Flavanols in somatic cell division and male meiosis of tea (Camellia sinensis) anthers.

Young anthers excised from closed tea flower buds ( Camellia sinensis L.) were stained as fresh tissues with p-dimethylaminocinnamaldehyde reagent to localize flavanols associated with nuclei and chromosomes, apart from those flavanols stored in vacuoles. This staining reagent yields a blue colour for flavanols. In the nonsporogenic somatic cells of developing anthers, flavanols were found to be attached to chromosomes at all mitotic stages. Male meiosis started at a bud size of about 3.5 mm in diameter in pollen mother cells which displayed generally more or less pronounced blue nuclei and cytoplasm. The meiotic divisions from prophase I to telophase II were characterized by blue stained nuclei and chromosomes, but within the cytoplasm there was, if any, a random and very poor reaction for flavanols. Metaphase and telophase of meiotic divisions showed maximally condensed chromosomes staining dark blue. Early in telophase II, the cytoplasm was again stained blue; this faded at late tetrad stage. Flavanols of young mitotic and older non-mitotic anthers were determined using high pressure liquid chromatography--chemical reaction detection (HPLC-CRD). Catechin, epicatechin, B2, and epigallocatechin were minor compounds, whereas epicatechin gallate and epigallocatechin gallate were found in higher amounts. The major flavanol compound of the anthers, epicatechin gallate, exhibited a significant affinity to histone sulphate, as shown by UV-VIS spectroscopic titration.

Developmental changes in the phenol concentrations of 'golden delicious' apple fruits and leaves.

The phenolic content of apple fruit skin and leaves was determined at the developmental stage of each organ. Phenolic levels decreased on a dry weight basis during the seasonal development of fruits and leaves with respect to their ontogenesis but the single compounds did not behave uniformly. A shift in flavanol pools from monomeric to oligomeric structures during fruit growth indicated the biosynthetic tendency towards the formation of procyanidins at the end of the growing period. Among the procyanidins identified epicatechin-(4 beta,6)-epicatechin-(4 beta,6)-epicatechin has not been reported previously from apple.

Nuclei of tea flowers as targets for flavanols.

The tea plant (Camellia sinensis L.) is famous for its flavanol-based constituents being valuable for human health. These flavanols associate with the nuclei of tea flowers, which is demonstrated histochemically by blue colouration using the selective staining reagent p-dimethylaminocinnamaldehyde (DMACA). Sepals, petals, stamens, pollen tubes, ovaries and ovules were studied. All these organs were shown to contain flavanols in vacuolar compartments, in nuclei and, exceptionally, also in the cytoplasm of pollen tubes. In all cells, even in those lacking vacuoles, the nuclei stained blue for flavanols. The extremely divergent development, shape and function of the diverse flower organs did not basically influence the nuclear flavanol association. Nevertheless, within the limits of this study, a few tissue-dependent differences in staining intensity were obvious. Interactions between epicatechin and nuclear histone proteins (histone sulphate) were studied by UV-VIS spectroscopic titration and by means of Mauser diagrams. The results show that the observed association equilibria are strongly dependent on pH (8.0 and 7.4) and on the buffer used (Tris, phosphate).

[Function and structure of families with an epileptic child]. / Funktion und Struktur von Familien mit einem epilepsiekranken Kind.

Although the impact of psycho-social factors on the individual patient with epilepsy has been widely investigated, the influence of the illness on the family as a whole is still underestimated. By means of the Family Assessment Measure (FAM III), a well-validated instrument, we investigated which measurable influence the epilepsy of one child had on the functioning of the families. Data from a group of 72 families with a child suffering from epilepsy (EG), but without any other handicap were analysed and compared with those of 75 families with a child with severe mental retardation (SMG) and 76 control families (CG). Data were gathered through home visits. Only complete families were studied. Results showed that the EG was similar to the SMG in all 3 scales of FAM III, but differed significantly from the CG. In 26% of the families in EG and in 19% of the SMG clear signs of family malfunctioning were found, as compared with 6.5% of the CG. Family disfunctioning in EG was of a greater variety that in SMG. We found that within the EG the type of seizures (41 patients with generalized tonic clonic fits, 10 with complex partial seizures and 21 with absences) did not have any impact on the results. In addition, neither the duration of the illness nor the absolute length of seizure-free periods seemed to matter. Only families who had the subjective feeling that their children were still suffering from epilepsy showed significantly higher rates of family malfunctioning. Summarizing, we found that epilepsy in a child can have a severe impact, not only on his individual life and on the mother-child relationship, but on the functioning of his family as the whole. This fact should be taken into account in the treatment of these children and should influence family counselling, as well.

Nuclei of plants as a sink for flavanols.

Onion cepa (L.) and Tsuga canadensis (L.) Carr. were investigated histochemically on the association of flavanols to nuclei. The young roots of Onion cepa are totally devoid of flavanol structures. Therefore, the excised roots tips were directly incubated into different solutions of flavanols. After 3 h of incubation a flavanol binding on the nuclei was recognizable, as seen by a yellowish-brown tanning reaction. Still to ensure the presence of flavanols on the nuclei, subsequent staining with the p-dimethylaminocinnamaldehyde reagent (DMACA) resulted in an intense blue colouration. Tsuga canadensis has significant amounts of vacuolar flavanol deposits in all parts of the tree as indicated by the DMACA reagent. It is obvious that also the nuclei were associated strongly with flavanols which can be demonstrated particularly elegant in the cells of the seed wings by histochemical methods. However, the mode of flavanol release from the original deposits is not yet clear.

Loss of nuclear flavanols during drought periods in Taxus baccata.

Normally, needles of Taxus baccata during the growth period prominently stain blue for nuclear flavanols with the histochemical DMACA procedure. However, under excess heat and drought conditions, nuclear flavanols of current-year needles decline to zero. Nevertheless, greenish-yellow-coloured flavonols (quercetin derivatives) were still observed in nuclei. All of these yellow nuclei were in a silenced state and without mitosis. This link between drought and loss of nuclear flavanols was found in 3 years, 2003, 2007 and 2010. In 2007, exceptional drought occurred in early spring, interrupted by short rains. This, in turn, led to flushing of new sprouts, a characteristic feature in which nuclei were overloaded with flavanols. By the end of three drought periods, all nuclei developed blue-coloured nuclear flavanols. The flavanols seem to be associated with the histone proteins of chromatin. The oxidative degradation of catechin in Tris buffer (pH 8.0) containing MgCl2 was studied in the presence of the H4-core fragment TYTEHAKRKTVTAMD, modified according to the epigenetic histone code. The results show that catechin degradation can be significantly inhibited by the non-modified peptides and the methylated peptides (methylation at both lysine residues). The acetylated and formylated peptides do not show this behaviour. These observations indicate that flavanol association at chromosomes appears to be regulated by the epigenetic histone code.

A new method for selective localization of flavan-3-ols in plant tissues involving glycolmethacrylate embedding and microwave irradiation.

A method for selective staining of flavan-3-ols in plant tissues fixed with glutaraldehyde is given. The use of glycolmethacrylate as embedding medium allows the sulphuric acid-containing staining solution to be heated without destroying the fine structure of the tissue. The distribution of flavan-3-ols and proanthocyanidins in different plant tissues is discussed.

Inhibition of growth and xylogenesis and promotion of vacuolation in prunus callus by the flavanone prunin.

Callus tissue of Prunus avium L. responded to supplied prunin (naringen in 7-glucoside) showing vaculoation and storage of oligomeric proanthocyanidins. In addition, prunin caused restricted callus initiation and/or less callus growth. When prunin was omitted from the medium numerous tracheids and more peroxidases were formed in the callus.

Localization and quantitative determination of catechins and proanthocyanidins in the phloem of elm and cherry.

The localization of catechins and proanthocyanidins in the phloem of elm (Ulmus minor Mill.) and cherry (Prunus avium L.) was determined histochemically by use of 4-dimethylaminocinnamaldehyde (DMACA) reagent. The two tree species showed a characteristic distribution of these phenols, which were most abundant in the phloem rays of cherry, but were largely confined to upright parenchyma cells of elm. Quantitative determination and qualitative separation of catechins and proanthocyanidins were performed using HPLC-CRD (chemical reaction detection), with 4-dimethylaminocinnamaldehyde as the staining reagent. Up to 14 different catechins and proanthocyanidins were found in both species.

Role of flavanols in yellowing beech trees of the Black Forest.

Beech leaves were sampled at the end of a prolonged hot dry period at a tree decline site in the Black Forest, Germany to investigate the potential role of flavanols in defense mechanisms against environmental stress. Green and yellowing leaves were harvested from the uppermost canopy of trees that were more than 200 years old and 30 m high. Yellowing leaves had a 7.4-fold higher concentration of total flavanols than green leaves. Green leaves contained flavanol inclusions, but during yellowing the inclusions disintegrated and the cells became filled with flavanols. Abscisic acid (ABA) stimulated the release of flavanols from intravacuolar inclusions of leaf petioles and flower pedicels. In addition, ABA caused flavanols to leach from the trichomes of beech galls. The antioxidative potential of leaf extracts, as estimated by indoleacetic acid (IAA) oxidation, was significantly higher in yellowing leaves than in green leaves. In vitro experiments revealed that (+)-catechin promoted growth of beech tissue.

Accumulation of flavanols in yellowing beech leaves from forest decline sites.

Beech leaves were sampled during two consecutive years from three sites characterized by forest decline. Both monomeric and oligomeric flavanols from green and yellowing leaves were determined quantitatively by reversed-phase, high-performance liquid chromatography (HPLC) combined with a chemical reaction detection technique (CRD). Yellowing leaves generally contained more than twice the quantity of flavanols than normal green leaves. The monomeric flavanols, epicatechin and (+)-catechin, comprised up to 80% of the total flavanol fraction. Histochemical staining with p-dimethylaminocinnamaldehyde was used to determine local deposition of flavanols, including insoluble oligomeric flavanols, in leaf tissues. The yellowing leaves stained intensely, whereas the green leaves stained lightly. Flavanol staining was strongest in the spongy parenchyma followed by the palisade cells. The upper epidermis stained more intensely than the lower epidermis. A 4-day treatment of small leaf pieces (5 x 5 mm) with 0.04 mM paraquat resulted in browning of the leaf pieces. The browning reaction was prevented when the paraquat treatment was carried out in the presence of 0.16 mM (+)-catechin, indicating an antioxidative property for this flavanol.

Flavanol binding of nuclei from tree species.

Light microscopy was used to examine the nuclei of five tree species with respect to the presence of flavanols. Flavanols develop a blue colouration in the presence of a special p-dimethylaminocinnamaldehyde (DMACA) reagent that enables those nuclei loaded with flavanols to be recognized. Staining of the nuclei was most pronounced in both Tsuga canadensis and Taxus baccata, variable in Metasequoia glyptostroboides, faint in Coffea arabica and minimal in Prunus avium. HPLC analysis showed that the five species contained substantial amounts of different flavanols such as catechin, epicatechin and proanthocyanidins. Quantitatively, total flavanols were quite different among the species. The nuclei themselves, as studied in Tsuga seed wings, were found to contain mainly catechin, much lower amounts of epicatechin and traces of proanthocyanidins. Blue-coloured nuclei located centrally in small cells were often found to maximally occupy up to 90% of a cell's radius, and the surrounding small rim of cytoplasm was visibly free of flavanols. A survey of 34 gymnosperm and angiosperm species indicated that the first group has much higher nuclear binding capacities for flavanols than the second group.