Molecular cloning and biochemical characterization of a novel anthocyanin 5-O-glucosyltransferase by mRNA differential display for plant forms regarding anthocyanin.
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UDP-glucose: anthocyanin 5-O-glucosyltransferase (5-GT) is responsible for the modification of anthocyanins to more stable molecules in complexes for co-pigmentation, supposedly resulting in a purple hue. The cDNA encoding 5-GT was isolated by a differential display applied to two different forms of anthocyanin production in Perilla frutescens var. crispa. Differential display was carried out for mRNA from the leaves of reddish-purple and green forms of P. frutescens, resulting in the isolation of five cDNA clones predominantly expressed in the red form. The cDNA encoded a polypeptide of 460 amino acids, exhibiting a low homology with the sequences of several glucosyltransferases including UDP-glucose: anthocyanidin 3-O-glucosyltransferase. By using this cDNA as the probe, we also isolated a homologous cDNA clone from a petal cDNA library of Verbena hybrida. To identify the biochemical function of the encoded proteins, these cDNAs were expressed in Saccharomyces cerevisiae cells. The recombinant proteins in the yeast extracts catalyzed the conversion of anthocyanidin 3-O-glucosides into the corresponding anthocyanidin 3,5-di-O-glucosides using UDP-glucose as a cofactor, indicating the identity of the cDNAs encoding 5-GT. Several biochemical properties (optimum pH, Km values, and sensitivity to inhibitors) were similar to those reported previously for 5-GTs. Southern blot analysis indicated the presence of two copies of 5-GT genes in the genome of both red and green forms of P. frutescens. The mRNA accumulation of the 5-GT gene was detected in the leaves of the red form but not in those of the green form and was induced by illumination of light, as observed for other structural genes for anthocyanin biosynthesis in P. frutescens. (+info)
Beneficial effects of thyme oil on age-related changes in the phospholipid C20 and C22 polyunsaturated fatty acid composition of various rat tissues.
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The aim of this study was to determine any age-related changes in phospholipid polyunsaturated fatty acid composition, in particular C20 and C22 fatty acids in rat liver, brain, kidney and heart, and to assess and compare the effects of dietary supplementation (42.5 mg/kg body weight/day) of the natural antioxidant thyme oil and its major component thymol throughout the rat life span. The fatty acid composition in the various tissues from young (7 months) and aged (28 months) rats was determined and compared. Livers from aged control, thyme oil and thymol treated rats exhibited an increase in 22:6(n-3). In contrast, 22:6(n-3) content of brain, kidney and heart declined in aged rats in all three dietary groups. However, aged rats treated with thyme oil and thymol displayed significantly higher levels of 22:6(n-3) than the respective age-matched controls. Tissue compositions of 20:4(n-6) were found to be significantly lower in the liver and kidney from aged control rats but not those fed either thyme oil or thymol. In aged rats, the composition of 20:4(n-6) in all tissues was highest in rats fed either thyme oil or thymol. These results show that dietary supplementation with thyme oil tended to maintain higher PUFA levels in all tissues studied. The majority of protection provided by thyme oil was by virtue of its thymol component, which comprises 49% of the whole oil. Thymol administered alone did not provide significantly higher protection than the whole oil, suggesting that other components within thyme oil are also contributing antioxidant activity. (+info)
The cyanogenic glucoside, prunasin (D-mandelonitrile-beta-D-glucoside), is a novel inhibitor of DNA polymerase beta.
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A DNA polymerase beta (pol. beta) inhibitor has been isolated independently from two organisms; a red perilla, Perilla frutescens, and a mugwort, Artemisia vulgaris. These molecules were determined by spectroscopic analyses to be the cyanogenic glucoside, D-mandelonitrile-beta-D-glucoside, prunasin. The compound inhibited the activity of rat pol. beta at 150 microM, but did not influence the activities of calf DNA polymerase alpha and plant DNA polymerases, human immunodefficiency virus type 1 reverse transcriptase, calf terminal deoxynucleotidyl transferase, or any prokaryotic DNA polymerases, or DNA and RNA metabolic enzymes examined. The compound dose-dependently inhibited pol. beta activity, the IC(50) value being 98 microM with poly dA/oligo dT(12-18) and dTTP as the DNA template and substrate, respectively. Inhibition of pol. beta by the compound was competitive with the substrate, dTTP. The inhibition was enhanced in the presence of fatty acid, and the IC(50) value decreased to approximately 40 microM. In the presence of C(10)-decanoic acid, the K(i) value for substrate dTTP decreased by 28-fold, suggesting that the fatty acid allowed easier access of the compound to the substrate-binding site. (+info)
Responses of Mentha suspension-cultured cells to 2,4-dichlorophenoxyacetic acid and accumulation of esterified phenolic acids in their cell walls.
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Two distinct types of cell growth of suspension-cultured Mentha were formed when the cells maintained in the medium containing 1000 micrograms l-1 2,4-D were subcultured into different 2,4-D concentrations. Few cell elongation of Mentha (average cell length: 34-40 microns) was observed after division in the medium containing 1-200 micrograms l-1 2,4-D; and significant cell elongation (average cell length: 95-130 microns) was observed after cell division in the medium containing 500-2000 micrograms l-1 2,4-D. A close correlation between culture medium and water content in the cells indicated that 2,4-D promoted cell elongation by water uptake. Amounts of phenolic acid in cell walls were much higher in unelongated cell walls than in elongated ones during the cultivation, and there was a close correlation between the amounts and the level of PAL activity in elongated and unelongated cells. However, there was no significant difference in cell wall components and its neutral sugar composition between elongated and unelongated cells. (+info)
Geranyl diphosphate synthase: cloning, expression, and characterization of this prenyltransferase as a heterodimer.
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Geranyl diphosphate synthase, which catalyzes the condensation of dimethylallyl diphosphate and isopentenyl diphosphate to geranyl diphosphate, the key precursor of monoterpene biosynthesis, was purified from isolated oil glands of spearmint. Peptide fragments generated from the pure proteins of 28 and 37 kDa revealed amino acid sequences that matched two cDNA clones obtained by random screening of a peppermint-oil gland cDNA library. The deduced sequences of both proteins showed some similarity to existing prenyltransferases, and both contained a plastid-targeting sequence. Expression of each cDNA individually yielded no detectable prenyltransferase activity; however, coexpression of the two together produced functional geranyl diphosphate synthase. Antibodies raised against each protein were used to demonstrate that both subunits were required to produce catalytically active native and recombinant enzymes, thus confirming that geranyl diphosphate synthase is a heterodimer. (+info)
Allergic alveolitis due to herb dust exposure.
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We report an episode of allergic alveolitis in a female farmer due to massive exposure to organic dust contaminated with microorganisms during threshing of herbs (thyme). The patient's medical history, the results of exposure test, inhalation challenge, and bronchoalveolar lavage suggested the diagnosis of allergic alveolitis (+info)
Tanshinone IIA, an ingredient of Salvia miltiorrhiza BUNGE, induces apoptosis in human leukemia cell lines through the activation of caspase-3.
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Tanshinone II-A is a derivative of phenanthrene-quinone isolated from Salvia miltiorrhiza BUNGE, a traditional herbal medicine that is known to induce antiinflammatory, anti-oxidative and cytotoxic activity. We have examined cellular effects of Tanshione II-A on HL60 human promyelocytic leukemic cells and K562 human erythroleukemic cells. Tanshione II-A induced a dose- and time-dependent DNA fragmentation into the multiples of 180 bp and specific proteolytic cleavage of poly(ADP-ribose) polymerase in both cell lines. PI-staining and flow cytometry analysis of K562 cells following Tanshione II-A treatment showed an increase of the cells possessing hypodiploid DNA indicative of apoptotic state of cells. Caspase-3 activity was significantly increased during Tanshinone II-A treatment of both HL60 and K562 cells, whereas caspase-1 activity was not changed. These results suggest that Tanshione II-A induced HL60 and K562 cellular apoptosis that may be associated with the selective members of caspase family. (+info)
Regulation of monoterpene accumulation in leaves of peppermint.
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Plants synthesize numerous classes of natural products that accumulate during development and are thought to function as constitutive defenses against herbivores and pathogens. However, little information is available about how the levels of such defenses are regulated. We measured the accumulation of monoterpenes, a model group of constitutive defenses, in peppermint (Mentha x piperita L.) leaves and investigated several physiological processes that could regulate their accumulation: the rate of biosynthesis, the rate of metabolic loss, and the rate of volatilization. Monoterpene accumulation was found to be restricted to leaves of 12 to 20 d of age, the period of maximal leaf expansion. The rate of monoterpene biosynthesis determined by (14)CO(2) incorporation was closely correlated with monoterpene accumulation, as determined by gas chromatographic analysis, and appeared to be the principal factor controlling the monoterpene level of peppermint leaves. No significant catabolic losses of monoterpenes were detected throughout leaf development, and monoterpene volatilization was found to occur at a very low rate, which, on a monthly basis, represented less than 1% of the total pool of stored monoterpenes. The composition of volatilized monoterpenes differed significantly from that of the total plant monoterpene pool, suggesting that these volatilized products may arise from a separate secretory system. With the demonstration that the rate of biosynthesis is the chief process that determines monoterpene accumulation in peppermint, efforts to improve production in this species can now focus on the genes, enzymes, and cell differentiation processes that regulate monoterpene biosynthesis. (+info)