[Alternative autologous arterial graft in cardiovascular surgery]. / Alternativní autologní tepenný step v kardiovaskulární chirurgii.

Arterial revascularization of myocardium is a method of choice for surgical treatment of ischemic heart disease. The descending branch of lateral femoral circumflex artery (DBLFCA) is one of possible alternative autologous arterial grafts. Up to now, since 2003, 12 of these grafts have been harvested and used for revascularization of myocardium at our department. All grafts have been used as a Y-graft to the left internal mammary artery for revascularization of target vessels on lateral and inferior heart wall. Graft patency was controlled by the angio-CT procedure after three months. One of the grafts was occluded at the control. It means patency rate after three months was more than 91%. No other complications (such as problematic wound healing, paraesthaesia of lower leg, bleeding, spasm of the graft, infection etc.) were observed with graft harvesting. DBLCFA is a high-quality and safe alternative arterial graft, available for arterial revascularization of myocardium.

Chromosome landmarks as tools to study the genome of Arabidopsis thaliana.

The model plant Arabidopsis thaliana has long been used for genetic, cellular and molecular studies. Whereas this plant was used as a model of genetics in the 1940's, the first cytogenetic observation of A. thaliana chromosomes was published in the beginning of the 20th century. Although Arabidopsis was not originally considered to be a good plant model for cytogenetics due to smallness of its genome, the number of published chromosome studies has expanded enormously in recent years. The advent of fluorescence in situ hybridization techniques on meiotic chromosomes together with indirect immuno-fluorescence localization of key chromosomal and nuclear proteins and wide accessibility of Arabidopsis mutants have resulted in a synergistic boost in Arabidopsis cytogenetics. In comparison to other plant species, the small genome with under-represented DNA repeats together with a small number of chromosomes makes this model plant easy to comprehend for a cytologist.

Cytogenetics for the study of telomere function in plants.

The ends of linear chromosomes of the vast majority of eukaryotic species possess specialized nucleo-protein structures called telomeres. Regardless of many exceptions, the structure and function of telomeres share high degrees of similarity between various eukaryotes. The underlying DNA structure of telomeres determines the particular setup of telomere chromatin and protein complexes as are telomere-associated proteins and a number of repair and cell cycle checkpoint agents. The structure of telomeres is highly dynamic during the cell's growth, replication, differentiation, senescence, or neoplastic transformation. Although the bulk of our knowledge about telomere function comes from molecular and biochemical studies in model organisms such as yeast and mouse, we want to show--with special emphasis on plants--in this short review that classical methods of plant cytogenetics can significantly complement the above experimental approaches and help in our understanding of telomere functions.

[Emergency surgical myocardial revascularization by means of a temporary arterial angioplasty]. / Urgentní chirurgická revaskularizace myokardu umoznená docasnou angioplastikou tepny.

The authors describe the case of a patient who was admitted for four-hour lasting acute myocardial infarction of the anterior wall with elevations of ST segments on ECG. The finding obtained in selective coronarography revealed an unsuitable condition for coronary intervention (a narrow stenosis of the stem, RIA occlusion, further two narrow stenoses in the coronary vascular bed). Since an operation room was not available at the moment the patient was indicated for palliative PTCA RIA (prevention of necrosis evolution) and subsequent urgent complete surgical revascularization of myocardium. The uncomplicated post-operation course and returned function of the affected myocardium indicates that the intervention may be considered as a suitable alternative for the treatment of acute myocardial infarction.

[Aortic connectors--the beginning or the end?]. / Aortální konektory--zacátek nebo konec?

In a group of 90 consecutive patients a total of 140 anastomoses were made, using the system of Aortic Connector Symmetry, and the results were compared with a control group of another 90 consecutive patients. Statistically significant differences were found in the period of extracorporeal circulation, the number of tight anastomoses and surgical revisions in favour of the investigated group. The other assessed parameters--period of cardiac ischaemia, number of peripheral anastomoses, mortality and blood losses were not significant.

Heterogeneity of rDNA distribution and genome size in Silene spp.

Genus Silene L. (Caryophyllaceae) contains about 700 species divided into 44 sections. According to recent taxonomic classification this genus also includes taxa previously classified in genera Lychnis and Melandrium. In this work, four Silene species belonging to different sections were studied: S. latifolia (syn. Melandrium album, Section Elisanthe), S. vulgaris (Inflatae), S. pendula (Erectorefractae), and S. chalcedonica (syn. Lychnis chalcedonica, Lychnidiformes). Flow cytometric analysis revealed a genome size of 2.25 and 2.35 pg/2C for S. vulgaris and S. pendula and of 5.73 and 6.59 pg/2C for S. latifolia and S. chalcedonica. All four species have the same chromosome number including the pair of sex chromosomes of the dioecious S. latifolia (2n = 2x = 24). Double target fluorescence in-situ hybridization revealed the chromosomal locations of 25S rDNA and 5S rDNA. A marked variation in number and localization of rDNA loci but no correlation between the numbers of rDNA clusters and genome size was found. FISH and genome size data indicate that nuclear genomes of Silene species are highly diversified as a result of numerous DNA amplifications and translocations.

Localization of male-specifically expressed MROS genes of Silene latifolia by PCR on flow-sorted sex chromosomes and autosomes.

The dioecious white campion Silene latifolia (syn. Melandrium album) has heteromorphic sex chromosomes, XX in females and XY in males, that are larger than the autosomes and enable their separation by flow sorting. The group of MROS genes, the first male-specifically expressed genes in dioecious plants, was recently identified in S. latifolia. To localize the MROS genes, we used the flow-sorted X chromosomes and autosomes as a template for PCR with internal primers. Our results indicate that the MROS3 gene is located in at least two copies tandemly arranged on the X chromosome with additional copy(ies) on the autosome(s), while MROS1, MROS2, and MROS4 are exclusively autosomal. The specificity of PCR products was checked by digestion with a restriction enzyme or reamplification using nested primers. Homology search of databases has shown the presence of five MROS3 homologues in A. thaliana, four of them arranged in two tandems, each consisting of two copies. We conclude that MROS3 is a low-copy gene family, connected with the proper pollen development, which is present not only in dioecious but also in other dicot plant species.

Euchromatic domains in plant chromosomes as revealed by H4 histone acetylation and early DNA replication.

Using specific polyclonal antisera raised against acetylated isoforms of histone H4, we have analyzed their distribution in the dioecious plant Silene latifolia (syn. Melandrium album) possessing heteromorphic sex chromosomes. Our previous studies on this species have shown that one of the two X chromosomes in homogametic female cells is heavily methylated and late replicating, as a possible consequence of dosage compensation. Here we report that there are no detectable differences in intensity and distribution of H4 acetylation between these two X chromosomes. In S. latifolia only distal-subtelomeric chromosome regions, on both the sex chromosomes and autosomes, display strong signals of H4 acetylation at N-terminal lysines 5, 8, and 12. These acetylated domains correspond to the very early replicating distal chromosome regions as revealed by 5-bromodeoxyuridine pulses followed by the indirect immunofluorescence microscopy. The distribution of H4 acetylated at lysine 16 was uniform along the chromosomes. The unique distal-subtelomeric H4 acetylation signals were also observed in three other Silene species (S. vulgaris, S. pendula, and S. chalcedonica), but not in two non-related plant species tested (Allium cepa and Nicotiana tobacum). The presented data as well as our recent studies on the structure of S. latifolia chromosome ends indicate that Silene species possess the specific distal-subtelomeric location of euchromatin, gene-rich regions on chromosomes.

DNA methylation patterns of Melandrium album chromosomes.

Melandrium album (syn. Silene latifolia) belongs to dioecious plant species possessing heteromorphic sex chromosomes, X and Y. Our previous experiments using in situ nick translation and replication kinetics analysis indicated structural and functional differences between the two X chromosomes in homogametic female cells. Here we show DNA methylation patterns of M. album root tip chromosomes using the indirect immunofluorescence approach with a monoclonal antibody raised against 5-methylcytosine (5-mC). In male cells, a more intensive 5-mC labelling on the shorter arm of the only X chromosome was observed in comparison with the longer X arm. A global hypermethylation of the male Y chromosome was not found, which indicates its prevalent euchromatic character. In female cells, the specific 5-mC pattern of the X chromosome was found on a single X chromosome, whereas the other X displayed an overall higher level of 5-mC labelling. Application of a hypomethylating drug, 5-azacytidine (5-azaC), during seed germination led to a deletion of any specific differences in the 5-mC distribution between the two X chromosomes. Confocal laser scanning microscopy analysis of DNA methylation in interphase nuclei showed hypermethylated domains that were efficiently decondensed and hypomethylated by 5-azaC treatment. The presented data show reproducible differences in the DNA methylation patterns between the two X chromosomes in M. album female somatic cells, which indicate their distinct transcriptional activities as a possible consequence of the negative dosage compensation of X-linked genes.

Histone H4 underacetylation in plant facultative heterochromatin.

It has been recently shown that facultative heterochromatin in some phyla is H4 and H3 histone underacetylated. Here we present H4 acetylation analyses in a monocotyledonous plant species, Gagea lutea, whose pentaploid endosperm nuclei possess prominent facultative heterochromatin regions. This heterochromatin is attributed to three chromosome sets originated from the chalazal polar nucleus of the embryo sac. We have previously shown that some parts of this heterochromatin contain heavily methylated DNA, but not all the heterochromatin is hypermethylated. In this report we demonstrate that this facultative heterochromatin is characterised by a conspicuous depletion of histone H4 acetylation at N-terminal lysine residues 5, 8, and 12, but not 16. Endosperm metaphases stained with antiserum against H4Ac5 indicated some heavily labelled chromosomes, while the others displayed no signal (presumably those coming from the three heterochromatinised chromosome sets). Western blotting analyses have shown that the antisera used, designed to detect human H4 histones, are suitable to recognise specific isoforms of acetylated H4 histones in plants and that the most abundant H4 in G. lutea leaves occurs in its diacetylated isoform. We conclude that flowering plants, similarly to protozoa, yeasts and animals, evolved core histone acetylation/deacetylation as a long-term transcriptional control mechanism to establish and/or transmit epigenetic information on gene expression.

Developmental control of telomere lengths and telomerase activity in plants.

Telomere lengths and telomerase activity were studied during the development of a model dioecious plant, Melandrium album (syn Silene latifolia). Telomeric DNA consisted of Arabidopsis-type TTTAGGG tandem repeats. The terminal positions of these repeats were confirmed by both Bal31 exonuclease degradation and in situ hybridization. Analysis of terminal restriction fragments in different tissues and ontogenetic stages showed that telomere lengths are stabilized precisely and do not change during plant growth and development. Telomerase activity tested by using a semiquantitative telomerase repeat amplification protocol correlated with cell proliferation in the tissues analyzed. Highest activity was found in germinating seedlings and root tips, whereas we observed a 100-fold decrease in telomerase activity in leaves and no activity in quiescent seeds. Telomerase also was found in mature pollen grains. Telomerase activity in tissues containing dividing cells and telomere length stability during development suggest their precise control during plant ontogenesis; however, the telomere length regulation mechanism could be unbalanced during in vitro dedifferentiation.

Structure and DNA methylation pattern of partially heterochromatinised endosperm nuclei in Gagea lutea (Liliaceae).

Pentaploid endosperm nuclei in certain Gagea species exhibit large masses of sticky and dense chromatin, not observed in somatic nuclei. These heterochromatin masses most probably stem from the triploid chalasal polar nucleus of the embryo sac, thus representing an example of facultative heterochromatinisation in plants. In the present investigation, we studied the nuclei in Gagea lutea (L.) Ker-Gawl, endosperm tissue. The position of the heterochromatin in interphase nuclei was observed by confocal laser scanning microscopy (CLSM) and the DNA methylation status of the euchromatin and heterochromatin was analysed by immunolabelling with an antibody raised against 5-methylcytosine (anti-5-mC). In young endosperms, heterochromatin was relatively dispersed, occupying some peripheral and inner parts of the nuclei. In a later endosperm development, the nuclei became smaller and more pycnotic, and the heterochromatin masses were placed predominantly near the nuclear periphery. The distribution of anti-5-mC labelling on the heterochromatic regions was unequal: some parts appeared hypermethylated while other parts were, like the euchromatin, not labelled. During mitosis, the labelling intensity of all the chromosomes was approximately the same, thus indicating that there are no cytologically detectable methylation differences among the individual sets of chromosomes. However, differences in the anti-5-mC signal intensity along individual chromosomes were observed, resulting in banding patterns with highly positive bands apparently representing constitutive heterochromatic regions. From these results it is obvious that facultative heterochromatinisation, in contrast to constitutive heterochromatinisation, need not be strictly accompanied by a prominent DNA hypermethylation.

A cytospin technique for spreading plant metaphases suitable for immunofluorescence studies.

Recent immunofluorescence techniques enable the localization of various cellular antigens, thus providing a powerful tool for cell and molecular biology research. Serious problems occur, however, when these techniques are applied to plant material. The presence of the cellulose wall can be a barrier to reproducible penetration of antibodies into cells and it often displays a confusing autofluorescence. A novel technique to prepare mitotic chromosome spreads from root tip meristems of germinating seeds is presented. Synchronous mitotic cells arrested in metaphase are converted into protoplasts using pectin and cellulose hydrolytic enzymes, and the purified protoplasts are fixed either in a methanol-acetic acid mixture to study DNA epitopes or in a nonextracting fixative to study chromosomal proteins. The latter fixative contains Triton X-100 to lyse the protoplasts and neutral formaldehyde to fix proteins by cross-linking. The protoplasts are immediately centrifuged onto microscopic slides as commonly done for mammalian cytogenetics. Using commercially available antibodies and both epifluorescence and confocal laser scanning microscopy, we demonstrated that the acid fixed chromosome slides are suitable for detection of DNA (anti-DNA antibody) or incorporated 5-bromodeoxyuridine (anti-BrdU antibody), while the cytospun formaldehyde and Triton X-100 fixed samples are convenient for detecting histones (antihistone antibody, pan). This technique should provide a general tool to study structural and functional domains of plant chromosomes.

Chromatin fragmentation associated with apoptotic changes in tobacco cells exposed to cold stress.

Programmed cell death (PCD) may be triggered by a variety of environmental stimuli. In this report we show that low temperature treatment of tobacco BY-2 cells results in specific chromatin changes. The early stage was characterised by chromatin condensation associated with specific endonucleolytic cleavage of the genome into fragments of 50-100 kbp in size. Later, after 2 weeks of the cold treatment, a ladder of nucleosomal units (178 bp) and their multiples occurred. Chromatin changes were accompanied by a general decrease in cell viability. However, the cell culture retained about 11% of living cells even after prolonged incubation in the cold suggesting the presence of a cold-resistant population of cells. The results support the view that PCD was activated by the cold stress. We suggest that cold-stressed tobacco BY-2 culture might be a useful system for investigation of PCD in plant cells.

Isolation and characterization of X chromosome-derived DNA sequences from a dioecious plant Melandrium album.

A number of X chromosome DNA sequences have been isolated from a dioecious plant, Melandrium album (syn. Silene latifolia), using chromosome microdissection followed by degenerate oligonucleotideprimed polymerase chain reaction (DOP-PCR) amplification. Six DNA clones were selected and further characterized by DNA/DNA hybridization techniques to check their copy numbers, sex-specific methylation patterns, species specificity and positions on chromosomes. These clones were moderately to highly repetitive (approximately 10(3)-10(5) copies per haploid genome) and none of them gave a positive signal on Northern blots. One of the clones yielded a sex-specific methylation pattern: its abundant non-methylated CCGG island was found only in males. All the clones also hybridized to two closely related dioecious Melandrium species (M. rubrum and M. dicline). Nucleotide sequences of two X-derived clones showed a number of internal short direct repeats; one of them strikingly resembled a plant conservative telomere sequence (TTTAGGG). None of the clones hybridized to the X chromosome only, but all were localized at the telomeric heterochromatic regions (DAPI C-bands) of both arms of a vast majority of M. album chromosomes using the fluorescence in situ hybridization (FISH) technique. However, the non-homologous arm of the Y chromosome (contrary to the arm homologous to the X chromosome, possessing the pseudoautosomal region) showed neither a DAPI C-banding-stained heterochromatin nor a FISH signal with any of the DNA probes tested, thus indicating its evolutionary diversification.

Isolation of Y chromosome-specific sequences from Silene latifolia and mapping of male sex-determining genes using representational difference analysis.

The genomic subtraction method representational difference analysis (RDA) was used to identify male-specific restriction fragments in the dioecious plant Silene latifolia. Male-specific restriction fragments are linked to the male sex chromosome (the Y chromosome). Four RDA-derived male-specific restriction fragments were used to identify polymorphisms in a collection of X-ray-generated mutant plants with either hermaphroditic or asexual flowers. Some of the mutants have cytologically detectable deletions in the Y chromosome that were correlated with loss of male-specific restriction fragments. One RDA-derived probe detected a restriction fragment present in all mutants, indicating that each has retained Y chromosomal DNA. The other three probes detected genomic fragments that were linked in a region deleted in some hermaphroditic and some asexual mutants. Based on the mutant phenotypes and the correlation of cytologically visible deletions with loss of male-specific restriction fragments, these markers were assigned to positions on the Y chromosome close to the carpel suppression locus. This RDA mapping also revealed a Y-linked locus, not previously described, which is responsible for early stamen development.

Epigenetic control of sexual phenotype in a dioecious plant, Melandrium album.

Melandrium album (syn. Silene latifolia) is a model dioecious species in which the Y chromosome, present only in heterogametic males, plays both a male-determining and a strict female-suppressing role. We showed that treatment with 5-azacytidine (5-azaC) induces a sex change to androhermaphroditism (an-dromonoecy) in about 21% of male plants, while no apparent phenotypic effect was observed in females. All of these bisexual androhermaphrodites (with the standard male 24, AA + XY karyotype) were mosaics possessing both male and hermaphrodite flowers and, moreover, the hermaphrodite flowers displayed various degrees of gynoecium development and seed setting. Southern hybridization analysis with a repetitive DNA probe showed that the 5-azacytidine-treated plants were significantly hypomethylated in CG doubles, but only to a minor degree in CNG triplets. The bisexual trait was transmitted to two successive generations, but only when androhermaphrodite plants were used as pollen donors. The sex reversal was inherited with incomplete penetrance and varying expressivity. Based on the uniparental inheritance pattern of androhermaphroditism we conclude that it originated either by 5-azaC induced inhibition of Y-linked female-suppressing genes or by a heritable activation of autosomal female-determining/promoting genes which can be reversed, on passage through female meiosis, by a genomic imprinting mechanism. The data presented indicate that female sex suppression in M. album XY males is dependent on methylation of specific DNA sequences and can be heritably modified by hypomethylating drugs.

Characterization of a new family of tobacco highly repetitive DNA, GRS, specific for the Nicotiana tomentosiformis genomic component.

Members of a new family of highly repetitive DNA sequences called GRS were isolated from Nicotiana tabacum L. genomic DNA and characterized. Cloned, sequenced monomeric units (180-182 bp) of GRS exhibit properties characteristic of molecules that possess a stable curvature. The GRS family represents about 0.15% of total genomic DNA (10(4) copies per haploid genome) and could be derived from either Nicotiana tomentosiformis or Nicotiana otophora, two possible ancestors of the T genome of the amphidiploid N. tabacum. Sequence homology between the HRS60 (Koukalová et al. 1989) and the GRS family has been estimated to be 57%. In situ hybridization was used to localize GRS on mitotic chromosomes. Hybridization signals were obtained on five pairs of chromosomes at intercalary sites of the longer chromosome arms. The majority of GRS sequences appeared to be organized in tandem arrays and a minority were found to be dispersed through the genome in short clusters, interspersed with other types of DNA repeats, including 25S rDNA sequences. Several loci containing both GRS and HRS60 were also found. Such hybrid loci may indicate intergenomic transfer of the DNA in the amphidiploid N. tabacum. GRS sequences, like HRS60 (Fajkus et al. 1992), were found to specify the location of nucleosomes. The position of the nucleosome core has been mapped with respect to a conserved Mbol site in the GRS sequence and an oligo A/T tract is a major centre of the DNA curvature.

Hybridization frequencies of different mammalian cell types by electrofusion.

The efficiency of electrofusion of four types of cells: CHO, HeLa, mouse melanoma cells and human skin fibroblasts has been studied. The frequencies of fusion products were determined 1) directly in a closed flow-through fusion chamber after dielectrophoresis and pulsation; 2) after short-term postfusion cultivation period of 5 to 10 minutes; and 3) in various intervals up to 30 hours after fusion induction. No substantial differences were found in the rates of formation of heterokaryons and synkaryons between the individual cell types, and this confirmed the uniformity of the effects of electric fields on diverse cell membranes. After 5 hours of culture the yield of fusion products reached 15 to 35% in various cell combinations and the frequencies of synkaryons reached up to 7% in almost all the combinations studied 24 to 30 hours after fusion.

Homokaryons from animal and plant cells generated by electrofusion.

A new apparatus was constructed which enables the use of the electrofusion method to obtain polynuclear cells of various mammalian cell lines, erythrocytes and plant protoplasts. This technique was applied to both suspensions and monolayers. Electrical and other physical parameters were monitored to find optimal conditions for mutual contact of cells (dielectrophoresis) and subsequent fusion. In the suspension technique, dielectrophoresis of mouse erythrocytes occurred at a field frequency of 20 kHz and a strength of 500 V.cm-1, whereas cultured mammalian cells and plant protoplasts required a frequency of 1-1.4 MHz and a strength of 250-800 V.cm-1. Fusion of cells was induced after the application of 1 to 10 high-voltage pulses of 1-5 kV.cm-1, 10-36 microseconds duration. After these high-voltage pulses were to the monolayer of mouse L cells, about 12% viable homokaryons were obtained.