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Biochemical Pharmacology (v.69, #2)

Editorial Advisory Board (pp. iii).

Ah receptor signals cross-talk with multiple developmental pathways by Alvaro Puga; Craig R. Tomlinson; Ying Xia (pp. 199-207).

For many years, the Ah receptor (AHR) has been a favorite of toxicologists and molecular biologists studying the connections between genes and the changes in the control of gene expression resulting from environmental exposures. Much of the attention given to the Ah receptor has focused on the nature of its ligands, many of which are known or suspected carcinogens, and on the role that its best studied regulatory product, the CYP1A1 enzyme, plays in toxic responses and carcinogen activation. This understandable bias has resulted in a disproportionate amount of Ah receptor research being directed at toxicological or adaptive end points. In recent times, it has become evident that Ah receptor functions are also involved in molecular cascades that lead to inhibition of proliferation, promotion of differentiation, or apoptosis, with an important bearing in development. Developmental and toxicological AHR functions may not always be related. The ancestral AHR protein in invertebrates directs the developmental fate of a few specific neurons and does not bind xenobiotic ligands. The mammalian AHR maintains normal liver function in the absence of exogenous ligands and, when activated by dioxin, cross-talks with morphogenetic and developmental signals. Toxic end points, such as the induction of cleft palate by dioxin in mice embryos, might be at the crossroads of these signals and provide important clues as to the developmental role of the AHR.

Keywords: Ah receptor; TGF-β; Retinoic acid; GABA; MAP kinases

Aminotransferase,l-amino acid oxidase and β-lyase reactions involvingl-cysteine S-conjugates found in allium extracts by Arthur J.L. Cooper; John T. Pinto (pp. 209-220).

Several cysteine S-conjugates that occur in extracts of garlic and other plants of the allium family possess anti-oxidant properties, and many, including S-allyl-l-cysteine (SAC) and S-allylmercapto-l-cysteine (SAMC), are promising anti-cancer agents. To understand possible biochemical mechanisms contributing to the protective effects, the ability of selected allium-derivedl-cysteine S-conjugates to undergo various enzyme-catalyzed transformations was investigated. SAC, SAMC, S-propylmercapto-l-cysteine and S-penta-1,3-dienylmercapto-l-cysteine were shown to be substrates of: (a) highly purified rat kidney glutamine transaminase K (GTK); (b) purified snake venoml-amino acid oxidase; and (c) a cysteine S-conjugate β-lyase present in rat liver cytosol. S-Methylmercapto-l-cysteine was shown to be a substrate of GTK andl-amino acid oxidase, but not of the cysteine S-conjugate β-lyase. Evidence is presented that a major enzyme responsible for the cysteine S-conjugate β-lyase reactions in the rat liver cytosol is γ-cystathionase. The possible role of γ-cystathionase in generating sulfane sulfur from the disulfide-containing cysteine S-conjugates present in allium extracts, and the possible role of this sulfane sulfur in enzyme regulation, targeting of cancer cells and detoxification reactions is discussed.An interesting side finding of the present work is that rat liver mitochondria are more active than rat liver cytosol in catalyzing a cysteine S-conjugate β-lyase reaction with the mitochondrial protoxicant S-(1,1,2,2-tetrafluoroethyl)-l-cysteine (TFEC) at physiological pH and at low substrate concentration.

Keywords: Abbreviations; DCVC; S; -(1,2-dichlorovinyl)-; l; -cysteine; GTK; glutamine transaminase K; GSH; glutathione; GSSG; glutathione disulfide; PLP; pyridoxal 5′-phosphate; PMP; pyridoxamine 5′-phosphate; SAC; S; -allyl-; l; -cysteine; SAMC; S; -allylmercapto-; l; -cysteine; TFEC; S; -(1,1,2,2-tetrafluoroethyl)-; l; -cysteine S; -Allyl-; l; -cysteine; S; -Allylmercapto-; l; -cysteine; l; -Amino acid oxidase; γ-Cystathionase; Cysteine; S; -conjugates; Glutamine transaminase K

Carnosol inhibits the invasion of B16/F10 mouse melanoma cells by suppressing metalloproteinase-9 through down-regulating nuclear factor-kappaB and c-Jun by Shiu-Chen Huang; Chi-Tang Ho; Shoei-Yn Lin-Shiau; Jen-Kun Lin (pp. 221-232).

Carnosol, a constant constituent of Rosmarinus officinalis extracts, is a phenolic diterpene shown to have antioxidant and anticarcinogen properties. In our studies, carnosol inhibited the invasion of highly metastatic mouse melanoma B16/F10 cells in vitro. First, the antimetastatic potentials of carnosol were examined by soft agar colony formation assay. Second, carnosol dose-dependently inhibited B16/F10 cell migration and invasion by in vitro transwell assay. Third, the decreasing activity of metalloproteinase was observed by zymographic assay. The result revealed that the treatment of carnosol could diminish the activity of MMP-9 more than MMP-2. Next, we analyzed the amounts of MMP-9 and MMP-2 proteins in the cells. The data indicated MMP-9 protein was also suppressed by carnosol in the same manner. In accordance with the above data, the results of reverse transcriptase polymerase chain reaction (RT–PCR) analysis showed a reduced level of MMP-9 mRNA. Furthermore, carnosol significantly inhibited the tyrosine phosphorylation of extracellular signal-regulated kinase (ERK) 1/2, AKT, p38, JNK and inhibition of activation of transcription factors NFκ-B and c-Jun. These results lead us to conclude that carnosol could restrict the invasive ability of B16/F10 mouse melanoma cells by reducing MMP-9 expression and activity through suppressing (ERK) 1/2, AKT, p38, and JNK signaling pathway and inhibition of NF-κB and AP-1 binding activity. Taken together, these results indicate that carnosol targets MMP-mediated cellular events in cancer cells and provides a new mechanism for its anticancer activity.

Keywords: Abbreviations; MMP; Matrix metalloproteinases; NF-κB; Nuclear factor-κB; AP-1; Activator protein-1; RT-PCR; Reverse transcriptase polymerase chain reaction; ERK; Extracellular signal-regulated kinaseCarnosol; Matrix metalloproteinases; Metastasis; Invasion; Nuclear factor-κB (NF-κB); c-Jun

Protective effect of dextromethorphan against endotoxic shock in mice by Guorong Li; Yuxin Liu; Nian-ssheng Tzeng; Gang Cui; Michelle L. Block; Belinda Wilson; Liya Qin; Tongguang Wang; Bin Liu; Jie Liu; Jau-Shyong Hong (pp. 233-240).

Dextromethorphan (DM) is a dextrorotatory morphinan and an over-the-counter non-opioid cough suppressant. We have previously shown that DM protects against LPS-induced dopaminergic neurodegeneration through inhibition of microglia activation. Here, we investigated protective effects of DM against endotoxin shock induced by lipopolysaccharide/d-galactosamine (LPS/GalN) in mice and the mechanism underlying its protective effect. Mice were given multiple injections of DM (12.5mg/kg, s.c.) 30min before and 2, 4h after an injection of LPS/GalN (20μg/700mg/kg). DM administration decreased LPS/GalN-induced mortality and hepatotoxicity, as evidenced by increased survival rate, decreased serum alanine aminotransferase activity and improved pathology. Furthermore, DM was also effective when it was given 30min after LPS/GalN injection. The protection was likely associated with reduced serum and liver tumor necrosis factor alpha (TNF-α) levels. DM also attenuated production of superoxide and intracellular reactive oxygen species in Kupffer cells and neutrophils. Real-time RT-PCR analysis revealed that DM administration suppressed the expression of a variety of inflammation-related genes such as macrophage inflammatory protein-2, CXC chemokine, thrombospondin-1, intercellular adhesion molecular-1 and interleukin-6. DM also decreased the expression of genes related to cell-death pathways, such as the DNA damage protein genes GADD45 and GADD153. In summary, DM is effective in protecting mice against LPS/GalN-induced hepatotoxicity, and the mechanism is likely through a faster TNF-α clearance, and decrease of superoxide production and inflammation and cell-death related components. This study not only extends neuroprotective effect of DM, but also suggests that DM may be a novel compound for the therapeutic intervention for sepsis.

Keywords: Abbreviations; DM; dextromethorphan; LPS; lipopolysaccharide; GalN; d; -galactosomine; ALT; alanine aminotransferas; TNF-α; tumor necrosis factor-alpha; ROS; reactive oxygen species; SOD; superoxide dismutase; MIP-2; macrophage inflammatory protein-2; TSP-1; thrombospondin1; mKC; a mouse CXC chemokine; ICAM-1; intercellular cell adhesion molecule-1; IL-6; interleukin-6; GADD45; growth arrest and DNA damage inducible protein 45; GADD153; growth arrest and DNA damage inducible protein 153DM; LPS/GalN; Liver injury; Inflammation; ROS; Gene expression

Decreased pro-inflammatory cytokine production by LPS-stimulated PBMC upon in vitro incubation with the flavonoids apigenin, luteolin or chrysin, due to selective elimination of monocytes/macrophages by Sander Hougee; Annemarie Sanders; Joyce Faber; Yvo M.F. Graus; Wim B. van den Berg; Johan Garssen; H. Friso Smit; Maarten A. Hoijer (pp. 241-248).

Apigenin and its structural analogues chrysin and luteolin were used to evaluate their capacity to inhibit the production of pro-inflammatory cytokines by lipopolysaccharide (LPS)-stimulated human peripheral blood mononuclear cells (PBMC). Furthermore, flowcytometric analysis was performed to compare the effects of apigenin, chrysin, luteolin, quercetin and naringenin on the different cell types present in PBMC.LPS-stimulated PBMC were cultured in the presence of the flavonoids and TNFα, IL-1β and IL-6 were measured in the supernatants. In parallel, metabolic activity of the PBMC was determined by measuring succinate dehydrogenase activity. Apigenin, chrysin and luteolin dose-dependently inhibited both pro-inflammatory cytokine production and metabolic activity of LPS-stimulated PBMC. With increasing concentration of apigenin, chrysin or luteolin the monocytes/macrophages disappeared as measured by flowcytometry. This also appeared to occur in the non-LPS-stimulated PBMC. At the same time there was an increase in dead cells. T- and B-lymphocytes were not affected. Quercetin and naringenin had virtually no effects on cytokines, metabolic activity or on the number of cells in the studied cell populations.In conclusion, monocytes were specifically eliminated in PBMC by apigenin, chrysin or luteolin treatment in vitro at low concentrations (around 8μM), in which apigenin appeared to be the most potent.

Keywords: Abbreviations; LPS; lipopolysaccharide; TNF; tumor necrosis factor; IL; interleukin; PBMC; human peripheral blood mononuclear cells; DMSO; dimethylsulfoxide; D-PBS; Dulbecco's phosphate-buffered salineFlavonoids; Apigenin; Pro-inflammatory; Metabolic activity; PBMC

FADD-dependent apoptosis induction in Jurkat leukemia T-cells by the resveratrol analogue, 3,4,5-trihydroxy- trans-stilbene by Yongbao Wang; Bo Wang; Jinchun Cheng; Li Yang; Zhong-Li Liu; Kannan Balan; Panayotis Pantazis; James H. Wyche; Zhiyong Han (pp. 249-254).

The plant-produced compound, resveratrol (3,5,4′-trihydroxy- trans-stilbene, 3,4,5-THS), induces apoptosis in various human leukemia cell types in vitro, and thus appears to be a promising anti-leukemia agent. In this study, we observed that treatment of resveratrol-resistant Jurkat cells with the resveratrol analogue, 3,4,5-trihydroxy- trans-stilbene (3,4,5-THS), rapidly induced extensive apoptosis, indicating that the apoptotic activity of the analogue differed from that of the parental compound resveratrol. Indeed, we found that treatment of Jurkat cells with 3,4,5-THS, unlike treatment with resveratrol, induced activation of caspase-8 and apoptosis by a Fas-associated death domain (FADD) protein-dependent mechanism without involving the known death ligands CD95 ligand (CD95L), tumor necrosis factor α (TNFα) and TNF-related apoptosis-inducing ligand (TRAIL). Therefore, 3,4,5-THS induced activation of a FADD-dependent apoptotic mechanism that was unresponsive to the parental compound resveratrol. Therefore, the ability of 3,4,5-THS, but not resveratrol, to induce apoptosis demonstrates a structure-associated apoptotic activity of the resveratrol analogue.

Keywords: Abbreviations; Resveratrol; 3,5,4′-trihydroxy-; trans; -stilbene; 3,4,5-THS; 3,4,5-tihydroxy-; trans; -stilbene; FADD; Fas-associated death domain; DN-FADD; dominant-negative FADD; PARP; poly(ADP-ribose) polymerase; CD95L; CD95 ligand; TNFα; tumor necrosis factor α; TRAIL; TNF-related apoptosis-inducing ligandResveratrol; Analogues; Apoptosis; FADD; Caspase-8; PARP

Pro-inflammatory properties for thiazolidinediones by Christophe Desmet; Barbara Warz�e; Philippe Gosset; Doroth�e M�lotte; Anthony Rongvaux; Laurent Gillet; Laurence Fi�vez; Gr�gory Seumois; Alain Vanderplasschen; Bart Staels; Pierre Lekeux; Fabrice Bureau (pp. 255-265).

Thiazolidinediones (TZDs) are pharmacological ligands of the peroxisome proliferator-activated receptor (PPAR)-γ that are extensively used in the treatment of type II diabetes. Recently, an anti-inflammatory potential for TZDs has been suggested, based on observations that these compounds may inhibit pro-inflammatory cytokine expression in vitro and may attenuate the inflammatory response in vivo. Here, we show that the TZDs rosiglitazone (RSG) and troglitazone (TRO) do not inhibit the inflammatory response to tumor necrosis factor (TNF)-α in various epithelial cell types. On the contrary, both RSG and TRO significantly potentiated TNF-α-induced production of granulocyte/macrophage-colony-stimulating factor, interleukin (IL)-6 and/or IL-8 in these cells. This increase in pro-inflammatory cytokine expression was functionally significant as supernatants from cells co-treated with TNF-α and TZDs displayed increased neutrophil pro-survival activity when compared with supernatants from cells treated with TNF-α alone. Additionally, it was shown that TZDs enhance cytokine expression at the transcriptional level, but that the pro-inflammatory effects of TZDs are independent on PPARγ, nuclear factor κB or mitogen-activated protein kinase activation. Our study shows that TZDs may potentiate the inflammatory response in epithelial cells, a previously unappreciated effect of these compounds.

Keywords: Abbreviations; AP-1; activator protein-1; HBEC; human bronchial epithelial cells; c/EBP; CAAT enhancer binding protein; CREB; cAMP responsive element binding protein; ERK; extracellular signal-regulated protein kinase; GM-CSF; granulocyte/macrophage-colony-stimulating factor; IL; interleukin; JNK; c-Jun N-terminal kinase; MAPK; mitogen-activated protein kinase; NF-AT; nuclear factor of activated T cells; NF-κB; nuclear factor-κB; PPARγ; peroxisome proliferator-activated receptor γ; RSG; rosiglitazone; TNF; tumor necrosis factor; TRO; troglitazone; TZD; thiazolidinedioneCytokines; Inflammation; Mitogen-activated protein kinases; Nuclear factor-κB; Peroxisome proliferator-activated receptor γ; Thiazolidinediones

Dexamethasone down-regulates cAMP-phosphodiesterase in human osteosarcoma cells by Mikael Ahlstr�m; Minna Pekkinen; Minna Huttunen; Christel Lamberg-Allardt (pp. 267-275).

Cyclic adenosine monophosphate (cAMP) is an important second messenger in the hormonal regulation of bone metabolism. cAMP is inactivated by the cyclic nucleotide phosphodiesterases (PDEs), a superfamily of enzymes divided into 11 known families, designated PDE1-11. Interference with the cAMP signaling pathway has been suggested as one mechanism causing glucocorticoid induced osteoporosis. We speculated that glucocorticoids could affect the cAMP pathway by a down-regulation of PDE-mediated cAMP hydrolysis. The main cAMP hydrolysing enzyme families of human MG-63 and SaOS-2 osteosarcoma cells were identified as PDE1 and PDE4 by assaying the PDE activity of Q-sepharose fractions and cell homogenates with selective inhibitors. Treatment with the glucocorticoid dexamethasone (Dex) decreased cAMP-PDE activity by up to 50%, without affecting cGMP-PDE activity. Dex treatment reduced the sensitivity of the total cAMP-PDE activity towards the PDE4 selective PDE inhibitor rolipram. Forskolin stimulated cAMP accumulation was increased 30–60-fold in the presence of rolipram. Treatment with Dex did not affect the basal or forskolin stimulated cAMP accumulation, but treatment resulted in a reduced effect of rolipram on cAMP accumulation. Expression of the following cAMP-PDE subtypes were detected by reverse transcriptase PCR (RT-PCR): PDE1A, PDE1C, PDE2A, PDE3A, PDE4A, PDE4B, PDE4C, PDE4D, PDE7A, PDE7B, PDE8A, PDE10A and PDE11A. Using semi-quantitative RT-PCR, we detected a 50–70% decrease in the mRNA of PDE4A and PDE4B subtypes following Dex treatment. Further analysis revealed that Dex reduced the PDE4A4 and PDE4B1 isoforms. PDE4A1 PDE4A, PDE4A7, PDE4A10, PDE4B2 were also expressed, but Dex did not affect the transcription of these isoforms. We conclude that Dex treatment could affect the cAMP signaling pathway of human osteosarcoma cells by reducing type 4 cAMP-phosphodiesterase (PDE4).

Keywords: Abbreviations; cAMP; cyclic adenosine 3′,5′monophosphate; Dex; dexamethasone; PDE; cyclic nucleotide phosphodiesterase; PTH; parathyroid hormone; BSA; bovine serum albumin; cGMP; cyclic guanosine 3′,5′monophosphateOsteoblast; Osteosarcoma; Dexamethasone; Glucocorticoids; Phosphodiesterase; cAMP

Role of adrenoceptor-linked signaling pathways in the regulation of CYP1A1 gene expression by Maria Konstandi; Dimitris Kostakis; Panagiotis Harkitis; Marios Marselos; Elizabeth Ourania Johnson; Konstantinos Adamidis; Matti Alarik Lang (pp. 277-287).

Alpha2-adrenoceptor agents as well as stress affect the activity of several hepatic monoxygenases including those related to CYP1A enzymes. This study was therefore designed to assess the role of central and/or peripheral catecholamines and, in particular, of adrenoceptors in the regulation of B(α)P-induced cytochrome CYP1A1 expression. In order to discriminate the role of central from that of peripheral catecholamines in the regulation of CYP1A1 induction, the effect of central and peripheral catecholamine depletion using reserpine versus only peripheral catecholamine depletion using guanethidine was assessed. By using selected agonists and antagonists, the role of alpha and beta-adrenoceptors in the regulation of CYP1A1 induction was evaluated. The results showed that the central catecholaminergic system has a negative regulatory effect on 7-ethoxyresorufin O-deethylase (EROD) inducibility by benzo(α)pyrene (B(α)P), and that this may be mediated via α1-, α2- and β-adrenoceptors. Specifically, stimulation of α2-adrenoceptors with dexmedetomidine and blockade of α1- or β-adrenoceptors with prazosin or propranolol respectively, resulted in a further increase of EROD inducibility. Adrenoceptors were found to be involved in the regulation of the CYP1A1 gene at mRNA level. Both, reduced noradrenaline release in central nervous system induced with dexmedetomidine and central catecholamine depletion, as well as blockade of central α1-adrenoceptors induced with prazosin, all were associated with up-regulation of CYP1A1 expression. In contrast, stimulation of central beta-adrenoceptors with isoprenaline resulted in a down-regulation of CYP1A1 expression. Our observations indicate that drugs, which stimulate or block adrenoceptors and catecholamine release may lead to complications in drug therapy and modulate the toxicity or carcinogenicity of drugs that are substrates for the CYP1A1.

Keywords: Abbreviations; EROD; 7-ethoxyresorufin O-deethylase; CYP1A1; cytochrome P4501A1 gene; B(α)P; benzo(α)pyrene; PAHs; polycyclic aromatic hydrocarbons; Ah; aromatic hydrocarbon; NA; noradrenaline; XREs; xenobiotic responsive elements; RIA; radioimmunoassayCatecholamines; Adrenoceptors; Benzo(α)pyrene; EROD; CYP1A1 gene; Rat

Thel-arginine/NO pathway in the early phases of platelet stimulation by collagen by Giuliana Leoncini; Debora Bruzzese; Maria Grazia Signorello (pp. 289-296).

Nitric oxide production,l-arginine transport and intracellular [Ca²⁺] changes in human platelets stimulated without stirring by low doses of collagen have been evaluated. Collagen decreased in a dose-dependent manner the nitric oxide formation. A reduction of about 30% of the basal level was produced by 5μg/mL. Aspirin did not change the collagen effect. The inhibition was reversed by EGTA. Moreover collagen reducedl-arginine uptake. The exposure of platelets to 5μg/mL collagen diminished of about 30%l-arginine transport. The specific involvement of the system y⁺ is suggested. In addition in FURA 2-loaded platelets collagen induced a dose-dependent slow sustained [Ca²⁺] rise that was almost completely cancelled by EGTA. Finally the treatment of whole platelets with collagen affected in a dose-dependent manner the maximal nitric oxide formation, suggesting a direct effect at the level of nitric oxide synthase enzyme. The phosphorylation of specific serine/threonine residues regulated by protein kinase C could be involved. In conclusion during the early phases of platelet stimulation with collagen nitric oxide formation is diminished. This reduction can be due to a lower availability ofl-arginine for cytosolic nitric oxide synthase and/or to a decreased activity related to modifications of the enzyme.

Keywords: Abbreviations; A23187; calcium ionophore A23187; cNOS; cytosolic nitric oxide synthase; NEM; N; -ethylmaleimide; NO; nitric oxide; NO; _x; nitrite; +; nitrate; Plts; platelets; WP; washed plateletsCollagen; Human platelets; Calcium; l; -Arginine; Transport; Nitric oxide; Nitric oxide synthase

Spin adduct formation from lipophilic EMPO-derived spin traps with various oxygen- and carbon-centered radicals by Klaus Stolze; Natascha Udilova; Thomas Rosenau; Andreas Hofinger; Hans Nohl (pp. 297-305).

Free radicals are involved in the onset of many diseases, therefore the availability of adequate spin traps is crucial to the identification and localization of free radical formation in biological systems. In recent studies several hydrophilic compounds of 2-ethoxycarbonyl-2-methyl-pyrroline- N-oxide (EMPO) have been found to form rather stable superoxide spin adducts with half-lives up to twenty minutes at physiological pH. This is a major improvement over DMPO ( t_1/2=ca. 45s), and even over DEPMPO ( t_1/2=ca. 14min), the best commercially available spin trap for the unambiguous detection of superoxide radicals. In order to allow the detection of superoxide and also other radicals in lipid environment a series of more lipophilic derivatives of EMPO was synthesized and their structure unambiguously characterized by¹H and¹³C NMR spectroscopy. In this way, six different compounds with a n-butyl group in position 5 and either an ethoxy- (EBPO), propoxy- (PBPO), iso-propoxy- ( iPBPO), butoxy- (BBPO), sec-butoxy- ( sBBPO) or tert-butoxycarbonyl group ( tBBPO) in position 5 of the pyrroline ring were obtained and fully analytically characterized (NMR, IR). The stability of the superoxide adducts of all investigated spin traps were comparable with EMPO ( t_1/2=ca. 8min), except for the two compounds bearing an additional methyl group in position 3 or 4 of the pyrroline ring, 5-butyl-5-ethoxycarbonyl-3-methyl-pyrroline- N-oxide (BEMPO-3) and 5-butyl-5-ethoxycarbonyl-4-methyl-pyrroline- N-oxide (BEMPO-4), of which the superoxide adducts were stable for more than 30min. Spin adducts of other carbon- and oxygen-centered radicals were also investigated.

Keywords: Abbreviations; BBPO; 5-(butoxycarbonyl)-5-butyl-1-pyrroline-; N; -oxide; DEPMPO; 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-; N; -oxide; DMPO; 5,5-dimethylpyrroline-; N; -oxide; DTPA; diethylenetriaminepentaacetic acid; EBPO; 5-(ethoxycarbonyl)-5-butyl-1-pyrroline-; N; -oxide; EMPO; 5-(ethoxycarbonyl)-5-methyl-1-pyrroline-; N; -oxide; BEMPO-3; 5-butyl-5-(ethoxycarbonyl)-3-methyl-1-pyrroline-; N; -oxide; BEMPO-4; 5-butyl-5-(ethoxycarbonyl)-4-methyl-1-pyrroline-; N; -oxide; EPR; electron paramagnetic resonance; HFS; hyperfine splitting; ^LO; lipoxyl radical; NMR; nuclear magnetic resonance; O; ₂; ⁻; superoxide anion radical; PBN; N; -; tert; -butyl-α-phenylnitrone; PBPO; 5-(propoxycarbonyl)-5-butyl-1-pyrroline-; N; -oxide; SOD; superoxide dismutaseSpin traps; EPR; Lipophilic EMPO derivatives; Superoxide; Linoleic acid hydroperoxide; Free radicals

The novel targets for anti-angiogenesis of genistein on human cancer cells by Shu-Jem Su; Trai-Ming Yeh; Woei-Jer Chuang; Chung-Liang Ho; Kee-Lung Chang; Hsiao-Ling Cheng; Hsiao-Sheng Liu; Hong-Lin Cheng; Pei-Yin Hsu; Nan-Haw Chow (pp. 307-318).

Genistein has been reported to be a natural chemopreventive in several types of human cancer. In our prior study, soy isoflavones were shown to induce cell cycle arrest and apoptosis of bladder cancer cells in the range of human urine excretion. This study was designed to identify the novel molecular basis underlying anti-angiogenic activities of soy isoflavones. An immortalized E6 and five human bladder cancer cell lines were studied by immunoassay, flow cytometry, functional activity, reverse transcription-polymerase chain reaction, immunoblotting, and transwell co-culture in vitro. The efficacy of soy isoflavones on angiogenesis inhibition in vivo was examined by nude mice xenograft and chick chorioallantoic membrane bioassay. Factors analyzed included angiogenic factors, matrix-degrading enzymes, and angiogenesis inhibitors. Genistein was the most potent inhibitor of angiogenesis in vitro and in vivo among the isoflavone compounds tested. It may also account for most of the reduced microvessel density of xenografts observed and the suppressed endothelial migration by soy isoflavones. Genistein exhibited a dose-dependent inhibition of expression/excretion of vascular endothelial growth factor₁₆₅, platelet-derived growth factor, tissue factor, urokinase plasminogen activator, and matrix metalloprotease-2 and 9, respectively. On the other hand, there was an up-regulation of angiogenesis inhibitors—plasminogen activator inhibitor-1, endostatin, angiostatin, and thrombospondin-1. In addition, a differential inhibitory effect between immortalized uroepithelial cells and most cancer cell lines was also observed. Altogether, we discovered that tissue factor, endostatin, and angiostatin are novel molecular targets of genistein. The current investigation provides further evidence in support of soy-based foods as natural dietary inhibitors of tumor angiogenesis.

Keywords: Abbreviations; CAM; chick chorioallantoic membrane; COX-2; cyclooxygenase-2; MMPs; matrix metalloproteinases; PAI-1; plasminogen activator inhibitor-1; PDGF; platelet-derived growth factor; RT-PCR; reverse transcription-polymerase chain reaction; TSP-1; thrombospondin-1; TF; tissue factor; TIMP-2; tissue inhibitor of metalloproteinase-2; tPA; tissue-type plasminogen activator; MT-MMP; transmembrane MMP; uPA; urokinase-type plasminogen; and VEGF; vascular endothelial growth factorBladder cancer; Genistein; Anti-angiognesis; Angiogenic factors; Angiogenesis inhibitors; Matrix-degrading enzymes

Calcium-pH crosstalks in rat mast cells: modulation by transduction signals show non-essential role for calcium in alkaline-induced exocytosis by A. Alfonso; M.R. Vieytes; L.M. Botana (pp. 319-327).

Alkalinization of cytosolic pH with ammonium chloride (NH₄Cl) was reported to be a stimulus for mast cell degranulation. This paper studied the modulatory role of drugs that target protein kinase C (PKC), adenosine 3′,5′-cyclic monophosphate (cAMP), tyrosine kinase (TyrK) and phosphatidylinositol 3-kinase (PI₃K) on this effect. We used Gö6976 (100nM) and low concentrations of GF109203X (Gf) (50nM) to inhibit calcium-dependent PKC isozymes. For calcium-independent isozymes, we used 500nM Gf, and 10μM rottlerin to specifically inhibit PKC δ, and chelerythrine as non-specific PKC inhibitor. Genistein (10μM) and lavendustin A (1μM) were used as unspecific TyrK inhibitors, and 10nM wortmannin as a PI₃K inhibitor. Chelerythrine and 50nM Gf inhibit histamine release in the presence of external calcium. The inhibition caused by wortmannin was strictly internal calcium-dependent. cAMP-active drugs did not modify the response to NH₄Cl. The effect of NH₄Cl on histamine release was triggered by a transient elevation on cytosolic pH, which was simultaneous to an elevation on cytosolic calcium and followed by a probable Ca²⁺–H⁺ exchange after addition of external calcium. EGTA inhibit the response to suboptimal concentrations of NH₄Cl, and BAPTA increased the effect of NH₄Cl. There is a clear relationship between NH₄Cl-mediated calcium release and histamine release, since those drugs that inhibit this release also inhibit NH₄Cl-mediated histamine release; nevertheless, NH₄Cl-mediated histamine release was possible in the absence of any calcium release, as shown with BAPTA. This data, in combination with the results with PKC inhibitors, suggest that calcium is not only unnecessary to trigger cell activation, but also that it may be a negative modulator of NH₄Cl-mediated exocytosis.

Keywords: Abbreviations; PKC; protein kinase C; pHi; cytosolic pH; Gf; GF 109203X; OA; okadaic acid; CT; cholera toxin; PT; pertussis toxin; PI; ₃; K; phosphatitilinositol 3 kinaseCytosolic pH; Cytosolic Ca; ²⁺; Mast cells; Transduction signal; Alkalinization

Quantitative structure–activity analyses of bufokinin and other tachykinins at bufokinin (bNK₁) receptors of the small intestine of the cane toad, Bufo marinus by Lu Liu; Michael Murray; J. Michael Conlon; Elizabeth Burcher (pp. 329-338).

The toad tachykinin, bufokinin (Lys-Pro-Arg-Pro-Asp-Gln-Phe-Tyr-Gly-Leu-Met amide; BUF), acts via tachykinin NK₁-like receptors to contract the intestine of the cane toad, Bufo marinus. In this structure–activity study, we used isolated segments of toad small intestine and performed binding studies with [¹²⁵I] Bolton–Hunter BUF in intestinal membranes to compare the contribution of individual amino acid residues to the potencies of 18 naturally occurring tachykinins and 13 BUF analogs. Potencies were similar ( r=0.94) in functional and binding studies, with BUF and ranakinin being most potent. Ranatachykinin A, physalaemin, hylambatin and cod, trout and mammalian SPs exhibited 10–60% of the potency of BUF. The Ala-substituted BUF analogs were 11–60% as potent as BUF in functional studies, with [Ala²]-BUF and [Ala⁴]-BUF the least efficacious, indicating the importance of both proline residues. QSAR equations were developed using 12 connectivity, shape and steric parameters for each of the 7 hypervariable amino acid residues in these peptides. For the binding data, the optimal regression equation explained 81% of the variance, and indicated the importance of the steric function at [Pro²] and simple connectivity functions at [Gln⁶] and [Tyr⁸]. The optimal functional regression equation (80% of variance) confirmed the importance of connectivity functions at [Gln⁶] and [Tyr⁸], as well as the shape of residues [Lys¹] and [Pro⁴]. The potencies of most full-length peptides were well predicted using the leave-one-out procedure, as were the potencies of a series of model Ala-substituted BUFs, thus emphasising the potential utility of these equations in the design of new ligands interacting with tachykinin receptors.

Keywords: Abbreviations; SP; substance P; NKA; neurokinin A; NKB; neurokinin B; BUF; bufokinin; BH; Bolton–Hunter; QSAR; quantitative structure–activity analysis; RTK; ranatachykininStructure–activity; Tachykinins; Bufokinin; Substance P; Tachykinin receptors; Non-mammalian; Amphibian; Intestine; Binding; Functional; QSAR

Dietary polyphenols protect dopamine neurons from oxidative insults and apoptosis: investigations in primary rat mesencephalic cultures by Linda D. Mercer; Belinda L. Kelly; Malcolm K. Horne; Philip M. Beart (pp. 339-345).

Naturally occurring polyphenols have the potential to prevent oxidative damage in various pathophysiological conditions. Various members of the flavonoid family were investigated to determine if they could protect mesencephalic dopamine (DA) neurones from injury and reduce apoptosis produced by oxidative stressors. Primary mesencephalic cultures were sensitive to oxidative insults (hydrogen peroxide, 4-hydroxynonenal, rotenone, 6-hydroxydopamine and N-methyl-4-phenyl-1,2,3,6-tetrahydropyridinium hydrochloride (MPP⁺)) which produced concentration-dependent decreases in cellular viability across an apoptotic-necrotic continuum of injury. Flavonoids (catechin, quercetin, chrysin, puerarin, naringenin, genestein) protected mesencephalic cultures from injury by MPP⁺, which was shown by DNA fragmentation studies and tyrosine hydroxylase (TH) immunocytochemistry of DA neurones to occur by apoptosis. Catechin also reduced injury produced by hydrogen peroxide, 4-hydroxynonenal, rotenone and 6-hydroxydopamine as shown by increases in cellular viability and [³H]DA uptake. When the neuroprotection of catechin against MPP⁺-induced injury was compared to that produced by the caspase-3 inhibitor, Z-DVED-FMK, both reduced DNA fragmentation and the injury patterns of TH-positive neurones. These data demonstrate the neuroprotective abilities of flavonoids which are able to attenuate the apoptotic injury of mesencephalic DA neurones. Since these DA neurones are under oxidative stress in Parkinsonism, our findings suggest that flavonoids could provide benefits along with other anti-oxidant therapies in Parkinson's disease.

Keywords: Oxidative stress; Flavonoids; Injury; Mesencephalic dopamine neurones; Neuroprotection; Cultures

Eukaryotic arylamine N-acetyltransferase by Akane Kawamura; James Graham; Adeel Mushtaq; Stefanos A. Tsiftsoglou; Gregory M. Vath; Patrick E. Hanna; Carston R. Wagner; Edith Sim (pp. 347-359).

Arylamine N-acetyltransferases (NAT; EC 2.3.1.5) catalyse the transfer of acetyl groups from acetylCoA to xenobiotics, including drugs and carcinogens. The enzyme is found extensively in both eukaryotes and prokaryotes, yet the endogenous roles of NATs are still unclear. In order to study the properties of eukaryotic NATs, high-throughput substrate and inhibitor screens have been developed using pure soluble recombinant Syrian hamster NAT2 (shNAT2) protein. The assay can be used with a wide range of compounds and was used to determine substrate specificity of shNAT2. We describe the expression and characterisation of shNAT2 and also purified recombinant human NAT1 and NAT2, including the use of the assay to explore the substrate specificities of each of the enzymes. Hamster NAT2 has similar substrate specificity to human NAT1, acetylating para-aminobenzoate but not arylhydrazine and hydralazine compounds. The overlapping but distinct substrate-specific activity profiles of human NAT1 and NAT2 were clearly observed from the screen. Naturally occurring compounds were tested as substrates or inhibitors of shNAT2 and succinylCoA was found to be a potent inhibitor of shNAT2.

Keywords: Abbreviations; NAT; arylamine; N; -acetyltransferase; shNAT2; Syrian hamster NAT2; INH; isoniazid; HDZ; hydralazine; PABA; para; -aminobenzoate; 4AS; 4-aminosalicylate; 5AS; 5-aminosalicylate; pABGlu; para; -aminobenzylglutamate; STNAT; Salmonella typhimurium; NAT; MSNAT; Mycobacterium smegmatis; NAT; DTNB; 5,5′-dithio-bis(2-nitrobenzoic acid); DLS; dynamic light scattering; IAM; iodoacetamide; CD; circular dichroism; DTT; dithiothreitolArylamine; N; -acetyltransferase; Xenobiotic metabolism; Recombinant protein; High-throughput screening; Arylamines; AcetylCoA/CoA derivatives

Corrigendum to “Geraniol and β-ionone inhibit proliferation, cell cycle progression, and cyclin-dependent kinase 2 activity in MCF-7 breast cancer cells independent of effects on HMG-CoA reductase activity�? [Biochem. Pharmacol. 68 (2004) 1739–1747] by Robin E. Duncan; Dominic Lau; Ahmed El-Sohemy; Michael C. Archer (pp. 361-361).

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Biochemical Pharmacology (v.69, #2)