BACKGROUND: Only fresh plant material is generally used for rapid DNA ploidy estimation by flow cytometry (FCM). This requirement, however, substantially limits convenient FCM application in plant biosystematics, population biology, and ecology. As desiccation is a routine way for sample preservation in field botany, potential utilization of dehydrated tissues of vascular plants in FCM research was examined. METHODS: Standard DAPI protocol was employed to evaluate the performance of 60 air-dried species, spanning more than 100-fold range of nuclear DNA amounts. Multiploid Vaccinium subg. Oxycoccus was selected as model taxon for detailed investigation and cytotype comparison. RESULTS: A majority of analyzed plants yielded distinct peaks with reasonable coefficients of variation after several months of storage at room temperature. Fluorescence intensity of nuclei isolated from desiccated tissues was highly comparable with that for fresh material, allowing reliable DNA ploidy estimation. Deep-freezer preservation substantially extended Vaccinium samples lifetime (at least to 3 years) and maintained high histogram resolution. CONCLUSIONS: The introduced approach eliminates the need for fresh material in many vascular plants and thus opens new prospects for plant FCM. Convenient cytotype investigation in field research and retrospective ploidy determination in already herbarized samples are among the principal advantages.
We applied the alkaline version of the single-cell gel electrophoresis (comet) assay to roots and leaves of tobacco (Nicotiana tabacum var. xanthi) seedlings or isolated leaf nuclei treated with: (1) the alkylating agent ethyl methanesulphonate, (2) necrotic heat treatments at 50 degrees C, and (3) DNase-I. All three treatments induced a dose-dependent increase in DNA migration, expressed as percentage of tail DNA. A comparison of the fluorochrome DNA dyes ethidium bromide, DAPI and YOYO-1 demonstrated that for the alkaline version of the comet assay in plants, the commonly used fluorescent dye ethidium bromide can be used with the same efficiency as DAPI or YOYO-1.
- MeSH
- Alkylating Agents toxicity MeSH
- Single-Cell Analysis MeSH
- Staining and Labeling methods MeSH
- Benzoxazoles chemistry MeSH
- Quinolinium Compounds chemistry MeSH
- Deoxyribonuclease I metabolism pharmacology MeSH
- DNA, Plant analysis chemistry MeSH
- Ethidium chemistry MeSH
- Ethyl Methanesulfonate toxicity MeSH
- Fluorescent Dyes MeSH
- Indoles chemistry MeSH
- Comet Assay MeSH
- Plant Roots chemistry drug effects MeSH
- Plant Leaves chemistry drug effects MeSH
- Seedlings chemistry drug effects MeSH
- Nicotiana chemistry drug effects MeSH
- Hot Temperature MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- MeSH
- Staining and Labeling MeSH
- Cell Nucleus * MeSH
- Fluorescent Dyes MeSH
- Fluorescent Antibody Technique MeSH
- Indoles * MeSH
- Humans MeSH
- Propidium MeSH
- Check Tag
- Humans MeSH
- Publication type
- Letter MeSH
- Research Support, Non-U.S. Gov't MeSH
Clorgylin je špecifický inhibitor enzýmu monoamínooxidáza A (MAO-A), ktorý je zodpovedný za metabolizmus noradrenalínu, serotonínu a dopamínu. Samice potkana Wistar sme náhodne rozdelili do skupín: 1) kontrolná, 2) nízka dávka clorgylínu, 3)vysoká dávka clorgylínu. Po ukončení aplikácie boli zvieratá pripúšťané so samcami a na 5. deň gravidity usmrtené letálnou dávkou tiopentalu. Embryá boli vypláchnuté z rohov maternice a zatriedené do jednotlivých vývinových skupín. Blastocysty a moruly sme dofarbili pomocou DAPI. Zvieratá po aplikácii nízkej dávky clorgylínu malí sice najvyšší počet izolovaných ale zároveň aj najvyšší počet degenerovaných embryí. U zvierat po vysokej dávke clorgylínu sme zase zistili najvyšší počet buniek v blastocystách. Zmeny hladín noradreninalínu. serotonínu a dopamínu môžu teda výrazne ovplyvňovať vývin nového jedinca už od jeho najranejšíďch štádií.
Clorgyline is a specific inhibitor of monoamine oxidase A (MAO-A), which is responsible for the degradation of the noradrenalin, serotonin and dopamine. Females of the Wistar rats were divided into the three groups: 1) controls, 2) animals after the low dose of the clorgyline, 3) animals after the high dose of the clorgyline. After the treatment were females mated with the males and on the 5th day of the pregnancy killed by the lethal dose of the tiopental. Embryos were flushed out from the horns of the uterus and divided into the developmental groups by the morphological examination. Blastocysts and morulae were stained with the DAPI. We have found that females from the group after the low clorgyline dose had the higher number of isolated embryos per female but also the higher number of the degenerated embryos. In the group with the high clorgyline dose we have found the higher numbers of cells in the blastocysts. From our results is clear that disturbed levels of noradrenalin, serotonin and dopamine can influence the new individual from its most early stages of the development.
- Keywords
- preimplantačné embryo, clorgyline, MAO,
- Publication type
- Abstracts MeSH
Contamination of cell cultures by mycoplasmas is a very common phenomenon. As they can substantially alter cell metabolism and potentially spread to all cell cultures in laboratory, their early detection is necessary. One of the fastest and cheapest methods of mycoplasma detection relies on the direct staining of mycoplasmas' DNA by DAPI or Hoechst dyes. Although this method is easy and fast to perform, it suffers from the low signal provided by these dyes compared to the nuclear DNA. Therefore, the reporter cell lines are used for cultivation of mycoplasmas before DAPI or the Hoechst staining step. In the study presented, we have developed and tested a new immunofluorescence assay for the detection of mycoplasmas. The method is based on the enzymatic labeling using DNA polymerase I and modified nucleotides utilizing nicks in the mycoplasmas' DNA. Modified nucleotides are incorporated into mycoplasmas' DNA and subsequently visualized by immunofluorescence microscopy. The developed approach is independent of the mycoplasma strain, does not intensely stain nuclear DNA, does not stain other bacteria, and provides higher sensitivity than the approach based on the direct labeling using DAPI or Hoechst dyes.
- MeSH
- Staining and Labeling MeSH
- A549 Cells MeSH
- DNA Polymerase I chemistry MeSH
- Microscopy, Fluorescence methods MeSH
- Humans MeSH
- Mycoplasma fermentans isolation & purification MeSH
- Mycoplasma hominis isolation & purification MeSH
- Mycoplasma isolation & purification MeSH
- Mycoplasma Infections microbiology MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Yeasts Cryptococcus humicola accumulated cadmium, cobalt, and iron (~ 50, 17, and 4% of the content in the medium, respectively) from the medium containing glucose, phosphate, and 2 mmol/L of metal salts. The effects of metal absorption on the levels of orthophosphate (Pi) and inorganic polyphosphate (polyP) varied for the metals under study. The levels of Pi and polyP increased in the case of cadmium and cobalt, respectively. In the case of iron, no changes in the levels of Pi and polyP were observed. Multiple DAPI-stained polyP inclusions were observed in the cytoplasm of cadmium-containing cells. The intensity of DAPI staining of the cell wall especially increased in case of cobalt and iron accumulation.
- MeSH
- Biomass MeSH
- Cryptococcus metabolism MeSH
- Nitrogen metabolism MeSH
- Cadmium chemistry metabolism pharmacokinetics MeSH
- Cobalt chemistry metabolism pharmacokinetics MeSH
- Polyphosphates chemistry metabolism pharmacokinetics MeSH
- Sorption Detoxification MeSH
- Iron chemistry metabolism pharmacokinetics MeSH
- Publication type
- Journal Article MeSH
Cellular senescence, an irreversible proliferation arrest evoked by stresses such as oncogene activation, telomere dysfunction, or diverse genotoxic insults, has been implicated in tumor suppression and aging. Primary human fibroblasts undergoing oncogene-induced or replicative senescence are known to form senescence-associated heterochromatin foci (SAHF), nuclear DNA domains stained densely by DAPI and enriched for histone modifications including lysine9-trimethylated histone H3. While cellular senescence occurs also in premalignant human lesions, it is unclear how universal is SAHF formation among various cell types, under diverse stresses, and whether SAHF occur in vivo. Here, we report that human primary fibroblasts (BJ and MRC-5) and primary keratinocytes undergoing replicative senescence, or premature senescence induced by oncogenic H-Ras, diverse chemotherapeutics and bacterial cytolethal distending toxin, show differential capacity to form SAHF. Whereas all tested cell types formed SAHF in response to activated H-Ras, only MRC-5, but not BJ fibroblasts or keratinocytes, formed SAHF under senescence induced by etoposide, doxorubicin, hydroxyurea, bacterial intoxication or telomere attrition. In addition, DAPI-defined SAHF were detected on paraffin sections of Ras-transformed cultured fibroblasts, but not human lesions at various stages of tumorigenesis. Overall, our results indicate that unlike the widely present DNA damage response marker γH2AX, SAHF is not a common feature of cellular senescence. Whereas SAHF formation is shared by diverse cultured cell types under oncogenic stress, SAHF are cell-type-restricted under genotoxin-induced and replicative senescence. Furthermore, while the DNA/DAPI-defined SAHF formation in cultured cells parallels enhanced expression of p16(ink4a) , such 'prototypic' SAHF are not observed in tissues, including premalignant lesions, irrespective of enhanced p16(ink4a) and other features of cellular senescence.
- MeSH
- Bacterial Toxins pharmacology MeSH
- Cell Line MeSH
- Genes, ras MeSH
- Heterochromatin chemistry MeSH
- Cyclin-Dependent Kinase Inhibitor p16 genetics metabolism physiology MeSH
- Humans MeSH
- DNA Damage MeSH
- Cell Proliferation MeSH
- Cellular Senescence drug effects genetics physiology MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
BACKGROUND: There are several cortical areas related to the limbic system that form the output from the hippocampal formation whose cellular and morphological features are important for the onset and progression of AD. We hypothesized that there would be a significant difference in the size of cortical pyramidal neurons and that there would also be a hemispheric asymmetry between Alzheimer disease patients and controls. These differences would potentially be accompanied by an increase in the numbers of Fluoro-Jade B-positive degenerating cortical neurons and a corresponding decrease in the numbers of DAPI-stained cortical neuronal nuclei in subjects with AD compared to controls. Such changes could potentially be used as another marker in postmortem neuropathological diagnosis of AD. METHODS: We measured absolute numbers of DAPI and Fluoro-Jade B stained cells in five cortical areas of the limbic system and four subareas of planum temporale in the post-mortem brains of subjects with Alzheimer disease. We also measured the size of pyramidal neurons in layer III in the five cortical areas of the limbic system in these subjects. All measurements were performed separately for the left and right hemisphere in order to identify asymmetries between the two hemispheres. RESULTS: We observed a significant decrease in numbers of DAPI stained cells in layers IV-VI of the anterior cingulate gyrus on the right side, in layers I-III of the posterior cingulate gyrus on the left side, in layers IV-VI in the transition region from superior temporal gyrus into planum temporale on the right and in layers IV-VI in the transition from planum temporale to insular cortex on the left. We also observed a significant increase in the numbers of Fluoro-Jade stained cells in layers I-III of the anterior cingulate gyrus and in layers I-III on the left and layers IV-VI of the right gyrus of Heschl. Shortening of the size of layer III pyramidal neurons in subjects with Alzheimer´s disease was found in the anterior cingulate gyrus on the right, in the posterior cingulate gyrus and entorhinal cortex on the left and on the right in the parahippocampal gyrus. CONCLUSION: Our study demonstrates asymmetries in different cortical regions of the temporal lobe that can be used as another marker in the postmortem diagnosis of AD.
- MeSH
- Alzheimer Disease pathology MeSH
- Gyrus Cinguli pathology MeSH
- Fluoresceins metabolism MeSH
- Functional Laterality physiology MeSH
- Middle Aged MeSH
- Humans MeSH
- Brain Mapping * MeSH
- Cell Count MeSH
- Pyramidal Cells pathology MeSH
- Aged, 80 and over MeSH
- Aged MeSH
- Temporal Lobe pathology MeSH
- Check Tag
- Middle Aged MeSH
- Humans MeSH
- Male MeSH
- Aged, 80 and over MeSH
- Aged MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
The phylogenetic composition, bacterial biomass, and biovolume of both planktonic and biofilm communities were studied in a low-order Bystřice stream near Olomouc City, in the Czech Republic. The aim of the study was to compare the microbial communities colonizing different biofilm substrata (stream aggregates, stream sediment, underwater tree roots, stream stones, and aquatic macrophytes) to those of free-living bacteria. The phylogenetic composition was analyzed using fluorescence in situ hybridization for main phylogenetic groups. All phylogenetic groups studied were detected in all sample types. The stream stone was the substratum where nearly all phylogenetic groups were the most abundant, while the lowest proportion to the DAPI-stained cells was found for free-living bacteria. The probe specific for the domain Bacteria detected 20.6 to 45.8 % of DAPI-stained cells while the probe specific for the domain Archaea detected 4.3 to 17.9 %. The most abundant group of Proteobacteria was Alphaproteobacteria with a mean of 14.2 %, and the least abundant was Betaproteobacteria with a mean of 11.4 %. The average value of the Cytophaga-Flavobacteria group was 10.5 %. Total cell numbers and bacterial biomass were highest in sediment and root biofilm. The value of cell biovolume was highest in stone biofilm and lowest in sediment. Overall, this study revealed relevant differences in phylogenetic composition, bacterial biomass, and biovolume between different stream biofilms and free-living bacteria.
- MeSH
- Archaea classification genetics isolation & purification MeSH
- Bacteria classification genetics isolation & purification MeSH
- Biofilms * MeSH
- Biomass MeSH
- Chemical Phenomena MeSH
- Phylogeny * MeSH
- Geologic Sediments microbiology MeSH
- In Situ Hybridization, Fluorescence MeSH
- Rivers microbiology MeSH
- Publication type
- Journal Article MeSH
- Comparative Study MeSH
- Geographicals
- Czech Republic MeSH
We explored the mechanism of human osteosarcoma MG-63 cell apoptosis induced by asta-xanthin. The MTT assay was used to detect the effect of astaxanthin on cell viability. Morphological changes associated with apoptosis were observed after DAPI staining. Early and late stages of apoptosis were detected by flow cytometry with annexin V-FITC/PI staining. Activation of caspases-8, -9 and -3 was detected by enzyme activity in vitro. Changes in the mitochondrial membrane potential were detected by MitoCapture staining. Western blot was used to detect the cleavage of PARP, which is a caspase-3 substrate, the release of cytochrome c and Smac into the cytosol, the translocation of pro-apoptotic proteins Bax and Bak, and the expression of mitochondrial pathway-related proteins. The translocation of Bax was also detected by immunofluorescence assay. Astaxanthin significantly inhibited the viability of human osteosarcoma MG-63 cells with an IC50 value of 12.36 μg/ml. The DAPI-stained cells showed characteristic apoptotic morphological changes - cell shrinkage, cell membrane blebbing, nuclear condensation, and apoptotic body formation. Cytochrome c and Smac were released from mitochondria to the cytosol. Pro-apoptotic proteins Bax and Bak were rapidly translocated to mitochondria after six hours of astaxanthin action. Caspases-9 and -3 were activated and PARP was cleaved. The expression of anti-apoptotic proteins Bcl-2, Bcl-xL and XIAP was significantly decreased. Astaxanthin induced human osteosarcoma MG-63 cell apoptosis through the mitochondria-mediated endogenous apoptosis pathway.
- MeSH
- Apoptosis MeSH
- Cytochromes c * metabolism MeSH
- Humans MeSH
- Osteosarcoma * drug therapy metabolism MeSH
- Poly(ADP-ribose) Polymerase Inhibitors pharmacology therapeutic use MeSH
- bcl-2-Associated X Protein metabolism MeSH
- Apoptosis Regulatory Proteins pharmacology therapeutic use MeSH
- Proto-Oncogene Proteins c-bcl-2 metabolism MeSH
- Xanthophylls MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
