxyuridine (FdUrd), 5-trifluorothymidine (TFT) and 5-halogenated 2′-deoxyuridines can be degraded by thymidine phosphorylase (TP) to their inactive bases. We found in M. hyorhinis-infected MCF-7 breast carcinoma cells (MCF-7/HYOR) a mycoplasma-encoded TP that dramatically (20–150-fold) reduces the cytostatic activity of these compounds. The reduction in cytostatic activity could be fully restored in the presence of TPI (5-chloro-6-[1-(2-iminopyrrolidinyl)methyl]uracil hydrochloride), a known inhibitor of human TP. This observation is in agreement with the markedly decreased formation of active metabolite (i.e. FdUMP for FdUrd) or diminished drug incorporation into nucleic acids (i.e. for TFT and 5-bromo-2′-deoxyuridine) in MCF-7/HYOR cells compared with uninfected MCF-7 cells. Antimetabolite formation is fully restored in the presence of TPI. In contrast, 5-fluoro-5′-deoxyuridine (5′DFUR), an intermediate metabolite of capecitabine, was markedly more cytostatic in MCF-7/HYOR cells than in uninfected cells, due to the activation of this prodrug by the mycoplasma-encoded TP. Thus, our data reveal that M. hyorhinis expresses a TP that activates 5′DFUR but inactivates FdUrd, TFT and 5-halogenated 2′-deoxyuridines, and that is highly sensitive to the inhibitory effect of the TP inhibitor TPI. Given the association of M. hyorhinis with several human cancers, our findings suggest that pyrimidine nucleoside-based but not 5FU-based anti-cancer therapy might be more effective when combined with a mycoplasmal TP inhibitor.

Keywords: Abbreviations; BrdUrd; 5-bromo-2′-deoxyuridine; CldUrd; 5-chloro-2′-deoxyuridine; 5′DFUR; 5-fluoro-5′-deoxyuridine; DPD; dihydropyrimidine dehydrogenase; dThd; thymidine; dUrd; 2′-deoxyuridine; FdUMP; 5-fluoro-2′-deoxyuridine-5′-monophosphate; FdUrd; 5-fluoro-2′-deoxyuridine; 5FU; 5-fluorouracil; IC; ₅₀; 50% inhibitory concentration; IdUrd; 5-iodo-2′-deoxyuridine; MCF-7/HYOR; MCF-7 cells infected with; Mycoplasma; hyorhinis; PD-ECGF; platelet-derived endothelial cell growth factor; TFT; 5-trifluorothymidine; Thy; thymine; TK; thymidine kinase; TP; thymidine phosphorylase; TPI; 5-chloro-6-(1-[2-iminopyrrolidinyl]methyl)uracil hydrochloride; TS; thymidylate synthase; Ura; uracilMycoplasma; Nucleoside analogues; Cytostatic activity; Thymidine phosphorylase; Capecitabine

Activated kRas protects colon cancer cells from cucurbitacin-induced apoptosis: The role of p53 and p21 by José M. Escandell; Pawan Kaler; M. Carmen Recio; Takehiko Sasazuki; Senji Shirasawa; Leonard Augenlicht; José-Luis Ríos; Lidija Klampfer (pp. 198-207).

Cucurbitacins have been shown to inhibit proliferation in a variety of cancer cell lines. The aim of this study was to determine their biological activity in colon cancer cell lines that do not harbor activated STAT3, the key target of cucurbitacin. In order to establish the role of activated kRas in the responsiveness of cells to cucurbitacins, we performed experiments in isogenic colon cancer cell lines, HCT116 and Hke-3, which differ only by the presence of an activated kRas allele. We compared the activity of 23, 24-dihydrocucurbitacin B (DHCB) and cucurbitacin R (CCR), two cucurbitacins that we recently isolated, with cucurbitacin I (CCI), a cucurbitacin with established antitumorigenic activity. We showed that cucurbitacins induced dramatic changes in the cytoskeleton (collapse of actin and bundling of tubulin microfilaments), inhibited proliferation and finally induced apoptosis of both HCT116 and Hke-3 cells. However, the presence of oncogenic kRas significantly decreased the sensitivity of cells to the three cucurbitacins tested, CCR, DHCB and CCI. We confirmed that mutational activation of kRas protects cells from cucurbitacin-induced apoptosis using nontransformed intestinal epithelial cells with inducible expression of kRasV12. Cucurbitacins induced the expression of p53 and p21 predominantly in HCT116 cells that harbor mutant Ras. Using HCT116 cells with targeted deletion of p53 or p21 we confirmed that p53 and p21 protect cells from apoptosis induced by cucurbitacins. These results demonstrated that sensitivity of human colon cancer cell lines to cucurbitacins depends on the kRas and p53/p21 status, and established that cucurbitacins can exert antitumorigenic activity in the absence of activated STAT3.

Keywords: Colon cancer; kRas; Cucurbitacin; Apoptosis; p53

MCF-7aro/ERE, a novel cell line for rapid screening of aromatase inhibitors, ERα ligands and ERRα ligands by Ki Lui; Takaya Tamura; Taisuke Mori; Dujin Zhou; Shiuan Chen (pp. 208-215).

We have previously generated a breast cancer cell line, MCF-7aro, which over-expresses aromatase and is also ER positive. Recently, this MCF-7aro cell line was stably transfected with a promoter reporter plasmid, pGL3-Luc, containing three repeats of estrogen responsive element (ERE). Experiments using MCF-7aro/ERE have demonstrated that it is a novel, non-radioactive screening system for aromatase inhibitors (AIs), ERα ligands and ERRα ligands. The screening is carried out in a 96-well plate format. To evaluate this system, the cells were cultured overnight in charcoal–dextran stripped FBS medium supplemented with 0.1nM testosterone or 17β-estradiol, and various concentrations of antiestrogens or AIs. We found that the luciferase activity was induced when the cells were cultured either in the presence of testosterone or 17β-estradiol. Furthermore, a 50% decrease in luciferase activity could be achieved when the cells were cultured in the presence of testosterone together with letrozole, anastrozole, tamoxifen or fulvestrant (concentrations being 75nM, 290nM, 100nM, and 5nM, respectively), compared to the testosterone-only cultured cells. Using this assay system, we confirmed that 3(2′-chlorophenyl)-7-methoxy-4-phenylcoumarin is an agonist of ER. Furthermore, genestein has been shown to be a ligand of ERRα because its binding could be blocked by an ERRα inverse agonist, XCT790. These results indicate that MCF-7aro/ERE is a novel cell line for rapid screening of AIs, ERα ligands and ERRα ligands.

Keywords: Abbreviations; AI; aromatase inhibitor; Cou; 3(2′-chlorphenyl)-7-methoxy-4-phenylcoumarin; E; ₂; 17β-estradiol; ER; estrogen receptor; ERE; estrogen responsive element; ERR; estrogen-related receptor; HTS; high-throughput screening; ICI; ICI 182, 780 or Faslodex; T; testosteroneAromatase; Aromatase inhibitor; ER agonist; ERRα ligands; MCF-7aro/ERE

Over-the-counter analgesics normalize blood glucose and body composition in mice fed a high fat diet by Eric L. Kendig; Scott N. Schneider; Deborah J. Clegg; Mary Beth Genter; Howard G. Shertzer (pp. 216-224).

Type 2 diabetes (noninsulin-dependent diabetes mellitus) develops from a pre-diabetic condition that is characterized by insulin resistance and glucose intolerance, and is exacerbated by obesity. In this study, we compared the ability of over-the-counter analgesic drugs (OTCAD) [acetaminophen (APAP); ibuprofen (IBU); naproxen (NAP); aspirin (ASA)], to protect against the development of a pre-diabetic state in mice fed a high fat diet. After 10 weeks on the high fat diet, mice had normal fasting blood glucose (FBG) levels, but exhibited impaired glucose tolerance. Treatment with 20mg OTCADs/kg body weight improved glucose tolerance, with the order of efficacy, APAP=ASA>IBU, while NAP proved ineffective. Mice fed the high fat diet also exhibited increases in weight gain associated with an increase in body fat. OTCADs prevented in part this increase in body fat, in the order of efficacy, APAP=IBU>NAP=ASA. In isolated liver mitochondria, OTCADs inhibited succinate-dependent H₂O₂ production, while in white adipose tissue, APAP inhibited NADPH-oxidase mediated H₂O₂ production and lipid peroxidation. Thus, OTCADs diminish pro-oxidant processes that might otherwise exacerbate inflammation and a pre-diabetic state. We conclude that OTCADs, especially APAP and IBU, may be valuable tools to delay or prevent the development of type 2 diabetes from a pre-diabetic condition.

Keywords: Abbreviations; APAP; acetaminophen,; N; -(4-hydroxyphenyl)acetamide; ASA; aspirin, 2-acetylsalicylic acid, 2-acetoxybenzoic acid; FBG; fasting blood glucose; IBU; ibuprofen, 2-[4-(2-methylpropyl)phenyl]propanoic acid; NAP; naproxen, (; S; )-6-methoxy-α-methyl-2-naphthaleneacetic acid; T2DM; type 2 diabetes mellitus; NSAID; non-steroidal anti-inflammatory drug; OTCAD; over-the-counter analgesic drugAnalgesic drugs; Diabetes; High fat diet; Mice; NADPH oxidase; Oxidative stress

PKA-mediated phosphorylation is a novel mechanism for levetiracetam, an antiepileptic drug, activating ROMK1 channels by Chien-Hsing Lee; Chun-Yao Lee; Ting-Shan Tsai; Horng-Huei Liou (pp. 225-235).

Levetiracetam (LEV) is an effective antiepileptic drug (AED) with distinct mechanism from the conventional AEDs. The major physiological function of ROMK1 channels is to maintain the resting membrane potential (RMP). In this study, we investigated the mechanisms underling LEV on ROMK1 channels. Xenopus oocytes were injected with mRNA to express the wild-type or mutant ROMK1 channels. Giant inside-out patch clamp recordings were performed to study the effect of LEV on these channels. LEV increased the activity of ROMK1 channels in a concentration-dependent manner and enhanced both wild-type and pH_i gating residue mutant (K80M) channels over a range of pH_i values. LEV activated the mutated channels at PIP₂-binding sites (R188Q, R217A and K218A) and PKC-phosphorylation sites channels (S4A, S183A, T191A, T193A, S201A and T234A). However, this drug failed to enhance the channel activity in the presence of PKA inhibitors and did not activate the mutants of PKA-phosphorylation sites on C-terminal (S219A, S313A) and the constructed mutants (S219D and S313D) that mimic the negative charge carried by a phosphate group bound to a serine. Our results demonstrated PKA-mediated phosphorylation is a novel mechanism for LEV activating ROMK1 channels. These findings show that LEV activates ROMK1 channels independently from pH_i and not via a PIP₂- or PKC-dependent pathway. The effects of LEV may come from the PKA-induced conformational change but not charge–charge interaction in ROMK1 channels. Enhancing the activity of ROMK1 channels may be an important molecular mechanism for the antiepileptic effects of LEV in restoring neuronal RMP to prevent seizure spreading.

Keywords: Epilepsy; Levetiracetam; PKA; ROMK1 channels

Extracts and constituents of Leontopodium alpinum enhance cholinergic transmission: Brain ACh increasing and memory improving properties by Ariane Hornick; Stefan Schwaiger; Judith M. Rollinger; Nguyen Phung Vo; Helmut Prast; Hermann Stuppner (pp. 236-248).

Leontopodium alpinum (‘Edelweiss’) was phytochemically investigated for constituents that might enhance cholinergic neurotransmission. The potency to increase synaptic availability of acetylcholine (ACh) in rat brain served as key property for the bioguided isolation of cholinergically active compounds using different chromatographic techniques. The dichlormethane (DCM) extract of the root, fractions and isolated constituents were injected i.c.v. and the effect on brain ACh was detected via the push–pull technique. The DCM extract enhanced extracellular ACh concentration in rat brain and inhibited acetylcholinesterase (AChE) in vitro. The extracellular level of brain ACh was significantly increased by the isolated sesquiterpenes, isocomene and 14-acetoxyisocomene, while silphiperfolene acetate and silphinene caused a small increasing tendency. Only silphiperfolene acetate showed in vitro AChE inhibitory activity, thus suggesting the other sesquiterpenes to stimulate cholinergic transmission by an alternative mechanism of action. Isocomene was further investigated with behavioural tasks in mice. It restored object recognition in scopolamine-impaired mice and showed nootropic effects in the T-maze alternation task in normal and scopolamine-treated mice. Additionally, this sesquiterpene reduced locomotor activity of untreated mice in the open field task, while the activity induced by scopolamine was abolished. The enhancement of synaptic availability of ACh, the promotion of alternation, and the amelioration of scopolamine-induced deficit are in accordance with a substance that amplifies cholinergic transmission. Whether the mechanism of action is inhibition of AChE or another pro-cholinergic property remains to be elucidated. Taken together, isocomene and related constituents of L. alpinum deserve further interest as potential antidementia agents in brain diseases associated with cholinergic deficits.

Keywords: Edelweiss; Isocomene; Acetylcholine release; T-maze; Object recognition

In vitro characterisation of human renal and hepatic frusemide glucuronidation and identification of the UDP-glucuronosyltransferase enzymes involved in this pathway by Oranun Kerdpin; Kathleen M. Knights; David J. Elliot; John O. Miners (pp. 249-257).

In order to gain insights into the renal and hepatic glucuronidation of frusemide (FSM), this study: (i) characterised the kinetics of FSM glucuronidation by human liver microsomes (HLM) and human kidney cortical- (HKCM) and medullary- (HKMM) microsomes, and (ii) identified the human UDP-glucuronosyltransferase enzyme(s) involved in this pathway. HLM, HKCM and HLMM efficiently glucuronidated FSM. FSM glucuronide (FSMG) formation followed Michaelis–Menten kinetics in all tissues. While the mean K_m for FSMG formation by HKMM (386±68μM) was lower than the K_m values for HLM (988±271μM) and HKCM (704±278μM), mean V_max/ K_m values were comparable for the three tissues. A panel of recombinant UGT enzymes was screened for the capacity to glucuronidate FSM. UGT 1A1, 1A3, 1A6, 1A7, 1A9, 1A10 and 2B7 metabolised FSM. Of the renally and hepatically expressed enzymes, comparison of kinetic parameters suggests a predominant role of UGT1A9 in FSM glucuronidation, although UGT1A1 may also contribute to FSMG formation by HLM. Consistent with these observations, the UGT1A selective inhibitors phenylbutazone and sulfinpyrazone decreased FSMG formation by HLM, HKCM and HKMM by 60–80%, whereas the UGT2B7 selective inhibitor fluconazole reduced FSM glucuronidation by ≤20%. The ability of HKCM and HKMM to form FSMG supports the proposition that the kidney is the main organ involved in FSM glucuronidation in vivo, although a role for hepatic metabolism remains a possibility in renal dysfunction. The data further demonstrate the potential importance of both the medulla and cortex in renal drug metabolism and detoxification.

Keywords: Frusemide; Glucuronidation; UDP-glucuronosyltransferase; Human kidney; Human liver

Regulation of liver cytochrome P450 by activation of brain dopaminergic system: Physiological and pharmacological implications by Jacek Wójcikowski; Krystyna Gołembiowska; Władysława Anna Daniel (pp. 258-267).

The aim of the present study was to investigate the influence of activation of brain dopaminergic system by different dopaminomimetics on the level and activity of liver cytochrome P450 (CYP) isoforms. Studies into the identification of hormones and cytokines which are known to mediate liver CYP expression were also simultaneously carried out.Stimulation of dopaminergic receptors in the pituitary, a target for the tuberoinfundibular pathway, by dopamine (a D₁/D₂ receptor agonist) administered intraperitoneally caused a significant increase in the activities and protein levels of CYP2B, CYP2C11 and CYP3A, a substantial increase in the blood plasma level of growth hormone (GH) and a significant decrease in triiodothyronine (T₃) level. Local stimulation of dopaminergic receptors in the nucleus accumbens, a target for the mesolimbic pathway, by apomorphine (a D₁/D₂ receptor agonist), amphetamine (an indirect D₁/D₂ dopaminemimetic) and quinpirole (a D₂ receptor agonist) produced a substantial rise in CYP3A activity and protein level, caused a large increase in corticosterone concentration and a moderate drop in T₃ level. SKF82958 (a D₁ receptor agonist) did not significantly affect the CYP isoforms or hormones studied. In both cases (activation of the tuberoinfundibular or mesolimbic pathway), the activity and the protein level of CYP1A considerably decreased. Plasma levels of thyroxine, testosterone, interleukin-2 and interleukin-6 were not changed after activation of the two pathways.The obtained results establish the brain dopaminergic system as a physiological centre regulating cytochrome P450 (engaging D₂ receptors and pituitary hormones) and demonstrate new pharmacological aspects of neuroactive drugs that affect this system.

Keywords: Brain dopaminergic system; Activation; Liver cytochrome P450; Plasma hormone levels

Differential regulation of human hepatic flavin containing monooxygenase 3 ( FMO3) by CCAAT/enhancer-binding protein β (C/EBPβ) liver inhibitory and liver activating proteins by David E. Klick; Jeff D. Shadley; Ronald N. Hines (pp. 268-278).

Flavin-containing monooxygenase 3 (FMO3) is important for oxidative xenobiotic metabolism, but regulation of the FMO3 gene remains poorly understood. FMO3 is not expressed in HepG2 cells, a commonly employed model for hepatic gene regulation studies. Transcription factor transient expression and treatment with histone deacetylase or DNA methylase inhibitors identified decreased hepatic nuclear factor (HNF) 4α levels and DNA hypermethylation as mechanisms suppressing HepG2 FMO3 expression. The absence of major deficiencies in transcriptional machinery suggested that within limits, the HepG2 model is suitable for the study of FMO3 regulation. DNA–protein binding studies with HepG2 cell and hepatic tissue nuclear protein extracts and reporter construct transient expression experiments were performed to characterize FMO3 sequences from position −494 to −439 (domain I), previously demonstrated to significantly impact promoter function. Although both HNF3β and CCAAT enhancer-binding protein (C/EBP) were observed to specifically interact with this element using HepG2 cell nuclear proteins, only C/EBP DNA–protein interactions were observed using adult liver nuclear proteins. No specific DNA/protein interactions were observed using fetal liver nuclear proteins. Mutation of a putative HNF3β element had no effect on FMO3 promoter activity, while mutagenesis of a distinct, but overlapping C/EBP element resulted in a 55% reduction in activity. Furthermore, promoter activity was regulated as a function of defined C/EBPβ liver activating protein:liver inhibitory protein ratios through this same element. Chromatin immunoprecipitation demonstrated C/EBPβ binding to the FMO3 domain I element in intact cells and adult liver tissue. These results are consistent with C/EBPβ being important for regulating hepatic FMO3 expression.

Keywords: Flavin-containing monooxygenase; FMO3; Gene regulation; CCAAT/enhancer-binding protein; HepG2 cells

RETRACTED: Antiviral and antiparasite properties of anl-amino acid oxidase from the Snake Bothrops jararaca: Cloning and identification of a complete cDNA sequence by Carolina D. Sant Ana; Danilo L. Menaldo; T ssia R. Costa; Harryson Godoy; Vanessa D.M. Muller; Victor H. Aquino; S rgio Albuquerque; Suely V. Sampaio; Marta C. Monteiro; Rodrigo G. St beli; Andreimar M. Soares (pp. 279-288).

This article has been retracted at the request of the Editor-in-Chief and Author. Please see Elsevier Policy on Article Withdrawal (http://www.elsevier.com/locate/withdrawalpolicy).Reason: The authors have plagiarized transmission electron microscopy figures published by others in Antimicrob. Agents Chemother., 47 (2003) 18951901; doi:10.1128/AAC.47.6.1895-1901.2003). As such this article represents a severe abuse of the scientific publishing system. The scientific community takes a very strong view on this matter and we apologize to readers of the journal for this incident.

Sections

Personal tools

Biochemical Pharmacology (v.76, #2)

Sections

Personal tools

Local resources

Biochemical Pharmacology (v.76, #2)