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Biochemical Pharmacology (v.69, #6)

Editorial Advisory Board (pp. iii).

Inhibitory effects on human eosinophil chemotaxis in vitro by BAY 41-2272, an activator of nitric oxide-independent site of soluble guanylate cyclase by Sara M. Thomazzi; Juliana Moreira; Gilberto De Nucci; Edson Antunes (pp. 875-882).

This study was designed to investigate the effects of the 5-cyclopropyl-2-[1-(2-fluoro-benzyl)-1 H-pyrazolo[3,4- b]pyridin-3-yl]-pyrimidin-4-ylamine (BAY 41-2272) on formyl-methionyl-leucyl-phenylalanine (fMLP; 10⁻⁷M)-induced human eosinophil chemotaxis, cyclic guanosine-3′,5′-monophosphate (cGMP) and cyclic adenosine-3′,5′-monophosphate (cAMP) levels. Human eosinophils were pretreated or not with 3-isobutyl-l-methyl-xanthine (IBMX; 500μM), and then exposed to BAY 41-2272 (0.1–10.0μM) for either short (10min) or prolonged (90min) time periods. Exposition of eosinophils with BAY 41-2272 for either 10min or 90min markedly inhibited the eosinophil chemotaxis, independently of IBMX pretreatment. Inhibition of fMLP-induced eosinophil chemotaxis by BAY 41-2272 (in absence of prior treatment with IBMX) was about of the same irrespective if cells were exposed for 10min or 90min with this compound. In IBMX-pretreated eosinophils, the inhibition of fMLP-induced chemotaxis by BAY 41-2272 in the 10-min exposure protocols was even higher in comparison with the 90-min protocols. Incubation of IBMX-treated eosinophils for 90min with BAY 41-2272 resulted in 2.0–2.5 times higher levels of cGMP and cAMP compared with the 10-min protocols. The BAY 41-2272-induced cGMP increases were abolished by pre-incubation of eosinophils with the soluble guanylate cyclase inhibitor 1 H-[1,2,4]-oxidiazolo[4,3- a] quinoxalin-1-one (ODQ). No eosinophil toxicity was observed in any experimental condition, according to 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide (MTT) assay. Our findings show that inhibitory effects of fMLP-induced human eosinophil chemotaxis by BAY 41-2272 at short-term or prolonged exposition time are accompanied by significant elevations of cGMP and cAMP, but we could not detect a clear correlation between chemotaxis inhibition and elevation of cyclic nucleotide levels.

Keywords: Abbreviations; BAY 41-2272; 5-cyclopropyl-2-[1-(2-fluoro-benzyl)-1; H; -pyrazolo[3,4-; b; ]pyridin-3-yl]-pyrimidin-4-ylamine; cAMP; cyclic adenosine-3′,5′-monophosphate; cGMP; cyclic guanosine-3′,5′-monophosphate; fMLP; formyl-methionyl-leucyl-phenylalanine; IBMX; 3-isobutyl-l-methyl-xanthine; MEM; minimum essential medium Eagle; MTT; 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide; NO; nitric oxide; ODQ; 1; H; -[1,2,4]-oxidiazolo[4,3-; a; ] quinoxalin-1-one; PDE; phosphodiesterase; sGC; soluble guanylate cyclase; SIN-1; 3-morpholinosydnonimine; SNAP; S; -nitroso-; N; -acetylpenicillamine; SNP; sodium nitroprusside; YC-1; 3-(5′-hydroxymethyl-2-furyl)-1-benzyl-indazoleBAY 41-2272; cAMP levels; cGMP levels; Chemotaxis; Cytotoxicity; Eosinophil

The role of adenosine A_2A and A_2B receptors in the regulation of TNF-α production by human monocytes by Jian G. Zhang; Lucy Hepburn; Gabriela Cruz; Richard A. Borman; Kenneth L. Clark (pp. 883-889).

Adenosine is an endogenous nucleoside that regulates many physiological processes through the activation of its four receptors: A₁, A_2A, A_2B and A₃. Previous studies have identified the involvement of A₂ receptors in the inhibitory activity of adenosine analogues on tumor necrosis factor-α (TNF-α) production by lipopolysaccharide (LPS) activated monocytes, but the relative contributions of A_2A versus A_2B receptors have not been determined in human primary monocytes. Nor has the role of A₁ and A₃ been clearly identified in the system. The lack of such information impacts on the selection of adenosine receptor agonists for disease intervention. Using LPS-stimulated human primary monocytes, we found that the adenosine receptor agonist, 5′- N-ethylcarboxamidoadenosine (NECA) or the A_2A receptor agonist, 4-[2-[[6-amino-9-( N-ethyl- b-d-ribofuranuronamidosyl)-9 H-purin-2-yl]amino]ethyl]benzenepropanoic acid hydrochloride (CGS21680) produced a concentration-dependent inhibition of TNF-α production, with IC₅₀s of 58.4nM (32.7–104.5nM, 95% confidence interval) and 49.2nM (22.7–105.9nM, 95% confidence interval), respectively. The selective A_2A receptor blocker, 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylaminso]ethyl)phenol (ZM241385, 30nM), antagonized the effects of NECA and CGS21680 ( pK_B estimates were 8.7±0.1 and 8.9±0.1, respectively), while the selective A_2B antagonist, N-(4-cyano-phenyl)-2-[4-(2,6-dioxo-1,3-dipropyl-2,3,4,5,6,7-hexahydro-1H-purin-8-yl)-phenoxy]-acetamide (MRS1754, 100nM), failed to antagonize the effects of either agonist. Furthermore, neither the A₁ receptor agonist, 2-chloro- N⁶-cyclopentyladenosine (CCPA) nor the A₃ receptor agonist, 1-[2-chloro-6-[[(3-iodophenyl)methyl]amino]-9H-purin-9-yl]-1-deoxy- N-methyl- b-d-ribofuranuronamide (2-Cl-IB-MECA) showed significant inhibitory activity at concentrations that effectively bind to their respective receptors. We conclude that A_2A receptor activation is predominantly responsible for the inhibitory effects of adenosine receptor agonists on TNF-α production from LPS-stimulated monocytes.

Keywords: Abbreviations; 2-Cl-IB-MECA; 1-[2-chloro-6-[[(3-iodophenyl)methyl]amino]-9H-purin-9-yl]-1-deoxy-; N; -methyl-b-; d; -ribofuranuronamide; CCPA; 2-chloro-; N; ⁶; -cyclopentyladenosine; CGS21680; 4-[2-[[6-Amino-9-(; N; -ethyl-b-; d; -ribofuranuronamidosyl)-9H-purin-2-yl]amino]ethyl]benzenepropanoic acid hydrochloride; LPS; Lipopolysaccharide; MRS1754; N; -(4-cyano-phenyl)-2-[4-(2,6-dioxo-1,3-dipropyl-2,3,4,5,6,7-hexahydro-1H-purin-8-yl)-phenoxy]-acetamide; NECA; 5′-; N; -ethylcarboxamidoadenosine; TNF-α; Tumor necrosis factor-α; ZM241385; 4-(2-[7-Amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenolAdenosine receptor; A; ₁; A; _2A; A; _2B; A; ₃; LPS

Sodium nitroprusside-induced osteoblast apoptosis is mediated by long chain ceramide and is decreased by raloxifene by Sabine Olivier; Marianne Fillet; Michel Malaise; Jacques Piette; Vincent Bours; Marie-Paule Merville; Nathalie Franchimont (pp. 891-901).

Release of high levels of nitric oxide (NO) is associated with osteoblastic cell death. The mechanisms of NO-induced cytotoxicity are not well documented and it is presently not known if estrogenic compounds prevent this effect. We studied the role of ceramides in cell death induced by the NO donor sodium nitroprusside (SNP) and we tested the possibility that 17β-estradiol, the anti-estrogen ICI 182.780 and two selective estrogen receptor modulators raloxifene and tamoxifen modify osteoblastic cell apoptosis. SNP dose-dependently decreased MC3T3-E1 osteoblast viability, increased NO production in the culture media and enhanced the release of intracellular ceramides C22 and C24. Cell death induced by SNP was partially inhibited when MC3T3-E1 cells were pretreated with raloxifene and tamoxifen but was not modified when the cells were pretreated with 17β-estradiol or ICI 182.780. Cell death induced by SNP resulted from apoptosis as demonstrated by Annexin-V and propidium iodide labeling and a reduction of SNP-induced MC3T3-E1 apoptosis was confirmed in the presence of raloxifene and tamoxifen. SNP induction of C22 and C24 production was inhibited by a pretreatment with raloxifene but not with 17β-estradiol. Moreover, the synthetic ceramide C24 (0.75 and 1μM) decreased MC3T3-E1 cell viability and osteoblast cell death induced by C24 was partially decreased by raloxifene and to a lesser extent by 17β-estradiol. These data demonstrate that SNP-induced cell death is mediated by the long chain ceramides C22 and C24 and that raloxifene protected osteoblast from apoptosis induced by SNP, an effect that might be relevant to its pharmacological properties on bone remodeling.

Keywords: Nitric oxide; Ceramides; Apoptosis; Raloxifene; Estrogen; Osteoblast

Antioxidant, prooxidant and cytotoxic activity of hydroxylated resveratrol analogues: structure–activity relationship by Marek Murias; Walter J�ger; Norbert Handler; Thomas Erker; Zsuzsanna Horvath; Thomas Szekeres; Hans Nohl; Lars Gille (pp. 903-912).

Resveratrol ( trans-3,4′,5-trihydroxystilbene), a naturally occurring hydroxystilbene, is considered an essential antioxidative constituent of red wine possessing chemopreventive properties. However, resveratrol and even more its metabolite piceatannol were reported to have also cytostatic activities. In order to find out whether this is related to antioxidative properties of those compounds, we synthesized five other polyhydroxylated resveratrol analogues and studied structure–activity relationships between pro-/antioxidant properties and cytotoxicity. Radical scavenging experiments with O₂⁻ (5,5-dimethyl-1-pyrroline- N-oxide/electron spin resonance (DMPO/ESR)) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) (photometry) revealed that 3,3′,4′,5-tetrahydroxystilbene (IC₅₀: 2.69μM; k₉: 443000M⁻¹s⁻¹), 3,4,4′,5-tetrahydroxystilbene (IC₅₀: 41.5μM; k₉: 882000M⁻¹s⁻¹) and 3,3′,4,4′,5,5′-hexahydroxystilbene (IC₅₀: 5.02μM), exerted a more than 6600-fold higher antiradical activity than resveratrol and its two other analogues. Furthermore, in HL-60 leukemic cells hydroxystilbenes with ortho-hydroxyl groups exhibited a more than three-fold higher cytostatic activity compared to hydroxystilbenes with other substitution patterns. Oxidation of ortho-hydroxystilbenes in a microsomal model system resulted in the existence of ortho-semiquinones, which were observed by ESR spectroscopy. Further experiments revealed that these intermediates undergo redox-cycling thereby consuming additional oxygen and forming cytotoxic oxygen radicals. In contrast to compounds with other substitution patterns hydroxystilbenes with one or two resorcinol groups (compounds1 and3) did not show an additional oxygen consumption or semiquinone formation. These findings suggest that the increased cytotoxicity of ortho-hydroxystilbenes is related to the presence of ortho-semiquinones formed during metabolism or autoxidation.

Keywords: Abbreviations; COX; cyclooxygenase; DMPO; 5,5-dimethyl-1-pyrroline-; N; -oxide; DPPH; 2,2-diphenyl-1-picrylhydrazyl; DTPA; diethylenetriaminepentaacetic acid; ESR; electron spin resonance; LPO; lipid peroxidation; O; ₂; ⁻; superoxide radical; PARP; poly(ADP-ribose) polymerase; ROS; reactive oxygen species; HO–Stilb–OH; hydroxystilbene; HO–Stilb–O; ⁻; deprotonated hydroxystilbene; [HO–Stilb–O–H–O; ₂; ⁻; ]; ^≠; adduct of hydroxystilbene with O; ₂; ⁻; HO–Stilb–O; phenoxyl radical; O=Stilb=O; two-electron oxidation product of hydroxystilbenes; ⁻; O–Stilb–O; semiquinone anion; SOD; superoxide dismutaseResveratrol; Hydroxystilbenes; Antioxidants; Prooxidants; Cytotoxicity; HL-60 cells

Mitochondrial-dependent, reactive oxygen species-independent apoptosis by myricetin: roles of protein kinase C, cytochrome c, and caspase cascade by Ching Huai Ko; Shing-Chuan Shen; Chun-Sen Hsu; Yen-Chou Chen (pp. 913-927).

Abrogation of mitochondrial permeability and induction of reactive oxygen species (ROS) production have been observed in chemical-induced apoptosis; however, the relationship between the mitochondria and intracellular ROS levels in apoptosis is still unclear. In the present study, myricetin (ME) but not its respective glycoside, myricitrin (MI; myricetin-3- O-rhamnose) reduced the viability of human leukemia HL-60 cells via apoptosis, characterized by the occurrence of DNA ladders and hypodiploid cells. Results of Western blotting and caspase activity assays showed that activation of caspases 3 and 9 but not caspases 1, 6 or 8 with cleavage of PARP and D4-GDI proteins is involved in ME-induced apoptosis. A reduction in mitochondrial functions characterized by a decrease in the Bcl-2/Bax protein ratio and translocation of cytochrome c (cyt c) from the mitochondria to the cytosol in accordance with a decrease in mitochondrial membrane potential were observed in ME-treated HL-60 cells. No significant induction of intracellular ROS levels by ME was observed by the DCHF-DA assay, DPPH assay or plasmid digestion assay, and antioxidants including N-acetyl-cysteine (NAC), catalase (CAT), superoxide dismutase (SOD), and tiron (TIR) showed no protective effects on ME-induced apoptosis. A PKC activator, 12- O-tetradecaoylphorbol-13-acetate (TPA) significantly attenuated ME-induced apoptosis via preventing cytochrome c release to the cytosol and maintaining the mitochondrial membrane potential by inhibiting the decrease in the Bcl-2/Bax protein ratio; these effects were blocked by protein kinase C (PKC) inhibitors including GF-109203X, H7, and staurosporin. Removing mitochondria by ethidium bromide (EtBr) treatment reduced the apoptotic effect of ME. Results of SAR studies showed that the presence of OH at C3′, C4′, and C5′ is important for the apoptosis-inducing activities of ME, and that ME induces apoptosis in another leukemia cell line, Jurkat cells, but not in primary human polymorphonuclear (PMN) cells or in murine peritoneal macrophages (PMs). The results of the present study suggest that apoptosis induced by ME occurs through a novel mitochondrion-dependent, ROS-independent pathway; TPA protects cells from ME-induced apoptosis via PKC activation which prevents the occurrence of mitochondrial destruction during apoptosis.

Keywords: Abbreviations; BCIP; 5-bromo-4-chloro-3-indolyl-phosphate; Bcl-2; B-cell lymphoma-2; CAT; catalase; cyt c; cytochrome c; DCHF-DA; dichlorodihydrofluorescein diacetate; GF; GF-109203X; H-7; isoquinoline-5-sulfonic 2-methyl-1-piperazide; ME; myricetin; MI; myricitrin; NAC; N; -acetyl-cysteine; NBT; nitro blue tetrazolium; PKC; protein kinase C; PMN cells; polymorphonuclear cells; PMs; murine peritoneal macrophages; ROS; reactive oxygen species; SOD; superoxide dismutase; ST; staurosporine; TIR; tiron; TPA; 12-; O; -Tetradecaoylphorbol-13-acetateMyricetin; Mitochondria; Reactive oxygen species; TPA; Cytochrome c

Suppression of Ca²⁺ influx by unfractionated heparin in non-excitable intact cells via multiple mechanisms by Kl�ra N�meth; Istv�n Kurucz (pp. 929-940).

Effect of unfractionated heparin (UFH), described as a cell-impermeant IP3 receptor antagonist, was studied on the capacitive Ca²⁺ entry in non-permeabilized, intact cells, measuring the intracellular Ca²⁺ levels using fluorescence microplate technique. Ca²⁺ influx induced via Ca²⁺ mobilization by histamine in Hela cells or evoked by store depletion with thapsigargin in RBL-2H3 cells was dose-dependently suppressed by UFH added either before or after the stimuli. UFH also prevented the spontaneous Ba²⁺ entry indicating that the non-capacitive Ca²⁺ channels may also be affected. In addition, UFH caused a significant and dose-dependent delay in Ca²⁺, and other bivalent cation inflow after treatment of the cells with Triton X-100, but it did not diminish the amount of these cations indicating that UFH did not act simply as a cation chelator, but modulated the capacitive Ca²⁺ entry possibly via store operated Ca²⁺ channels (SOCCs). Inhibitory activities of UFH and 2-aminoethyl diphenyl borate on the capacitive Ca²⁺ influx was found reversible, but the time courses of their actions were dissimilar suggesting distinct modes of action. It was also demonstrated using a fluorescence potentiometric dye that UFH had a considerable hyperpolarizing effect and could alter the changes of membrane potential during Ca²⁺ influx after store depletion by thapsigargin. We presume that the hyperpolarizing property of this agent might contribute to the suppression of Ca²⁺ influx. We concluded that UFH can negatively modulate SOCCs and also other non-capacitive Ca²⁺ channels and these activities might also account for its multiple biological effects.

Keywords: Abbreviations; 2-APB; 2-aminoethyl-diphenyl-borate; CRAC; Ca; ²⁺; release activated Ca; ²⁺; channel; IP3; inositol-triphosphate; SOCC; store operated Ca; ²⁺; channel; TG; thapsigargin; TRP proteins; transient receptor potential proteins; UFH; unfractionated heparinUnfractionated heparin; Intracellular Ca; ²⁺; Store operated calcium channels; Membrane potential; 2-Aminoethyl diphenyl borate; Bivalent cation entry

Involvement of SULT1A3 in elevated sulfation of 4-hydroxypropranolol in Hep G2 cells pretreated with β-naphthoflavone by Junko Miyano; Shigeo Yamamoto; Nobumitsu Hanioka; Shizuo Narimatsu; Tsutomu Ishikawa; Kenichiro Ogura; Tadashi Watabe; Masuhiro Nishimura; Nobuhiko Ueda; Shinsaku Naito (pp. 941-950).

Pretreatment of Hep G2 cells with β-naphthoflavone (BNF 1–25μM) significantly increased cytosolic sulfation activities of 4-hydroxypropranolol (4-OH-PL) racemate. The profile was similar to those of sulfations towards dopamine and triiodothyronine in the same cytosolic fractions. Kinetic studies of 4-OH-PL sulfation in Hep G2 cytosolic fractions revealed that V_max values increased but apparent K_m values remained unchanged following the BNF pretreatment. Among five recombinant human SULT isoforms (SULT1A1, -1A3, -1B1, -1E1 and -2A1) examined, only SULT2A1 did not show 4-OH-PL sulfation activities under the conditions used. SULT1A3 and -1E1 exhibited an enantioselectivity of 4-OH- R-PL sulfation>4-OH- S-PL sulfation, which agreed with that of BNF-pretreated Hep G2 cells as well as of nontreated cells, whereas SULT1A1 and -1B1 showed a reversed enantioselectivity ( R< S). In kinetic studies of 4-OH-PL sulfations by four kinds of human SULT isoforms, apparent K_m values for SULT1A3 were the lowest, and the parameters were close to those of Hep G2 cytosolic fractions. Real time RT-PCR using TaqMan probes demonstrated that the mRNA levels of SULT1A3 increased following BNF pretreatment, which paralleled the results from Western blotting showing the elevated levels of SULT1A3 proteins. These results suggest that the induction of SULT1A3 is mainly responsible for the elevated 4-OH-PL sulfation activities following the pretreatment of Hep G2 cells with BNF.

Keywords: Abbreviations; BNF; β-naphthoflavone; CYP; cytochrome P450; 4-OH-BTL; 4-hydroxybunitrolol; DHEA; dehydroepiandrosterone; 4-OH-PL; 4-hydroxypropranolol; IPTG; isopropyl-β-d-thiogalactopyranoside; NDP; N; -desisopropyl propranolol; PL; propranolol; PVDF; polyvinylidene difluoride; SDS-PAGE; sodium dodecyl sulfate-polyacrylamide gel electrophoresisSULT1A3; Hep G2 cell line; 4-Hydroxypropranolol; β-Naphthoflavone

Activity profiles of deoxynucleoside kinases and 5′-nucleotidases in cultured adipocytes and myoblastic cells: insights into mitochondrial toxicity of nucleoside analogs by Svetlana N. Rylova; Freidoun Albertioni; Gunnar Flygh; Staffan Eriksson (pp. 951-960).

Nucleoside reverse transcriptase inhibitor (NRTI) treatment of HIV is associated with complications, including lipodystrophy (LD) and myopathy. Inhibition of mitochondrial DNA polymerase and depletion of mtDNA by NRTI triphosphates are believed to be key mechanisms in NRTI toxicity. Here, we determined the activities and mRNA levels of deoxynucleoside kinases (dNK) and 5′-nucleotidases (5′-NT) controlling the rate-limiting step in intracellular phosphorylation of NRTIs in cell models representing adipose, muscle tissue and peripheral blood cells using specific assays and Taqman RT-PCR. In vitro phosphorylation of 3′-azido-2′,3′-dideoxythymidine (AZT) and 2′,3′-didehydro-2′,3′-dideoxythymidine (d4T) in extracts was also determined. 3T3-L1 adipocytes showed similar activity of mitochondrial thymidine kinase-2 (TK2) and deoxyguanosine kinase (dGK) but 3- to 36-fold lower levels of cytosolic deoxycytidine kinase (dCK), thymidine kinase-1 (TK1) and thymidine monophosphate kinase (TMPK) and higher levels of deoxyribonucleotidase activity compared to proliferating 3T3-L1. dCK, dGK and TK2 activities correlated with their mRNA levels in proliferating, resting and differentiating 3T3-L1. Differentiated L6 myoblasts had lower activities of cytosolic dNK's and TMPK, higher dGK and similar TK2 and deoxyribonucleotidases (dNT) activities compared to proliferating myoblasts. TK2 was the limiting dNK activity while dGK was predominant in adipocytes and myocytes. Activity profiles revealed limited capacity to phosphorylate dThd and dCyd in adipocytes and myocytes compared to proliferating cells and CEM lymphocytes. Phosphorylation of AZT and d4T was low in adipocytes and myocytes, and the presence of these analogs inhibited the phosphorylation of dThd by TK2 suggesting that mitochondrial toxicity of some NRTIs in adipocytes and myocytes is due to the depletion of normal mitochondrial dNTP pools.

Keywords: Abbreviations; AZT; 3′-azido-2′,3′-dideoxythymidine; cN-1b; cytosolic 5′-nucleotidase 1b; cN-2; cytosolic 5′-nucleotidase; dCK; deoxycytidine kinase; dGK; deoxyguanosine kinase; DMEM; Dubelcco's modified Eagle's medium; DNC; deoxynucleotide carrier; dNK; deoxynucleoside kinase; d4T; 2′,3′-didehydro-2′,3′-dideoxythymidine; dNT-1; deoxynucleotidase 1; HIV; human immunodeficiency virus; LD; lipodystrophy; mtDNA; mitochondrial DNA; NRTI; nucleoside reverse transcriptase inhibitor; NRTI-DP; NRTI diphosphate; NRTI-MP; NRTI monophosphate; NRTI-TP; NRTI triphosphate; 5′-NT; 5′-nucleotidases; TK1; thymidine kinase 1; TK2; thymidine kinase 2; TMPK; thymidine monophosphate kinaseNRTI; Mitochondrial toxicity; Adipocytes; Myoblasts; Deoxynucleoside kinases; 5′-Nucleotidases

Mechanism for the inhibition of transglutaminase 2 by cystamine by Thomas M. Jeitner; E. James Delikatny; Jenny Ahlqvist; Hugh Capper; Arthur J.L. Cooper (pp. 961-970).

Cystamine is neuroprotective in a number of models of neurodegeneration. The therapeutic benefit of cystamine has been attributed, in part, to its inhibition of transglutaminase activity. Cystamine [β-mercaptoethanolamine (MEA) disulfide] is reduced within cells to MEA which is largely responsible for the in vivo effects of its disulfide precursor. In the current study, the amine group of MEA was shown to act as a transglutaminase (TG) substrate resulting in the formation of N^β-(γ-l-glutamyl)-MEA bonds. The formation of such bonds would compete with the generation of other TG-catalyzed reactions that may contribute to neurodegeneration such as polyamination, protein cross-linking, deamination and the covalent attachment of ceramide to proteins. The demonstration that cystamine-derived MEA can form N^β-(γ-l-glutamyl)-MEA bonds offers a unique tool for identifying the TG substrates that occur in diseased brains in vivo. Structure-function studies also indicated that the mercapto group of MEA significantly influences the substrate behavior of this compound. These structure-function studies also identified the following hierarchy of physico-chemical characteristics: hydrophobicity > S as the group VIII atom > distance separating the N and group VIII atom, as the major determinants contributing to the substrate behavior for low-molecular weight amine substrates of TG 2.

Keywords: Abbreviations; DMC; N,N; -dimethylcasein; DTT; dithiothreitol; GSH; glutathione; GSSG; glutathione disulfide; MEA; β-mercaptoethanolamine; TG; transglutaminase

l-Arginine regulates neuronal nitric oxide synthase production of superoxide and hydrogen peroxide by Pei Tsai; John Weaver; Guan Liang Cao; Sovitj Pou; Linda J. Roman; Anatoly A. Starkov; Gerald M. Rosen (pp. 971-979).

Tetrahydrobiopterin (H₄B) in the absence ofl-arginine has been shown to be an important factor in promoting the direct formation of hydrogen peroxide (H₂O₂) at the expense of superoxide (O₂⁻) by neuronal nitric oxide synthase (NOS1) [Rosen GM, Tsai P, Weaver J, Porasuphatana S, Roman LJ, Starkov AA, et al. Role of tetrahydrobiopterin in the regulation of neuronal nitric-oxide synthase-generated superoxide. J Biol Chem 2002;277:40275–80]. Based on these findings, it is hypothesized thatl-arginine also shifts the equilibrium between O₂⁻ and H₂O₂. Experiments were designed to test this theory. As the concentration ofl-arginine and N^ω-hydroxyl-l-arginine increases, the rate of NADPH consumption for H₄B-bound NOS1 decreased resulting in lower rates of both O₂⁻ and H₂O₂ generation, while increasing the rate of nitric oxide (NO) production. At saturating concentrations ofl-arginine or N^ω-hydroxyl-l-arginine (50μM), NOS1 still produced O₂⁻ and H₂O₂. Bothl-arginine and N^ω-hydroxyl-l-arginine have greater impact on the rate of generation of O₂⁻ than on H₂O₂.

Keywords: Abbreviations; Amplex Red; 10-acetyl-3,7-dihydroxyphenoxazine; BMPO; 5-; tert; -butoxycarbonyl-5-methyl-1-pyrroline; N; -oxide; CaCl; ₂; calcium chloride; HbO; ₂; oxyhemoglobin; H; ₄; B; (6R)-5,6,7,8-tetrahydro-L-biopterin; HRP; horseradish peroxidase; H; ₂; O; ₂; hydrogen peroxide; NOS1; neuronal nitric oxide synthase; NO; nitric oxide; O; ₂; ⁻; superoxide; SOD; superoxide dismutase; SPER-NO; (Z)-1-{; N; -(3-aminopropyl)-; N; -[4-(3-amino propylammonio)- butyl]amino}diazen-1-ium-1,2-diolatel; -Arginine; N; ^ω; -Hydroxyl-; l; -arginine; superoxide; Nitric oxide; Hydrogen peroxide; Neuronal nitric oxide synthase

Biodistribution and metabolism of immunostimulatory oligodeoxynucleotide CPG 7909 in mouse and rat tissues following subcutaneous administration by Bernhard O. Noll; Michael J. McCluskie; Tanja Sniatala; Angela Lohner; Stephanie Yuill; Arthur M. Krieg; Christian Schetter; Heather L. Davis; Eugen Uhlmann (pp. 981-991).

To evaluate pharmacokinetics (PK) and biodistribution, CPG 7909, a 24-mer immunostimulatory fully phosphorothioated oligodeoxynucleotide (PS-ODN), was administered by subcutaneous injection at 2, 5 and 12.5mg/kg to mice and at 9mg/kg to rats. Parent compound and metabolites were isolated from plasma and tissues and quantified by capillary gel electrophoresis with UV detection (CGE-UV) and molecular masses were determined by matrix-assisted-laser-desorption-ionization time of flight detection (MALDI-TOF). An established method for PS-ODN isolation from plasma and tissue was modified to prevent oxidation of the phosphorothioate bonds during the extraction process, significantly increasing sensitivity in the subsequent MALDI-TOF analysis. Concentrations of CPG 7909 and metabolites were highest at the injection site (>600mg/kg at 4h). Maximal concentrations in local (draining) lymph nodes (LLN), kidney and liver were 10–15% of that at the injection site. The highest total amount of PS-ODN (percentage of administered dose) was found in the liver (32% at 4h), followed closely by the injection site (23% at 4h). Only very low levels of CPG 7909 and metabolites were found in plasma and only during the first hours. Metabolites identified by MALDI-TOF were similar for both species and all analyzed tissues, although the relative amounts of the different metabolites varied with tissue and over time. Degradation of CPG 7909 in vivo occurred predominantly by 3′exonucleases with additional cleavage by endonucleases.

Keywords: Abbreviations; ODN; oligodeoxynucleotide; PS-ODN; fully phosphorothioated oligodeoxynucleotide; PK; pharmacokinetics; IV; intravenous; SC; subcutaneous; CGE-UV/LIF; capillary gel electrophoresis with UV or laser induced fluorescence detection; MALDI-TOF; matrix-assisted-laser-desorption-ionization time of flight detection; SPE; solid phase extraction; HPLC/ESI-MS; high-performance-liquid-chromatography/electro-spray-ionization-mass-spectrometry; LLN; local (draining) lymph nodes; DTT; 1,4-dithio-; dl; -threitolOligonucleotide; Metabolites; Pharmacokinetics; Capillary gel electrophoresis; MALDI-TOF; Solid phase extraction

Restored expression and activity of organic ion transporters rOAT1, rOAT3 and rOCT2 after hyperuricemia in the rat kidney by Yasushi Habu; Ikuko Yano; Masahiro Okuda; Atsushi Fukatsu; Ken-ichi Inui (pp. 993-999).

We previously reported that in hyperuricemic rats, renal impairment occurred and organic ion transport activity decreased, accompanied with a specific decrease in the expression of rat organic anion transporters, rOAT1 and rOAT3, and organic cation transporter, rOCT2. In the present study, we investigated the reversibility of the organic ion transport activity and expression of organic ion transporters (slc22a) during recovery from hyperuricemia. Hyperuricemia was induced by the administration of a chow containing uric acid and oxonic acid, an inhibitor of uric acid metabolism. Four days after discontinuance of the chow, the plasma uric acid concentration returned to the normal level, and renal functions such as creatinine clearance and BUN levels were restored, although the recovery of tubulointerstitial injury was varied in sites of the kidney. Basolateral uptake of p-aminohippurate (PAH) and tetraethylammonium (TEA), and both protein and mRNA levels of rOAT1, rOAT3 and rOCT2 in the kidney gradually improved during 14 days of recovery from hyperuricemia. Basolateral PAH transport showed a higher correlation with the protein level of rOAT1 ( r²=0.80) than rOAT3 ( r²=0.34), whereas basolateral TEA transport showed a strong correlation with rOCT2 protein ( r²=0.91). The plasma testosterone concentration, which is a dominant factor in the regulation of rOCT2, was gradually restored during the recovery from hyperuricemia, but the correlation between the plasma testosterone level and rOCT2 protein expression in the kidney was not significant. These results suggest that the regulation of organic ion transporters, rOAT1, rOAT3 and rOCT2, by hyperuricemia is reversible, and the organic ion transport activity restores according to the expression levels of these transporters.

Keywords: Abbreviations; PAH; p; -aminohippurate; TEA; tetraethylammoniumRenal transport; Organic anion transporter; Organic cation transporter; slc22a; Rats; Hyperuricemia

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Biochemical Pharmacology (v.69, #6)

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Biochemical Pharmacology (v.69, #6)