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Biochemical Pharmacology (v.69, #10)

Editorial Advisory Board (pp. iii).

Ah receptor- and TCDD-mediated liver tumor promotion: clonal selection and expansion of cells evading growth arrest and apoptosis by Karl Walter Bock; Christoph K�hle (pp. 1403-1408).

The Ah receptor (AhR) has been characterized as a ligand-activated transcription factor which belongs to the bHLH/PAS (basic helix-loop-helix/Per-Arnt-Sim) family of chemosensors. Transgenic mouse models revealed adaptive and developmental functions of the AhR in the absence of exogenous ligands. Use of persistent agonists such as dioxins including 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) and related compounds demonstrated that the AhR mediates a plethora of species- and tissue-dependent toxicities, including chloracne, wasting, teratogenicity, immunotoxicity, liver tumor promotion and carcinogenicity. However, molecular mechanisms underlying most aspects of these toxic responses as well as biological functions of the AhR are currently unknown. Previous studies of liver tumor promotion in the two-stage hepatocarcinogenesis model indicated that TCDD mediates clonal expansion of ‘initiated’ preneoplastic hepatocytes, identified as enzyme-altered foci (EAF) by inhibiting apoptosis and bypassing AhR-mediated growth arrest. In contrast, the Ah receptor has been shown in cell models to stimulate growth arrest and apoptosis. Possible underlying mechanisms of these AhR responses are discussed, including enhanced metabolism of retinoic acid which attenuates TGFβ-mediated apoptosis and interaction of the Ah receptor with the hypophosphorylated retinoblastoma tumor suppressor protein. The discrepancy between in vivo findings in EAF and AhR functions may be solved by hypothesizing that sustained activation of the Ah receptor generates a strong selective pressure in liver treated with genotoxic carcinogens leading to selection and expansion of clones evading growth arrest and apoptosis. Models are discussed which may facilitate verification of this hypothesis.

Keywords: Abbreviations; AhR; aryl hydrocarbon receptor; RA; retinoic acids; CYP; cytochrome P450; EAF; enzyme-altered foci; Rb; retinoblastoma protein; TCDD; 2,3,7,8-tetrachlorodibenzo-; p; -dioxinAh receptor; Apoptosis; Cell contact inhibition; Liver tumor promotion; Retinoblastoma protein; Retinoids

6-Benzylthioinosine analogues as subversive substrate of Toxoplasma gondii adenosine kinase: Activities and selective toxicities by Reem H. Rais; Omar N. Al Safarjalani; Vikas Yadav; Vincenzo Guarcello; Marion Kirk; Chung K. Chu; Fardos N.M. Naguib; Mahmoud H. el Kouni (pp. 1409-1419).

Toxoplasma gondii adenosine kinase (EC.2.7.1.20) is the major route of adenosine metabolism in this parasite. The enzyme is significantly more active than any other enzyme of the purine salvage in T. gondii and has been established as a potential chemotherapeutic target for the treatment of toxoplasmosis. Certain 6-substituted purine nucleosides act as subversive substrates of T. gondii, but not the human, adenosine kinase. Therefore, these compounds are preferentially metabolized to their respective nucleotides and become selectively toxic against the parasites but not their host. Herein, we report the testing of newly synthesized 6-benzylthioinosine analogues with various substituents on the phenyl ring of their benzyl group as subversive substrates of T. gondii adenosine kinases. The binding affinity of these compounds to T. gondii adenosine kinase and their efficacy as antitoxoplasmic agents varied depending on the nature and position of the various substituents on the phenyl ring of their benzyl group. p- Cyano-6- benzylthioinosine and 2,4-dichloro-6-benzylthioinosine were the best ligands. In general, analogues with substitution at the para position of the phenyl ring were better ligands than those with the same substitutions at the meta or ortho position. The better binding of the para-substituted analogues is attributed to the combined effect of hydrophobic as well as van der Waals interactions. The 6-benzylthioinosine analogues were devoid of host-toxicity but all showed selective anti-toxoplasmic effect in cell culture and animal models. These results further confirm that toxoplasma adenosine kinase is an excellent target for chemotherapy and that 6-substituted purine nucleosides are potential selective antitoxoplasmic agents

Keywords: Abbreviations; FBS; fetal bovine serum; HPMC; hydroxypropylmethylcellulose; MTT; microculture tetrazolium test; PBS; phosphate buffered salineToxoplasma; Adenosine kinase; Subversive substrates; 6-Benzylthioinosine analogues; Chemotherapy

Dietary flavonoids as proteasome inhibitors and apoptosis inducers in human leukemia cells by Di Chen; Kenyon G. Daniel; Marina S. Chen; Deborah J. Kuhn; Kristin R. Landis-Piwowar; Q. Ping Dou (pp. 1421-1432).

It has been shown that proteasome activity is required for cancer cell survival and consumption of fruits and vegetables is associated with decreased cancer risk. Previously, we reported that grape extract could inhibit proteasome activity and induce apoptosis in tumor cells. In this study, we examined the flavonoids apigenin, quercetin, kaempferol and myricetin for their proteasome-inhibitory and apoptosis-inducing abilities in human tumor cells. We report that apigenin and quercetin are much more potent than kaempferol and myricetin at: (i) inhibiting chymotrypsin-like activity of purified 20S proteasome and of 26S proteasome in intact leukemia Jurkat T cells; (ii) accumulating putative ubiquitinated forms of two proteasome target proteins, Bax and Inhibitor of nuclear factor κβ-α in Jurkat T cells and (iii) inducing activation of caspase-3 and cleavage of poly(ADP-ribose) polymerase in Jurkat T cells. The proteasome-inhibitory abilities of these compounds correlated with their apoptosis-inducing potencies. Results from computational modeling of the potential interactions of these flavonoids to the chymotrypsin site (β5 subunit) of the proteasome were consistent with the obtained proteasome-inhibitory activities. We found that the C₄ carbon may be a site of nucleophilic attack by the OH group of N-terminal threonine of proteasomal β5 subunit and that the C₃ hydroxyl may alter the ability of these flavonoids to inhibit the proteasome. Finally, apigenin neither effectively inhibited the proteasome activity nor induced apoptosis in non-transformed human natural killer cells. Our results suggested that the proteasome may be a target of these dietary flavonoids in human tumor cells and that inhibition of the proteasome by flavonoids may be one of the mechanisms responsible for their cancer-preventive effects.

Keywords: Abbreviations; AMC; 7-amido-4-methyl-coumarin; BSA; bovine serum albumin; (−)-EGCG; (−)-epigallocatechin-3-gallate; FBS; fetal bovine serum; FITC; fluorescein isothiocyanate; IκB-α; Inhibitor of Nuclear Factor κB-α; PARP; poly(ADP-ribose) polymeraseFlavonoids; Chemoprevention; Chemotherapy; Proteasome inhibitors; Apoptosis; Computer modelling

Protection against 2,4,6-trinitrobenzenesulphonic acid-induced colonic inflammation in mice by the marine products bolinaquinone and petrosaspongiolide M by Jérôme Busserolles; Miguel Payá; Maria Valeria D’Auria; Luigi Gomez-Paloma; Maria José Alcaraz (pp. 1433-1440).

Proinflammatory mediators, namely eicosanoids, reactive oxygen and nitrogen species and cytokines, are clearly involved in the pathogenesis of intestinal bowel disease. bolinaquinone (BQ) and petrosaspongiolide M (PT), two marine products with potent anti-inflammatory action, have been shown to control the production of mediators in acute and chronic inflammatory processes. Hence, we have tested here the hypothesis that BQ and PT could ameliorate inflammation and oxidative stress parameters in 2,4,6-trinitrobenzenesulphonic acid (TNBS)-induced colitis in Balb/c mice. BQ and PT were given orally in doses of 10 or 20mg/kg/day. Treatment of the animals with BQ or PT at the highest dose significantly protected against TNBS-induced inflammation, as assessed by a reduced colonic weight/length ratio and histological scoring. Neutrophilic infiltration, interleukin (IL)-1β and prostaglandin E₂ (PGE₂) levels, as well as cyclooxygenase-2 (COX-2) protein expression were inhibited by both compounds. Colonic nitrite and nitrate levels and protein expression of inducible nitric oxide synthase (iNOS) were also lower in the treated groups in comparison to the TNBS control. BQ and PT reduced nitrotyrosine immunodetection and colonic superoxide anion production. Neither compound inhibited the expression of the protective protein heme oxygenase-1 (HO-1), although they reduced the extension of apoptosis. Our study also indicated that PT could interfere with the translocation of p65 into the nucleus, a key step in nuclear factor-κB (NF-κB) activation. Altogether, the results suggest that BQ and PT can have potential protective actions in intestinal inflammatory diseases.

Keywords: Abbreviations; BQ; bolinaquinone; COX-2; cyclooxygenase-2; Dex; dexamethasone; HO-1; heme oxygenase-1; IBD; intestinal bowel disease; IL; interleukin; MPO; myeloperoxidase; NF-κB; nuclear factor-κB; NO; nitric oxide; NO; _x; ⁻; NO; ₂; ⁻; +; NO; ₃; ⁻; iNOS; inducible nitric oxide synthase; PT; petrosaspongiolide M; PGE; ₂; prostaglandin E2; PLA; ₂; phospholipase A2; ROS; reactive oxygen species; Sh; Sham; TNBS; 2,4,6-trinitrobenzenesulphonic acid; TUNEL; terminal deoxynucleotidyl transferase dUTP nick-end labelingBolinaquinone; Petrosaspongiolide M; Intestinal inflammation; 2,4,6-Trinitrobenzenesulphonic acid; Balb/c mice; Nuclear factor-κB

Representative aminopeptidases and prolyl endopeptidase from murine macrophages: Comparative activity levels in resident and elicited cells by Renata do Amaral Olivo; Catarina de F�tima Pereira Teixeira; Paulo Fl�vio Silveira (pp. 1441-1450).

Macrophages are considered the main effector cells of immune system. Under stimulation these cells are known to be activated by a process involving morphological, biochemical and functional changes. Since altered peptidase activities could be among the factors leading to the differentiation and activation of these cells, in the present work seven naphthylamide derivative substrates were employed to assess representative aminopeptidase and prolyl endopeptidase activities in resident and elicited macrophages of mice. Soluble basic aminopeptidase and prolyl endopeptidase and soluble and particulate neutral and prolyl dipeptidyl aminopeptidase IV activities were present at measurable levels while particulate prolyl endopeptidase and basic aminopeptidase, and particulate and soluble cystyl and pyroglutamyl aminopeptidases were not detectable. Kinetic parameters, chloride activation and the inhibitory effects of puromycin, bestatin, amastatin and diprotin A characterized differential properties of these peptidase activities. The observed increment (about 6–17-fold) of the soluble basic aminopeptidase and prolyl endopeptidase and soluble and particulate neutral and prolyl dipeptidyl aminopeptidase IV activities in elicited macrophages was particularly relevant, as these might contribute to an increased ability of this cell to inactivate several susceptible substrates known to be inflammatory and/or immunological mediators.

Keywords: Macrophages; Peptidases; Peptides; Mediators; Inflammation; Immune system

Regulation of dense core vesicle release from PC12 cells by interaction between the D2 dopamine receptor and calcium-dependent activator protein for secretion (CAPS) by Alicia V. Binda; Nadine Kabbani; Robert Levenson (pp. 1451-1461).

We identified CAPS1 (calcium-dependent activator protein for secretion) as a D2 dopamine receptor interacting protein (DRIP) in a yeast two-hybrid screen of a human brain library using the second intracellular domain of the human D2 receptor (D2IC2). CAPS1 is an evolutionarily conserved calcium binding protein essential for late-stage exocytosis of neurotransmitters from synaptic terminals. CAPS1 interaction was confirmed for both the long and short isoforms of the D2 receptor, but not with any other dopamine receptor subtype. Interaction between CAPS1 and the D2 receptor was validated using both pulldown and coimmunoprecipitation assays. Deletion mapping localized the D2 receptor binding site to a segment located within the C-terminal region of CAPS1 as well CAPS2. In PC12 cells, CAPS1 and D2 receptors were found to colocalize within both cytosolic and plasma membrane compartments. Overexpression of a truncated D2 receptor fragment caused a significant decrease in K⁺-evoked dopamine release from PC12 cells, whereas no effect on norepinephrine or BDNF release was observed. These results suggest that D2 dopamine receptors may modulate vesicle release from neuroendocrine cells via direct interaction with components of the exocytotic machinery

Keywords: Abbreviations; BDNF; brain-derived neurotrophic factor; CAPS; calcium-dependent activator protein for secretion; D2L; dopamine receptor D2 long isoform; D2S; dopamine receptor D2 short isoform; LDCV; large dense core vesicle; DRIP; dopamine receptor interacting protein; EGFP; enhanced green fluorescent protein; FBS; fetal bovine serum; GFP; green fluorescent protein; GST; glutathione-S-transferase; HEK; human embryonic kidney; IC2; second intracellular domain; KCl; potassium chloride; MHD; munc homology domain; NE; norepinephrine; PKA; protein kinase A; PKC; protein kinase C; RT-PCR; reverse transcriptase-polymerase chain reactionCAPS; Dopamine; Dopamine receptor; Exocytosis; G-protein coupled receptor; Neurotransmission

Differential inhibition of cellular glutathione reductase activity by isocyanates generated from the antitumor prodrugs Cloretazine™ and BCNU by Kevin P. Rice; Philip G. Penketh; Krishnamurthy Shyam; Alan C. Sartorelli (pp. 1463-1472).

The antitumor, DNA-alkylating agent 1,3-bis[2-chloroethyl]-2-nitrosourea (BCNU; Carmustine), which generates 2-chloroethyl isocyanate upon decomposition in situ, inhibits cellular glutathione reductase (GR; EC 1.8.1.7) activity by up to 90% at pharmacological doses. GR is susceptible to attack from exogenous electrophiles, particularly carbamoylation from alkyl isocyanates, rendering the enzyme unable to catalyze the reduction of oxidized glutathione. Evidence implicates inhibition of GR as a cause of the pulmonary toxicity often seen in high-dose BCNU-treated animals and human cancer patients. Herein we demonstrate that the prodrug Cloretazine™ (1,2-bis[methylsulfonyl]-1-[2-chloroethyl]-2-[(methylamino)carbonyl]hydrazine; VNP40101M), which yields methyl isocyanate and chloroethylating species upon activation, did not produce similar inhibition of cellular GR activity, despite BCNU and Cloretazine™ being equally potent inhibitors of purified human GR (IC₅₀ values of 55.5μM and 54.6μM, respectively). Human erythrocytes, following exposure to 50μM BCNU for 1h at 37°C, had an 84% decrease in GR activity, whereas 50μM Cloretazine™ caused less than 1% inhibition under the same conditions. Similar results were found using L1210 murine leukemia cells. The disparity between these compounds remained when cells were lysed prior to drug exposure and were partially recapitulated using purified enzyme when 1mM reduced glutathione was included during the drug exposure. The superior antineoplastic potential of Cloretazine™ compared to BCNU in animal models could be attributed in part to the contribution of the methyl isocyanate, which is synergistic with the co-generated cytotoxic alkylating species, while at the same time unable to significantly inhibit cellular GR.

Keywords: Abbreviations; BCNU; 1,3-bis[2-chloroethyl]-2-nitrosourea; GR; glutathione reductase; MiC; methyl isocyanate; CEiC; 2-chloroethyl isocyanate; AGT; O; ⁶; -alkylguanine-DNA alkyltranferaseCloretazine™; BCNU; Glutathione reductase; DNA alkylating agents; Methyl isocyanate; 2-Chloroethyl isocyanate

Protective effect of FK506 against apoptosis of SH-SY5Y cells correlates with regulation of the serum inducible kinase gene by Masakazu Muramoto; Takao Yamazaki; Noriyuki Morikawa; Osamu Okitsu; Takeyuki Nagashima; Tomoya Oe; Shintaro Nishimura; Yasuhiro Kita (pp. 1473-1481).

Recently, we established an in vitro model of apoptosis induced by exposure of neuroblastoma SH-SY5Y cells to thapsigargin, an endoplasmic reticular calcium-ATPase inhibitor, and demonstrated that FK506 (tacrolimus) protected against apoptosis. The purpose of this paper was to investigate a possible correlation between the protective effect of FK506 against apoptosis and the regulation of the serum inducible kinase (SNK) and fibroblast growth factor inducible kinase (FNK) genes—which are polo-like kinases expressed abundantly in the brain by FK506. Thapsigargin increased the mRNA level of SNK and FNK in SH-SY5Y cells. FK506 inhibited the increase in SNK mRNA but not FNK mRNA. Deletion analysis of the SNK promoter showed that the promoter site, which was regulated by thapsigargin and FK506 in a calcineurin-dependent manner, is a cAMP response element (CRE)/activating transcription factor (ATF)-like element located 84 base pairs (bp) proximal to the transcriptional initiation site. Although transcription of the SNK gene was also regulated by tunicamycin, etoposide, or staurosporine, FK506 did not show any effects on these regulations. We recently reported that FK506 did not protect against apoptosis induced by these agents. These results indicate that the induction of SNK mRNA by thapsigargin in SH-SY5Y cells is regulated by FK506 via an inhibition of calcineurin at the transcriptional stage, and the transcriptional regulation of the SNK gene by FK506 was well correlated with the protective effect of the compound against apoptosis. Thus, transcriptional regulation of the SNK gene may be a biological marker for analysis of apoptosis of SH-SY5Y cells.

Keywords: Abbreviations; FK506; the immunosuppressive macrolactamlactone tacrolimus; SNK; serum inducible kinase; FNK; fibroblast growth factor inducible kinase; Plk; polo-like kinase; NF-IL3; nuclear factor interleukin-3; CRE; cAMP response element; CREB; cAMP response element binding protein; ATF; activating transcription factorFK506; Thapsigargin; Apoptosis; SH-SY5Y cells; Serum inducible kinase; Biological marker

Ca²⁺ sensitization and the regulation of contractility in rat anococcygeus and retractor penis muscle by Cleber E. Teixeira; Liming Jin; Zhekang Ying; Trenis Palmer; R. Clinton Webb (pp. 1483-1492).

Stimulation of the RhoA/Rho-kinase (ROK) signaling represents a key step in the maintenance of agonist-induced contraction of smooth muscle. We aimed to demonstrate Ca²⁺ sensitization in rat anococcygeus and retractor penis muscles and to identify the molecular expression of major components of this pathway. Both anococcygeus and retractor penis showed a similar expression of RhoA, ROKα, and ROKβ at the protein level as well as the mRNA for RhoGEFs. Cumulative addition of the ROK inhibitors H-1152 (0.001–3μM), Y-27632 (0.01–30μM) or HA-1077 (0.01–30μM) caused sustained relaxations of precontracted smooth muscle strips. Ca²⁺ sensitization induced by phenylephrine, norepinephrine and carbachol was markedly antagonized by all three ROK inhibitors. In addition, the contractile response to KCl-induced depolarization was highly sensitive to these ROK inhibitors. H-1152 was approximately 8–20 more potent than Y-27632 and HA-1077 to inhibit contraction. Electrical field stimulation (EFS, 1–32Hz) caused transient contractions in both anococcygeus and retractor penis muscle, which were blocked by tetrodotoxin (1μM), phentolamine (1μM) or bretylium tosylate (30μM). Similarly, H-1152 (0.1–1μM), Y-27632 (1–10μM) or HA-1077 (1–10μM) significantly reduced EFS-evoked contractions in a concentration-dependent manner. The results indicate that the RhoA/ROK-mediated Ca²⁺ sensitization pathway is expressed in anococcygeus and retractor penis muscles and enhances contractions produced by receptor-dependent and independent mechanisms.

Keywords: Abbreviations; CCh; carbachol; EFS; electrical field stimulation; GAPDH; glyceraldehyde-3-phosphate dehydrogenase; GEFs; guanine nucleotide exchange factors; GPCR; G protein coupled-receptor; HA-1077; (5-isoquinolinesulfonyl)homopiperazine; H-1152; (; S; )-(+)-2-methyl-1-[(4-methyl-5-isoquinolinyl)sulfonyl]homopiperazine; LARG; leukemia-associated RhoGEF; MLC; myosin light chain; NE; norepinephrine; PE; phenylephrine; RGS; regulator of G protein signaling domain; ROK; Rho-kinase; Y-27632; (; R; )-(+)-; trans; -; N; -(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamideAnococcygeus muscle; Retractor penis muscle; Rho-kinase; RhoGEFs; RhoA; Ca; ²⁺; sensitization

Stimulation of neuropeptide Y-mediated calcium responses in human SMS-KAN neuroblastoma cells endogenously expressing Y₂ receptors by co-expression of chimeric G proteins by Frank M. Dautzenberg (pp. 1493-1499).

Human SMS-KAN neuroblastoma cells endogenously express the neuropeptide Y (NPY) type 2 (Y₂) receptor. Although ligand binding and GTPγS binding studies supported high functional Y₂ receptor expression, only weak coupling to the natural second messenger cyclic AMP was observed. The main reason was the low responsiveness of SMS-KAN cells to forskolin, a direct activator of adenylyl cyclases. In order to obtain a cell-based functional assay for the Y₂ receptor in SMS-KAN cells, the transient calcium (Ca²⁺) mobilization assay in the fluorimetric imaging plate reader (FLIPR) format was established by stably expressing a chimeric G protein Gq_i9. This manipulation resulted in robust mobilization of Ca²⁺ after challenge with various NPY-related agonists in a 384-well format. The sensitivity of the FLIPR readout was in the low nanomolar range for NPY agonists and comparable to that of the recombinant Y₂ receptor. The selective Y₂ antagonist BIIE0246 competitively inhibited NPY-mediated Ca²⁺ transients in SMS-KAN/Gq_i9 cells with a p A₂ value of 7.39±0.1. This is the first evidence that an endogenously expressed G protein-coupled receptor couples to an overexpressed chimeric G protein, thereby functionally responding in the FLIPR readout.

Keywords: Abbreviations; NPY; neuropeptide Y; Y; _1–5; NPY type 1–5 receptor; y; ₆; NPY type 6 receptor; GPCR; G protein-coupled receptor; G; _o; and G; _i; protein; inhibitory G proteins that are negatively coupled to adenylate cyclase; Gq protein; phosphoinositide- and calcium stimulating G protein; Gq; _o5; Gq; _i5; and Gq; _i9; chimeric G proteins carrying the last 5–9 amino acids of G; _o; and G; _i; proteins linked to the Gq proteinLigand binding; cAMP inhibition; GTPγS binding; Ca; ²⁺; stimulation; Chimeric G protein

Histone deacetylases inhibition and tumor cells cytotoxicity by CNS-active VPA constitutional isomers and derivatives by Sara Eyal; Boris Yagen; Jakob Shimshoni; Meir Bialer (pp. 1501-1508).

The tumor cells toxicity of the antiepileptic drug valproic acid (VPA) has been associated with the inhibition of histone deacetylases (HDACs). We have assessed, in comparison to VPA, the HDACs inhibition and tumor cells cytotoxicities of CNS-active VPA's constitutional isomers, valnoctic acid (VCA), propylisopropylacetic acid (PIA), diisopropylacetic acid (DIA), VPA's cyclopropyl analogue 2,2,3,3-tetramethylcyclopropanecarboxylic acid (TMCA) and VPA's metabolites, 2-ene-VPA and 4-ene-VPA, all possessing, as does VPA, eight carbon atoms in their structures. The aim was to define structural components of the VPA molecule that are involved in HDACs inhibition and tumor cells cytotoxicity.HDACs inhibition by the above-mentioned compounds was estimated using an acetylated lysine substrate and HeLa nuclear extract as a HDACs source. SW620 cells were used for assessing HDACs inhibition in vivo. The cytotoxicity of these compounds was assessed in SW620 and 1106mel cells.HDAC inhibition potency was the highest for VPA and 4-ene-VPA (IC₅₀=1.5mM each). 2-Ene-VPA inhibited HDACs with IC₅₀=2.8mM. IC₅₀ values of the other tested compounds for HDACs inhibition were higher than 5mM, 4-ene-VPA and VPA induced histone hyperacetylation in SW620 cells. 4-Ene-VPA and VPA at 2mM each were also most potent in reducing cell viability, to 59�2.0% and 67.3�5.4%, respectively, compared to control. VCA, PIA, DIA, TMCA, 2-ene-VPA and valpromide (VPD) did not reduce viability to less than 80%. All tested compounds did not significantly affect the cell cycle of SW620 cells.In conclusion, in comparison to the VPA derivatives and constitutional isomers tested in this study, VPA had the optimal chemical structure in terms of HDACs inhibition and tumor cells cytotoxicity.

Keywords: JEL classification; Molecular pharmacologyAbbreviations; AED; antiepileptic drug; BuA; butyric acid; HDAC; histone deacetylase; DIA; diisopropylacetic acid; PI; propidium iodide; PIA; propylisopropylacetic acid; TMCA; 2,2,3,3-tetramethylcyclopropanecarboxylic acid; TSA; trichostatin A; VCA; valnoctic acid; VPA; valproic acid; VPD; valpromideValproic acid; Valproic acid constitutional isomers; Anticancer drugs; Histone deacetylase; Antiepileptic drugs

Binding of [³H]MK-801 in subcellular fractions of Schistosoma mansoni: Evidence for interaction with nicotinic receptors by Renata Fittipaldi Pess�a; Newton Gon�alves Castro; Fran�ois No�l (pp. 1509-1516).

Several studies have suggested thatl-glutamate is a putative neurotransmitter in Schistosoma mansoni. Recently, we detected the presence of low-affinity binding sites for [³H]kainic acid in the heterogeneous (P₁) subcellular fraction of S. mansoni. In an attempt to characterize N-methyl-d-aspartate (NMDA) receptors in this worm, we performed binding assays with [³H]MK-801, a NMDA non-competitive antagonist, in the P₁ fraction of adult S. mansoni. In competition experiments, MK-801 (IC₅₀ ∼200μM) and ketamine (IC₅₀ ∼500μM) exhibited a low affinity for the sites labeled with [³H]MK-801. Along with the lack of modulation of this binding by glutamatergic agonists and antagonists and the absence of stereoselectivity for MK-801 isomers, these results suggest that [³H]MK-801 could label a site different from the classical NMDA receptor in S. mansoni. Based on the evidences that MK-801 interacts with mammalian muscle and central nervous system nicotinic receptors as a low-affinity noncompetitive antagonist, we have investigated the effects of MK-801 on the nicotine-induced flaccid paralysis of the worm, in vivo. The motility of S. mansoni was quantified by image analysis through a measure of displacement of the worm's extremities. In the presence of (−)-nicotine (10–100μM), we observed an immediate paralysis of the worms, that was inhibited by 1mM MK-801. Besides nicotine, choline (10–50mM) was also able to inhibit the worm's motility. As a conclusion, we suggest that [³H]MK-801 binds to nicotinic receptors, and not NMDA receptors, in subcellular fractions of S. mansoni.

Keywords: Abbreviations; AMPA; α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid; AP5; 2-amino-5-phosphonopentanoic acid; DNQX; 6,7-dinitroquinoline-2,3-dione; EDTA; ethylenediaminetetraacetic acid; 5-HT; 5-hydroxytryptamine creatine sulfate; NMDA; N; -methyl-; d; -aspartate; NMDG; N; -methyl-; d; -glucamine; PMSF; phenylmethylsulfonyl fluoride; POPOP; 1,4-bis-[2-(5-phenyloxazolyl)]-benzene; PPO; 2,5-diphenyloxazole; Tris; tris(hydroxymethyl) aminomethane Schistosoma mansoni; Dizocilpine; Glutamate; Platyhelminth; MK-801; Nicotine

Inhibition of thymidine phosphorylase (PD-ECGF) from SD-lymphoma by phosphonomethoxyalkyl thymines by Ivan Votruba; Karel Pomeisl; Eva Tloušt’ová; Antonín Holý; Berta Otová (pp. 1517-1521).

A series of thymine phosphonomethoxyalkyl derivatives were evaluated for their ability to inhibit thymidine phosphorylase (dThdPase) purified from rat spontaneous T-cell lymphoma. A kinetic study of thymidine phosphorolysis catalyzed by dThdPase was performed with thymidine and/or inorganic phosphate as substrates. Data show that the substantial inhibitory effect of these acyclic nucleotide analogues is decreasing in the order of ( R)-FPMPT>( S)-FPMPT≥( R)-HPMPT>( S)-PMPT>( S)-HPMPT>PMET≥( R)-PMPT. The inhibitory potency ( K_i/^dThd K_m) of the most efficient inhibitors from this series against T-cell lymphoma enzyme is 0.0026 for ( R)-FPMPT and 0.0048 for ( S)-FPMPT. The studied compounds do not inhibit Escherichia coli and human enzyme and possess lower inhibitory potency against rat liver thymidine phosphorylase.

Keywords: Abbreviations; (; S; )-HPMPC; 1-(; S; )-[3-hydroxy-2-(phosphonomethoxy)propyl]-cytosine; (; R; )-PMPA; 9-(; R; )-[2-(phosphonomethoxy)propyl]adenine; PME; 9-[2-(phosphonomethoxy)ethyl]; PMEDAP; 9-[2-(phosphonomethoxy)ethyl]-2,6-diamino-purine; PMEG; 9-[2-(phosphonomethoxy)ethyl]guanine; PMET; 1-[2-(phosphono-methoxy)ethyl]thymine; (; S; )-HPMPT; (; S; )-1-[3-hydroxy-2-(phosphonomethoxy)-propyl]thymine; (; R; )-HPMPT; (; R; )-1-[3-hydroxy-2-(phosphonomethoxy)propyl]-thymine; (; S; )-PMPT; (; S; )-1-[2-(phosphonomethoxy)propyl]thymine; (; R; )-PMPT; (; R; )-1-[2-(phosphonomethoxy)propyl]thymine; (; S; )-FPMPT; (; S; )-1-[3-fluoro-2-(phosphono-methoxy)propyl]thymine; (; R; )-FPMPT; (; R; )-1-[3-fluoro-2-(phosphonomethoxy)-propyl]thymine; dThd; thymidine (2′-deoxythymidine); dTMP; 2′-deoxythymidine 5′-phosphate; dThdPase; thymidine phosphorylase; Pi; inorganic phosphate; ANP; acyclic nucleoside phosphonateThymidine phosphorylase inhibition; Thymine acyclic nucleoside phosphonates; FPMPT

Inhibition of human liver catechol- O-methyltransferase by tea catechins and their metabolites: Structure–activity relationship and molecular-modeling studies by Dapeng Chen; Ching Y. Wang; Joshua D. Lambert; Ni Ai; William J. Welsh; Chung S. Yang (pp. 1523-1531).

(–)-Epigallocatechin-3-gallate (EGCG) is the major polyphenol present in green tea. We previously demonstrated that EGCG was both a substrate and potent inhibitor of human liver cytosolic catechol- O-methyltransferease (COMT). We now report the structure–activity relationship for the inhibition of COMT-catalyzed O-methylation of catecholestrogens in human liver cytosol by tea catechins and some of their metabolites. The most potent inhibitors were catechins with a galloyl-type D-ring, including EGCG (IC₅₀=0.07μM), 4″- O-methyl-EGCG (IC₅₀=0.10μM), 4′,4″-di- O-methyl-EGCG (4′,4″-DiMeEGCG) (IC₅₀=0.15μM), and (–)-epicatechin-3-gallate (ECG) (IC₅₀=0.20μM). Catechins without the D-ring showed two to three orders of magnitude less inhibitory potency. Enzyme kinetic analyses revealed that EGCG behaved as a mixed inhibitor, whereas 4′,4″-di- O-methyl-EGCG exhibited competitive kinetics for the S-adenosylmethionine (SAM), and noncompetitive kinetics for the catechol binding site. These compounds may represent a new type of COMT inhibitor. In silico molecular-modeling studies using a homology model of human COMT were conducted to aid in the understanding the catalytic and inhibitory mechanisms. Either D-ring or B-ring of EGCG could be accommodated to the substrate binding pocket of human COMT. However, the close proximity (2.6Å) of 4″-OH to the critical residue Lys144, the higher acidity of the hydroxyl groups of the D-ring, and the hydrophobic interactions between the D-ring and residues in the binding pocket greatly facilitated the interaction of the D-ring with the enzyme, and resulted in increased inhibitory potency. These results provide mechanistic insight into the inhibition of COMT by commonly consumed tea catechins.

Keywords: Abbreviations; EGCG; (–)-epigallocatechin-3-gallate; ECG; (–)-epicatechin-3-gallate; EGC; (–)-epigallocatechin; EC; (–)-epicatechin; EGCG-7-Gluc; (–)-EGCG-7-; O; -glucuronide (and similar abbreviations for other glucuronidates); 4′-MeEGC; 4′-; O; -methyl-EGC; 4″-MeEGCG; 4″-; O; -methyl-EGCG; 4′,4″-DiMeEGCG; 4’,4″-di-; O; -methyl-EGCG; 2-OH-E; ₂; 2-hydroxyestradiol; 4-OH-E; ₂; 4-hydroxyestradiol; COMT; catechol-; O; -methyltransferase; SAM; S; -adenosylmethionine; SAH; S; -adenosyl-; l; -homocysteine; L-DOPA; 3,4-dihydroxy-; l; -phenylalanine; HPLC–ECD; high-performance liquid chromatography–electrochemical detectorCatechol-; O; -methyltransferase; Tea catechins; Epigallocatechin-3-gallate; Catecholestrogens; Methylation; Molecular modeling

Electrochemical characterisation of the human cytochrome P450 CYP2C9 by D.L. Johnson; B.C. Lewis; D.J. Elliot; J.O. Miners; L.L. Martin (pp. 1533-1541).

The electrochemistry of human cytochrome P4502C9 (CYP2C9) was characterised using purified His-tagged enzyme. The His-tagged enzyme was shown to have similar functional characteristics to native CYP2C9 heterologously expressed in Escherichia coli and to the CYP2C9 activity of human liver microsomes. Evidence was observed for a reversible one-electron transfer between the P450 heme and the electrode. Both pH and ionic strength influenced the electrochemical behaviour of CYP2C9. A range of substrates was investigated to determine the effect of the heme–substrate interaction on CYP2C9 redox potential. In the absence of oxygen, tolbutamide, diclofenac, warfarin and sulfaphenazole did not alter the redox potential of the iron heme. In contrast, torsemide, carbon monoxide and oxygen led to an anodic shift in redox potential. These results suggest alternative mechanisms by which CYP2C9 (and by inference other P450 enzymes) may alter redox potential to facilitate electron delivery from physiological donors.

Keywords: Cytochrome P450; CYP2C9; Electrochemistry; Substrate shift; Midpoint potential; DDABAbbreviations; CYP; cytochrome P450; CYP2C9; cytochrome P4502C9; DDAB; didodecyldimethylammonium bromide; E; _mid; midpoint potential; Δ; E; peak separation; I; ionic strength; i; _pc; /; i; _pa; cathodic peak current divided by anodic peak current; k; _s; electron transfer rate constant; NHE; Normal Hydrogen Electrode; OxR; cytochrome P450 oxidoreductase; PGE; edge-oriented pyrolytic graphite

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Biochemical Pharmacology (v.69, #10)