Lactococcus lactis KR-050L extract suppresses house dust mite induced-atopic skin inflammation through inhibition of keratinocyte and mast cell activation.

AIMS: Atopic dermatitis (AD) is a chronic inflammatory skin disease, with a steadily increasing prevalence. Lactic acid bacteria (LAB) have been widely used in the food industry and are an attractive option for preventing and treating allergic skin diseases. We previously isolated new LABs including Lactococcus lactis KR-050L from Gajuknamu kimchi, and showed the anti-inflammatory effects of extract of L. lactis KR-050L culture broth (LLK). In this study, we investigated the effects of LLK on AD. METHODS AND RESULTS: For the in vitro study, we used human keratinocytes (HaCaT) and mast cells (RBL-2H3). In vivo study, we investigated the effects of LLK on Dermatophagoides farinae extract (DFE) and 2,4-dinitrochlorobenzene (DNCB)-induced atopic skin inflammation in mice. LLK suppressed expression of pro-inflammatory cytokines and chemokines by down-regulation of p38 MAPK, STAT1 and nuclear translocation of NF-κB in keratinocytes. Topical application of LLK suppressed AD symptoms based on reduction in ear thickness, serum IgE levels and immune cell infiltration. Furthermore, LLK inhibited serum histamine levels and mast cells infiltration in vivo, and reduced mast cells activation in vitro. CONCLUSIONS: These results suggest that LLK inhibits AD symptoms through inhibition of keratinocytes and mast cells activation. SIGNIFICANCE AND IMPACT OF THE STUDY: LLK is a potential therapeutic candidate for AD treatment.

Lactococcus lactis KR-050L inhibit IL-6/STAT3 activation.

AIMS: The purpose of this study was to investigate IL-6/STAT3 inhibitory activity using lactic acid bacteria (LABs) isolated from Gajuknamu kimchi. METHODS AND RESULTS: Six LABs were isolated from Gajuknamu kimchi and identified through 16S rRNA sequencing. Among them, the culture broth of Lactococcus lactis KR-050L inhibited IL-6-induced STAT3 luciferase activity. Fifteen compounds were isolated from the EtOAc extract of culture broth though column chromatography and preparative high-performance liquid chromatography, and they were identified as 2,5-diketopipperazine structures by spectroscopic analyses (MS, 1 H- and 13 C-NMR). They also showed inhibitory activities on IL-6-induced STAT3 activation, and showed the different in activity according to the presence of a phenylalanine residue, hydroxyl groups and isometric structure. CONCLUSIONS: The six new LABs isolated from Gajuknamu kimchi, and Lc. lactis KR-050L was selected as candidate IL-6/STAT3 inhibitors. The activity levels of 15 2,5-DKPs isolated from Lc. lactis KR-050L were verified. SIGNIFICANCE AND IMPACT OF THE STUDY: This study constitutes the first attempt to isolate various LABs from Gajuknamu kimchi and to discover IL-6/STAT3 inhibitors in the EtOAc extract of Lc. lactis KR-050L culture broth. Moreover, our data provide useful biochemical information regarding the commercialization of Lc. lactis isolated from Gajuknamu kimchi as an approach to use functional foods for the treatment of various diseases via IL-6/STAT3 activation.

Purification and functional characterization of the first stilbene glucoside-specific ß-glucosidase isolated from Lactobacillus kimchi.

This study aimed to develop viable enzymes for bioconversion of resveratrol-glucoside into resveratrol. Out of 13 bacterial strains tested, Lactobacillus kimchi JB301 could completely convert polydatin into resveratrol. The purified enzyme had an optimum temperature of 30-40°C and optimum pH of pH 5.0 against polydatin. This enzyme showed high substrate specificities towards different substrates in the following order: isorhaponticin>>polydatin>>mulberroside A>oxyresveratrol-3-O-glucoside. Additionally, it rarely hydrolyzed astringin and desoxyrhaponticin. Based on these catalytic specificities, we suggest this enzyme be named stilbene glucoside-specific ß-glucosidase. Furthermore, polydatin extracts from Polygonum cuspidatum were successfully converted to resveratrol with a high yield (of over 99%). Stilbene glucoside-specific ß-glucosidase is the first enzyme isolated from lactic acid bacteria capable of bio-converting various stilbene glucosides into stilbene.

Reversal of P-glycoprotein associated multidrug resistance by new isoprenoid derivatives.

To find a drug to overcome P-glycoprotein associated multidrug resistance, we synthesized 43 new isoprenoid derivatives. Ten compounds were effective in an in vitro assay with the human MDR-type resistant carcinoma KB/VJ-300 and MRP-type KB/VP-4 cell lines. One of the most effective compounds, N-5228 [trans-N,N'-bis(3,4-dimethoxybenzyl)-N-solanesyl-1,2-diaminocyclohexane, mol. wt 1100.481, was tested in P388/VCR-bearing mice. It showed a antitumor effect on MDR-type resistant tumor cells. Moreover, N-5228 potentiated the accumulation of [3H]vincristine in drug-resistant cells and blocked [3H]azidopine photoaffinity labeling of P-glycoprotein molecules in MDR-type resistant cell membranes. We think that N-5228 is promising as a lead compound in the screening of resistance reversing drugs for multidrug resistant cancers.

Reversal of multidrug resistance by 7-O-benzoylpyripyropene A in multidrug-resistant tumor cells.

7-O-Benzoylpyripyropene A (7-O-BzP), a semi-synthetic analog of pyripyropene, was investigated for its reversing effect on multidrug-resistant (MDR) tumor cells. 7-O-BzP (6.25 microg/ml) completely reversed resistance against vincristine and adriamycin in vincristine-resistant KB cells (VJ-300) and adriamycin-resistant P388 cells (P388/ADR), respectively. 7-O-BzP alone had no effect on the growth of drug sensitive and drug-resistant cells. 7-O-BzP (6.25 microg/ml) significantly enhanced accumulation of [3H]vincristine in VJ-300 cells and completely inhibited the binding of [3H]azidopine to the P-glycoprotein in VJ-300 cells and P388/ADR cells. The result suggests that 7-O-BzP effectively reverses P-glycoprotein-related MDR by interacting directly with P-glycoprotein in drug resistant VJ-300 and P388/ADR cells.

Reversal of multidrug resistance by kopsiflorine isolated from Kopsia dasyrachis.

Kopsiflorine, an indole alkaloid of the aspidofractinine-type isolated from Kopsia dasyrachis, was examined for its effect in enhancing drug cytotoxicity in multidrug-resistant tumor cells. The cytotoxicity of vincristine was enhanced in a concentration-dependent manner by kopsiflorine in drug-resistant KB cells (VJ-300). Kopsiflorine alone had no effect on the growth of drug sensitive or resistant cells, but the intracellular accumulation of vincristine was enhanced by kopsiflorine in VJ-300 cells. Kopsiflorine (10 micrograms/ml) significantly inhibited the binding of [3H]azidopine to P-glycoprotein in VJ-300 cells. The results suggest that kopsiflorine interacts directly with P-glycoprotein and inhibits the efflux of antitumor agents in drug-resistant cells.

Enhancement of drug accumulation by andrastin A produced by Penicillium sp. FO-3929 in vincristine-resistant KB cells.

In the course of our screening for compounds that reverse multidrug resistance, we found that the cytotoxicity of vincristine was enhanced 1.5-20-fold depending on the concentration of andrastin A in vincristine-resistant KB cells (VJ-300). Andrastin A alone had no effect on the growth of drug sensitive KB cells and VJ-300 cells. On the other hand, andrastin A (25 and 50 micrograms/ml) significantly enhanced accumulation of [3H]vincristine in VJ-300 cells. Andrastin A (50 micrograms/ml) completely inhibited the binding of [3H]azidopine to the P-glycoprotein in VJ-300 cells. The result suggests that andrastin A directly interacts with P-glycoprotein and inhibits the efflux of antitumor agents in drug resistant cells.

Two distinct effects of 12S-hydroxyeicosatetraenoic acid on platelet aggregation by zooxanthellatoxin-A and ionomycin.

The marine toxin zooxanthellatoxin-A (ZT-A) and the Ca2+ ionophore ionomycin caused aggregation in rabbit platelets. While ZT-A-induced platelet aggregation was inhibited by indomethacin, ionomycin-induced aggregation was potentiated by the drug. In contrast, both ZT-A- and ionomycin-induced accumulations of thromboxane B2 (TXB2), a stable metabolite of thromboxane A2 (TXA2), were completely inhibited by indomethacin. 12S-Hydroxyeicosatetraenoic acid (12-HETE), which could be accumulated in the presence of indomethacin, inhibited ZT-A-induced aggregation, but it potentiated the ionomycin-induced one. These results suggest that the different effects of indomethacin may be attributed to the distinct effects of 12-HETE on ZT-A- and ionomycin-induced platelet aggregations.

Reversal of multidrug resistance (MDR) by aspidofractinine-type indole alkaloids.

A series of indole alkaloids of the aspidofractinine-type was assessed for their potential in reversing MDR in vincristine-resistant KB cells. Of the compounds tested, kopsiflorine, kopsamine, pleiocarpine, 11-methoxykopsilongine, lahadinine A and N-methoxycarbonyl-11,12-methylenedioxy-delta 16,17-kopsinine were found to show appreciable activity.

Involvement of phospholipase C-gamma2 in activation of mitogen-activated protein kinase and phospholipase A2 by zooxanthellatoxin-A in rabbit platelets.

Zooxanthellatoxin-A (ZT-A), a polyhydroxypolyene isolated from a symbiotic dinoflagellate Symbiodinium sp., caused thromboxane A2-(TXA2) dependent and genistein-sensitive aggregation in rabbit platelets. Our study was performed to clarify the mechanism of the action of ZT-A. ZT-A caused an increase in tyrosine phosphorylation of 42-kDa protein, which is defined as p42 mitogen-activated protein kinase (MAPK) by immunoprecipitation. Although indomethacin (10 microM) completely inhibited ZT-A-induced TXB2 release, it partially inhibited the MAPK activation. The remained MAPK activation was completely inhibited by genistein (50 microM). Genistein (50 microM), by itself, abolished TXB2 release induced by ZT-A. ZT-A (2 microM) stimulated liberation of arachidonic acid and the subsequent metabolites such as TXB2 and 12-hydroperoxyeicosatetraenoic acid. However, ZT-A-stimulated phosphoinositide hydrolysis which was due to an increase in tyrosine phosphorylation of phospholipase C-(PLC)gamma2. The phosphorylation of PLC-gamma2 and the phosphoinositide hydrolysis were also partially inhibited by indomethacin (10 microM), and were abolished by a combined treatment of indomethacin (10 microM) and genistein (50 microM). ZT-A- (2 microM) induced MAPK activation in the presence of indomethacin (10 microM) was concentration-dependently inhibited by staurosporine and calphostin C, protein kinase C inhibitors. PD98059 (50 microM), a MAPK kinase inhibitor, also inhibited ZT-A-induced TXB2 release. Depletion of external Ca++ abolished ZT-A- (2 microM) induced MAPK activation, phosphoinositide hydrolysis, arachidonic acid liberation and TXB2 release. These results suggest that ZT-A stimulates a protein tyrosine kinase in the presence of external Ca++, resulting in the activation of MAPK probably via PLC-gamma2 and protein kinase C. The MAPK stimulated a liberation of arachidonic acid that is rapidly converted to TXA2. The released TXA2 causes aggregation accompanied with second stimulation of MAPK cascade.

Tyrphostin 23 blocks phosphorylation of p42 but not p38 mitogen-activated protein kinase by zooxanthellatoxin-A.

Zooxanthellatoxin-A isolated from a symbiotic dinoflagellate, caused aggregation in rabbit platelets that was inhibited by genistein (50 microM) and tyrphostin 23 (500 microM). Zooxanthellatoxin-A increased tyrosine phosphorylation of 42-kDa proteins which were identified as p42 and p38 mitogen-activated protein kinase (MAPK) by immunoprecipitation. Tyrphostin 23 inhibited the tyrosine phosphorylation of p42 MAPK but not p38 MAPK. In contrast, genistein abolished zooxanthellatoxin-A-induced tyrosine phosphorylation of both p42 and p38 MAPK. The results suggest that tyrphostin 23 selectively inhibits tyrosine phosphorylation of p42 MAPK. The p38 MAPK tyrosine phosphorylation is not involved in zooxanthellatoxin-A-induced platelet aggregation.

A sulfonoglycolipid with Na+,K+-ATPase inhibitory activity, produced by a cultured unique diatom symbiont isolated from a larger foraminifera.

A unique diatom (Entomoneis sp.) was isolated from a larger foraminifera (Marginopora vertebralis) and was successfully mass cultured. 6-Sulfo-O-alpha-D-quinovopyranosyldiacylglycerol was isolated from this diatom as an Na+,K+-ATPase inhibitor.

A novel inhibitor of platelet aggregation from the Cyanophyceae Oscillatoria rosea (NIES-208)

(2S)-1-O-Palmitoleyl-3-O-beta-D-galactopyranosylglycerol was isolated from the marine alga Oscillatoria rosea. It caused a concentration-dependent inhibition of platelet aggregation induced by U46619. Its structure was elucidated by spectroscopic analysis and chemical evidence.

A novel monogalactosylacylglycerol with inhibitory effect on platelet aggregation from the cyanophyceae Oscillatoria rosea.

(2S)-1-O-Palmitoyl-3-O-beta-D-galactopyranosylglycerol, isolated from the marine alga Oscillatoria rosea, inhibited platelet aggregation induced by U46619, a thromboxane A2 analogue. Its structure was elucidated by spectroscopic analysis and chemical evidence.

The mode of rabbit platelet shape change and aggregation induced by theonezolide-A, a novel polyketide macrolide, isolated from the Okinawan marine sponge Theonella sp.

Theonezolide-A (TZ-A), a novel polyketide macrolide, isolated from the Okinawan marine sponge Theonella sp., caused a marked platelet shape change at low concentrations (0.2-0.6 microM). Increasing concentrations of TZ-A to 6 microM or more caused shape change followed by a small but sustained aggregation. In a Ca(2+)-free solution, TZ-A-induced aggregation was markedly inhibited, although the marked shape change was still observed. Aggregation stimulated by TZ-A increased in a linear fashion with increasing Ca2+ concentrations from 0.1 to 3.0 mM. Furthermore TZ-A markedly enhanced 45Ca2+ uptake into platelets. Aggregation induced by TZ-A was inhibited by Arg-Gly-Asp-Ser, an inhibitor of fibrinogen binding to glycoprotein IIb-IIIa, H-7 and staurosporine, protein kinase C inhibitors, or genistein and tyrphostin A23, protein tyrosine kinase inhibitors, whereas shape change was blocked by genistein and tyrphostin A23. H-7 or staurosporine did not affect the TZ-A-induced shape change. These results suggest that TZ-A-induced platelet shape change is not dependent on external Ca2+, whereas TZ-A-induced aggregation is caused by an increase in Ca2+ permeability of the plasma membrane. It is also suggested that both aggregation and shape change induced by TZ-A are associated with protein phosphorylation by protein kinase C and tyrosine kinase.

Activation of rabbit platelets by Ca2+ influx and thromboxane A2 release in an external Ca(2+)-dependent manner by zooxanthellatoxin-A, a novel polyol.

1. Zooxanthellatoxin-A (ZT-A), a novel polyhydroxylated long chain compound, isolated from a symbiotic marine alga Simbiodinium sp., caused aggregation in rabbit washed platelets in a concentration-dependent manner (1-4 microM), accompanied by an increase in cytosolic Ca2+ concentration ([Ca2+]i). 2. ZT-A did not cause platelet aggregation or increase [Ca2+]i in a Ca(2+)-free solution, and Cd2+ (0.1-1 mM), Co2+ (1-10 mM) and Mn2+ (1-10 mM) inhibited ZT-A-induced aggregation. SK&F96365 (1-100 microM), a receptor operated Ca2+ channel antagonist, and mefenamic acid (0.1-10 microM), a non-specific divalent cation channel antagonist, inhibited platelet aggregation and the increase in [Ca2+]i induced by ZT-A. 3. Indomethacin (0.1-10 microM), a cyclo-oxygenase inhibitor, and SQ-29548 (0.1-10 microM), a thromboxane A2 (TXA2) receptor antagonist, inhibited platelet aggregation and the increase in [Ca2+]i induced by ZT-A. 4. Methysergide (0.01-1 microM), a 5-HT2 receptor antagonist, inhibited ZT-A-induced platelet aggregation but did not affect the increase in [Ca2+]i induced by ZT-A. 5. Tetrodotoxin (1 microM), a Na+ channel blocker and chlorpheniramine (1 microM), a H1-histamine receptor antagonist, neither affected ZT-A-induced platelet aggregation nor the increase in [Ca2+]i induced by ZT-A. 6. Genistein (1-100 microM), a protein tyrosine kinase inhibitor, and staurosporine (0.01-1 microM), a protein kinase C inhibitor, also inhibited ZT-A-induced platelet aggregation. 7. The present results suggest that ZT-A elicits Ca(2+)-influx from platelet plasma membranes. The resulting increase in [Ca2+]i subsequently stimulates the secondary release of TXA2 from platelets. Furthermore, the response to ZT-A may be associated with tyrosine phosphorylation.