Integrative Analysis of Metabolomics and Transcriptomics Reveals Molecular Mechanisms of Anthocyanin Metabolism in the Zikui Tea Plant (Camellia sinensis cv. Zikui).

In this study, we performed an association analysis of metabolomics and transcriptomics to reveal the anthocyanin biosynthesis mechanism in a new purple-leaf tea cultivar Zikui (Camellia sinensis cv. Zikui) (ZK). Three glycosylated anthocyanins were identified, including petunidin 3-O-glucoside, cyanidin 3-O-galactoside, and cyanidin 3-O-glucoside, and their contents were the highest in ZK leaves at 15 days. This is the first report on petunidin 3-O-glucoside in purple-leaf tea. Integrated analysis of the transcriptome and metabolome identified eleven dependent transcription factors, among which CsMYB90 had strong correlations with petunidin 3-O-glucoside, cyanidin 3-O-galactoside, and cyanidin 3-O-glucoside (PCC > 0.8). Furthermore, we also identified key correlated structural genes, including two positively correlated F3'H (flavonoid-3'-hydroxylase) genes, two positively correlated ANS (anthocyanin synthase) genes, and three negatively correlated PPO (polyphenol oxidase) genes. Overexpression of CsMYB90 in tobacco resulted in dark-purple transgenic calluses. These results showed that the increased accumulation of three anthocyanins in ZK may promote purple-leaf coloration because of changes in the expression levels of genes, including CsMYB90, F3'Hs, ANSs, and PPOs. These findings reveal new insight into the molecular mechanism of anthocyanin biosynthesis in purple-leaf tea plants and provide a series of candidate genes for the breeding of anthocyanin-rich cultivars.

Screening and identification of bioactive components resistant to metallo-beta-lactamase from Schisandra chinensis (Turcz.) Baill. by metalloenzyme-immobilized affinity chromatography.

The screening and identification of bioactive components, which are effectively resistant to metallo-beta-lactamase (MßL), were studied in the alcohol extract of Schisandra chinensis (Turcz.) Baill. by metalloenzyme-immobilized affinity chromatography. Taking bizinc metalloenzyme beta-lactamase II from Bacillus cereus (Bc II) and monozinc metalloenzyme CphA from aeromonas hydrophila (CphA) as examples, we studied the feasibility of this scheme based on the construction of metalloenzyme-immobilized chromatographic model. It was found that the Bc II- and CphA-immobilized chromatographic column could be used not only to explore the interaction between the MßL and their specific ligands, but also to screen the bioactive components from traditional Chinese medicine. The Bc II-and CphA-immobilized columns were used to screen the bioactive components from the alcohol extract of Schisandra chinensis (Turcz.) Baill. Time-of-flight tandem mass spectrometry analysis and molecular docking revealed that isobutyl 3-O-sulfo-ß-D-galactopyranoside is the effective bioactive components that could bind with metalloenzyme Bc II. It is believed that our current work may provide a methodological reference for screening MßL inhibitors from traditional Chinese medicine.

Consumption evaluation of one apple flesh a day in the initial phases prior to adenoma/adenocarcinoma in an azoxymethane rat colon carcinogenesis model.

Colorectal cancer (CRC) is the fourth cancer with the most new cases reported in 2018 worldwide. Consumption of fruit and vegetables is a protective factor against the risk of CRC. Beyond this, flavonoids could orchestrate these healthy effects. Apart from containing the typical apple flavonoids, red-fleshed apples also contain anthocyanins, mainly cyanidin-3-O-galactoside (Cy3Gal). Through an azoxymethane rat carcinogenesis model, a study was carried out in order to assess the possible protective effects of apple polyphenols, with special attention to anthocyanins. In addition, apart from negative and positive controls, a group with chemotherapy with 5-fluorouracil (5FU) was included to compare their performance against the output collected from the animal treatments with white-fleshed apple (WF), red-fleshed apple (RF) and Cy3Gal (AE). Although the 5FU group presented the best performance towards aberrant crypt foci (ACF) inhibition (70.1%), rats fed with white-fleshed apples ('Golden Smoothee') were able to achieve 41.3% ACF inhibition, while none of the challenged treatments (WF, RF and AE) suffered mucin depletion in their colonocytes. Expression changes of 17 genes related to CRC were assessed. In detail, the ACF inhibition phenotype detected in 5FU and WF groups could be explained through the expression changes detected in the apoptosis-related genes of Aurka, p53 and Cox2. Moreover, in the apple consumption groups (WF and RF), a reduced protein expression of matrix metalloproteinases with gelatinase activity (MMP-2 and 9) was detected. Overall, our study suggests an effect of apple polyphenols and apple anthocyanin Cy3Gal against colon carcinogenesis, retarding/diminishing the appearance of the precancerous markers studied.

ACTIVITY OF BLACKCURRANT AND CHOKEBERRY EXTRACTS AND TWO MAJOR CYANIDIN GLYCOSIDES AGAINST LIPID MEMBRANE OXIDATION AND THEIR BINDING PROPERTIES TO ALBUMIN.

The purpose of this study was to explain how extracts from chokeberry and blackcurrant interact with the lipid phase of biological membrane and with human albumin - the main protein of blood. Aiming at better understanding of the observed biological activity of the extracts, we also conducted experiments with their main components: cyanidin-3-0-galactoside and cyanidin-3-0-ruthinoside. Antioxidant activities of extracts and cyanidin derivatives were investigated with phosphatidy1choline liposomes and AAPH as oxidation inducing factor. Fluorescent probes (merocyanin and N-phenyl-1-naphthylamine) that were located at different depths within the membrane lipid bilayer were also used. The interaction between the compounds and human serum albumin was investigated using natural fluorescence quenching. According to our study it is highly likely that the significant antioxidant activity of chokeberry and blackcurrant extracts (IC50chokeberry = 4.92 pg/mL; IC50blackbcurrant = 7.04 µg/mL) is probably due to cyjanidin's main derivatives, which protect the lipid membrane more than the extracts. In addition, it has been suggested that the compounds are anchored mainly on the membrane surface and rigidify/order the lipids in the membrane. That rigidifying effect is the key factor for understanding their antioxidant properties. Experimental results have proved that all the study compounds quench the fluorescence of HSA through a static mechanism and the main interaction forces are the Van der Waals and hydrogen bonding interactions. The results of the study have improved our knowledge on how to protect membranes against lipid peroxidation using extracts rich in anthocyanins. The results can be relevant to pharmacists and nutritionists.

Effects of anthocyans on the expression of organic anion transporting polypeptides (SLCOs/OATPs) in primary human hepatocytes.

Anthocyans (anthocyanins and their aglycones anthocyanidins) are colorful pigments, naturally occurring in fruits. They exhibit many biological effects and have potent health benefits. Anthocyans are widely used as dietary supplements and the safety of products containing them is of great importance. To investigate whether anthocyans influence the expression of hepatic uptake transporters from the organic anion transporting polypeptide (SLCO gene/OATP protein) family, we carried out studies on primary cultures of human hepatocytes. The hepato-cellular accumulation of widely used drugs such as statins and some anticancer drugs is mediated by the liver-specific OATP1B1 and OATP1B3, thus any interference with expression of these particular transporters might influence therapeutic outcomes. We evaluated the effects of 21 anthocyanins and their corresponding 6 anthocyanidins on the expression levels of SLCO1B1/SLCO1B3 by RT-qPCR. Changes in OATP protein levels were confirmed by western blotting. Our data show that OATP1B1 responds differently to anthocyans compared with OATP1B3. We observed the induction of SLCO1B1 gene and OATP1B1 protein in four hepatocyte samples by the anthocyanins malvin chloride, malvidin-3-O-galactoside chloride and cyanidin-3-O-sophoroside chloride. For SLCO1B3, a reduction in the expression levels was seen with delphin chloride and the anthocyanidin pelargonidin. Although the values varied considerably between primary hepatocyte isolates from different individuals, a mean induction of SLCO1B1 (up to 60%) and reduction of SLCO1B3 (by less than 25%) were detected. We propose that the effects of anthocyans derived from high dose dietary supplements may have to be taken into account in patients undergoing a therapy with drugs transported by OATP1B1 and OATP1B3.

Selective inhibition of the cytochrome P450 isoform by hyperoside and its potent inhibition of CYP2D6.

Hyperoside, quercetin-3-O-galactoside, is a flavonoid isolated from Oenanthe javanica. In the present study, we investigated potential herb-drug inhibitory effects of hyperoside on nine cytochrome P450 (CYP) isoforms in pooled human liver microsomes (HLMs) and human recombinant cDNA expressed CYP using a cocktail probe assay. Hyperoside strongly inhibited CYP2D6-catalyzed dextromethorphan O-demethylation, with IC50 values of 1.2 and 0.81 µM after 0 and 15 min of preincubation, and a Ki value of 2.01 µM in HLMs, respectively. Hyperoside strongly decreased CYP2D6 activity dose-, but not time-, dependently in HLMs. In addition, the Lineweaver-Burk and Secondary plots for the inhibition of CYP2D6 in HLMs fitted a competitive inhibition mode. Furthermore, hyperoside decreased CYP2D6-catalyzed dextromethorphan O-demethylation activity of human recombinant cDNA-expressed CYP2D6, with an IC50 value of 3.87 µM. However, other CYPs were not inhibited significantly by hyperoside. In conclusion, our data demonstrate that hyperoside is a potent selective CYP2D6 inhibitor in HLMs, and suggest that hyperoside might cause herb-drug interactions when co-administrated with CYP2D substrates.

Developmental variation of phenolic compounds in fruit tissue of two apple cultivars.

BACKGROUND: Qualitative and quantitative analyses of 'Zonouz' and 'Gala' apples phenolic compounds were accomplished by HPLC. MATERIAL AND METHODS: Samples for phenolics study were taken at three different growing stages [1 - fruit early growing stage 2 - mid-stage of fruit development (80 days after full bloom) 3 - during commercial harvest time]. RESULTS: The results showed qualitative differences between two apple cultivars regarding phenolic compounds. The highest amounts of total phenols, flavonoids, flavonols and phenolic acids content in both cultivars were recorded during the fruit early growing stage. The high phenolic acids content was due to increasing in chlorogenic acid content in fruits during early growing stage. The highest amount for flavanol content was recorded in 'Zonouz' peel at the harvest time. Descending pattern was recorded for phloridzin dihydrate content during the season. 'Gala' peel had the greatest amounts for cyanidin-3-galactoside at harvest time. Increasing in total flavonoid content was due to the great amounts of cyanidine-3-galactoside and epicatechin (in 'Gala' peel), rutin hydrate (in 'Zonouz' pulp) at fruit early growing stage, catechin (in 'Zonouz' peel) during mid-stage of fruit development and cyanidin-3-galactoside and quercetin-3-D-galactoside (in 'Gala' peel) at the harvest time in both apple cultivars. CONCLUSIONS: There were meaningful quantitative differences between two cultivars, and 'Gala' was richer in phenolics than 'Zonouz'.

Selenium induces cholinergic motor neuron degeneration in Caenorhabditis elegans.

Selenium is an essential micronutrient required for cellular antioxidant systems, yet at higher doses it induces oxidative stress. Additionally, in vertebrates environmental exposures to toxic levels of selenium can cause paralysis and death. Here we show that selenium-induced oxidative stress leads to decreased cholinergic signaling and degeneration of cholinergic neurons required for movement and egg-laying in Caenorhabditis elegans. Exposure to high levels of selenium leads to proteolysis of a soluble muscle protein through mechanisms suppressible by two pharmacological agents, levamisole and aldicarb which enhance cholinergic signaling in muscle. In addition, animals with reduction-of-function mutations in genes encoding post-synaptic levamisole-sensitive acetylcholine receptor subunits or the vesicular acetylcholine transporter developed impaired forward movement faster during selenium-exposure than normal animals, again confirming that selenium reduces cholinergic signaling. Finally, the antioxidant reduced glutathione, inhibits selenium-induced reductions in egg-laying through a cellular protective mechanism dependent on the C. elegans glutaredoxin, GLRX-21. These studies provide evidence that the environmental toxicant selenium induces neurodegeneration of cholinergic neurons through depletion of glutathione, a mechanism linked to the neuropathology of Alzheimer's disease, amyotrophic lateral sclerosis, and Parkinson's disease.

The DC2.3 gene in Caenorhabditis elegans encodes a galectin that recognizes the galactoseß1→4fucose disaccharide unit.

Galectins comprise a large family of ß-galactoside-binding proteins in animals and fungi. We previously isolated cDNAs of 10 galectin and galectin-like genes (lec-1 to lec-6 and lec-8 to lec-11) from Caenorhabditis elegans and characterized the carbohydrate-binding properties of their recombinant proteins. In the present study, we isolated cDNA corresponding to an open reading frame of the DC2.3a gene from C. elegans total RNA; this cDNA encodes another potential galectin. A recombinant DC2.3a protein was expressed in Escherichia coli and used for analysis. The protein displayed hemagglutinating activity against rabbit erythrocytes, bound to an asialofetuin-Sepharose column, and was eluted with lactose. Furthermore, frontal affinity chromatography (FAC) analysis confirmed that DC2.3a recognized oligosaccharides with a non-reducing terminal galactose. According to these results, we designated DC2.3 as lec-12. The carbohydrate-binding property of the recombinant DC2.3a/LEC-12a was essentially similar to that of LEC-6. Additionally, DC2.3a/LEC-12a and LEC-6 showed higher affinities for the galactoseß1→4fucose (Galß1→4Fuc) disaccharide than for N-acetyllactosamine. This suggests that the principal recognition unit is the Galß1→4Fuc disaccharide as in the case of the C. elegans galectins. However, the recombinant DC2.3a/LEC-12a showed weak affinity for N-glycan E3, which was previously shown to be a preferential endogenous ligand for LEC-6. The DC2.3a/LEC-12a endogenous ligand structures appear to be somewhat different but contain the same galactose-fucose recognition motif.

Comparison of metabolic profiling of cyanidin-3-O-galactoside and extracts from blueberry in aged mice.

The metabonomics changes of plasma and brain tissue after dietary supplementation with blueberry extracts (BBE) and cyanidin-3-O-galactoside from blueberry (BBM) in aged mice were investigated by (1)H NMR technique. The mice received intragastric administration of BBE (200 mg/kg/day), BBM (50 mg/kg/day), and saline water (0.9%) for 6 weeks, respectively, in the BBE, BBM, and control groups. At the end of the experiment, plasma and brain samples were collected for NMR analysis. The results demonstrated that the level of choline in plasma from BBE and BBM groups were obviously elevated relative to the control group, whereas the levels of lactate and phosphocholine in plasma were remarkably reduced. Compared with those in the control group, the levels of choline and GABA in the brain from the BBE group were obviously increased, whereas glutamate and phosphocholine in the BBE group were significantly decreased. The level of taurine in the brain from the BBM group was particularly higher than that in the control group. These results indicated supplementation with BBE or BBM might induce similar changes of endogenous plasma and brain metabolic profiles in aged mice.

Inhibitors of oxidation and matrix metalloproteinases, floridoside, and D-isofloridoside from marine red alga Laurencia undulata.

In the exploration of abundant marine biological resources, edible red alga Laurencia undulata led to two bioactive isolates: floridoside (1) and D-isofloridoside (2). For the first time, the antioxidant properties of both derivatives (1 and 2) were characterized via free radical scavenging using the ESR technique, reactive oxygen species (ROS) inhibition, membrane protein oxidation, myeloperoxidase (MPO) inhibition, gene expression levels of glutathione (GSH) and superoxide dismutase (SOD), and protein expression of MMP-2 and MMP-9. The results demonstrate that floridoside and D-isofloridoside possess significant antioxidant capacity and are potential inhibitors of MMP-2 and MMP-9. These results clarified that these components may be responsible for the relative activities of crude extract from this genus, which is used as folk medicine. Furthermore, the structure-activity relationships were also suggested. Both isomers could be effective candidates for applications in food and pharmaceutical fields as natural marine antioxidants.

AB-type lectin (toxin/agglutinin) from mistletoe: differences in affinity of the two galactoside-binding Trp/Tyr-sites and regulation of their functionality by monomer/dimer equilibrium.

Viscumin of mistletoe (Viscum album L.) has a concentration-dependent activity profile unique to plant AB-toxins. It starts with lectin-dependent mitogenicity and then covers toxicity and cell agglutination, associated with shifts in the monomer/dimer equilibrium. Each lectin subunit harbors two sections for ligand contact. In the dimer, the B-chain sites in subdomain 2 gamma (designated as the Tyr-sites) appear fully accessible, whereas Trp-sites in subdomain 1 alpha are close to the dimer interface. It is unclear whether both types of sites operate similarly in binding glycoligands in solution. By systematically covering a broad range of lactose/lectin ratio in isothermal titration calorimetry, we obtained evidence for two sites showing dissimilar binding affinity. Intriguingly, the site with higher affinity was only partially occupied. To assign the observed properties to the Trp/Tyr-sites, we next performed chemically induced dynamic nuclear polarization measurements of Trp and Tyr accessibility. A Tyr signal, but not distinct Trp peaks, was recorded when testing the dimer. Lactose-quenchable Trp peaks became visible on the destabilization of the dimer by citraconylation, intimating Trp involvement in ligand contact in the monomer. Fittingly, Tyr acetylation but not mild Trp oxidation reduced the dimer hemagglutination activity and the extent of binding to asialofetuin-Sepharose 4B. Altogether, the results attribute lectin activity in the dimer primarily to Tyr-sites. Full access to Trp-sites is gained on dimer dissociation. Thus, the monomer/dimer equilibrium of viscumin regulates the operativity of these sites. Their structural divergence affords the possibility for differences in ligand selection when comparing monomers (Tyr- and Trp-sites) with dimers (primarily Tyr-sites).

Physicochemical characterization and gene transfection efficiency of lipid emulsions with various co-emulsifiers.

Transfection systems based on complexes of DNA and lipid emulsions were evaluated with respect to their effectiveness, toxicity, physicochemical characteristics, and cell-type dependence. The potential of a series of co-emulsifiers to serve as vectors was investigated. The co-emulsifiers examined included 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), Tween, cholesterol, stearylamine, and polyethylenimine (PEI). Squalane and 1,2-dioleoyl-sn-glycero-3-trimethylammonium-propane (DOTAP), respectively, were the main oil phase and cationic lipid added to the lipid emulsions. Cell viability was reduced after inclusion of either of the two cationic components of stearylamine and PEI. DOPE and cholesterol showed both higher transfection activity and cell viability as compared to the other co-emulsifiers. The incorporation of DOPE and cholesterol also prevented droplet aggregation of the emulsions after long-term storage. Results of the transfection of COS-1, A549, or HaCat cell lines with lipid emulsions indicated differences in transfection activities of each formulation for the different cell lines. It is concluded that DOPE and cholesterol as co-emulsifiers for DOTAP were preferable for stability and DNA transfection of emulsions.

Lack of nociceptin receptor alters body temperature during resting period in mice.

The role of nociceptin (NOC) receptor on body core temperature (Tcore) control was examined using NOC receptor knockout mice. In homozygote NOC receptor-knockout, wild-type, and control C57BL/6J and 129/SV mice, Tcore was continuously recorded under 12:12 h light:dark (LD) and conditions of constant darkness (DD). The Tcore values during the resting period were higher in the NOC receptor-knockout mice than in both wild-type and control mice under both LD and DD conditions. Spontaneous activity during the resting period and plasma cortisol levels were not different between the NOC receptor-knockout and control mice. The findings herein indicate that the NOC receptor is involved in the control of Tcore during the resting period and is independent of light, physical activity and/or cortisol regulation.

Overexpression of the secreted factor Mig30 expressed in the Spemann organizer impairs morphogenetic movements during Xenopus gastrulation.

The Spemann organizer secretes several antagonists of growth factors during gastrulation. We describe a novel secreted protein, Mig30, which is expressed in the anterior endomesoderm of the Spemann organizer. Mixer-inducible gene 30 (Mig30) was isolated as a target of Mixer, a homeobox gene required for endoderm development. The Mig30 gene encodes a secreted protein containing a cysteine-rich domain and an immunoglobulin-like domain that belongs to the insulin-like growth factor-binding protein family. Overexpression of Mig30 in the dorsal region results in the retardation of morphogenetic movements during gastrulation and leads to microcephalic embryos. Overexpression of Mig30 also inhibits activin-induced elongation of ectodermal explants without affecting gene expression patterns in mesoderm and endoderm. These results suggest that Mig30 is involved in the regulation of morphogenetic movements during gastrulation in the extracellular space of the Spemann organizer.

Involvement of EphA2 in the formation of the tail notochord via interaction with ephrinA1.

Eph receptors have been implicated in cell-to-cell interaction during embryogenesis. We generated EphA2 mutant mice using a gene trap method. Homozygous mutant mice developed short and kinky tails. In situ hybridization using a Brachyury probe found the notochord to be abnormally bifurcated at the caudal end between 11.5 and 12.5 days post coitum. EphA2 was expressed at the tip of the tail notochord, while one of its ligands, ephrinA1, was at the tail bud in normal mice. In contrast, EphA2-deficient notochordal cells were spread broadly into the tail bud. These observations suggest that EphA2 and its ligands are involved in the positioning of the tail notochord through repulsive signals between cells expressing these molecules on the surface.

Galactosides in the rhizosphere: utilization by Sinorhizobium meliloti and development of a biosensor.

Identifying the types and distributions of organic substrates that support microbial activities around plant roots is essential for a full understanding of plant-microbe interactions and rhizosphere ecology. We have constructed a strain of the soil bacterium Sinorhizobium meliloti containing a gfp gene fused to the melA promoter which is induced on exposure to galactose and galactosides. We used the fusion strain as a biosensor to determine that galactosides are released from the seeds of several different legume species during germination and are also released from roots of alfalfa seedlings growing on artificial medium. Galactoside presence in seed wash and sterile root washes was confirmed by HPLC. Experiments examining microbial growth on alpha-galactosides in seed wash suggested that alpha-galactoside utilization could play an important role in supporting growth of S. meliloti near germinating seeds of alfalfa. When inoculated into microcosms containing legumes or grasses, the biosensor allowed us to visualize the localized presence of galactosides on and around roots in unsterilized soil, as well as the grazing of fluorescent bacteria by protozoa. Galactosides were present in patches around zones of lateral root initiation and around roots hairs, but not around root tips. Such biosensors can reveal intriguing aspects of the environment and the physiology of the free-living soil S. meliloti before and during the establishment of nodulation, and they provide a nondestructive, spatially explicit method for examining rhizosphere soil chemical composition.

Long-term administration of galactoside-specific mistletoe lectin in an animal model: no protection against N-butyl-N-(4-hydroxybutyl)-nitrosamine-induced urinary bladder carcinogenesis in rats and no induction of a relevant local cellular immune response.

Aqueous extracts from leaves of the European mistletoe (Viscum album L.) are postulated to exert an anticancer efficacy by cytotoxic and/or immunological mechanisms of action. Although popular as an unconventional therapy modality, no controlled randomized clinical trials are available, reliably documenting a clinically beneficial antineoplastic potential of the various commercial mistletoe preparations. Since previous investigations have focused on the purified galactoside-specific lectin (Viscum album L. agglutinin, VAA) as major biological response modifier in the low-dose range, the objective of the present experimental study was to examine its effect on N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN)-induced carcinogenesis in the urinary bladder of rats, a suitable animal model for human disease. The carcinogen was fed by gavage in three fractionated low doses (150 mg/kg body weight each) to obtain low-grade and low-stage transitional cell carcinomas. From the onset of the experiment VAA was injected subcutaneously twice a week (1 ng/kg body weight) continuously for either 6 or 15 months. Following an experimental period of 6 months the incidence of bladder carcinomas was 10.2% in rats given exclusively BBN and 6.7% in those additionally treated with VAA. After an experimental time of 15 months 25.8% of the rats fed BBN only and 19.7% of the animals additionally receiving VAA had developed urothelial carcinomas. The differences of the tumor incidences did not reach the level of statistical significance, neither after an experimental duration of 6 (P = 0.88) nor of 15 months (P = 0.71). A difference was found in the size of the transitional cell carcinomas. They proved to be significantly larger (P = 0.02) in the rats additionally treated with VAA for 15 months (mean maximum diameter: 3.31 mm) than in those without lectin treatment (mean maximum diameter: 1.88 mm). Quantitative immunocytochemistry analyzing a panel of immune cells yielded no evidence for the ability of the lectin to provoke a substantial, biologically relevant local cellular immune response in the wall of tumor-free and tumor-bearing bladders. From the current experiment it is obvious that galactoside-specific mistletoe lectin failed to protect against, inhibit, delay or reduce development of chemically induced urothelial carcinomas of the urinary bladder even after long-term administration in the clinically recommended schedule. It seems highly unlikely that adjuvant treatment with mistletoe extracts or VAA might favorably influence bladder cancer in patients by immunological effector mechanisms.

Transcriptional activation of Cu/Zn superoxide dismutase and catalase genes by panaxadiol ginsenosides extracted from Panax ginseng.

Superoxide dismutase (SOD) converts superoxide radical to H(2)O(2), which is in turn broken down to water and oxygen by catalase. Thus, SOD and catalase constitute the first coordinated unit of defence against reactive oxygen species. A wide variety of chemical and environmental factors are known to induce these antioxidant enzymes. Here, we examined the effect of ginseng saponins on the induction of SOD and catalase gene expression. To explore this possibility, the upstream regulatory promoter region of Cu/Zn superoxide dismutase (SOD1) and catalase genes were linked to the chloramphenicol acetyltransferase (CAT) structural gene and introduced into human hepatoma HepG2 cells. Total saponin and panaxatriol did not activate the transcription of SOD1 and catalase genes but panaxadiol increased the transcription of these genes about 2-3 fold. Among the panaxadiol ginsenosides, the Rb(2) subfraction appeared to be a major inducer of SOD1 and catalase genes. The specificity of the Rb(2) effect was further confirmed by time course- and dose-dependent induction experiments. These results suggest that the panaxadiol fraction and its ginsenosides could induce the antioxidant enzymes which are important for maintaining cell viability by lowering the level of oxygen radical generated from intracellular metabolism.

Isolation and characterization of beta-galactoside specific lectin from Korean mistletoe (Viscum album var. coloratum) with lactose-BSA-sepharose 4B and changes of lectin conformation.

Lectins and its A- and B-chains from Korean mistletoe (Viscum album var. coloratum) were isolated by affinity chromatography on the Sepharose 4B modified by lactose-BSA conjugate synthesized by reductive amination of ligand (lactose) to epsilon-amino groups of lysine residues of spacer (BSA) after reduction by NaCNBH3. The lactose-BSA conjugate was coupled to Sepharose 4B activated by cyanogen bromide. The molecular weight determined by SDS-PAGE were a 31 kD of A-chain and a 35 kD of B-chain. Amino acid analysis and N-terminal sequencing were performed. The effects of pH, temperature and guanidine chloride on the conformation of the lectin were investigated by measuring its intrinsic fluorescence and compared with its hemagglutinating activities. Blue shift was detected on the acidic pH and there was a close relationship between activities and conformation of the lectin. Under denaturing conditions, the tryptophan emission profile of lectin showed typical denaturational red shift which also correspond to the conformations and activity of lectin.