Flavonoids in plant nuclei: detection by laser microdissection and pressure catapulting (LMPC), in vivo staining, and uv–visible spectroscopic titration

Previous studies in our laboratory have indicated that the nuclei of a number of trees are associated with flavonoids, especially flavan‐3‐ols. In the present study, three techniques were applied to verify that flavonoids are naturally incorporated into nuclei. These were histochemistry, UV–visible (UV‐VIS) titration and laser microdissection. Nuclei from intact seed wings of Tsuga canadensis were isolated from their cells using laser microdissection and pressure catapulting (LMPC). Thereafter, the excised nuclei were stained with p‐dimethylamino‐cinnamaldehyde (DMACA), which resulted in a blue coloration due to the presence of flavanols. Thus, there is no doubt that the nuclei were, prior to staining, associated with flavanols. The nuclei of the coniferous species Abies lasiocarpa, Cedrus deodara, Cedrus libani, Juniperus communis, Picea abies, Picea orientalis and Pseudotsuga menziessii(Douglas fir) showed a yellow fluorescence typical for flavonols from the beginning of bud break over the entire growing season. However, after the bud‐breaking period, the nuclei of all species, except for Cedrus deodara, showed additionally a blue reaction for flavanols. Rather late, in midsummer, blue‐stained flavanols in nuclei were found in Picea orientalis. Generally, zeatin intensified the flavanol association with the nuclei. The main components of nucleosomes are DNA and the histone proteins. The nature of their association with the flavonols quercetin and rutin was investigated by UV‐VIS spectroscopic titration. The data were evaluated by means of the Mauser (A and AD) diagrams. The results indicate that DNA shows largely no spectroscopically detectable association equilibria under the experimental conditions chosen. However, association (aggregation) equilibria can be observed with rutin or quercetin and histone sulphate in Tris buffer (pH 8.0, 7.4 and 7.0). In phosphate buffer, rutin shows spectroscopically no or only weak association with histone sulphate, in contrast to its behaviour towards quercetin.     

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).     

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 cells 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.Abbreviations DMACA p-Dimethylaminocinnamaldehyde Communicated by W. Barz     

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.     

Chromatin decondensation and nuclear reprogramming by nucleoplasmin

Somatic cell nuclear cloning has repeatedly demonstrated striking reversibility of epigenetic regulation of cell differentiation. Upon injection into eggs, the donor nuclei exhibit global chromatin decondensation, which might contribute to reprogramming the nuclei by derepressing dormant genes. Decondensation of sperm chromatin in eggs is explained by the replacement of sperm-specific histone variants with egg-type histones by the egg protein nucleoplasmin (Npm). However, little is known about the mechanisms of chromatin decondensation in somatic nuclei that do not contain condensation-specific histone variants. Here we found that Npm could widely decondense chromatin in undifferentiated mouse cells without overt histone exchanges but with specific epigenetic modifications that are relevant to open chromatin structure. These modifications included nucleus-wide multiple histone H3 phosphorylation, acetylation of Lys 14 in histone H3, and release of heterochromatin proteins HP1beta and TIF1beta from the nuclei. The protein kinase inhibitor staurosporine inhibited chromatin decondensation and these epigenetic modifications with the exception of H3 acetylation, potentially linking these chromatin events. At the functional level, Npm pretreatment of mouse nuclei facilitated activation of four oocyte-specific genes from the nuclei injected into Xenopus laevis oocytes. Future molecular elucidation of chromatin decondensation by Npm will significantly contribute to our understanding of the plasticity of cell differentiation.     

Epigenetic marks in the <Emphasis Type="Italic">Hyacinthus orientalis</Emphasis> L. mature pollen grain and during in vitro pollen tube growth

During the sexual reproduction of flowering plants, epigenetic control of gene expression and genome integrity by DNA methylation and histone modifications plays an important role in male gametogenesis. In this study, we compared the chromatin modification patterns of the generative, sperm cells and vegetative nuclei during Hyacinthus orientalis male gametophyte development. Changes in the spatial and temporal distribution of 5-methylcytosine, acetylated histone H4 and histone deacetylase indicated potential differences in the specific epigenetic state of all analysed cells, in both the mature cellular pollen grains and the in vitro growing pollen tubes. Interestingly, we observed unique localization of chromatin modifications in the area of the generative and the vegetative nuclei located near each other in the male germ unit, indicating the precise mechanisms of gene expression regulation in this region. We discuss the differences in the patterns of the epigenetic marks along with our previous reports of nuclear metabolism and changes in chromatin organization and activity in hyacinth male gametophyte cells. We also propose that this epigenetic status of the analysed nuclei is related to the different acquired fates and biological functions of these cells.     

Interactive effects of ozone and drought stress on pigments and activities of antioxidative enzymes in Pinus halepensis

The aim of this study was to investigate the interactive effects of ozone (O₃) and drought on pigments and antioxidant enzymes of Aleppo pine (Pinus halepensis). Two‐year‐old seedlings were exposed in open‐top chambers to charcoal‐filtered air or non‐filtered air plus an additional 40 nL L^?1 of ozone. After 20 months of O₃ exposure, a subset of plants was subjected to drought stress by withholding water supply for 11 d. Ozone induced higher guaiacol peroxidase, catalase and KCN‐resistant superoxide dismutase (SOD) activities in young needles, while drought stress increased glutathione reductase and CuZnSOD. One‐year‐old needles showed lower capacity to activate these enzymes in response to stress. Both ozone and drought activated the xanthophyll cycle pool and reduced chlorophyll contents in both current and 1‐year‐old needles. The combined effects of ozone and drought decreased antioxidant enzyme activities and the capacity of recovering after re‐watering. Similarly, interactive effects of O₃ and drought reduced xanthophyll‐mediated photoprotection capacity in 1‐year‐old needles but induced a higher conversion of the cycle in current‐year needles. These results showed that ozone modified the Aleppo pine response to drought stress, suggesting that this pollutant might be reducing the ability of this species to withstand other environmental stresses.     

Recent advances in the development of peptide-based inhibitors targeting epigenetic readers of histone lysine acetylation and methylation marks

Inhibitors for epigenetic readers of histone modifications are useful chemical probes to interrogate the functional roles played by their cognate targets in epigenetic regulation and can even serve as drugs for the treatment of diseases associated with the dysregulated targets. However, many epigenetic readers are intractable to small molecules, as the recognition of modified histone peptides commonly involves flat and extended protein surfaces. In contrast, the relatively large sizes and structural complexity of peptides help them achieve tight and specific binding to the target proteins. Increasing efforts have been made to target epigenetic readers using peptide-based inhibitors that can complement small molecules. In this review, we discuss the recent advances in the development of peptide-based inhibitors of lysine acetylation and methylation readers.     

Histone modifications and nuclear architecture: a review.

Epigenetic modifications, such as acetylation, phosphorylation, methylation, ubiquitination, and ADP ribosylation, of the highly conserved core histones, H2A, H2B, H3, and H4, influence the genetic potential of DNA. The enormous regulatory potential of histone modification is illustrated in the vast array of epigenetic markers found throughout the genome. More than the other types of histone modification, acetylation and methylation of specific lysine residues on N-terminal histone tails are fundamental for the formation of chromatin domains, such as euchromatin, and facultative and constitutive heterochromatin. In addition, the modification of histones can cause a region of chromatin to undergo nuclear compartmentalization and, as such, specific epigenetic markers are non-randomly distributed within interphase nuclei. In this review, we summarize the principles behind epigenetic compartmentalization and the functional consequences of chromatin arrangement within interphase nuclei.     

Colour perception of single and mixed monochromatic lights in the blowfly Lucilia cuprina

Colour perception of spectral lights and mixtures of two monochromatic lights of blue and yellow wavelengths was studied in the blowfly Lucilia cuprina by using a generalization test in which the fly had to compare these lights in memory with coloured papers (blue, green, yellow and red) represented in the test array. Flies trained to a monochromatic light in the wavelength range of 429–491 nm responded to blue; those trained to 502–511 nm to green; and those trained to 522–582 nm to yellow. The maximal generalization for blue was found at 429 nm and that for yellow at 543 nm. Flies trained to the mixtures responded neither to blue, green nor yellow, when the blue component was mixed with the yellow component in a ratio of approximately 1 3. It seems that the fly perceives the mixtures as a neutral or an achromatic light. Colour loci of coloured papers, spectral lights and mixtures of two monochromatic lights used formed blue, yellow and neutral clusters in a colour triangle with respect to generalization responses to test colours.     

A simple polychrome stain for conventionally fixed Epon-embedded tissues.

The described technique, based upon a one-step Mallory-Heidenhain stain, can be applied as a routine stain for glutaraldehyde or OsO4 fixed, Epon embedded tissues of various organs. The technique consists of a short treatment of the sections with H2O2, a nuclear staining with celestine blue B and a final staining in a modified Cason's solution. The different tissue and cell components are displayed as follows: dark brown nuclei, yellow cytoplasm, red collagen fibers and blue elastic fibers. Intracytoplasmic components as glycogen and mucus are stained respectively blue and violet, whereas other inclusions such as leucocyte granules are colored orange to red.     

Epigenetic marks in melanoma

Melanoma is a highly heterogeneous cancer that comes in different flavors (lentigo maligna melanoma, superficial spreading melanoma, nodular melanoma, acral lentiginous/mucosal melanoma and other less common subtypes including malignant cellular blue nevus, desmoplastic melanoma, nevoid melanoma, and animal‐type melanoma) and colors (black/bluish or unpigmented). Pathologists have known for many years that melanoma displays notable changes in the nuclear architecture including thick chromatic rims, presence of mitosis, nuclear grooves, and more. It is now evident from other cancers that such changes reflect not only genomic alterations but also non‐genomic changes in both the structure of DNA and the structure of chromatin to which the DNA is bound (nucleosomes). Although aberrant gene expression resulting from DNA methylation has been known for many years, genome alterations resulting from histone modifications became evident in the current decade. In prostate and other cancers, some histone marks have clinical diagnostic and/or prognostic value. Here, we review the current data on epigenetic research in melanoma skin cancers, discuss ways to modify the epigenetic landscape of melanoma for inhibiting its growth, and propose strategies for identifying novel melanoma markers.     

A Simple Polychrome Stain for Conventionally Fixed Epon-Embedded Tissues

The described technique, based upon a one-step Mallory-Heidenhain stain, can be applied as a routine stain for glutaraldehyde or OsO₄ fixed, Epon embedded tissues of various organs. The technique consists of a short treatment of the sections with H₂O₂, a nuclear staining with celestine blue B and a final staining in a modified Cason's solution. The different tissue and cell components are displayed as follows: dark brown nuclei, yellow cytoplasm, red collagen fibers and blue elastic' fibers. Intra cytoplasmic components as glycogen and mucus are stained respectively blue and violet, whereas other inclusions such as leucocyte granules are colored orange to red.     

Are histones the targets for flavan-3-ols (catechins) in nuclei?

Binding of flavan-3-ols to nuclei is characteristic of Tsuga canadensis (coniferous tree). This is achieved with the DMACA reagent (p-dimethylamino-cinnamaldehyde), which stains almost exclusively monomeric and oligomeric flavan-3-ols with an intense blue colour. Deep flavanol staining also occurred when calf cells of small intestine were enriched with added catechins. In order to detect the components of nuclei that may associate with catechins, the principal components of chromatin (DNA, histones) were subjected to UV-VIS spectroscopic titration. DNA or histone sulphate containing the histones H1, H2A, H2B, H3 and H4 were dissolved in cationic and anionic buffers (Tris, phosphate) at different pH values (pH 8.0, 7.4 and 7.0) and titrated with EGCG (epigallocatechin gallate) or catechin. The results show that DNA of calf thymus and the catechins investigated form no spectroscopically detectable association equilibria. However, strong association complexes are found between histone sulphate and EGCG or catechin by means of the Mauser diagrams (A and AD diagrams). The association equilibria can be accompanied by aggregation (precipitation) of histone proteins, especially initiated by EGCG. The titration equilibria are spectroscopically more pronounced in Tris buffers at higher pH values than at lower values, whereas in phosphate buffers the opposite trend is found. Surprisingly, catechin shows nearly no interactions with histone sulphate in phosphate buffers in the pH range 7.0-8.0, which is in contrast to EGCG. Fundamentally, the targets of chromosomes for catechins seem to be the histone proteins of chromatin.     

Chromosome diminution in Ascaris suum. Two-fold increase of nucleosomal histone to DNA ratios during development

The swine intestinal nematode, Ascaris suum, eliminates chromatin material from its primordial somatic cells during early embryogenesis. A technique for isolation of nuclei from pre- and post-diminution stage embryos has been developed and these isolated nuclei were used in investigations of nuclear events during diminution. The amount of DNA per nucleus determined by diphenylamine assays and isotope dilutions was 0.66 pg and 0.29 pg in pre- and post-diminution nuclei, respectively. Thus, A. suum loses 56% of its nuclear DNA during diminution. The loss of nuclear DNA enabled in vivo examination of histone to DNA ratios as a function of changes in DNA quantities. Ascaris histones were identified by acid extractability and tryptic fingerprint comparison with rat liver histones. Measurement of histone quantities was accomplished using linearity of Coomassie blue binding to histones separated in dodecyl sulfate gels. Ascaris nucleosomal histones levels were relatively constant in pre- and post-diminution nuclei. However, nucleosomal histone to DNA ratios approximately doubled during diminution.