Kyung Rak Min

Inhibitory action of novel aromatic diamine compound on lipopolysaccharide-induced nuclear translocation of NF-κB without affecting IκB degradation

4‐Methyl‐N 1‐(3‐phenyl‐propyl)‐benzene‐1,2‐diamine (JSH‐23) is a novel chemically synthetic compound. The aromatic diamine JSH‐23 compound exhibited inhibitory effect with an IC50 value of 7.1 μM on nuclear factor (NF)‐κB transcriptional activity in lipopolysaccharide (LPS)‐stimulated macrophages RAW 264.7, and interfered LPS‐induced nuclear translocation of NF‐κB without affecting IκB degradation. This mechanism of action is very rare for controlling NF‐κB activation. Furthermore, the compound inhibited not only LPS‐induced expressions of tumor necrosis factor‐α, interleukin (IL)‐1β, IL‐6 and inducible nitric oxide synthase and cyclooxygenase‐2 but also LPS‐induced apoptosis of the RAW 264.7 cells.

Anti-inflammatory effects of fangchinoline and tetrandrine.

Fangchinoline and tetrandrine are the major alkaloids from Stephania tetrandrae S. Moore which has been used traditionally for the treatment of inflammatory diseases in oriental countries including Korea. Both fangchinoline and tetrandrine showed anti-inflammatory effects on mouse ear edema induced by croton oil. In addition, the effects of fangchinoline and tetrandrine on cyclooxygenase, murine interleukin-5 (mIL-5) and human interleukin-6 (hIL-6) were examined in vitro to investigate the anti-inflammatory action mechanisms. One hundred micromolar of fangchinoline showed 35% of inhibition on cyclooxygenase, but the same concentration of tetrandrine did not show any inhibition. On the other hand, 12.5 microM of tetrandrine exhibited 95% of inhibition on mIL-5 activity, while fangchinoline did not show any effects. However, 4 microM of fangchinoline and 6 microM of tetrandrine showed 63 and 86% of inhibitions on hIL-6 activity, respectively. These results suggest that biochemical mechanisms of fangchinoline and tetrandrine on anti-inflammation are significantly different even though they are similar in chemical structure.

Artemisolide from Artemisia asiatica: nuclear factor-kappaB (NF-kappaB) inhibitor suppressing prostaglandin E2 and nitric oxide production in macrophages.

Aerial parts ofArtemisia asiatica (Compositae) have been traditionally used as an oriental medicine for the treatment of inflammatory and ulcerogenic diseases. In the present study, artemisolide was isolated as a nuclear factor (NF)-κB inhibitor fromA. asiatica by activity-guided fractionation. Artemisolide inhibited NF-κB transcriptional activity in lipopolysaccharide (LPS)-stimulated macrophages RAW 264.7 with an IC50 value of 5.8 μM. The compound was also effective in blocking NF-κB transcriptional activities elicited by the expression vector encoding the NF-κB p65 or p50 subunits bypassing the inhibitory kB degradation signaling NF-κB activation. The macrophages markedly increased their PGE2 and NO production upon exposure to LPS alone. Artemisolide inhibited LPS-induced PGE2 and NO production with IC50 values of 8.7 μM and 6.4 μM, respectively, but also suppressed LPS-induced synthesis of cyclooxygenase (COX)-2 or inducible NO synthase (iNOS). Taken together, artemisolide is a NF-κB inhibitor that attenuates LPS-induced production of PGE2 or NOvia down-regulation of COX-2 or iNOS expression in macrophages RAW 264.7. Therefore, artemisolide could represent and provide the anti-inflammatory principle associated with the traditional medicine,A. asiatica.

Differential inhibitory effects of sophoricoside analogs on bioactivity of several cytokines

Effects of sophoricoside and its analogs on proinflammatory cytokines have been investigated. Sophoricoside, genistein and orobol exhibited inhibitory effects on IL-5, IL-3, GM-CSF and IL-6 bioactivities. Genistin showed inhibitory effects on IL-5 and IL-3 bioactivities, but did not inhibit GM-CSF and IL-6 bioactivities. None of the sophoricoside analogs showed inhibitory effects on both IL-1beta and TNF-alpha bioactivities. Among the compounds, sophoricoside exhibited the highest inhibitory effects on IL-5, IL-3 and IL-6 bioactivities with IC50 values of 1.9 microM, 6.9 microM and 6.0 microM, respectively and orobol did show on GM-CSF bioactivity with an IC50 value of 18.0 microM. The result would provide an additional mechanism by which the compounds exert immunosuppressive and anti-inflammatory effects.

Anti-inflammatory mode of isoflavone glycoside sophoricoside by inhibition of interleukin-6 and cyclooxygenase-2 in inflammatory response.

Soy, high dietary intake for the oriental population, is a main source of isoflavonoids. Sophoricoside (SOP) an isoflavone glycoside was isolated from immature fruits ofSophora japonica (Leguminosae family) and its inhibitory effect on chemical mediators involved in inflammatory response was investigated in this study. SOP inhibited the interleukin (IL)-6 bioactivity with an IC50 value of 6.1 μM whereas it had no effects on IL-1(3 and TNF-α bioactivities. SOP was identified as a selective inhibitor of cyclooxygenase (COX)-2 activity with an IC50 value of 4.4 μM, but did not show inhibitory effect on the synthesis of COX-2. However, SOP had no effect on the production of reactive oxygen species including superoxide anions and nitric oxide. These results revealed thatin vitro anti-inflammatory action of SOP is significantly different from that of genistein known as a phytoestrogen of soy products. This experimental study has documented an importance of dietary soy isoflavonoids as multifunctional agents beneficial to human health, and will help to clarify protective mechanisms of SOP against inflammatory con-ditions.

Characterization of catechol 2,3-dioxygenases☆

Three catechol 2,3-dioxygenases for biphenyl, naphthalene/salicylate, and toluene/xylene oxidation were cloned from Achromobacter xylosoxidans KF701, Pseudomonas putida (NAH7), and Pseudomonas sp. (pWWO). The cloned catechol 2,3-dioxygenases were identified by enzymatic activity assay in addition to yellow bands on polyacrylamide gel after electrophoresis and activity staining. All of the cloned catechol 2,3-dioxygenases exhibited their highest activities on catechol as a substrate compared with catechol derivatives including 4-chlorocatechol, 3-methylcatechol, and 4-methylcatechol. The cloned catechol 2,3-dioxygenases are not fused proteins but were significantly different from one another in their electrophoretic mobilities on nondenaturing 7.5%-polyacrylamide gel.

Molecular cloning and characterization of catechol 2,3-dioxygenases from biphenyl/polychlorinated biphenyls-degrading bacteria

Catechol 2,3-dioxygenases were cloned from Alcaligenes sp. KF711, Pseudomonas putida KF715, and Achromobacter xylosoxidans KF701 which are biphenyl/polychlorinated biphenyls-degrading bacteria. All of the cloned enzymes were purified by preparative polyacrylamide gel electrophoresis (PAGE). The purified catechol 2,3-dioxygenases were significantly different from one another in ring-fission activities to catechol and its derivatives. The catechol 2,3-dioxygenase from Alcaligenes sp. KF711 exhibited higher ring-fission activity to 4-chlorocatechol than those from P. putida KF715 and A. xylosoxidans KF701. In electrophoretic mobilities, the three enzymes were different from one another on nondenaturing PAGE but the same on SDS-PAGE.

Anti-inflammatory effects of N1-Benzyl-4-methylbenzene-1,2-diamine (JSH-21) analogs on nitric oxide production and nuclear factor-kappa B transcriptional activity in lipopolysaccharide-stimulated macrophages RAW 264.7

N1-Benzyl-4-methylbenzene-1,2-diamine (JSH-21) and its analogs were chemically synthesized and their anti-inflammatory potentials investigated. JSH-21 inhibited nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated macrophages RAW 264.7 in a dose-dependent manner, with an IC50 value of 9.2 μM, where pyrrolidine dithiocarbamate and parthenolide as positive controls exhibited IC50 values of 29.3 and 3.6 μM, respectively. The inhibitory effect of JSH-21 on the NO production was attributable to its down-regulatory action on LPS-induc-ible NO synthase (iNOS), which was documented by iNOS promoter activity. In the mechanism of the anti-inflammatory action, JSH-21 exhibited inhibitory effects on LPS-induced DNA binding activity and transcriptional activity of nuclear factor-kappa B(NF-kB). Structural analogs of JSH-21 also inhibited both the LPS-induced NO production andNF-kB transcriptional activity, where diamine substitution at positions 1 and 2 of JSH-21 seems to play an important role in the anti-inflammatory activity.

Suppressive effects of furonaphthoquinone NFD-37 on the production of lipopolysaccharide-inducible inflammatory mediators in macrophages RAW 264.7

Abstract2-Methyl-2-(2-methylpropenyl)-2,3-dihydronaphthoquinone[2,3-b]furan-4,9-dione (NFD-37) is a synthetic furonaphthoquinone compound. In this study, we determined that NFD-37 could inhibit the lipopolysaccharide (LPS)-induced production of inflammatory mediators in macrophages RAW 264.7. This compound inhibited LPS-induced nitric oxide (NO) or prostaglandin (PG) E2 production in dose-dependent manners, with IC50 values of 7.2 μM and 5.3 μM, respectively. As the positive controls, pyrrolidine dithiocarbamate (30 μM) exhibited a 57% inhibition of NO production, and NS-398 (1 μM) manifested a 48% inhibition of PGE2 production. The inhibitory effects of NFD-37 on NO and PGE2 production were determined to occur in conjunction with the suppression of inducible NO synthase or cyclooxygenase-2 expression. NFD-37 also inhibited the production of LPS-inducible tumor necrosis factor-α, interleukin (IL)-1β and IL-6, at IC50 values of 4.8–8.9 μM. We also determined the anti-inflammatory efficacy of NFD-37 using carrageenin-induced paw edema in experimental mice.

Characterization of thepcbE gene encoding 2-hydroxypenta-2,4-dienoate hydratase inPseudomonas sp. DJ-12

Nucleotide sequence extending 2,3-dihydroxybiphenyl 1,2-dioxygenase gene (pcbC) and 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate hydrolase gene (pcbD) ofPseudomonas sp. DJ-12 was previously analyzed and the two genes were present in the order ofpcbD-pcbC preceded by a promoter fromPseudomonas sp. DJ-12. In this study, a 3.8-kb nucleotide sequence located downstream of thepcbC gene was analyzed to have three open reading frames (ORFs) that are designated asorf1, pcbE andorf2 genes. All of the ORFs were preceded by each ribosome-binding sequence of 5-GGAXA-3 (X=G or A). However, no promoter-like sequence and transcription terminator sequence were found in the analyzed region, downstream ofpcbC gene. Therefore, the gene cluster appeared to be present in the order ofpcbD-pcbC-orf1-pcbE-orf2 as an operon, which is unique organization characterized so far in biphenyl- and PCB-degrading bacteria. Theorf1 gene was composed of 1,224 base pairs which can encode a polypeptide of molecular weight 44,950 containing 405 amino acid residues. A deduced amino acid sequence of theorf1 gene product exhibited 21–33% identity with those of indole dioxygenase and phenol hydroxylase components. ThepcbE gene was composed of 783 base pairs encoding 2-hydroxypenta-2,4-dienoate hydratase involved in the 4-chlorobiphenyl catabolism. Theorf2 gene was composed of 1,017 base pairs encoding a polypeptide of molecular weight 37,378 containing 338 amino acid residues. A deduced amino acid sequence of theorf2 gene product exhibited 31% identity with that of a nitrilotriacetate monooxygenase component.