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Antidotal effect of grape juice (Vitis vinifera) on ochratoxin A caused hepatorenal carcinogenesis in mice (Mus musculus).

Oral administration of ochratoxin A to young weanling mice (Mus musculus) caused several haematological changes and induced hepatoma and renal carcinoma. Concurrent administration of berry and leaf juice of the common grape (Vitis vinifera) to mice together with ochratoxin A significantly reduced the hepatic and renal damage caused by ingestion of this mycotoxin. None of the animals receiving berry/leaf juice of V. vinifera showed the formation of hepatorenal carcinoma whereas 25% of animals receiving only ochratoxin A developed well differentiated renal carcinoma and hepatic lesions.

Animals↗

Microsatellite analysis of ancient alpine grape cultivars: pedigree reconstruction of Vitis vinifera L. 'Cornalin du Valais'.

Ancient and closely related grape cultivars from the Alps were analyzed with 50 microsatellite markers: 'Cornalin', 'Humagne Rouge' and 'Goron' from Valais (Switzerland); 'Cornalin', 'Petit Rouge' and 'Mayolet' from the Aosta Valley (Italy). Our results confirmed previous studies showing that the 'Cornalin' cultivars from Switzerland and Italy are distinct, and that 'Humagne Rouge' is identical to 'Cornalin' from the Aosta Valley. We propose the nomenclature 'Cornalin du Valais' and 'Cornalin d'Aoste' in order to prevent further confusion. At each locus, 'Goron', 'Petit Rouge', 'Mayolet' and 'Cornalin d'Aoste' all share at least one allele with 'Cornalin du Valais', strongly suggesting parent/offspring relationships. Alleles at 49 out of 50 microsatellite loci are consistent with 'Cornalin du Valais' being the progeny of 'Petit Rouge' and 'Mayolet'. The exception is a 10-base pair discrepancy at one locus, most likely the result of somatic mutation in one of the parents, since this parentage is supported by high likelihood ratios and historical data. We hypothesize that 'Cornalin du Valais' originated in the Aosta Valley through a natural cross and was then introduced into Valais centuries ago, probably via the Great St. Bernard Pass. Furthermore, 'Cornalin du Valais' is likely to be one of the parents of both 'Goron' and 'Cornalin d'Aoste', the respective second parents remaining unknown. This pedigree provides a convincing explanation for the allele-sharing patterns and is strongly supported by historical data. The present work is the first grapevine parentage study to deal with a multiple repeat unit discrepancy at a microsatellite locus. We suggest that the use of increasingly large numbers of loci in making parentage determinations leads to a corresponding increase in the probability of encountering a locus with intra-cultivar variability during the analysis. We therefore assume that a sole multiple repeat unit discrepancy is not sufficient to discard a parentage hypothesis.

Gene Frequency↗

Evidence on the evolution of polymorphism of microsatellite markers in varieties of Vitis vinifera L.

This paper contributes a description of four mutations in microsatellite allele-length found within strains of each of the varieties known as Muscat d'Alsace, Greco di Tufo, Primitivo and Corvina veronese: the comparison of microsatellite sequences of wild and mutated strains showed in all cases an increase of one or more repeats of the core GA sequence. Whereas studies on animals reported mutations which originated in gametes, in grapevines somatic cell-mutations take place and are eventually fixed and transmitted to new individuals through vegetative propagation. The mutations detected in 1998 were found again 3 years later in seven out of eight plants. A chimeric situation was clearly noted and resolved in three of the four cases described. Considerations about the genesis of microsatellite alleles and the reliability of fingerprinting with these markers are provided.

DNA, Plant↗

Quantification of abscisic acid in grapevine leaf (Vitis vinifera) by isotope-dilution liquid chromatography-mass spectrometry.

A specific, sensitive, precise, and accurate method for the determination of abscisic acid (ABA) in grapevine leaf tissues is described. The method employs high-performance liquid chromatography and electrospray ionization-mass spectrometry (LC-ESI-MS) in selected ion monitoring mode (SIM) to analyze ABA using a stable isotope-labeled ABA as an internal standard. Absolute recoveries ranged from 72% to 79% using methanol/water pH 5.5 (50:50 v/v) as an extraction solvent. The best efficiency was obtained when the chromatographic separation was carried out by using a porous graphitic carbon (PGC) column. The statistical evaluation of the method was satisfactory in the work range. A relative standard deviation (RDS) of < 5.5% and < 6.0% was obtained for intra-batch and inter-batch comparisons, respectively. As for accuracy, the relative error (%Er) was between -2.7 and 4.3%, and the relative recovery ranged from 95% to 107%.

Abscisic Acid↗

Transgenic plants of Vitis vinifera cv. Seyval blanc.

Leaf discs of grapevine cv. Seyval blanc originating from in vitro cultures were transformed with Agrobacterium tumefaciens strain LBA 4404 harbouring the vector pGJ42 carrying genes for chitinase and RIP (ribosome-inactivating protein) in an attempt to improve fungal resistance. The gene for neomycin phosphotransferase II (nptII) was used as the selectable marker gene. The explants were cocultivated for 2 days with recombinant Agrobacteria and then submitted to selection on NN69 medium containing 100 mg/l kanamycin. Successful regeneration and conversion of transgenic plantlets were obtained. Stable integration of foreign DNA was confirmed by PCR and Southern blot analyses, and protein expression was detected by Western blot. The regenerated transgenic plants were adapted to the greenhouse and showed no evidence of phenotypical alterations. The foreign genes introduced into the transformed plants did not effect the expected improvement in fungal disease resistance under field conditions for the major pests Uncinula necator and Plasmopara viticola.

Agrobacterium tumefaciens↗

Antiviral activity of tiazofurin and mycophenolic acid against Grapevine leafroll-associated virus 3 in Vitis vinifera explants.

The ability to control plant viral diseases with chemicals has great potential value for agriculture, but few chemicals are available to date due to the difficulty in obtaining effective drugs. IMP dehydrogenase is an enzyme which catalyzes the conversion of inosine 5'-monophosphate to xanthosine 5'-monophosphate in the de novo purine nucleotide synthetic pathway, and is considered a sensitive target for antiviral drugs. Two IMPDH inhibitors, tiazofurin (TR) and mycophenolic acid (MPA), were tested for their inhibitory effect on Grapevine leafroll-associated virus 3 (GLRaV-3) in in vitro grapevine explants. TR administration produced plantlets characterized by negative ELISA readings. No PCR products were obtained from these samples. This was confirmed by the absence of viral particles. MPA was essentially ineffective against GLRaV-3 replication in Sangiovese explants. This is the first report of GLRaV-3 eradication in grapevine explants following TR administration.

Closteroviridae↗

Transient effect of the herbicide flazasulfuron on carbohydrate physiology in Vitis vinifera L.

In the Champagne vineyard, most of the areas treated in early 2000 with the newly approved herbicide flazasulfuron had vines with altered growth and yellow leaves throughout the growing season. In order to clarify the physiological perturbations caused on the non-target grapevine and their potential consequences, C nutrition of grape plants grown in vineyards treated or not with flazasulfuron in 2000 was characterized during the following season. Vines from treated areas exhibited yellow leaves and an alteration of photosynthetic activity, characterized by declines in leaf gas exchanges (by 85%) and photosynthetic pigment concentrations (by 88%), and a marked disorganization of the leaf plastids. The herbicide also caused a decrease in leaf starch and soluble carbohydrate levels (-74% and -90%, respectively). Surprisingly, some vines re-greened after bloom, then exhibiting similar carbohydrate physiology to those grown in a non-treated area. Thus, recovery of CO(2) fixation rates, plastid ultra-structure, pigment concentrations and carbohydrate levels was found in re-greening leaves. Unlike the informations available in the literature, our results showed that flazasulfuron may be phytotoxic for grapevine. However, this toxicity was overcome the following year, indicating that vines have the potential to recover from this herbicide stress after one season.

Carbohydrate Metabolism↗

Partitioning and mobilization of starch and N reserves in grapevine (Vitis vinifera L.).

We followed C and N reserves of grapevines grown in trenches under semi-controlled conditions over a 3-year period after planting. Temporal mobilization of stored C and N and subsequent distribution of reserve materials within the vines were described in parallel with 15N uptake, particularly during the third growing season. Storage C in the perennial tissues (roots, trunk, canes) was mainly made of starch, which accumulated in the ray parenchyma of the wood. In the permanent tissues, starch and total nitrogen contents were found to decrease early in the development (bleeding sap, budbreak) whereas, on a concentration basis, they decreased only after stage 7 (first leaf fully expanded). Starch started to accumulate again in the perennial tissues during flowering. The same observation was made with total nitrogen, although N levels were much lower than those of starch. The 15N study showed that N uptake by the roots started at budbreak and increased with vine development, becoming predominant over reserve mobilization only after the onset of flowering. Taken together, these results indicate that the spring growth period can be divided into three main phases: In the first (dormancy to budbreak), significant losses of C and N proceed mainly via root necrosis. In the second period (first leaf to the onset of bloom), a strong mobilization of starch (and, to a lower extent, of N) occurred for supporting vegetative and reproductive growth. At that point, most of the C and N reserves used on the spring flush were those of the roots, rather than those of the old wood (trunk, canes). In the third period (bloom and early berry development), the mobilization process became low and was relieved by N uptake (and CO2 assimilation) supplying nutrients to the sink structures.

Nitrates↗

Monitoring the stability of Rubisco in micropropagated grapevine (Vitis vinifera L.) by two-dimensional electrophoresis.

Plants cultured in vitro suffer from several physiological and biochemical impairments due to the artificial conditions of growth, namely the composition of the heterotrophic media. Upon transfer to ex vitro, the higher irradiances, compared to in vitro, can lead to oxidative stress symptoms, which can be counteracted by CO2 concentrations above atmospheric levels. Here we analyse the stability of Rubisco in in vitro grapevine plantlets, and after transfer to ex vitro under four acclimatization treatments: low irradiance (LL, 150 micromol m(-2)s(-1)) and high irradiance (HL, 300 micromol m(-2)s(-1)) in association with CO2 concentrations of 350 (LCO2) and 700 (HCO2) microL L(-1). Proteins were separated with SDS polyacrylamide gel electrophoresis and two-dimensional electrophoresis and Rubisco degradation peptides were analysed by immunoblotting with anti-LSU antibodies. These degradation products were present in the leaves of plantlets under both in vitro and ex vitro treatments. Under LCO2 they were maintained for almost all of the 28 days of the acclimatization period, while becoming scarcely detected after 14 days under HCO2 and after 7 days when HCO2 was associated with HL. These results appear to confirm the counteraction of HCO2 concentrations over the oxidative stress eventually caused by HL. The patterns of soluble sugars in acclimatizing leaves under HLHCO2 also gave an indication of a faster acquisition of autotrophic characteristics.

Carbon Dioxide↗

Effect of procyanidolic oligomers of Vitis vinifera on the biosynthesis and excretion of corneal glycosaminoglycans.

Glycosaminoglycans (GAG) as hyaluronan and proteoglycans (PG) as dermatan sulfate (DSPG) and keratan sulfate (KSPG) play important role in the structure and physiological functions of cornea. Several corneal pathologies are associated with qualitative and quantitative alterations of GAG and/or PG biosynthesis. For these reasons we investigated the effect of procyanidolic oligomers (PCO) on the biosynthesis and excretion of GAG-s in bovine corneal explant cultures. This was accomplished by the determination of their chemical components, hexoses, hexosamines and uronic acids as well as the incorporation of (35)S-sulfate in the presence and without PCO in the culture medium. In presence of 1 mg/ml PCO total hexoses, hexosamines and uronic acids increased at 5 and 24 h of incubation, hexoses less than the two other components. Stimulation of biosynthesis concerned essentially the uronic acid containing GAG-s, DS and hyaluronan. (35)S-sulfate incorporation decreased in presence of PCO, showing a decreased sulfation of GAG-s. The most striking effect was however the strongly increased excretion of neosynthesized GAG-s in the culture medium. This effect could be beneficial in decreasing corneal hydration in inflammatory conditions, together with the previously demonstrated protection of corneal macromolecules against proteolytic degradation.

Animals↗

Carbon-14 biolabelling of wine polyphenols in Vitis vinifera cell suspension cultures.

14C-L-phenylalanine is incorporated into a range of polyphenolic compounds when fed to grape cell cultures. Optimisation of several parameters such as the quantity of precursor applied and the duration of metabolism led to incorporation yields of 15% and to specific activities of 875 mu Ci g(-1) in stilbenes. Purification of the products by several chromatographic steps is reported. Both trans- and cis-resveratrols were easily obtained by enzymatic hydrolysis of their corresponding glucosides, with specific activity of 1200-1400 mu Ci g(-1). The specific radioactivity obtained for all the compounds is suitable for in vivo feeding trials to trace their metabolic fate when consumed by animals and for in vitro activity mechanism studies. Indeed, these polyphenols seem to be implicated in the health benefits associated with regular and moderate wine consumption but little is known about their pharmacokinetics and cellular uptake.

Anthocyanins↗

A study on the interplay between energy and matter transformation: the effect of elevated temperatures on the leaf morphology of Vitis vinifera var. Merlot.

This investigation explores the relationship between increased energy levels and leaf morphology. It tests the idea that the causal agent of development is the dissipation of energy into transformed matter. The energy under which leaves developed was modified by increasing temperatures in grape cordons through wrapping them in clear plastic sleeves in the early spring. At the higher temperatures, and energy levels, there was a small but statistically significant decrease in leaf size and a change in organization the leaves. The decrease in leaf size may be due to a reallocation of resources, either to greater shoot growth as a previous study demonstrated or to the appearance of more vectors of development in the leaves, i.e. the appearance of more developmental subsystems. The leaves that grew under the higher temperature regime were more complex, perhaps indicating that the grapes on those same vines may produce more complex juice, another expression of more developmental subsystems. The change in organization in these leaves that developed at higher temperature argues that the causal agent in plant development is energy dissipation and the concomitant transformation of matter, the latter expressed in the appearance of more growth vectors.

Plant Leaves↗

[Impact of subculture cycles and inoculum sizes on suspension cultures of Vitis vinifera].

The commercial application of plant cell cultures is often hindered by the instability of secondary metabolite biosynthesis, where the metabolite yield fluctuates and decline dramatically over subcultures. This study proposed that such instability is due to the fluctuations of culture variables. To validate this hypothesis, the effects of the fluctuations of two culture variables (subculture cycle and inoculum size) on the biomass, anthocyanin biosynthesig, intracellular carbon, nitrogen and phosphate during continuous 10 subculture cycles were investigated. The subculture cycle was fluctuated for 12h in a 7 day cycle (6.5, 7 and 7.5 d), and the inoculum size was fluctuated by 20% on basis of 2.00 g (1.60, 2.00 and 2.40 g). It was found that all the measured culture parameters fluctuated over the 10 subculture cycles. The fluctuation in terms of inoculum sizes had a greater effect on the stability of anthocyanin biosynthesis in suspension cultures of V. vinifera. Among all the subculture conditions investigated, 7d-subculture cycle and 1.60 g-inoculum size was the best one to hold the relatively stable anthocyanin production. The anthocyanin yield presented a negative correlation with intracellular sucrose content or intracellular total phosphate content.

Anthocyanins↗

Systematic reappraisal of Coniella and Pilidiella, with specific reference to species occurring on Eucalyptus and Vitis in South Africa.

The genus Pilidiella, including its teleomorphs in Schizoparme, has a cosmopolitan distribution and is associated with disease symptoms on many plants. In the past, conidial pigmentation has been used as a character to separate Pilidiella (hyaline to pale brown conidia) from Coniella (dark brown conidia). In recent years, however, the two genera have been regarded as synonymous, the older name Coniella having priority. To address the generic question, sequences of the internal transcribed spacer region (ITS1, ITS2), 5.8S gene, large subunit (LSU) and elongation factor 1-alpha gene (EF 1-alpha) were analysed to compare the type species of Pilidiella and Coniella. All three gene regions supported the separation of Coniella from Pilidiella, with the majority of taxa residing in Pilidiella. Pilidiella is characterised by having species with hyaline to pale brown conidia (avg. length:width > 1.5), in contrast to the dark brown conidia of Coniella (avg. length:width < or = 1.5). Pilidiella diplodiella, which is a pathogen associated with white rot of grapevines, was shown to be an older name for C. petrakii. To delineate species in the P. diplodiella species complex, isolates were also compared based on histone (H3) gene sequences. Analyses derived from these sequence data separated P. diplodiella from a newly described species, P. diplodiopsis. The new species P. eucalyptorum sp. nov. is proposed for isolates formerly treated as C. fragariae and associated with leaf spots of Eucalyptus spp. This species clustered basal to Pilidiella, and may represent yet a third genus within this complex. Pilidiella destruens sp. nov. is newly described as anamorph of Schizoparme destruens, which is associated with twig dieback of Eucalyptus spp. in Hawaii. A key based on morphological characteristics is provided to separate the taxa treated in this study.

Ascomycota↗

New flavanol derivatives from grape (Vitis vinifera) byproducts. Antioxidant aminoethylthio-flavan-3-ol conjugates from a polymeric waste fraction used as a source of flavanols.

A new family of antioxidants has been obtained from a residual fraction of polymeric polyphenols of grape origin. The integral exploitation of resources is important in any sustainable production scheme. Many byproducts and residues generated by the agroindustries contain polyphenols with potential application as preventative agents against cancer and cardiovascular diseases. Among these polyphenols oligomeric proanthocyanidins are particularly significant. The polymeric forms, considered of less interest because of their astringent properties, constitute the largest portion of the biologically active plant proanthocyanidins. The new compounds described here result from the breakdown of polymeric flavanols in the presence of cysteamine and bear an amino function, which facilitates their isolation from complex mixtures by cation-exchange gels or resins. In this way, valuable antioxidant molecules can be efficiently obtained from otherwise wasted polymers. The new molecules appear to be as effective as their underivatized counterparts (flavan-3-ols) as antioxidants.

Antioxidants↗

Determination of carotenoid profiles in grapes, musts, and fortified wines from Douro varieties of Vitis vinifera.

beta-Carotene and six xanthophylls (lutein, neoxanthin, violaxanthin, luteoxanthin, cryptoxanthin, and echinenone) have been identified and semiquantitatively or quantitatively determined in musts and port wines for the first time. An HPLC method was developed and compared with that of one based on thin layer cromatography with scanning densitometry. The most abundant carotenoids present in red grape varieties are beta-carotene and lutein. In wines, significant quantities of violaxanthin, luteoxanthin, and neoxanthin were found. This study was done with berries (skin and pulp), musts, and fortified wines. Some experiments were performed to follow carotenoid content from grapes to wines. Although the levels of beta-carotene and lutein found in fortified wines were lower than those found in musts, other xanthophylls, such as neoxanthin, violaxanthin, and luteoxanthin, exist in appreciable amounts in young ports.

Carotenoids↗

Analysis of grape Vitis vinifera L. DNA in must mixtures and experimental mixed wines using microsatellite markers.

Because wine quality highly relies on the varietal composition of the must, the development of methods allowing the authentication of varieties in musts and wines would be of great value as a guarantee of quality. Microsatellite markers have already been applied to the authentication of grape juices (Faria, M. A.; Magalhães, R.; Ferreira, M. A.; Meredith, C. P.; Ferreira Monteiro, F. J. Agric. Food Chem. 2000, 48, 1096-1100) and to the analysis of experimental wines (Siret, R.; Boursiquot, J. M.; Merle, M. H.; Cabanis, J. C.; This, P. J. Agric Food Chem. 2000, 48, 5035-5040). In the present paper, we accessed the usefulness of this technology for the analysis of must and wine mixtures. The detection limit of DNA mixtures was first estimated on DNA extracted from leaves: 4% of a foreign DNA can be detected. Analysis of must and wine mixtures (Chardonnay B/Clairette B and Syrah N/Grenache N) was performed on experimental fermentations. DNA was extracted along the fermentation process and analyzed using five microsatellite loci. The 70:30 (v/v) mixtures were successfully analyzed until the end of the fermentation. The applications of these results to commercial purposes are discussed.

DNA, Plant↗