Physicochemical parameters influencing coaggregation between the freshwater bacteria Sphingomonas natatoria 2.1 and Micrococcus luteus 2.13.

The aim of this study was to explore the physicochemical parameters that influence coaggregation between the freshwater bacteria Sphingomonas natatoria 2.1 and Micrococcus luteus 2.13. Using visual coaggregation assays, the effect of different buffers, solutions of differing ionic strength, pH, temperature, and viscosity on the degree of coaggregation was assessed. Coaggregation occurred maximally in distilled water but was inhibited when coaggregates were suspended in a commonly-used oral bacterial coaggregation buffer, saline solutions, and Tris-Cl buffers. Coaggregation was weakly expressed in standard laboratory buffers. The ionic strength of inorganic salt solutions required to inhibit coaggregation depended upon the inorganic salt being tested. Coaggregation occurred at a pH of 3-10, between 5 and 80°C and was inhibited in solutions with a viscosity of 22.5 centipoises at 20°C. Inhibition of coaggregation with NaCl impaired biofilm development. When developing buffers to test for coaggregation, the natural liquid environment should be considered. Coaggregation between S. natatoria 2.1 and M. luteus 2.13 is only affected by physicochemical conditions beyond those typically found in natural freshwater ecosystems. Such a robust ability to coaggregate may enhance the ability of S. natatoria 2.1 and M. luteus 2.13 to develop a niche in freshwater biofilms.

Coaggregation by the freshwater bacterium Sphingomonas natatoria alters dual-species biofilm formation.

Coaggregation is hypothesized to enhance freshwater biofilm development. To investigate this hypothesis, the ability of the coaggregating bacterium Sphingomonas natatoria to form single- and dual-species biofilms was studied and compared to that of a naturally occurring spontaneous coaggregation-deficient variant. Attachment assays using metabolically inactive cells were performed using epifluorescence and confocal laser scanning microscopy. Under static and flowing conditions, coaggregating S. natatoria 2.1gfp cells adhered to glass surfaces to form diaphanous single-species biofilms. When glass surfaces were precoated with coaggregation partner Micrococcus luteus 2.13 cells, S. natatoria 2.1gfp cells formed densely packed dual-species biofilms. The addition of 80 mM galactosamine, which reverses coaggregation, mildly reduced adhesion to glass but inhibited the interaction and attachment to glass-surface-attached M. luteus 2.13 cells. As opposed to wild-type coaggregating cells, coaggregation-deficient S. natatoria 2.1COGgfp variant cells were retarded in colonizing glass and did not interact with glass-surface-attached M. luteus 2.13 cells. To determine if coaggregation enhances biofilm growth and expansion, viable coaggregating S. natatoria 2.1gfp cells or the coaggregation-deficient variant S. natatoria 2.1COGgfp cells were coinoculated in flow cells with viable M. luteus 2.13 cells and allowed to grow together for 96 h. Coaggregating S. natatoria 2.1gfp cells outcompeted M. luteus 2.13 cells, and 96-h biofilms were composed predominantly of S. natatoria 2.1gfp cells. Conversely, when coaggregation-deficient S. natatoria 2.1COGgfp cells were coinoculated with M. luteus 2.13 cells, the 96-h biofilm contained few coaggregation-deficient S. natatoria 2.1 cells. Thus, coaggregation promotes biofilm integration by facilitating attachment to partner species and likely contributes to the expansion of coaggregating S. natatoria 2.1 populations in dual-species biofilms through competitive interactions.

Inhibitory effects of anti-inflammatory drugs on interleukin-6 bioactivity.

Interleukin-6 (IL-6) is known as a proinflammatory cytokine involved in immune response, inflammation, and hematopoiesis. Inhibitory effects of anti-inflammatory drugs on IL-6 bioactivity using IL-6-dependent hybridoma have been evaluated. Three out of 16 nonsteroidal anti-inflammatory drugs (NSAIDs) showed IC50 values of less than 100 microM, which were in the order of oxyphenylbutazone hydrate (IC50=7.5 microM)>meclofenamic acid sodium salt (31.9 microM)>sulindac (74.9 microM). Steroidal anti-inflammatory drugs (SAIDs) exhibited significant inhibitory effects at 100 microM on the IL-6 bioactivity, and their inhibitory potencies were in the order of budesonide (IC50=2.2 microM)>hydrocortisone 21-hemisuccinate (6.7 microM), prednisolone (7.5 microM), betamethasone (10.9 microM)>dexamethasone (18.9 microM) and triamcinolone acetonide (24.1 microM). The results would provide an additional mechanism by which anti-inflammatory drugs display their anti-inflammatory and immunosuppressive effects at higher concentrations.

Effect of genistein analogs on oxygen radical production in leukocytes stimulated by unopsonized zymosan.

Effects of genistein analogs on oxygen radical production have been analyzed in human neutrophils, human monocytes or murine macrophages Raw264.7 stimulated with unopsonized zymosan by lucigenin- and luminol-enhanced chemiluminescence assays. Genistein exhibited IC50 values of 10.7-11.5 microM on the oxygen radical production in human neutrophils, 10.9-11.0 microM in human monocytes, and 14.8-27.3 microM in Raw264.7 cells. Orobol, a genistein analog with an additional hydroxy group at the 3' position, exhibited IC50 values of 3.0-3.3 microM on the oxygen radical production in human neutrophils, 2.8-3.1 microM in human monocytes, and 1.5-3.9 microM in Raw264.7 cells. Genistin and sophoricoside are genistein glycosides with a glucose moiety at 7 or 4' position, respectively. The genistein glycosides exhibited 23-37% inhibitory effects at 100 microM on the oxygen radical production.

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.

Characterization of the pcbE gene encoding 2-hydroxypenta-2,4-dienoate hydratase in Pseudomonas 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) of Pseudomonas sp. DJ-12 was previously analyzed and the two genes were present in the order of pcbD-pcbC preceded by a promoter from Pseudomonas sp. DJ-12. In this study, a 3.8-kb nucleotide sequence located downstream of the pcbC gene was analyzed to have three open reading frames (ORFs) that are designated as orf1, pcbE and orf2 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 of pcbC gene. Therefore, the gene cluster appeared to be present in the order of pcbD-pcbC-orf1-pcbE-orf2 as an operon, which is unique organization characterized so far in biphenyl- and PCB-degrading bacteria. The orf1 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 the orf1 gene product exhibited 21-33% identity with those of indole dioxygenase and phenol hydroxylase components. The pcbE gene was composed of 783 base pairs encoding 2-hydroxypenta-2,4-dienoate hydratase involved in the 4-chlorobiphenyl catabolism. The orf2 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 the orf2 gene product exhibited 31% identity with that of a nitrilotriacetate monooxygenase component.

Flavonol glycoside gallate and ferulate esters from Persicaria lapathifolia as inhibitors of superoxide production in human monocytes stimulated by unopsonized zymosan.

Aerial parts of Persicaria lapathifolia S.F. Gray (Polygonaceae) exhibited an inhibitory effect on superoxide production in unopsonized zymosan-stimulated human monocytes. Two known compounds, quercetin 3-O-beta-(2"-galloyl)-glucopyranoside and quercetin 3-O-beta-(2"-galloyl)-rhamnopyranoside, and a new compound, quercetin 3-O-beta-(6"-feruloyl)-galactopyranoside, were isolated as the inhibitors of superoxide production by activity-guided fractionation. IC50 values were shown at the concentrations of 2.1 microM by quercetin 3-O-beta-(2"-galloyl)-glucopyranoside, 1.9 microM by quercetin 3-O-beta-(2"-galloyl)-rhamnoypranoside, and 3.5 microM by quercetin 3-O-beta-(6"-feruloyl)-galactopyranoside whose inhibitory potencies were similar to oxyphenylbutazone (IC50 = 1.9 microM) as a positive control.

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.

Sophoricoside analogs as the IL-5 inhibitors from Sophora japonica.

Fruits of Sophora japonica L. (Leguminosae) exhibited an inhibitory effect in the IL-5 bioassay of mIL-5-dependent Y16 proliferation. The isoflavonoids of sophoricoside, genistein, orobol, and genistin were isolated as the IL-5 inhibitors from fresh fruits of the plant by activity-guided fractionation. Among the IL-5 inhibitors, sophoricoside exhibited the highest inhibitory effect with 89% inhibition at 12.5 microM, 82% at 6.3 microM, 72% at 3.1 microM, 59% at 1.6 microM, and 24% at 0.8 microM where the 50% inhibition (IC50) was shown at the concentration of 1.5 microM. Oxyphenylbutazone as the positive control exhibited the IC50 value at the concentration of 31.7 microM. In the order of IC50 values the inhibitory potency in the IL-5 bioassay was sophoricoside > orobol (9.8 microM) approximately equal to genistin (10.6 microM) > genistein (51.9 microM). The position of O-glycosylation and number of hydroxy groups in the isoflavonoids seem to play an important role in the inhibitory effect in the IL-5 bioassay.

Inhibitory effect of luteolin 4'-O-glucoside from Kummerowia striata and other flavonoids on interleukin-5 bioactivity.

Interleukin (IL)-5 is a chemotactic factor of eosinophils, and promotes the growth and survival of eosinophils, which plays an important role in the eosinophilia-associated allergic inflammation. In this study, luteolin 4'-O-glucopyranoside was identified as the IL-5 inhibitor from Kummerowia striata Thunb. (Leguminosae) by activity-guided fractionation followed by structural analysis compared with reported spectral data. The flavone compound exhibited dose-dependent inhibitory effect on IL-5 bioactivity with 95% inhibition at 30 microM, 79% at 15 microM, 60% at 7.5 microM, 54% at 3.8 microM and 29% at 1.9 microM, where 50% of inhibition (IC50) value was shown at the concentration of 3.7 microM. Furthermore, the inhibitory effect on IL-5 bioactivity by other flavonoid compounds available was estimated. In view of the IC50 values, the inhibitory potency on IL-5 bioactivity was in order of luteolin 4'-O-glucopyranoside > cosmosiin (14.2 microM) approximately equal to apigenin (16.4 microM) approximately equal to luteolin (18.7 microM) > quercimeritrin (27.3 microM) approximately equal to kaempferol (30.0 microM).

(-)-Epiafzelechin: cyclooxygenase-1 inhibitor and anti-inflammatory agent from aerial parts of Celastrus orbiculatus.

An inhibitor of cyclooxygenase (COX)-1 activity of prostaglandin H2 synthase was isolated from aerial parts of Celastrus orbiculatus Thunb. (Celastraceae), an oriental folk medicine for rheumatoid arthritis by activity-guided column chromatographic methods. The COX inhibitor was identified as (-)-epiafzelechin, a member of flavan-3-ols by the structural analysis with HR-EI-mass, 1H-NMR and 13C-NMR spectral data. The compound exhibited a dose-dependent inhibition on the COX activity with an IC50 value of 15 microM. (-)-Epiafzelechin exhibited about 3-fold weaker inhibitory potency on the enzyme activity than indomethacin as a positive control. (-)-Epiafzelechin exhibited significant anti-inflammatory activity on carrageenin-induced mouse paw edema when the compound (100 mg/kg) was orally administrated at 1 h before carrageenin treatment.

Inhibitory effects of resveratrol analogs on unopsonized zymosan-induced oxygen radical production.

Resveratrol, a natural hydroxystilbene, has been reported to have anti-inflammatory and anticarcinogenic activities. Inhibitory effects of resveratrol and its analogs on reactive oxygen species (ROS) production in unopsonized zymosan-stimulated murine macrophage Raw264.7 cells, human monocytes, and neutrophils were analyzed to investigate if the anti-inflammatory and anticarcinogenic activities of resveratrol are related to the inhibition of ROS production. Resveratrol was a potent inhibitor of ROS production in both unopsonized zymosan-stimulated Raw264.7 cells and human monocytes and neutrophils. Resveratrol exhibited 50% inhibition values (IC50) of 17 microM in activated Raw264.7 cells, 18 microM in human monocytes, and 23 microM in human neutrophils. 3,5-Dihydroxy-4'-methoxystilbene or 3,4'-dimethoxy-5-hydroxystilbene exhibited IC50 values of 63 or 73 microM in Raw264.7 cells, 51 or >100 microM in human monocytes, and 10 or 37 microM in human neutrophils, respectively. Trimethylresveratrol, piceid, and 3,5-dihydroxy-4'-methoxystilbene-3-O-beta-D-glucoside were weak inhibitors of ROS production. Thus, resveratrol was identified as a potent inhibitor of ROS production, which might be one biochemical mechanism related to its anti-inflammatory and anticarcinogenic activities. The number and position of hydroxy substituents in resveratrol analogs seem to play an important role in the inhibitory potency of ROS production.

Barbarin as a new tyrosinase inhibitor from Barbarea orthocerus.

A tyrosinase inhibitor was isolated from the whole plant of Barbarea orthocerus Led. (Brassicaceae) by activity-guided fractionation, and identified as (R)-5-phenyl-2-oxazolidinethione (barbarin) by structural analysis followed by comparison with reported spectral data. The compound exhibited significant inhibitory effects on mushroom and murine tyrosinases at more than 1.6 x 10(-5) M. Barbarin exhibited IC50 values of 4.2 x 10(-5) M on mushroom tyrosinase and of 4.8 x 10(-5) M on murine tyrosinase. Kojic acid as a positive control exhibited IC50 values of 3.4 x 10(-5) M and 6.0 x 10(-5) M on mushroom and murine tyrosinases, respectively. Therefore, barbarin exhibited a similar level of inhibitory potency with kojic acid used as a positive control. In a kinetic study with various concentrations of L-dopa as the substrate, barbarin was identified as an uncompetitive inhibitor and kojic acid as a mixed inhibitor of both mushroom and murine tyrosinases. Barbarin exhibited KEIS values of 3.3 x 10(-5) M and 3.6 x 10(-5) M on mushroom and murine tyrosinases, respectively. Kojic acid exhibited KEIS and KEI values of 2.4 x 10(-5) M and 2.2 x 10(-5) M on mushroom tyrosinase and those of 8.9 x 10(-5) M and 7.2 x 10(-5) M on murine tyrosinase, respectively.

Reynosin from Sassurea lappa as inhibitor on CINC-1 induction in LPS-stimulated NRK-52E cells.

An inhibitor on CINC-1 (cytokine-induced neutrophil chemoattractant-1) induction in LPS-stimulated rat kidney epithelioid NRK-52E cells was purified from the roots of Sassurea lappa Clarke, a herbal medicine used in Korean traditional prescriptions for gastric intestinal diseases by a variety of column chromatographic procedures. The inhibitor was identified as reynosin, a sesquiterpene lactone isolated and characterized previously from Ambrosia confertiflora DC., and Magnolia grandiflora L. Reynosin exhibited a dose-dependent inhibition on CINC-1 induction in LPS-stimulated NRK-52E cells, where 50% of inhibitory effect was shown at the concentration of about 1 microM.

Genetic structure of the bphG gene encoding 2-hydroxymuconic semialdehyde dehydrogenase of Achromobacter xylosoxidans KF701.

2-Hydroxymuconic semialdehyde dehydrogenase catalyzes the conversion of 2-hydroxymuconic semialdehyde (HMS) to an enol form of 4-oxalocrotonate which is a step in the catechol meta-cleavage pathway. A bphG gene encoding HMS dehydrogenase of A. xylosoxidans KF701, a soil bacterium degrading biphenyl, was identified at between catechol 2,3-dioxygenase gene and HMS hydrolase gene, and its sequence was analyzed. An open reading frame (ORF) corresponding to bphG gene was consisted of 1461 nucleotides with ATG initiation codon and TGA termination codon. The ORF exhibited 66% of G + C content, and a putative ribosome-binding sequence, AGAGA, was identified at about 10 nucleotides upstream initiation codon of the bphG gene. The bphG gene can encode a polypeptide of molecular weight 52 kDa containing 486 amino acid residues. A deduced amino acid sequence of HMS dehydrogenase encoded in bphG gene from A. xylosoxidans KF701 exhibited the highest 94% homology with that of corresponding enzyme encoded in xylG from P. putida mt-2, 63% to 90% homology with those of other reported HMS dehydrogenases, and 29% to 42% homology with those of betaine aldehyde dehydrogenase, 5-carboxy-HMS dehydrogenase, aldehyde dehydrogenase, indole-3-acetaldehyde dehydrogenase, succinic semialdehyde dehydrogenase, methylmalonate semialdehyde dehydrogenase, and succinylglutamate 5-semialdehyde dehydrogenase. From an alignment of amino acid sequence of HMS dehydrogenase from A xylosoxidans KF701 with other reported dehydrogenases, putative cofactor NAD(+)-binding regions and catalytic residues were identified.

Alpha-viniferin: a prostaglandin H2 synthase inhibitor from root of Carex humilis.

An inhibitor on cyclooxygenase activity of prostaglandin H2 synthase was purified from the root of Carex humilis Leyss (Cyperaceae) by a variety of column chromatographic methods. As a result of the structure analysis by FAB-mass, 1H-NMR, and 13C-NMR spectral data, the active compound was identified as (+)-alpha-viniferin, an oligomeric stilbene characterized originally from Caragana chamlagu Lamarck (Leguminosae). (+)-alpha-Viniferin exhibited a dose-dependent inhibition on cyclooxygenase activity, where 50% of inhibition (IC50) was shown at a final concentration of about 7 microM. Resveratrol, a putative building block of oligomeric stilbenes, also inhibited the cyclooxygenase activity. The inhibitory potency of (+)-alpha-viniferin was about 3- to 4-fold stronger than that of resveratrol on cyclooxygenase activity of prostaglandin H2 synthase partially purified from sheep seminal vesicles.

Structure of catechol 2,3-dioxygenase gene from Alcaligenes eutrophus 335.

Catechol 2,3-dioxygenase (C23O), one of extradiol-type dioxygenases cleaving aromatic C-C bond at meta position of dihydroxylated aromatic substrates, catalyzes the conversion of catechol to 2-hydroxymuconic semialdehyde. As our ongoing study to characterize biochemical and genetic properties of the extradiol-type dioxygenases at molecular level, a C23O gene encoded in chromosomal DNA of Alcaligenes eutrophus 335, a strain degrading phenol and p-cresol, was cloned. The C23O gene was localized in an 1.4-kb PstI fragment from A. eutrophus 335, and was expressed in E. coli HB101. The C23O exhibited the highest aromatic ring-fission activity to catechol as a substrate, and its relative activity to other dihydroxylated aromatic substrates was in order of catechol >> 4-methylcatechol > 3-methylcatechol, protocatechuate, 4-chlorocatechol > 3,4-dihydroxy-phenylacetate > 2,3-dihydroxybiphenyl. Nucleotide sequence of the 1.4-kb fragment has revealed that an open reading frame (ORF) corresponding to the C23O gene was composed of 930 base pairs. A putative ribosome-binding sequence of AGGAG was found at about 10 nucleotides upstream the ORF which can encode a polypeptide of molecular weight 34 kDa consisting of 309 amino acid residues. The deduced amino acid sequence of C23O from A. eutrophus 335 exhibited the highest 59% identity with those of corresponding enzymes from Pseudomonas sp. CF600 (p VI150), P. putida HS1 (pDK1), and P. putida PpG7 (NAH7). An alignment of amino acid sequences of extradiol-type dioxygenases including C23O from A. eutrophus 335 has revealed that catalytically and structurally important amino acid residues of the enzymes were conserved during evolution.

Oxyresveratrol as the potent inhibitor on dopa oxidase activity of mushroom tyrosinase.

Oxyresveratrol (2,3',4,5'-tetrahydroxystilbene), a naturally occurring compound particularly found in Morus alba L., exhibited a potent inhibitory effect on dopa oxidase activity of tyrosinase which catalyzes rate-limiting steps of melanin biosynthesis. Oxyresveratrol with 0.3 to 5 microM exhibited potent and dose-dependent inhibitions (25 to 84%) on the enzyme activity, where 50% of inhibition was shown at the concentration of about 1 microM. Oxyresveratrol seemed to inhibit the dopa oxidase activity of tyrosinase via a noncompetitive manner (Ki = 9.1 x 10(-7) M) when L-dopa was used as a substrate. Oxyresveratrol exhibited about a 150-fold more potent inhibitory effect than resveratrol (3,4',5-trihydroxystilbene). The more hydroxy groups of the hydroxystilbenes are methylated to be methoxy groups, while the less inhibitory effects on the enzyme activity were exhibited. The results indicate that both the number and positions of hydroxy groups in oxyresveratrol seem to play a critical role in exerting the inhibitory effect on dopa oxidase activity of mushroom tyrosinase.

Characterization of the gene encoding catechol 2,3-dioxygenase from Achromobacter xylosoxidans KF701.

Catechol 2,3-dioxygenase (C23O) catalyzes a meta cleavage of the aromatic ring in catechol to form 2-hydroxymuconic semialdehyde. A C23O gene was cloned from chromosomal DNA of A. xylosoxidans KF701, a soil bacterium degrading biphenyl, and expressed in E. coli HB101. In substrate specificity to catechol and its analogs, the C23O exhibited the highest aromatic ring-fission activity to catechol, and its relative activity to other dihydroxylated aromatics was 4-chlorocatechol > 4-methylcatechol > 3-methylcatechol >> 2, 3-dihydroxybiphenyl. Aromatic ring-fission activity of the C23O to catechol was about 40-fold higher than that to 2,3-dihydroxybiphenyl. Nucleotide sequence analysis of the C23O gene from A. xylosoxidans KF701 revealed an open reading frame consisting of 924 base pairs, and identified a putative ribosome-binding sequence (AGGTGA) at about 10 nucleotides upstream from the initiation codon. The open reading frame can encode a polypeptide chain with molecular weight of 34 kDa containing 307 amino acid residues. The deduced amino acid sequence of the C23O exhibited the highest homology with that of C23O from Pseudomonas sp. IC with 96% identity, and the least homology with that of C23O from P. putida F1 with 22% identity among reported C23O sequences. Furthermore, comparison of the C23O sequence with other extradiol dioxygenases has led to identification of evolutionally conserved amino acid residues whose possible catalytic and structural roles are proposed.