Extracellular production of azurin from Pseudomonas aeruginosa in the presence of Triton X-100 or Tween 80.

Azurin which is a bacterial secondary metabolite has attracted much attention as potential anticancer agent in recent years. This copper-containing periplasmic redox protein supresses the tumor growth selectively. High-level secretion of proteins into the culture medium offers a significant advantage over periplasmic or cytoplasmic expression. The aim of this study was to investigate the effect of nonionic surfactants on the expression of the Pseudomonas aeruginosa azurin. Different concentrations of Triton X-100 and Tween 80 were used as supplements in growth media and extracellular azurin production was stimulated by both surfactants. According to western blot analysis results, in the presence of Triton X-100, maximum azurin expression level was achieved with 96 h of incubation at 1% concentration, and 48 h at 2% concentration. On the other hand, maximum azurin expression level was achieved in the presence of 1% Tween 80 at 72 h incubation. This study suggested for the first time a high level of azurin secretion from P. aeruginosa in the presence of Triton X-100 or Tween 80, which would be advantageous for the purification procedure.

Eleutheroside K isolated from Acanthopanax henryi (Oliv.) Harms suppresses methicillin resistance of Staphylococcus aureus.

Acanthopanax (A.) henryi (Oliv.) Harms contain many bioactive compounds commonly used in traditional Chinese medicine. The objective of the present study was to investigate the antibacterial activity of the single constituent, Eleutheroside K (ETSK) isolated from the leaves of A. henryi (Oliv.) Harms, against methicillin-resistant Staphylococcus (S.) aureus (MRSA). Broth microdilution assay was used to measure the minimal inhibitory concentration (MIC) and the MIC values of ETSK against eight clinical S. aureus strains were all 50 µg ml-1 . At sub-inhibitory concentrations, a synergistic effect between oxacillin (OXA) and ETSK was confirmed using checkerboard dilution assay and time-kill curve analysis. The bacteriostatic effect became more pronounced when ETSK was used in combination with detergent (Triton X-100) or ATPase inhibitor (N, N'-dicyclohexylcarbodiimide). According to western blot analysis, the down-regulated expression of Penicillin-binding protein 2a (PBP2a) further validated that the bacterial activity was inhibited when treated with ETSK in a dose-dependent manner. Results based on our study verified that ETSK significantly suppressed MRSA infections and emphasized the potential application of ETSK as a novel anti-MRSA natural drug.

Characterization of an efficient estrogen-degrading bacterium Stenotrophomonas maltophilia SJTH1 in saline-, alkaline-, heavy metal-contained environments or solid soil and identification of four 17ß-estradiol-oxidizing dehydrogenases.

The efficient bioremediation of estrogen contamination in complex environments is of great concern. Here the strain Stenotrophomonas maltophilia SJTH1 was found with great and stable estrogen-degradation efficiency even under stress environments. The strain could utilize 17ß-estradiol (E2) as a carbon source and degrade 90% of 10 mg/L E2 in a week; estrone (E1) was the first degrading intermediate of E2. Notably, diverse pH conditions (3.0-11.0) and supplements of 4% salinity, 6.25 mg/L of heavy metal (Cd2+ or Cu2+), or 1 CMC of surfactant (Tween 80/ Triton X-100) had little effect on its cell growth and estrogen degradation. The addition of low concentrations of copper and Tween 80 even promoted its E2 degradation. Bioaugmentation of strain SJTH1 into solid clay soil achieved over 80% removal of E2 contamination (10 mg/kg) within two weeks. Further, the whole genome sequence of S. maltophilia SJTH1 was obtained, and a series of potential genes participating in stress-tolerance and estrogen-degradation were predicted. Four dehydrogenases similar to 17ß-hydroxysteroid dehydrogenases (17ß-HSDs) were found to be induced by E2, and the four heterogenous-expressed enzymes could oxidize E2 into E1 efficiently. This work could promote bioremediation appliance potential with microorganisms and biodegradation mechanism study of estrogens in complex real environments.

Consortia of bioactives in supercritical carbon dioxide extracts of mustard and small cardamom seeds lower serum cholesterol levels in rats: new leads for hypocholesterolaemic supplements from spices.

Melatonin-rich and 1,8-cineole-rich extracts have been successfully obtained from yellow mustard (YM) and small cardamom (SC) seeds, respectively, employing green technology of supercritical CO2 (SC-CO2) extraction. Chemical profiling confirmed the presence of melatonin and 1,8-cineole and co-extractants in the respective extracts. Electron paramagnetic resonance spectroscopy attested strong antioxidant activities of the extracts foregoing pan-assay interference compounds involved in spectroscopic analysis. These extracts also exhibited synergistic efficacies greater than unity confirming antioxidant synergy among the co-extracted bioactives therein. To ascertain hypocholesterolaemic efficacies, these extracts were co-administered orally with Triton X (at the pre-optimised dose of 175 mg/kg body weight (BW)) to Wistar albino rats at doses of 550, 175 and 55 mg/kg BW. Serum total cholesterol levels in the rats were monitored on days 3, 7, 15 and 21. On day 21, total cholesterol level reduced appreciably by 49·44 % in rats treated with YM seed extract and by 48·95 % in rats treated with SC seed extract, comparable with atorvastatin-administered rats (51·09 %). Either extract demonstrated inhibitory effects on hepatic 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase activity. A molecular docking exercise identified specific compounds in the extracts which possessed binding affinities comparable with therapeutically used HMG-CoA reductase inhibitors. In silico and in vivo studies concertedly concluded that the consortium of bioactive components in the extracts cannot be considered as invalid metabolic panaceas and therefore these 'green' extracts could be safely subjected to clinical studies as preventive biotherapeutics for hypercholesterolaemia. These extracts could be consumed per se as hypocholesterolaemic supplements or could be ingredients of new spice-based therapeutic foods.

Heterologous production of free dihomo-γ-linolenic acid by Aspergillus oryzae and its extracellular release via surfactant supplementation.

Free dihomo-γ-linolenic acid (DGLA) and its desaturated form, free arachidonic acid (ARA) are polyunsaturated free fatty acids (FFAs). They are useful raw materials to produce eicosanoid pharmaceuticals. In this study, we aimed at their production by the oleaginous filamentous fungus Aspergillus oryzae via metabolic engineering. Three genes encoding enzymes involved in the synthesis of DGLA and ARA, were isolated from the filamentous fungus Mortierella alpina that produces ARA in a triacylglycerol form. These genes were concatenated to promoters and terminators of highly expressed genes of A. oryzae, and the concatenated DNA fragments were further concatenated with each other to generate a single DNA fragment in the form of a biosynthetic gene cluster. By homologous recombination, the resulting DNA fragment was integrated to the chromosome of the A. oryzae acyl-CoA synthetase gene disruptant whose FFA productivity was enhanced at 9.2-fold more than the wild-type strain. The DNA-integrated disruptant produced free DGLA but did not produce free ARA. Thus, focusing on free DGLA, after removal of the gene for converting DGLA to ARA, the constructed strain produced free DGLA at 145 mg/l for 5 d. Also, by supplementing Triton X-100 surfactant at 1% to the culture, over 80% of free DGLA was released from cells without inhibiting the growth. Consequently, the constructed strain will be useful for attempting production of free DGLA-derived eicosanoids because it bypasses excision of free DGLA from triacylglycerols by lipase. To our knowledge, this is the first report on microbial production of free DGLA and its extracellular release.

Improvement of the Culture Medium for the Dichlorvos-Ammonia (DV-AM) Method to Selectively Detect Aflatoxigenic Fungi in Soil.

The dichlorvos-ammonia (DV-AM) method is a simple but sensitive visual method for detecting aflatoxigenic fungi. Here we sought to develop a selective medium that is appropriate for the growth of aflatoxigenic fungi among soil mycoflora. We examined the effects of different concentrations of carbon sources (sucrose and glucose) and detergents (deoxycholate (DOC), Triton X-100, and Tween 80) on microorganisms in soils, using agar medium supplemented with chloramphenicol. The results demonstrated that 5⁻10% sucrose concentrations and 0.1⁻0.15% DOC concentrations were appropriate for the selective detection of aflatoxigenic fungi in soil. We also identified the optimal constituents of the medium on which the normal rapid growth of Rhizopus sp. was completely inhibited. By using the new medium along with the DV-AM method, we succeeded in the isolation of aflatoxigenic fungi from non-agricultural fields in Fukui city, Japan. The fungi were identified as Aspergillus nomius based on their calmodulin gene sequences. These results indicate that the new medium will be useful in practice for the detection of aflatoxigenic fungi in soil samples including those from non-agricultural environments.

Releasing intracellular product to prepare whole cell biocatalyst for biosynthesis of Monascus pigments in water-edible oil two-phase system.

Selective releasing intracellular product in Triton X-100 micelle aqueous solution to prepare whole cell biocatalyst is a novel strategy for biosynthesis of Monascus pigments, in which cell suspension culture exhibits some advantages comparing with the corresponding growing cell submerged culture. In the present work, the nonionic surfactant Triton X-100 was successfully replaced by edible plant oils for releasing intracellular Monascus pigments. High concentration of Monascus pigments (with absorbance nearly 710 AU at 470 nm in the oil phase, normalized to the aqueous phase volume approximately 142 AU) was achieved by cell suspension culture in peanut oil-water two-phase system. Furthermore, the utilization of edible oil as extractant also fulfills the demand for application of Monascus pigments as natural food colorant.

High level extracellular production of a recombinant alkaline catalase in E. coli BL21 under ethanol stress and its application in hydrogen peroxide removal after cotton fabrics bleaching.

The effects of induction parameters, osmolytes and ethanol stress on the productivity of the recombinant alkaline catalase (KatA) in Escherichia coli BL21 (pET26b-KatA) were investigated. The yield of soluble KatA was significantly enhanced by 2% ethanol stress. And a certain amount of Triton X-100 supplementation could markedly improved extracellular ratio of KatA. A total soluble catalase activity of 78,762U/mL with the extracellular ratio of 92.5% was achieved by fed-batch fermentation in a 10L fermentor, which was the highest yield so far. The purified KatA showed high stability at 50°C and pH 6-10. Application of KatA for elimination of H2O2 after cotton fabrics bleaching led to less consumption of water, steam and electric power by 25%, 12% and 16.7% respectively without productivity and quality losing of cotton fabrics. Thus, the recombinant KatA is a promising candidate for industrial production and applications.

Investigation of relationship between lipid and Monascus pigment accumulation by extractive fermentation.

Fermented Monascus pigments have been utilized as traditional Chinese medicine and food colorant for thousands of years. Under the limited nitrogen concentration and/or low initial pH 2.5 conditions, it was observed that production of intracellular pigments and accumulation of microbial lipids (high content reaching to approximately 50% in dry cell weight) by edible Monascus anka exhibited a positive correlated relationship. Extractive fermentation in nonionic surfactant micelle aqueous solution selectively exported the intracellular Monascus pigments into its extracellular broth, in which the concentration of intracellular pigments was negligible while the extracellular one was enhanced. The extractive fermentation provides a novel strategy for shifting of the metabolic channeling from intracellular lipid accumulation to Monascus pigment production. High pigment concentration, i.e., approximately 40 AU of extracellular Monascus pigments, was achieved by extractive fermentation at a relatively high nonionic surfactant concentration 10 g/l. This phenomenon might be attributed to the nonionic surfactant micelles acting as pigment reservoirs by biomimetic of intracellular lipids.

Secretion of recombinant archeal lipase mediated by SVP2 signal peptide in Escherichia coli and its optimization by response surface methodology.

Towards the targeting of recombinant Thermoanaerobacter thermohydrosulfuricus lipase (TtL) for secretion into the culture medium of Escherichia coli, we have investigated a combination of the archeal lipase gene with a Salinovibrio metalloprotease (SVP2) signal peptide sequence. The SVP2 signal peptide has shown all necessary features of a leader sequence for high level secretion of a recombinant target protein in E. coli. Two sets of primers were designed for amplification of the corresponding gene fragments by PCR. Firstly, the PCR product of the TtL gene with designed restriction sites of SacI and HindIII was cloned into pQE-80L plasmid, named as pQE80L-TtL. Afterwards, the amplified fragment of SVP2 signal peptide with EcoRI and SacI restriction sites was also cloned into pQE80L-TtL and the final construct pQE-STL was obtained. A study on the extracellular expression of recombinant STL revealed that most of the enzyme activity was located in the periplasmic space. Glycine and Triton X-100 were investigated to determine whether the leakage of recombinant STL from the outer membrane was promoted, and it was revealed that glycine has a positive effect. Statistical media optimization design was then applied to optimize the effect of seven factors including glycine, Triton X-100, IPTG, yeast extract concentration, incubation time, induction time, and temperature on the extracellular expression of STL. The optimum conditions for the secretion of the lipase was obtained by incubating recombinant E. coli BL21 cells in the medium supplemented by 1.27% glycine and 24h of incubation in the presence of 0.2mM IPTG concentration.

Cellular uptake of Nigella sativa oil-PLGA microparticle by PC-12 cell line.

The aim of this study is to investigate the cell uptake of Nigella sativa oil (NSO)-PLGA microparticle by neuron-like PC-12 cells in comparison to surfactants; hydrophilic (Tween 80 & Triton X100) and hydrophobic (Span 80). Solvent evaporation was used to precisely control the size, zeta potential and morphology of the particle. The results revealed varying efficiencies of the cell uptake by PC-12 cells, which may be partially attributed to the surface hydrophobicity of the microparticles. Interestingly, the uptake efficiency of PC-12 cells was higher with the more hydrophilic microparticle. NSO microparticle showed evidence of being preferably internalised by mitotic cells. Tween 80 microparticle showed the highest cell uptake efficiency with a concentration-dependent pattern suggesting its use as uptake enhancer for non-scavenging cells. In conclusion, PC-12 cells can take up NSO-PLGA microparticle which may have potential in the treatment of neurodegenerative disease.

[Effect of Triton X-100 on genetic segregation and associated monocotyledonous and dicotyledonous traits in sugarbeet (Beta vulgaris L.)].

The effect of Triton X-100 (TX-100) on the ratio of phenotypic classes and the expression of morphological traits in the progeny of sugar beet hybrids (N12 and N2) was investigated. It was shown that the TX-100 exposition on the unopened flower buds of sugar beets has different effects on hybrid progenies. In agamospermic progeny of hybrid plant No 12km-4, a significant decrease in the heteroallelic (heterozygous) phenotypic classes of alcohol dehydrogenase (ADH1) fraction was determined in the nonagamospermic progeny of hybrid plant No 2km-2 appearance of sugar beet seedlings with one cotyledon leaf was detected. The obtained results indicate the high efficiency of the epimutagenic effect of TX-100 on the early stages of plant ontogenesis.

Pre-harvest methyl jasmonate treatment enhances cauliflower chemoprotective attributes without a loss in postharvest quality.

Methyl jasmonate (MeJA) treatment can significantly increase glucosinolate (GS) concentrations in Brassica vegetables and potentially enhance anticancer bioactivity. Although MeJA treatment may promote ethylene biosynthesis, which can be detrimental to postharvest quality, there are no previous reports of its effect on cauliflower postharvest quality. To address this, cauliflower curds in field plots were sprayed with either 0.1 % Triton X-100 (control) or 500 µM MeJA solutions four days prior to harvest, then stored at 4 °C. Tissue subsamples were collected after 0, 10, 20, and 30 days of postharvest storage and assayed for visual color change, ethylene production, GS concentrations, and extract quinone reductase inductive activity. MeJA treatment increased curd GS concentrations of glucoraphanin, glucobrassicin, and neoglucobrassicin by 1.5, 2.4, and 4.6-fold over controls, respectively. MeJA treated cauliflower showed significantly higher quinone reductase activity, a biomarker for anticancer bioactivity, without reducing visual color and postharvest quality for 10 days at 4 °C storage.

Antimicrobial activity of nanoemulsion in combination with cetylpyridinium chloride in multidrug-resistant Acinetobacter baumannii.

Acinetobacter baumannii has emerged as a serious problematic pathogen due to the ever-increasing presence of antibiotic resistance, demonstrating a need for novel, broad-spectrum antimicrobial therapeutic options. Antimicrobial nanoemulsions are emulsified mixtures of detergent, oil, and water (droplet size, 100 to 800 nm) which have broad antimicrobial activity against bacteria, enveloped viruses, and fungi. Here, we screened the antimicrobial activities of five nanoemulsion preparations against four Acinetobacter baumannii isolates to identify the most suitable preparation for further evaluation. Among them, N5, which contains 10% (vol/vol) Triton X-100, 25% (vol/vol) soybean oil, and 1% (wt/vol) cetylpyridinium chloride (CPC), showed the best efficacy against A. baumannii in both its planktonic and biofilm forms and was selected for further study. Our data demonstrate that, while the killing of planktonic forms of A. baumannii was due to the 1% CPC component of our nanoemulsions, the breakdown of biofilms was achieved via the emulsified oil and detergent fractions. Furthermore, we documented the effect of ethanol and NaCl in combination with N5 on planktonic A. baumannii. In killing curves of N5 combined with other agents (ethanol or NaCl), a synergistic effect of a ≥ 2-log decrease in CFU/ml was observed. The antibiofilm activity of N5 was confirmed via a cell proliferation test and scanning electron microscopy. The effects of exposure to severe environmental conditions, which simulates the field conditions in Iraq and Afghanistan, were evaluated, and this exposure did not affect the overall antimicrobial activity of N5. These studies lay a solid foundation for the utilization of nanoemulsions against the antibiotic-resistant forms of A. baumannii.

Effects of surfactants on rhizodegradation of oil in a contaminated soil.

The effects of nonionic surfactants on degradation of engine oil in metal contaminated soil using Indian mustard (Brassica juncea) were investigated. Triton X-100 and Tween 80 were individually applied to test pots in which the soil had been earlier spiked with 500 mg kg(-1) of used engine oil, 500 mg kg(-1) of PbCl(2) and 50 mg kg(-1) of CdCl(2). For he application of Tween 80 to the soil, the fractions of rhizodegraded oil and the fractions of removed metals from the soil were well correlated. On the other hand, such a correlation did not exist between the fractions of rhizodegraded oil and the fractions of removed metals for the application of Triton X-100 to the soil. It was observed that Triton X-100 caused a significant decrease in basal soil respiration (BSR) which can be attributed to a reduction in microbial activity. This, in turn, resulted in a reduction of the rhizodegraded oil fraction. Tween 80 proved to be effective in the rhizodegradation of oil under aerobic conditions. Further, this surfactant seems to have the positive effect on the soil microbial population when viewed in terms of BSR.

Flip-flop of phospholipids in proteoliposomes reconstituted from detergent extract of chloroplast membranes: kinetics and phospholipid specificity.

Eukaryotic cells are compartmentalized into distinct sub-cellular organelles by lipid bilayers, which are known to be involved in numerous cellular processes. The wide repertoire of lipids, synthesized in the biogenic membranes like the endoplasmic reticulum and bacterial cytoplasmic membranes are initially localized in the cytosolic leaflet and some of these lipids have to be translocated to the exoplasmic leaflet for membrane biogenesis and uniform growth. It is known that phospholipid (PL) translocation in biogenic membranes is mediated by specific membrane proteins which occur in a rapid, bi-directional fashion without metabolic energy requirement and with no specificity to PL head group. A recent study reported the existence of biogenic membrane flippases in plants and that the mechanism of plant membrane biogenesis was similar to that found in animals. In this study, we demonstrate for the first time ATP independent and ATP dependent flippase activity in chloroplast membranes of plants. For this, we generated proteoliposomes from Triton X-100 extract of intact chloroplast, envelope membrane and thylakoid isolated from spinach leaves and assayed for flippase activity using fluorescent labeled phospholipids. Half-life time of flipping was found to be 6 ± 1 min. We also show that: (a) intact chloroplast and envelope membrane reconstituted proteoliposomes can flip fluorescent labeled analogs of phosphatidylcholine in ATP independent manner, (b) envelope membrane and thylakoid reconstituted proteoliposomes can flip phosphatidylglycerol in ATP dependent manner, (c) Biogenic membrane ATP independent PC flipping activity is protein mediated and (d) the kinetics of PC translocation gets affected differently upon treatment with protease and protein modifying reagents.

[Effect of colchicine and Triton X-100 on expression of the enzyme-encoding genes in nongerminating seeds of sugarbeet (Beta vulgaris L.)].

The expression of the enzyme-coding genes, controlling glucose-phosphate isomerase (GPI), malate dehydrogenase (MDH), and alcohol dehydrogenase (ADH), was examined in nongerminating seeds of sugarbeet after Triton X-100 (TX-100) and colchicine treatment. Two types of changes revealed included modification of the enzymatic loci expression (change of the isozyme electrophoretic mobility) and inactivation of standard profiles. In the MDH and GPI systems, these processes were found to be associated. Complete isozyme modification was accompanied with the disappearance of standard profiles. In the ADH system, the treatment with TX-100 and colchicine gave rise to two independent processes, including silencing of the Adh1 locus and the appearance of the ADH isozymes with abnormal electrophoretic mobility, which were probably the products of the Adh2 locus. It was suggested that the effect of TX-100 and colchicine on the expression of the enzyme-encoding genes examined depended on the intracellular localization of the encoded enzymes.

Antimicrobial activity of nanoemulsion on cariogenic Streptococcus mutans.

OBJECTIVE: To assess antimicrobial activities of nanoemulsion (NE) to control the adhesion and biofilm formation by Streptococcus mutans by in vitro. DESIGN: In vitro antimicrobial susceptibility of nanoemulsion was determined as per National Committee for Clinical Laboratory Standards guidelines and agar diffusion, serial dilution technique for the determination of minimum inhibitory concentration and minimum bactericidal concentration (MIC/MBC). Efficacy was tested by kinetics of killing, biofilm assay and scanning electron microscopy. RESULTS: : NE concentrations ranging from 1:100 to 1:10,000 dilutions were effective against S. mutans as shown through MIC/MBC assays. NE showed antimicrobial activity against planktonic cells at high dilutions, confirmed by time kill studies. 4-day-old S. mutans biofilms were treated with NE; subsequent reductions of bacterial cell counts were noticed with decreasing dilutions. Staining of NE-treated biofilms with LIVE/DEAD BacLight resulted in dead cell areas of up to 48% in 1 min, 84% at 1h and significant (<0.05) increases in dead cell counts at all time points. Damage to cell membranes and cell walls of S. mutans by NE was demonstrated using scanning electron microscopy (SEM). CONCLUSION: These results suggest that nanoemulsion has effective antibacterial activity against S. mutans and may be a useful medication in the prevention of dental caries.

Novel method of gene transfer in birds: intracytoplasmic sperm injection for green fluorescent protein expression in quail blastoderms.

This study was conducted to establish a new method of avian transgenesis by intracytoplasmic sperm injection (ICSI). First, we evaluated the fertilization ability of quail oocytes after microinjection of Triton X-100 (TX-100)-treated quail sperm with PLCZ cRNA. The quail oocytes were cultured for 24 h, and blastoderm development was examined by histological observation. The TX-100 treatment induced damage to the quail sperm membrane and interfered with fertilization of oocytes injected with sperm. On the other hand, when quail oocytes were injected with TX-100-treated sperm and PLCZ cRNA simultaneously, 43.5% (10/23) of the oocytes developed into blastoderms. This rate of development was comparable to that for oocytes injected with sperm without TX-100 treatment but with PLCZ cRNA (6 [42.9%] of 14). Second, we evaluated the rate of transduction of the enhanced green fluorescent protein (EGFP) gene in quail oocytes injected with TX-100-treated sperm and PLCZ cRNA. The EGFP expression was assessed by histological observation of fluorescence emission in the embryos. The intracytoplasmic injection of sperm without TX-100 treatment but with PLCZ cRNA and EGFP vector induced blastoderm development in 40% (4/10) of the oocytes, but those oocytes showed no fluorescence emission. In contrast, the intracytoplasmic injection of TX-100-treated sperm and PLCZ cRNA induced blastoderm development in 43.8% (7/16) of the oocytes, and, importantly, 85.7% (6/7) of oocytes showed fluorescence emission. In addition, PCR analysis detected GFP fragments in 50% (3/6) of GFP-expressing blastoderms. These results indicate that this ICSI method with additional treatments described herein may be the first step toward the production of transgenic birds.

The polyphenol epigallocatechin-3-gallate affects lipid rafts to block activation of the c-Met receptor in prostate cancer cells.

The HGF/c-Met pathway is an important regulator of signaling pathways responsible for invasion and metastasis of most human cancers, including prostate cancer. Exposure of DU145 prostate tumor cells to HGF stimulates the PI3-kinase and MAPK pathways, leading to increased scattering, motility, and invasion, which was prevented by the addition of EGCG. EGCG acted at the level of preventing phosphorylation of tyrosines 1234/1235 in the kinase domain of the c-Met receptor without effecting dimerization. HGF-induced changes were independent of the formation of reactive oxygen species, suggesting that EGCG functioned independent of its antioxidant ability. ECG, another tea polyphenol, was as effective as EGCG, while EGC and EC were less effective. EGCG added up to 4 h after the addition of HGF still blocked cell scattering and reduced the HGF-induced phosphorylation of c-Met, Akt, and Erk, suggesting that EGCG could act both by preventing activation of c-Met by HGF and by attenuating the activity of pathways already induced by HGF. HGF did not activate the MAPK and PI3-K pathways in cells treated with methyl-beta-cyclodextrin (mCD) to remove cholesterol. Furthermore, subcellular fractionation approaches demonstrated that only phosphorylated c-Met accumulated in Triton X-100 membrane insoluble fractions, supporting a role for lipid rafts in regulating c-Met signaling. Finally, EGCG treatment inhibited DiIC16 incorporation into membrane lipid ordered domains, and cholesterol partially inhibited the EGCG effects on signaling. Together, these results suggest that green tea polyphenols with the R1 galloyl group prevent activation of the c-Met receptor by altering the structure or function of lipid rafts.