Labelling of DNA in replicating cells using 5-bromo-2'- deoxyuridine (BrdU) is widely used, however the rapid clearance and metabolisation of BrdU in the living organism is a critical issue. Although the pharmacokinetic of BrdU in experimental animals is empirically approximated, the exact time-curve remains unknown. Here we present novel method for estimation of the BrdU content in the blood serum. The application is based on the in vitro cocultivation of tumour cells with the examined serum and the subsequent quantification of the incorporated BrdU in the DNA using flow cytometry analysis. Our results demonstrate that this approach can quantify the BrdU concentration in serum at 1 μmol.dm-3 and might represent an attractive alternative to conventional chromatographic analysis. The employment of tumour cells as "detectors" of the BrdU content in serum provides an advantage over high pressure liquid chromatography (HPLC), as this approach allows us to approximate not only the concentration of BrdU, but also to determine, whether BrdU is present in the blood serum in effective concentration to reliable label all cells undergoing the S-phase of the cell cycle. The presented application might be a helpful tool for studies on pharmacokinetics of BrdU or other thymidine analogues when testing various administration routes or protocols., J. Mikeš, J. Ševc, J. Košuth, A. Matiašová, Z. Daxnerová, P. Fedoročko., and Obsahuje bibliografii
The agamosporous and taxonomically critical Dryopteris affinis group was investigated as part of a cytogeographic and morphometric study of ferns in Central Europe. Material from 27 localities in the Czech Republic, Slovakia, Poland and Austria was sampled and evaluated using both morphometric multivariate and karyological analyses. Chromosome counts and flow cytometric analyses revealed the existence of two distinct triploid taxa (2n = 123) of differing genome size, which correspond to D. borreri and D. cambrensis, and of a rare pentaploid hybrid (2n = 205) D. ×critica (D. borreri × D. filix-mas). Morphometric analyses confirmed a clear separation between both triploid taxa. New quantitative characters were selected based on a discriminant analyses, and a key for the identification of the species is presented.
DAPI and propidium iodide flow cytometry were used to determine the variation in genome size in 166 samples and of all taxa and ploidy levels of Fallopia section Reynoutria (knotweeds) recorded in the Czech Republic. Significant differences were detected in the amount of nuclear DNA, associated with the ploidy levels and taxonomic identity of the material. At each ploidy level, F. sachalinensis showed the lowest and F. japonica the highest fluorescence intensities. The fluorescence values for the hybridogenous F. ×bohemica were located in-between these two levels. In most cases, there was at least a four-percent gap in fluorescence values between the nearest neighbours belonging to a different taxon. Intraspecific variation in genome size was very low in all taxa except hexaploid F. ×bohemica; this could be due to the complex evolutionary history of this taxon. Our results indicate that the amount of nuclear DNA can be used as a reliable marker for the identification of homoploid knotweed species and their hybrids. Different evolutionary pathways for the origin of high polyploids and/or hybridogenous taxa are proposed based on genome size.
Variation in genome size in a particular taxonomic group can reflect different evolutionary processes including polyploidy, hybridization and natural selection but also neutral evolution. Using flow cytometry, karyology, ITS sequencing and field surveys, the causes of variation in genome size in the ecologically and morphologically diverse high-Andean genus Lasiocephalus (Asteraceae, Senecioneae) were examined. There was a 1.64-fold variation in holoploid genome size (C-values) among 189 samples belonging to 20 taxa. The most distinct was a group of plants with large genomes corresponding to DNA triploids. Disregarding the DNA triploids, the remaining samples exhibited a pronounced (up to 1.32-fold) and rather continuous variation. Plants with the smallest genomes most likely represent intergeneric hybrids with the closely related and sympatric Culcitium nivale, which has a smaller genome than Lasiocephalus. The variation in genome size in samples of diploid Lasiocephalus was strongly correlated with several environmental and life history traits (altitude, habitat and growth form). However, all these factors, as well as genome size itself, were correlated with phylogeny (main split into the so-called ‘forest’ and ‘páramo’ clades), which most probably represents the true cause of the differentiation in intrageneric genome size. In contrast, relationships between genome size and phylogeny were not apparent at lower divergence levels. Instead, here we suggest that ecological conditions have played a role in driving shifts in genome size between closely related species inhabiting different environments. Collectively, this study demonstrates that various evolutionary forces and processes have shaped the variation in genome size and indicates that there is a need for multi-approach analyses when searching for the causes and consequences of changes in genome size.
Liverworts are poorly represented in the record of DNA C-values. Data for not more than nine species are reported in the literature. Here we present flowcytometric measurements of genome size for 32 foliose and 11 thallose species from 22 out of 83 families. The main method used in this study was flow cytometry using propidium iodide as the DNA stain. Feulgen densitometrywas applied as a supplementary method but it proved less suitable because the rigid cellwalls of liverwort tissue are resistant to maceration and apparently often inhibit the diffusion of reagents, which results in low estimates of DNA content. The precise or approximate number of chromosomes were counted, where possible. Among the thallose liverworts, the lowest 1C-value was recorded for Marchantia polymorpha (0.293 pg) and the highest for diploid Pellia epiphylla (7.401 pg). Haploid P. epiphylla (1C = 3.803 pg) had the largest genome among the haploid thalloid liverworts. Among the foliose liverworts, Lejeunea cavifolia with a value of 0.211 pg (1C) was ranked the lowest and Mylia taylorii, a haploid with 7.966 pg (1C) and a large amount of dense heterochromatin, concentrated in one big spherical chromocentre, the highest. This 38-fold variation covers the extremes of the whole sample and exceeds the ca 12-fold variation recorded in mosses (0.174–2.160 pg, 1C). This variation is nevertheless low compared to the 2000-fold interspecific variation found in angiosperms. Several instances of intraspecific variation in DNA ploidy (x and 2x) were found – in Radula complanata, Pellia epiphylla and Metzgeria furcata. In Lophocolea heterophylla, accessions differed 3.37-fold in C-value at haploid chromosome number. This points to cryptic taxonomic differentiation and warns against premature statements about ploidy levels based only on DNA measurements. Significant intraspecific intraploidal variation in C-value was also observed in certain instances. In Frullania dilatata, female plants with two large heterochromatic sex-chromosomes have a 1.35-fold higher C-value than male plants with only one sex chromosome. In most other cases of intraspecific variation the role of sex differences remains to be clarified.
A detailed cytogeographic and morphometric study of the Asplenium trichomanes group in the Czech Republic is presented. We detected diploid (2n = 72), tetraploid (2n = 144) and hybrid triploid plants (2n = 108). Based on the morphometric study, four intraspecific taxa are recognized. These taxa correspond to the four subspecies of A. trichomanes (A. t.subsp. trichomanes, <I.A. t. subsp. quadrivalens, A. t. subsp. pachyrachis and A. t. subsp. hastatum) distinguished in the floras of western, southern and northern Europe. Triploid plants were determined as A. t. nothosubsp. lusaticum (A. t. subsp. trichomanes × A. t. subsp. quadrivalens). The individual morphological characters used for determining subspecies are evaluated and a determination key presented.
Genome size has been suggested as one of the traits associated with invasiveness of plant species. To provide a quantitative insight into the role of this trait, we estimated nuclearDNAcontent in 93 alien species naturalized in the Czech Republic, belonging to 32 families, by using flow cytometry, and compared it with the values reported for non-invading congeneric and confamilial species from the Plant DNA C-values database. Species naturalized in the Czech Republic have significantly smaller genomes than their congeners not known to be naturalized or invasive in any part of the world. This trend is supported at the family level: alien species naturalized in the Czech flora have on average a smaller genome than is the mean value for non-invading confamilials. Moreover, naturalized and non-invading species clearly differed in the frequency of five genome size categories; this difference was mainly due to very small genomes prevailing and intermediate to very large genomes underrepresented in the former group. Our results provide the first quantitative support for association of genome size with invasiveness, based on a large set of alien species across a number of plant families. However, there was no difference in the genome size of invasive species compared to naturalized but non-invasive. This suggests that small genome size provides alien plants with an advantage already at the stage of naturalization and need not be necessarily associated with the final stage of the process, i.e. invasion.
Flow cytometry measurements confirmed the occurrence of Polypodium ×mantoniae (P. interjectum × P. vulgare) at three localities in the eastern part of the Czech Republic (Blansko and Rudice N of Brno and Javoříčko WNW of Olomouc). Nuclear DNA contents (± Sx) were determined for P. vulgare (2C = 29.00 ± 0.32 pg), P. ×mantoniae (2C = 37.18 ± 0.38 pg) and P. interjectum (2C = 45.24 ± 0.31 pg) using a PAS Partec GmbH flow cytometer (PI staining / standard Vicia faba, 2C = 26.9 pg). The relative DNA content ratio was measured in all pairs of taxa (± Sx range), i.e. P. ×mantoniae : P. vulgare = 1.340 ± 0.008; P. interjectum : P. vulgare = 1.681 ± 0.003; P. interjectum : P. ×mantoniae = 1.255 ± 0.008. Six new localities for Polypodium interjectum were found in the region of Moravský Kras (= Moravian Karst, N of Brno). From the PI/DAPI index it can be inferred that the AT/GC ratio (or heterochromatin occurrence) is 1.05× bigger in P. ×mantoniae than in P. vulgare and 1.08× bigger in P. interjectum than in P. vulgare. Anatomical data (number of thick- walled cells in the anulus, spore length and stomata length) of selected specimens and live samples from the Czech Republic were in good agreement with the range of variation of these features published by earlier authors from other European countries. A brief historical survey of the knowledge of P. interjectum in the Czech Republic is included.
Plasma levels of circulating platelet extracellular vesicles (PEVs) are an emerging marker of platelet activation, thrombosis, inflammation, and endothelial dysfunction. Analysis of PEVs in cord blood of preterm newborns may reflect the underlying pathology and possibly serve as a new diagnostic and prognostic tool. However, collection, preparation and analysis of cord blood samples in clinical settings is a logistically complex process. We have studied the effect of delay in sample preparation and sample freezing on the PEV analysis by flow cytometry. PEVs in the cord blood plasma were identified after double labelling with monoclonal antibodies CD36+CD41 or CD41+CD62. Both, the delay and the freezing significantly affected the count and often also fluorescence of the detected PEVs. Additionally, our pilot study utilizing fresh cord blood samples of term and preterm newborns demonstrated significantly decreased CD36 and CD62 PEV fluorescence in preterm newborns. Our data highlight the importance of pre-analytical steps in the analysis of cord blood PEVs and suggest that not only the count, but also the level of PEV fluorescence may have possible diagnostic potential.