The 21-aminosteroid U74389G prevents the down-regulation and decrease in activity of CYP1A1, 1A2 and 3A6 induced by an inflammatory reaction

In vivo, the 21-aminosteroid U74389G prevents the decrease in cytochrome P450 (P450) activity produced by a turpentine-induced inflammatory reaction (TIIR). To investigate the underlying mechanism of action, four groups of rabbits were used, controls receiving or not U74389G, and rabbits with the inflammatory reaction receiving or not U74389G. Hepatocytes were isolated 48h later and incubated for 4 and 24h with the serum of the rabbits. In vivo, the TIIR diminished CYP1A1/2 and 3A6 expression, and enhanced hepatic malondialdehyde (MDA) and nitric oxide (NO*) concentrations (p<0.05). U74389G prevented the increase in MDA, as well as the decrease in CYP1A1/2 amounts and activity, but increased CYP3A6 expression by 40% (p<0.05). In vitro, compared with serum from control rabbits (S(CONT)), incubation of serum from rabbits with TIIR (S(TIIR)) for 4 and 24h with hepatocytes from rabbits with TIIR (H(TIIR)) reduced CYP1A2 and CYP3A6 activity (p<0.05) and increased the formation of NO* and MDA. In rabbits with TIIR pretreated with U74389G, the S(TIIR+U) failed to reduce CYP1A2 activity or to increase MDA, although increased NO* and further reduced CYP3A6 activity. On the other hand, in hepatocytes harvested from rabbits with TIIR pretreated with U74389G, S(TIIR) did not decrease CYP1A2 activity and did not enhance MDA, but still increased NO*. In vitro, the reduction of CYP1A2 and CYP3A6 activity by S(TIIR) is not associated to NF-kappaB activation. In conclusion, U74389G prevents CYP1A1/2 down-regulation and decrease in activity by a double mechanism: hindering the release of serum mediators and by averting intracellular events, effect possibly associated with its antioxidant activity. On the other hand, U74389G up-regulates CYP3A6 but inhibits its catalytic activity.     

Metabolism of 26,26,26,27,27,27-F6-1alpha,25-dihydroxyvitamin D3 by CYP24: species-based difference between humans and rats

The compound 26,26,26,27,27,27-F(6)-1alpha,25(OH)(2)D(3) is a hexafluorinated analog of the active form of Vitamin D(3). The enhanced biological activity of F(6)-1alpha,25(OH)(2)D(3) is considered to be related to a decreased metabolic inactivation of the compound in target tissues such as the kidneys, small intestine, and bones. Our previous study demonstrated that CYP24 is responsible for the metabolism of F(6)-1alpha,25(OH)(2)D(3) in the target tissues. In this study, we compared the human and rat CYP24-dependent metabolism of F(6)-1alpha,25(OH)(2)D(3) by using the Escherichia coli expression system. In the recombinant E. coli cells expressing human CYP24, bovine adrenodoxin and NADPH-adrenodoxin reductase, F(6)-1alpha,25(OH)(2)D(3) was successively converted to F(6)-1alpha,23S,25(OH)(3)D(3), F(6)-23-oxo-1alpha,25(OH)(2)D(3), and the putative ether compound with the same molecular mass as F(6)-1alpha,25(OH)(2)D(3). The putative ether was not observed in the recombinant E. coli cells expressing rat CYP24. These results indicate species-based difference between human and rat CYP24 in the metabolism of F(6)-1alpha,25(OH)(2)D(3). In addition, the metabolite with a cleavage at the C(24)z.sbnd;C(25) bond of F(6)-1alpha,25(OH)(2)D(3) was detected as a minor metabolite in both human and rat CYP24. Although F(6)-1alpha,23S,25(OH)(3)D(3) and F(6)-23-oxo-1alpha,25(OH)(2)D(3) had a high affinity for Vitamin D receptor, the side-chain cleaved metabolite and the putative ether showed extremely low affinity for Vitamin D receptor. These findings indicate that human CYP24 has a dual pathway for metabolic inactivation of F(6)-1alpha,25(OH)(2)D(3) while rat CYP24 has only one pathway. Judging from the fact that metabolism of F(6)-1alpha,25(OH)(2)D(3) in rat CYP24-harboring E. coli cells is quite similar to that in the target tissues of rat, the metabolism seen in human CYP24-harboring E. coli cells appear to exhibit the same metabolism as in human target tissues. Thus, this recombinant system harboring human CYP24 appears quite useful for predicting the metabolism and efficacy of Vitamin D analogs in human target tissues before clinical trials.     

Associations of ABCB1, NFKB1, CYP3A,and NR1I2 polymorphisms with cyclosporine trough concentrations in Chinese renal transplant recipients

Aim:

Cyclosporine requires close therapeutic drug monitoring because of its narrow therapeutic index and marked inter-individual pharmacokinetic variation. In this study, we investigated the associations of CYP3A4, CYP3A5, ABCB1, NFKB1, and NR1I2 polymorphisms with cyclosporine concentrations in Chinese renal transplant recipients in the early period after renal transplantation.

Methods:

A total of 101 renal transplant recipients receiving cyclosporine were genotyped for CYP3A4^*1G, CYP3A5^*3, ABCB1 C1236T, G2677T/A, C3435T, NFKB1 −94 ins/del ATTG, and NR1I2 polymorphisms. Cyclosporine whole blood levels were measured by a fluorescence polarization immunoassay. Trough concentrations of cyclosporine were determined for days 7-18 following transplantation.

Results:

The dose-adjusted trough concentration (C₀) of cyclosporine in ABCB1 2677 TT carriers was significantly higher than that in GG carriers together with GT carriers [90.4±24.5 vs 67.8±26.8 (ng/mL)/(mg/kg), P=0.001]. ABCB1 3435 TT carriers had a significantly higher dose-adjusted C₀ of cyclosporine than CC carriers together with CT carriers [92.0±24.0 vs 68.4±26.5 (ng/mL)/(mg/kg), P=0.002]. Carriers of the ABCB1 1236TT-2677TT-3435TT haplotype had a considerably higher CsA C₀/D than carriers of other genotypes [97.2±21.8 vs 68.7±26.9 (ng/mL)/(mg/kg), P=0.001]. Among non-carriers of the ABCB1 2677 TT and 3435 TT genotypes, patients with the NFKB1 −94 ATTG ins/ins genotype had a significantly higher dose-adjusted C₀ than those with the −94 ATTG del/del genotype [75.9±32.9 vs 55.1±15.1 (ng/mL)/(mg/kg), P=0.026].

Conclusion:

These results illustrate that the ABCB1 and NFKB1 genotypes are closely correlated with cyclosporine trough concentrations, suggesting that these SNPs are useful for determining the appropriate dose of cyclosporine.     

Nicotine and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone induce cyclooxygenase-2 activity in human gastric cancer cells: Involvement of nicotinic acetylcholine receptor (nAChR) and beta-adrenergic receptor signaling pathways

Induction of cyclooxygenase-2 (COX-2) associates with cigarette smoke exposure in many malignancies. Nicotine and its derivative, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), are the two important components in cigarette smoke that contributes to cancer development. However, the molecular mechanism(s) by which nicotine or NNK promotes gastric carcinogenesis remains largely unknown. We found that nicotine and NNK significantly enhanced cell proliferation in AGS cells that expressed both alpha7 nicotinic acetylcholine receptor (α7 nAChR) and β-adrenergic receptors. Treatment of cells with α-bungarotoxin (α-BTX, α7nAChR antagonist) or propranolol (β-adrenergic receptor antagonist) blocked NNK-induced COX-2/PGE₂ and cell proliferation, while nicotine-mediated cell growth and COX-2/PGE₂ induction can only be suppressed by propranolol, but not α-BTX. Moreover, in contrast to the dependence of growth promoting effect of nicotine on Erk activation, inhibitor of p38 mitogen-activated protein kinase (MAPK) repressed NNK-induced COX-2 upregulation and resulted in suppression of cell growth. In addition, nicotine and NNK mediated COX-2 induction via different receptors to modulate several G1/S transition regulatory proteins and promote gastric cancer cell growth. Selective COX-2 inhibitor (SC-236) caused G1 arrest and abrogated nicotine/NNK-induced cell proliferation. Aberrant expression of cyclin D1 and other G1 regulatory proteins are reversed by blockade of COX-2. These results pointed to the importance of adrenergic and nicotinic receptors in gastric tumor growth through MAPK/COX-2 activation, which may perhaps provide a chemoprevention strategy for cigarette smoke-related gastric carcinogenesis.     

Inhibition of arachidonic acid release and cytosolic phospholipase A2 alpha activity by D-erythro-sphingosine

Sphingolipid metabolites such as sphingosine 1-phosphate (S1P) and ceramide can mediate many cellular events including apoptosis, stress responses and growth arrest. Although ceramide stimulates arachidonic acid metabolism in several cells, the effects of sphingosine and its endogenous analogs have not been established. We investigated the effects of D-erythro-sphingosine and its metabolites on arachidonic acid release in the two cells and on the activity of cytosolic phospholipase A2alpha. C2-Ceramide (N-acetyl-D-erythro-sphingosine, 100 microM) alone stimulated [3H]arachidonic acid release and enhanced the ionomycin-induced release from the prelabeled PC12 cells and L929 cells. In contrast, exogenous addition of D-erythro-sphingosine inhibited the responses in a concentration-dependent manner in the two cell lines. D-erythro-sphingosine, D-erythro-N,N-dimethylsphingosine (D-erythro-DMS) and D-erythro-dihydrosphingosine (D-erythro-DHS) significantly inhibited mastoparan-, but not Na3VO4-, stimulated arachidonic acid release in PC12 cells. D-erythro-S1P and DL-threo-DHS showed no effect on the responses. Production of prostaglandin F2alpha was also enhanced by C2-ceramide (20 microM) and suppressed by D-erythro-sphingosine (10 microM) in PC12 cells. An in vitro study revealed that D-erythro-sphingosine, D-erythro-DMS and D-erythro-DHS directly inhibited cytosolic phospholipase A2alpha activity. These findings suggest that ceramide and D-erythro-analogs of sphingosine have opposite effects on phospholipase A2 activity and thus regulate arachidonic acid release from cells.     

Inhibition of CYP2E1 catalytic activity in vitro by S-adenosyl-L-methionine

The objective of this work was to evaluate the possible in vitro interactions of S-adenosyl-l-methionine (SAM) and its metabolites S-(5'-Adenosyl)-l-homocysteine (SAH), 5'-Deoxy-5'-(methylthio)adenosine (MTA) and methionine with cytochrome P450 enzymes, in particular CYP2E1. SAM (but not SAH, MTA or methionine) produced a type II binding spectrum with liver microsomal cytochrome P450 from rats treated with acetone or isoniazid to induce CYP2E1. Binding was less effective for control microsomes. SAM did not alter the carbon monoxide binding spectrum of P450, nor denature P450 to P420, nor inhibit the activity of NADPH-P450 reductase. However, SAM inhibited the catalytic activity of CYP2E1 with typical substrates such as p-nitrophenol, ethanol, and dimethylnitrosamine, with an IC(50) around 1.5-5mM. SAM was a non-competitive inhibitor of CYP2E1 catalytic activity and its inhibitory actions could not be mimicked by methionine, SAH or MTA. However, SAM did not inhibit the oxidation of ethanol to alpha-hydroxyethyl radical, an assay for hydroxyl radical generation. In microsomes engineered to express individual human P450s, SAM produced a type II binding spectrum with CYP2E1-, but not with CYP3A4-expressing microsomes, and SAM was a weaker inhibitor against the metabolism of a specific CYP3A4 substrate than a specific CYP2E1 substrate. SAM also inhibited CYP2E1 catalytic activity in intact HepG2 cells engineered to express CYP2E1. These results suggest that SAM interacts with cytochrome P450s, especially CYP2E1, and inhibits the catalytic activity of CYP2E1 in a reversible and non competitive manner. However, SAM is a weak inhibitor of CYP2E1. Since the K(i) for SAM inhibition of CYP2E1 activity is relatively high, inhibition of CYP2E1 activity is not likely to play a major role in the ability of SAM to protect against the hepatotoxicity produced by toxins requiring metabolic activation by CYP2E1 such as acetaminophen, ethanol, carbon tetrachloride, thioacetamide and carcinogens.     

Regulation of sterol synthesis by histamine in human mononuclear leukocytes: Roles of H1- and H2-receptors

Summary The effect of histamine on sterol synthesis has been investigated in freshly isolated human mononuclear leukocytes from healthy subjects.Incubation of cells for 6 h in a medium containing lipid depleted serum led to a threefold increase in the incorporation of (¹⁴C)-acetate or tritiated water into sterols. Histamine 0.3 µM added to the incubation medium at zero time inhibited this induction by 35% with a sigmoidal log dose-effect curve.The receptors mediating this action were characterised pharmacologically by using selective H₁- and H₂-agonists and -antagonists. The H₂-agonists impromidine and 4-methylhistamine mimicked the effect of histamine on sterol synthesis, the suppression being 42% and 31%, respectively, at a concentration of 1 µM. In contrast, the H₁-agonist 2-pyridylethylamine did not affect the pathway. The H₂-antagonist cimetidine (10 µM) but not the H₁-antagonist mepyramine (10 µM) totally reversed the inhibition of sterol synthesis by histamine.The results provide evidence that sterol synthesis in human mononuclear leukocytes is regulated by histamine, which appears to act predominantly via H₂-receptors.     

Inhibition of C6 glioma cell proliferation by anandamide, 1-arachidonoylglycerol,and by a water soluble phosphate ester of anandamide: variability in response and involvement of arachidonic acid

It has previously been shown that the endocannabinoids anandamide and 2-arachidonoylglycerol (2-AG) inhibit the proliferation of C6 glioma cells in a manner that can be prevented by a combination of capsazepine (Caps) and cannabinoid (CB) receptor antagonists. It is not clear whether the effect of 2-AG is due to the compound itself, due to the rearrangement to form 1-arachidonoylglycerol (1-AG) or due to a metabolite. Here, it was found that the effects of 2-AG can be mimicked with 1-AG, both in terms of its potency and sensitivity to antagonism by Caps and CB receptor antagonists. In order to determine whether the effect of Caps could be ascribed to actions upon vanilloid receptors, the effect of a more selective vanilloid receptor antagonist, SB366791 was investigated. This compound inhibited capsaicin-induced Ca(2+) influx into rVR1-HEK293 cells with a pK(B) value of 6.8+/-0.3. The combination of SB366791 and CB receptor antagonists reduced the antiproliferative effect of 1-AG, confirming a vanilloid receptor component in its action. 1-AG, however, showed no direct effect on Ca(2+) influx into rVR1-HEK293 cells indicative of an indirect effect upon vanilloid receptors. Identification of the mechanism involved was hampered by a large inter-experimental variation in the sensitivity of the cells to the antiproliferative effects of 1-AG. A variation was also seen with anandamide, which was not a solubility issue, since its water soluble phosphate ester showed the same variability. In contrast, the sensitivity to methanandamide, which was not sensitive to antagonism by the combination of Caps and CB receptor antagonists, but has similar physicochemical properties to anandamide, did not vary between experiments. This variation greatly reduces the utility of these cells as a model system for the study of the antiproliferative effects of anandamide. Nevertheless, it was possible to conclude that the antiproliferative effects of anandamide were not solely mediated by either its hydrolysis to produce arachidonic acid or its CB receptor-mediated activation of phospholipase A(2) since palmitoyltrifluoromethyl ketone did not prevent the response to anandamide. The same result was seen with the fatty acid amide hydrolase inhibitor palmitoylethylamide. Increasing intracellular arachidonic acid by administration of arachidonic acid methyl ester did not affect cell proliferation, and the modest antiproliferative effect of umbelliferyl arachidonate was not prevented by a combination of Caps and CB receptor antagonists.     

The lathyrus toxin, beta-N-oxalyl-L-alpha,beta-diaminopropionic acid (ODAP), and homocysteic acid sensitize CA1 pyramidal neurons to cystine and L-2-amino-6-phosphonohexanoic acid

A brief exposure of hippocampal slices to L-quisqualic acid (QUIS) sensitizes CA1 pyramidal neurons 30- to 250-fold to depolarization by certain excitatory amino acids analogues, e.g., L-2-amino-6-phosphonohexanoic acid (L-AP6), and by the endogenous compound, L-cystine. This phenomenon has been termed QUIS sensitization. A mechanism similar to that previously described for QUIS neurotoxicity has been proposed to describe QUIS sensitization. Specifically, QUIS has been shown to be sequestered into GABAergic interneurons by the System x(c)(-) and subsequently released by heteroexchange with cystine or L-AP6, resulting in activation of non-NMDA receptors. We now report two additional neurotoxins, the Lathyrus excitotoxin, beta-N-oxalyl-L-alpha,beta-diaminopropionic acid (ODAP), and the endogenous compound, L-homocysteic acid (HCA), sensitize CA1 hippocampal neurons >50-fold to L-AP6 and >10-fold to cystine in a manner similar to QUIS. While the cystine- or L-AP6-mediated depolarization can be inhibited by the non-NMDA receptor antagonist CNQX in ODAP- or QUIS-sensitized slices, the NMDA antagonist D-AP5 inhibits depolarization by cystine or L-AP6 in HCA-sensitized slices. Thus, HCA is the first identified NMDA agonist that induces phosphonate or cystine sensitization. Like QUIS sensitization, the sensitization evoked by either ODAP or HCA can be reversed by a subsequent exposure to 2 mM alpha-aminoadipic acid. Finally, we have demonstrated that there is a correlation between the potency of inducers for triggering phosphonate or cystine sensitivity and their affinities for System x(c)(-) and either the non-NMDA or NMDA receptor. Thus, the results of this study support our previous model of QUIS sensitization and have important implications for the mechanisms of neurotoxicity, neurolathyrism and hyperhomocystinemia.     

Stereoselectivity in metabolism of ifosfamide by CYP3A4 and CYP2B6

The aim was to identify the hepatic cytochromes P450 (CYPs) responsible for the enantioselective metabolism of ifosfamide (IFA). The 4-hydroxylation, N²- and N³-dechloroethylation of IFA enantiomers were monitored simultaneously in the same metabolic systems using GC/MS and pseudoracemate techniques. In human and rat liver microsomes, (R)-IFA was preferentially metabolized via 4-hydroxylation, whereas its antipode was biotransformed in favour of N-dechloroethylation. CYP3A4 was the major enzyme responsible for metabolism of IFA enantiomers in human liver. The study also revealed that CYP3A (human CYP3A4/5 and rat CYP3A1/2) and CYP2B (human CYP2B6 and rat CYP2B1/2) enantioselectively mediated the 4-hydroxylation, N²- and N³-dechloroethylation of IFA. CYP3A preferentially supported the formation of (R)-4-hydroxyIFA (HOIF), (R)-N²-dechloroethylIFA (N2D) and (R)-N³-dechloroethylIFA (N3D), whereas CYP2B preferentially mediated the generation of (S)-HOIF, (S)-N2D and (S)-N3D. The enantioselective metabolism of IFA by CYP3A4 and CYP2B1 was confirmed in cDNA transfected V79 cells.     

Effects of synthetic sphingosine-1-phosphate analogs on arachidonic acid metabolism and cell death

Sphingolipid metabolites such as sphingosine regulate cell functions including cell death and arachidonic acid (AA) metabolism. D-erythro-C18-Sphingosine-1-phosphate (D-e-S1P), a sphingolipid metabolite, acts as an intracellular messenger in addition to being an endogenous ligand of some cell surface receptors. The development of S1P analogs may be useful for studying and/or regulating S1P-mediated cellular responses. In the present study, we found that several synthetic S1P analogs at pharmacological concentrations stimulated AA metabolism and cell death in PC12 cells. D-erythro-N,O,O-Trimethyl-C18-S1P (D-e-TM-S1P), L-threo-O,O-dimethyl-C18-S1P (L-t-DM-S1P) and L-threo-O,O-dimethyl-3O-benzyl-C18-S1P (L-t-DMBn-S1P) at 100 microM stimulated [(3)H]AA release from the prelabeled PC12 cells. L-t-DMBn-S1P at 20 microM increased prostanoid formation in PC12 cells. L-t-DMBn-S1P-induced AA release was inhibited by D-e-sphingosine, but not by the tested PLA(2) inhibitors. L-t-DMBn-S1P did not stimulate the activity of cytosolic phospholipase A(2alpha) (cPLA(2alpha)) in vitro and the translocation of cPLA(2alpha) in the cells, and caused AA release from the cells lacking cPLA(2alpha). These findings suggest that L-t-DMBn-S1P stimulated AA release in a cPLA(2alpha)-independent manner. In contrast, D-e-S1P and D-erythro-N-monomethyl-C18-S1P caused cell death without AA release in PC12 cells, and the effects of D-e-TM-S1P, L-t-DM-S1P and L-t-DMBn-S1P on cell death were limited. Synthetic S1P analogs may be useful tools for studying AA metabolism and cell death in cells.     

Role of Ca2+-independent phospholipase A2 in cell growth and signaling

Phospholipase A₂ (PLA₂) are esterases that cleave glycerophospholipids to release fatty acids and lysophospholipids. Several studies demonstrate that PLA₂ regulate growth and signaling in several cell types. However, few of these studies have focused on Ca²⁺-independent phospholipase A₂ (iPLA₂ or Group VI PLA₂). This class of PLA₂ was originally suggested to mediate phospholipid remodeling in several cell types including macrophages. As such, it was labeled as a housekeeping protein and thought not to play as significant of roles in cell growth as its older counterparts cytosolic PLA₂ (cPLA₂ or Group IV PLA₂) and secretory PLA₂ (sPLA₂ or Groups I-III, V and IX-XIV PLA₂). However, several recent studies demonstrate that iPLA₂ mediate cell growth, and do so by participating in signal transduction pathways that include epidermal growth factor receptors (EGFR), mitogen activated protein kinases (MAPK), mdm2, and even the tumor suppressor protein p53 and the cell cycle regulator p21. The exact mechanism by which iPLA₂ mediates these pathways are not known, but likely involve the generation of lipid signals such as arachidonic acid, lysophosphatidic acid (LPA) and lysophosphocholines (LPC). This review discusses the role of iPLA₂ in cell growth with special emphasis placed on their role in cell signaling. The putative lipid signals involved are also discussed.     

Lactonase and lactonizing activities of human serum paraoxonase (PON1) and rabbit serum PON3

Human paraoxonase (PON1) was previously shown to hydrolyze over 30 different lactones (cyclic esters). In the present study purified human PON1 was found to catalyze the reverse reaction (lactonization) of a broad range of hydroxy acids. Hydroxy acid lactonization or lactone hydrolysis is catalyzed until equilibrium between the open and closed forms is reached. Lactonization by PON1 was calcium-dependent, had a pH optimum of 5.5-6 and could be stimulated with dilauroylphosphatidylcholine. Rabbit serum PON3 and a serine esterase in mouse plasma, presumably a carboxylesterase, also catalyzed hydroxy acid lactonization. Two endogenous oxidized unsaturated fatty acids, (+/-)4-hydroxy-5E,7Z,10Z,13Z,16Z,19Z-docosahexaenoic acid (4-HDoHE) and (+/-)5-hydroxy-6E,8Z,11Z,14Z-eicosatetraenoic acid (5-HETE) lactone, were very efficiently lactonized and hydrolyzed, respectively, by PON1. Human and mouse plasma samples also catalyzed 4-HDoHE lactonization and 5-HETE lactone hydrolysis. Studies with the PON1 inhibitor EDTA and the serine esterase inhibitor phenylmethylsulfonylfluoride suggest that about 80-95% of both activities can be attributed to PON1 in the human samples. In the mouse sample, PON1 accounted for about 30% of the 4-HDoHE lactonizing activity and 72% of the 5-HETE lactonase activity. Our results demonstrate that PON1 can lactonize the hydroxy acid form of its lactone substrates and that reversible hydrolysis of lactones may be a property of lactonases that is not generally considered. Also, the high activity of PON1 towards 4-HDoHE and 5-HETE lactone suggests that oxidized eicosanoids and docosanoids may be important physiological substrates for PON1.     

Synthesis and Antimicrobial Activity of Some New 2-(3-(4-Aryl)-1-phenyl-1H-pyrazol-4-yl) Chroman-4-ones

Seven new 2-(3-(4-aryl)-1-phenyl-1H-pyrazol-4-yl) chroman-4-ones (4a-4g) have been synthesized by cyclization of 2-hydroxychalcone analogues of pyrazole 3a-3g using conc. HCl in acetic acid. The structures of the compounds 4a-4g were established by the combined use of (1)HNMR, IR and mass spectra. All the seven compounds were tested in vitro for their antibacterial activity against two Gram positive bacteria namely Staphylococcus aureus and Bacillus subtilis and two Gram negative bacteria Escherichia coli and Pseudomonas aeruginosa. The compounds 4b, 4c, 4e, 4f, 4g have displayed good antibacterial activity when compared with commercially available antibiotic, ciprofloxacin. These compounds also were screened for their antifungal activity against two ear pathogenic fungi, namely Aspergillus Niger and A. flavus. The compounds 4a, 4c, 4d, 4g exhibited good antifungal activity when compared with commercially available antifungal, fluconazole.     

Antioxidant properties of Thonningianin A,isolated from the African medicinal herb,Thonningia sanguinea

The antioxidant properties of Thonningianin A (Th A), an ellagitannin, isolated from the methanolic extract of the African medicinal herb, Thonningia sanguinea were studied using the NADPH and Fe2+/ascorbate-induced lipid peroxidation (LPO), electron spin resonance spectrometer and the deoxyribose assay. Th A at 10 microM inhibited both the NADPH and Fe2+/ascorbate-induced LPO in rat liver microsomes by 60% without inhibitory effects on cytochrome P450 activity. Th A was similar to the synthetic antioxidant, tannic acid, as an inhibitor of both the NADPH and Fe2+/ascorbate-induced LPO but potent than gallic acid, vitamin C and vitamin E. While Th A poorly scavenged the hydroxyl radical generated by the Fenton reaction it dose-dependently scavenged 1,1-diphenyl-2-picrylhydrazyl, superoxide anion and peroxyl radicals with IC50 of 7.5, 10 and 30 microM, respectively. Furthermore, Th A showed inhibitory effects on the activity of xanthine oxidase with an IC50 of 30 microM. In the deoxyribose assay both T. sanguinea and its methanolic component Th A showed only site-specific (Fe3+ + H2O2) but not non-site-specific (Fe3+ + EDTA + H2O2) hydroxyl radical scavenging suggesting chelating ability for iron ions. Spectroscopic studies showed that Th A enhanced absorbance in the visible region in the presence of Fe2+ ions. These results indicate that the antioxidant properties of Th A involve radical scavenging, anti-superoxide formation and metal chelation.     

Vanadate-induced activation of cytosolic phospholipase A2alpha in L929 cells: Roles of tyrosine kinase, protein kinase C, and extracellular signal-regulated kinase

Orthovanadate (Na3VO4), which acts as an inhibitor of protein tyrosine phosphatases, has a various pharmacological effects including the release of arachidonic acid (AA) from cells. We investigated roles of alpha-type cytosolic phospholipase A2 (cPLA2alpha), Src family kinases (Src) and protein kinase C (PKC) in the release of AA induced by Na3VO4 from a murine fibroblast cell line, L929. C12 cells, a variant of L929 that lacks expression of cPLA2alpha, were used along with a clone of C12 cells that are stably expressing cPLA2alpha (C12-cPLA2alpha cells). In the presence of a Ca2+ ionophore (10 microM A23187), 5 and 10mM Na3VO4 synergistically stimulated AA release from L929 and C12-cPLA2alpha cells, and to a much lesser extent from control C12 cells. The release of AA by Na3VO4/A23187 was inhibited by a selective cPLA2alpha inhibitor (3 microM pyrrophenone). The release of AA by Na3VO4/A23187 was significantly inhibited by a PKC inhibitor (10 microM GF109203X), in PKC-depleted cells, by a Src inhibitor (2 microM PP2) and by an inhibitor of extracellular signal-regulated kinase 1/2 (ERK1/2) kinase (10 microM U0126). The phosphorylation of ERK1/2 was stimulated by Na3VO4, and the response was significantly decreased by inhibitors of Src, PKC and ERK1/2 kinase. Our data show that Na3VO4 stimulates AA release largely via cPLA2alpha activation in Ca2+-dependent manner, and the cross-talk between Src and PKC and the ERK-dependent pathways are involved in Na3VO4-induced AA release from L929 cells.     

The role of reactive oxygen species in arsenite and monomethylarsonous acid-induced signal transduction in human bladder cells: acute studies

Arsenicals are known to induce ROS, which can lead to DNA damage, oxidative stress, and carcinogenesis. A human urothelial cell line, UROtsa, was used to study the effects of arsenicals on the human bladder. Arsenite [As(III)] and monomethylarsonous acid [MMA(III)] induce oxidative stress in UROtsa cells after exposure to concentrations as low as 1 microM and 50 nM, respectively. Previous research has implicated ROS as signaling molecules in the MAPK signaling pathway. As(III) and MMA(III) have been shown to increase phosphorylation of key proteins in the MAPK signaling cascade downstream of ErbB2. Both Src phosphorylation (p-Src) and cyclooxygenase-2 (COX-2) are induced after exposure to 50 nM MMA(III) and 1 microM As(III). These data suggest that ROS production is a plausible mechanism for the signaling alterations seen in UROtsa cells after acute arsenical exposure. To determine importance of ROS in the MAPK cascade and its downstream induction of p-Src and COX-2, specific ROS antioxidants (both enzymatic and non-enzymatic) were used concomitantly with arsenicals. COX-2 protein and mRNA was shown to be much more influenced by altering the levels of ROS in cells, particularly after MMA(III) treatment. The antioxidant enzyme superoxide dismutase (SOD) effectively blocked both As(III)-and MMA(III)- associated COX-2 induction. The generation of ROS and subsequent altered signaling did lead to changes in protein levels of SOD, which were detected after treatment with either 1 microM As(III) or 50 nM MMA(III). These data suggest that the generation of ROS by arsenicals may be a mechanism leading to the altered cellular signaling seen after low-level arsenical exposure.