Identification and characterisation of proteinase inhibitors and their genes from seeds of apple (Malus domestica).
(41/489)
Trypsin and papain proteinase inhibitors have been identified and purified from aqueous extracts of apple seeds (Malus domestica). Superdex G75 gel filtration chromatography identified a higher molecular weight (HMW) papain inhibitory fraction (22-26 kDa) and a lower molecular weight papain and trypsin inhibitory fraction (6-12 kDa). The lower molecular weight fraction was separated into a trypsin inhibitor (designated Trp1) and early (designated Pap1) and late (designated Pap2) eluting papain inhibitors after anion exchange (Hitrap SP) chromatography. For Pap2, two inhibitory peaks (designated Pap2-1 and Pap2-2) were identified after further anion exchange (Resource S) chromatography. Each of these lower molecular weight inhibitors was purified by reverse phase HPLC to homogeneity as determined by SDS-PAGE and by mass spectrometry. The HMW papain inhibitory fraction was purified further by anion-exchange (Hitrap Q followed by Resource Q) column chromatography where a minor inhibitor (HMWPap1) and major inhibitor (HMWPap2) fraction were identified. The relative abundance in seeds of apple and the spectrum of proteinase inhibition has been determined for all of these inhibitors. Reverse-phase HPLC separated HMWPap2 into a minor (HMWPap2-1) and a major (HMWPap2-2) inhibitory fraction, and SDS-PAGE and mass spectrometry confirmed that HMWPap2-2 was purified to homogeneity. Amino acid composition data were obtained from Trp1, Pap1, Pap2-2, and HMWPap2-2, and N-terminal sequence data from Trp1, Pap2-1, Pap2-2, and HMWPap2-2, with two of these sequences (Pap2-2 and HMWPap2-2) perfectly matching predicted protein sequences based on EST sequences from an apple database. The relationship of these inhibitors with those of other species is discussed. (+info)
Deuterium-labeled phaseic acid and dihydrophaseic acids for internal standards.
(42/489)
The concentration of abscisic acid in plants is regulated not only by biosynthesis, but also by metabolism. Abscisic acid is metabolized to phaseic acid via 8'-hydroxyabscisic acid, and phaseic acid is then converted to dihydrophaseic acid and its epimer. A quantitative analysis of these metabolites is important as well as that of abscisic acid to understand changes in the concentration of abscisic acid in plants. However, no internal standards of the metabolites suitable for quantitative analysis have been reported. We prepared 7'-deuterium-labeled phaseic acid with a deuterium content of 86%, using the equilibrium reaction between phaseic acid and 8'-hydroxyabscisic acid. 7'-Deuterium-labeled dihydrophaseic acids were obtained by reducing 7'-deuterium-labeled phaseic acid. The levels of the metabolites in plant organs were determined by using the deuterated metabolites as internal standards. (+info)
Inhibition of AP-1 and neoplastic transformation by fresh apple peel extract.
(43/489)
Consumption of fruits and vegetables has been associated with a low incidence of cancers and other chronic diseases. Previous studies suggested that fresh apples inhibit tumor cell proliferation. Here we report that oral administration of apple peel extracts decreased the number of nonmalignant and malignant skin tumors per mouse induced by 12-O-tetradecanolyphorbol-13-acetate (TPA) in 7,12-dimethylbenz(a)anthracene-initiated mouse skin. ESR analysis indicated that apple extract strongly scavenged hydroxyl (OH) and superoxide (O(2)(-)) radicals. Mechanistic studies showed that pretreatment with apple peel extract inhibited AP-1 transactivation induced by ultraviolet B irradiation or TPA in JB6 cells and AP-1-luciferase reporter transgenic mice. This inhibitory effect appears to be mediated by the inhibition of ERKs and JNK activity. The results provide the first evidence that an extract from fresh apple peel extract may inhibit tumor promoter-induced carcinogenesis and associated cell signaling, and suggest that the chemopreventive effects of fresh apple may be through its antioxidant properties by blocking reactive oxygen species-mediated AP-1-MAPK activation. (+info)
The phytoalexin-inducible multidrug efflux pump AcrAB contributes to virulence in the fire blight pathogen, Erwinia amylovora.
(44/489)
The enterobacterium Erwinia amylovora causes fire blight on members of the family Rosaceae, with economic importance on apple and pear. During pathogenesis, the bacterium is exposed to a variety of plant-borne antimicrobial compounds. In plants of Rosaceae, many constitutively synthesized isoflavonoids affecting microorganisms were identified. Bacterial multidrug efflux transporters which mediate resistance toward structurally unrelated compounds might confer tolerance to these phytoalexins. To prove this hypothesis, we cloned the acrAB locus from E. amylovora encoding a resistance nodulation division-type transport system. In Escherichia coli, AcrAB of E. amylovora conferred resistance to hydrophobic and amphiphilic toxins. An acrB-deficient E. amylovora mutant was impaired in virulence on apple rootstock MM 106. Furthermore, it was susceptible toward extracts of leaves of MM 106 as well as to the apple phytoalexins phloretin, naringenin, quercetin, and (+)-catechin. The expression of acrAB was determined using the promoterless reporter gene egfp. The acrAB operon was up-regulated in vitro by the addition of phloretin and naringenin. The promoter activity of acrR, encoding a regulatory protein involved in acrAB expression, was increased by naringenin. In planta, an induction of acrAB was proved by confocal laser scanning microscopy. Our results strongly suggest that the AcrAB transport system plays an important role as a protein complex required for virulence of E. amylovora in resistance toward apple phytoalexins and that it is required for successful colonization of a host plant. (+info)
The HcrVf2 gene from a wild apple confers scab resistance to a transgenic cultivated variety.
(45/489)
The Vf gene from the wild species Malus floribunda 821 is the most studied apple scab resistance gene. Several molecular markers mapping around this gene were the starting point for a positional cloning project. The analysis of the bacterial artificial chromosome clones spanning the Vf region led to the identification of a cluster of genes homologous to the Cladosporium fulvum resistance gene family of tomato. One of these genes, HcrVf2 (homologue of the C. fulvum resistance genes of the Vf region), was used to transform the susceptible apple cultivar Gala. Four independent transformed lines resistant to apple scab were produced, proving that HcrVf2 is sufficient to confer scab resistance to a susceptible cultivar. The results show that direct gene transfer between cross-compatible species can be viable when, as in apple, the use of backcrosses to introduce resistance genes from wild species cannot exactly reconstitute the heterozygous genotype of clonally propagated cultivars. (+info)
Preimaginal learning determines adult response to chemical stimuli in a parasitic wasp.
(46/489)
The behavioural responses of parasitic wasps to chemical cues from their hosts and host plants are known to be affected by genetic and environmental components. In a previous study of the codling moth ectoparasitoid Hyssopus pallidus, we found that the response of adult parasitoids to the frass of their host caterpillars depended on a learning process involving plant cues. In the present study, we investigated how and when learning takes place. A series of experiments was conducted involving exposure of parasitoids to fruit cues at different developmental stages. While parasitoids were not able to learn the fruit cues in the adult stage, exposure to fruit odour at early preimaginal stages significantly increased the adult response to frass from fruit-fed caterpillars. The olfactory memory persisted through metamorphosis, with a retention time of 14 days. Preimaginal learning was not confined to fruit cues but was also demonstrated for a host- and fruit-independent cue, menthol. Parasitoids exposed to menthol odour at the egg and larval stages no longer showed negative responses as adults. Sensitization to fruit cues and habituation to menthol are considered to be the mechanisms involved. This study provides evidence of true preimaginal learning of olfactory cues in a parasitic wasp. (+info)
Reducing the 137Cs-load in the organism of "Chernobyl" children with apple-pectin.
(47/489)
As a complement of standard radioprotective measures, apple-pectin preparations are given, especially in the Ukraine, to reduce the 137Cs uptake in the organism of children. The question has been raised: is oral pectin also useful when children receive radiologically clean food, or does this polysaccharide only act in binding 137Cs in the gut, blocking its intestinal absorption? In this case, pectin would be useless if radiologically clean food could be given. The study was a randomised, double blind placebo-controlled trial comparing the efficacy of a dry and milled apple-extract containing 15-16% pectin with a similar placebo-powder, in 64 children originating from the same group of contaminated villages of the Gomel oblast. The average 137Cs load was of about 30 Bq/kg bodyweight (BW). The trial was conducted during the simultaneous one-month stay in the sanatorium Silver Spring. In this clean radiological environment only radiologically "clean" food is given to the children. The average reduction of the 137Cs levels in children receiving oral pectin powder was 62.6%, the reduction with "clean" food and placebo was 13.9%, the difference being statistically significant (p <0.01). The reduction of the 137Cs load is medically relevant, as no child in the placebo group reached values below 20 Bq/kg BW (which is considered by Bandazhevsky as potentially associated with specific pathological tissue damages), with an average value of 25.8 +/- 0.8 Bq/kg. The highest value in the apple-pectin group was 15.4 Bq/kg, the average value being 11.3 +/- 0.6 Bq/kg BW. (+info)
NorM, an Erwinia amylovora multidrug efflux pump involved in in vitro competition with other epiphytic bacteria.
(48/489)
Blossoms are important sites of infection for Erwinia amylovora, the causal agent of fire blight of rosaceous plants. Before entering the tissue, the pathogen colonizes the stigmatic surface and has to compete for space and nutrient resources within the epiphytic community. Several epiphytes are capable of synthesizing antibiotics with which they antagonize phytopathogenic bacteria. Here, we report that a multidrug efflux transporter, designated NorM, of E. amylovora confers tolerance to the toxin(s) produced by epiphytic bacteria cocolonizing plant blossoms. According to sequence comparisons, the single-component efflux pump NorM is a member of the multidrug and toxic compound extrusion protein family. The corresponding gene is widely distributed among E. amylovora strains and related plant-associated bacteria. NorM mediated resistance to the hydrophobic cationic compounds norfloxacin, ethidium bromide, and berberine. A norM mutant was constructed and exhibited full virulence on apple rootstock MM 106. However, it was susceptible to antibiotics produced by epiphytes isolated from apple and quince blossoms. The epiphytes were identified as Pantoea agglomerans by 16S rRNA analysis and were isolated from one-third of all trees examined. The promoter activity of norM was twofold greater at 18 degrees C than at 28 degrees C. The lower temperature seems to be beneficial for host infection because of the availability of moisture necessary for movement of the pathogen to the infection sites. Thus, E. amylovora might employ NorM for successful competition with other epiphytic microbes to reach high population densities, particularly at a lower temperature. (+info)