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Biomedical subjects

J Malínský

Publications and source records attributed to J Malínský.

At least 19 recordsLinked to original sources
PubMed2006

NORs and their transcription competence during the cell cycle.

In human cells ribosomal genes are organized as clusters, NORs, situated on the short arms of acrocentric chromosomes. It was found that essential components of the RNA polymerase I transcription machinery, including UBF, can be detected on some NORs, termed "competent" NORs, during mitosis. The competent NORs are believed to be transcriptionally active during interphase. However, since individual NORs were not observed in the cell nucleus, their interphase status remains unclear. To address this problem, we detected the competent NORs by two commonly used methods, UBF immunofluorescence and silver staining, and combined them with FISH for visualization of rDNA and/or specific chromosomes. We found that the numbers of competent NORs on specific chromosomes were largely conserved in the subsequent cell cycles, with certain NOR-bearing homologues displaying a very stable pattern of competence. Importantly, those and only those NORs that were loaded with UBF incorporated bromo-uridine in metaphase after stimulation with roscovitine and in telophase, suggesting that competent and only competent NORs contain ribosomal genes transcriptionally active during interphase. Applying premature chromosome condensation with calyculin A, we visualized individual NORs in interphase cells, and found the same pattern of competence as observed in the mitotic chromosomes.

Cell Cycle↗
PubMed2005

Dynamics of replication foci in early S phase as visualized by cross-correlation function.

To monitor gradual changes in the replication foci distribution during early S phase, different segments of newly synthesized DNA were visualized by immunocytochemical mapping of two consecutively incorporated deoxythymidine analogs in pulse-chase-pulse experiments in HeLa cells. The resulting dual-labeled fluorescence images were evaluated using cross-correlation function (CCF) analysis. General changes of CCF shape due to image deterioration caused by blur, noise, and lateral sampling (pixel size) were also discussed. Using CCF analysis of model images simulating either random initiation of new replication foci, or the firing of new foci in close proximity to completed ones, we were able to ascribe the changes in the early S replication foci distribution to the latter mechanism. In contrast to the data published previously, we monitored the dynamics of all replication foci for up to 3 h. In addition, we showed that the replication foci dynamics is well described by random walk model, so that the average de-localization of individual foci is proportional to square root of the applied chase.

DNA↗
PubMed2001

Non-isotopic mapping of ribosomal RNA synthesis and processing in the nucleolus.

The precise location of ribosomal RNA (rRNA) synthesis within the nucleolus is the subject of recent controversy; some investigators have detected nascent RNA in the dense fibrillar components (DFCs) while others have localized transcription to the fibrillar centers (FCs). We endeavored to resolve this controversy by applying a new technique for non-isotopic labeling of RNA and examined the synthesis and movement of non-isotopically labeled rRNA within the nucleolus. We found that rRNA is synthesized only in a restricted area of DFCs, also involving the boundary region with FCs. We traced a movement of RNA from transcription sites through DFCs to granular components. Our results indicate functional compartmentalization of DFCs with respect to the synthesis and processing of precursor rRNA. In situ mapping of the 5' leader sequence of the 5' external transcribed spacer together with transcription labeling indicated that transcription and the first steps in processing of precursor rRNA are spatially separated. Surprisingly, the results also pointed to a partially extended conformation of newly synthesized precursor rRNA transcripts.

Animals↗
PubMed2000

In situ fluorescence visualization of bromouridine incorporated into newly transcribed nucleolar RNA.

Bromouridine-triphosphate is commonly used for in situ immunocytochemical labeling of newly synthesized RNA in living cells. While extranucleolar transcripts do not require special conditions for visualization, special treatment prior to fixation (e.g. incubation with alpha-amanitine) is necessary for immunofluorescence detection of bromouridine-labeled nucleolar RNA in previous studies. We show in the present investigation that bromouridine-triphosphate is efficiently used by both extranucleolar and nucleolar RNA polymerases in living cultured cells. The failure to detect incorporated bromouridine within nucleoli is entirely due to improper treatment of cells after bromouridine incorporation. When methanol/acetone fixation is used, fluorescence signals within nucleoli can be routinely found.

Animals↗
PubMed2000

Nuclear pre-mRNA compartmentalization: trafficking of released transcripts to splicing factor reservoirs.

In the present study, the spatial organization of intron-containing pre-mRNAs of Epstein-Barr virus (EBV) genes relative to location of splicing factors is investigated. The intranuclear position of transcriptionally active EBV genes, as well as of nascent transcripts, is found to be random with respect to the speckled accumulations of splicing factors (SC35 domains) in Namalwa cells, arguing against the concept of the locus-specific organization of mRNA genes with respect to the speckles. Microclusters of splicing factors are, however, frequently superimposed on nascent transcript sites. The transcript environment is a dynamic structure consisting of both nascent and released transcripts, i.e., the track-like transcript environment. Both EBV sequences of the chromosome 1 homologue are usually associated with the track, are transcriptionally active, and exhibit in most cases a polar orientation. In contrast to nascent transcripts (in the form of spots), the association of a post-transcriptional pool of viral pre-mRNA (in the form of tracks) with speckles is not random and is further enhanced in transcriptionally silent cells when splicing factors are sequestered in enlarged accumulations. The transcript environment reflects the intranuclear transport of RNA from the sites of transcription to SC35 domains, as shown by concomitant mapping of DNA, RNA, and splicing factors. No clear vectorial intranuclear trafficking of transcripts from the site of synthesis toward the nuclear envelope for export into the cytoplasm is observed. Using Namalwa and Raji cell lines, a correlation between the level of viral gene transcription and splicing factor accumulation within the viral transcript environment has been observed. This supports a concept that the level of transcription can alter the spatial relationship among intron-containing genes, their transcripts, and speckles attributable to various levels of splicing factors recruited from splicing factor reservoirs. Electron microscopic in situ hybridization studies reveal that the released transcripts are directed toward reservoirs of splicing factors organized in clusters of interchromatin granules. Our results point to the bidirectional intranuclear movement of macromolecular complexes between intron-containing genes and splicing factor reservoirs: the recruitment of splicing factors to transcription sites and movement of released transcripts from DNA loci to reservoirs of splicing factors.

Biological Transport↗
PubMed1999

Nuclear organization studied with the help of a hypotonic shift: its use permits hydrophilic molecules to enter into living cells.

A new procedure for introduction of hydrophilic molecules into living cells based on efficient uptake of these molecules into the cells during hypotonic treatment is presented and its use is demonstrated by a variety of applications. Experiments with cultured vertebrate and Drosophila cells and various animal tissues demonstrated that the increase in cell membrane permeability under hypotonic conditions is a general phenomenon in all animal cells tested. The efficiency of the method depends on the composition and temperature of the hypotonic buffer, the duration of the hypotonic treatment and the molecular weight of the molecules introduced into living cells. The versatility of this approach is demonstrated with various types of molecules such as modified nucleotides, nucleotides with conjugated fluorochrome, peptides, phosphatase substrates and fluorescent dyes. The method opens new possibilities for the direct investigation of a variety of biological problems as documented here with data on the functional organization of the cell nucleus.

Amanitins↗
PubMed1999

An ultrastructural study of the biostimulative effect of He-Ne laser radiation on the early postnatal differentiation of the rat submandibular gland.

Submandibular gland of the rat during the first week of postnatal development was used for the study of the ultrastructural changes after the He-Ne laser radiation. In the experimental group we observed an increase of the dark cells with greater quantity of the granular endoplasmic reticulum and an increased number of specific secretory granules. These changes show positive biostimulative effect of He-Ne laser radiation on the morphologic differentiation of the submandibular gland of the rat in the early postnatal period.

Aging↗
PubMed1999

Nuclear channel system (NCS) in normal endometrium and after hormonal stimulation.

The ultrastructure of NCS was investigated in two groups of patients (15 in each) during the secretory phase of the normal menstrual cycle and after the hormonal stimulation performed in the IVF programme by GnRH-a, FSH and hCG. Three developmental stages of the NCS were classified. The most numerous were the NCS in the mature stage (type 2), less in the early developmental stage (type 1) and least in the regression stage (type 3). The simultaneous occurrence of all three types does not conform the statement of Spornitz. The proportions of the three types were identical in both investigated groups of patients. The number of NCS (per 100 of nuclei) was higher in the group after hormonal stimulation than in the normal cycle. The quantitative measurement of the NCS showed its larger size in the group after hormonal stimulation than in the normal cycle, but the differences in the shape were not significant. In the serial ultrathin sections we demonstrated the relation of NCS to the nucleolus and to the invaginations of the nuclear envelope, the formation of the electron lucent center and the spiral arrangement of the tubules. In the early regression stage we described the relation of the internal row of tubules to the formed myelin figures. We suppose that the higher number and larger size of NCS can be taken as a sign of the increased activity of the endometrium during the early secretory phase after the hormonal stimulation.

Chorionic Gonadotropin↗
PubMed1999

Ultrastructure of endometrial surface relief in normal menstrual cycle and after hormonal stimulation.

Ultrastructure of the superficial relief of the endometrial epithelium was investigated under the scanning (SEM) and transmission electron microscopes (TEM) in a group of patients after the hormonal stimulation, performed in the program of in vitro fertilization and embryotransfer. The results were compared with those obtained in a group of patients studied during the secretory phase of normal menstrual cycle. Total 26 bioptic samples were subjected to the investigation, 13 in each group. Different types of the epithelial surface were classified in TEM and SEM and their relative amount was evaluated quantitatively in TEM. Special attention was paid to the pinopodes, considered as specific markers of the nidation window. Differences in the number of pinopodes were observed under the SEM in two groups of patients after different schemes of the hormonal treatment. These differences were not manifested under the TEM due to the small frequency of pinopodes in the ultrathin sections. The ultrastructural changes observed in the two groups of patients after different schemes of hormonal stimulation can be considered as signs of the changed metabolic activity of the endometrial epithelium and they can reflect the different endometrial receptivity for nidation.

Cell Membrane↗
PubMed1998

Fluorescent probing of membrane potential in walled cells: diS-C3(3) assay in Saccharomyces cerevisiae.

Membrane-potential-dependent accumulation of diS-C3(3) in intact yeast cells in suspension is accompanied by a red shift of the maximum of its fluorescence emission spectrum, lambda max, caused by a readily reversible probe binding to cell constituents. Membrane depolarization by external KCl (with or without valinomycin) or by ionophores causes a fast and reproducible blue shift. As the potential-reporting parameter, the lambda max shift is less affected by probe binding to cuvette walls and possible photobleaching than, for example, fluorescence intensity. The magnitude of the potential-dependent red lambda max shift depends on relative cell-to-probe concentration ratio, a maximum shift (572-->582 nm) being found in very thick suspensions and in cell lysates. The potential therefore has to be assessed at reasonably low cell (< or = 5 x 10(6) cells/ml) and probe (10(-7)M) concentrations at which a clearly defined relationship exists between the lambda max shift and the potential-dependent accumulation of the dye in the cells. The redistribution of the probe between the medium and yeast protoplasts takes about 5 min, but in intact cells it takes 10-30 min because the cell wall acts as a barrier, hampering probe penetration into the cells. The barrier properties of the cell wall correlate with its thickness: cells grown in 0.2% glucose (cell wall thickness 0.175 +/- 0.015 micron, n = 30) are stained much faster and the lambda max is more red-shifted than in cells grown in 2% glucose (cell wall thickness 0.260 +/- 0.043 micron, n = 44). At a suitable cell and probe concentration and under standard conditions, the lambda max shift of diS-C3(3) fluorescence provides reliable information on even fast changes in membrane potential in Saccharomyces cerevisiae.

Carbocyanines↗
PubMed1994

Effect of the new fluorescent brightener Rylux BSU on morphology and biosynthesis of cell walls in Saccharomyces cerevisiae.

Rylux BSU, a new fluorescent brightener from the family of 4,4'-diaminostilbene-2,2'disulfonic acid derivatives, inhibited growth and cytokinesis of the yeast Saccharomyces cerevisiae. In the presence of 0.1-1 mg/ml Rylux BSU the cells grew in clumps, had irregular shape and were larger than controls. They formed apparently normal primary septa but their secondary septa and lateral cell walls, especially those in older cells, were abnormally thick with large deposits of amorphous wall material in the periplasmic spaces all over the cell surface. Chitin content in the cell walls of cells grown in the presence of Rylux BSU was increased 2 to 5 times in comparison to that of the controls and glucan content was reduced by up to 30%. In the in vitro assays with particulate membrane fractions, Rylux BSU acted as a non-competitive inhibitor of beta-1,3-glucan synthase with inhibitory constant Ki = 1.75 mg/ml whereas the chitin synthase was inhibited to a much lesser extent. From the difference of the effects of Rylux BSU on the synthesis of chitin in vivo and in vitro it is concluded that the brightener interacts with chitin synthase only indirectly, possibly by influencing the properties of integral plasma membrane.

Benzenesulfonates↗
PubMed1992

The nucleus raphe magnus of the cat. I. Quantitative analysis of neurons.

According to the shape of the perikarya six different types of the neurons in the nucleus raphe magnus of the cat were classified: spherical, oval, fusiform, pyramidal, piriform and stellate. According to size small, medium, large and giant cells were described. The spherical neurons are small and the most uniform in the shape and the stellate perikarya are the most variable in the shape and size.

Animals↗
PubMed1992

The nucleus raphe magnus of the cat. II. Quantitative analysis of axosomatic synapses.

Quantitative analysis of axosomatic synapses in the nucleus raphe magnus of the cat showed great variations in their number, shape and size. There exist some relations between the size of perikarya and the number and size of the synaptic knobs. In the small neurons the number and size of synapses was smaller than in the large neurons, but there exist some exception from this rule. The observed morphological differences can be ascribed to different functional significance of various neurons.

Animals↗
PubMed1991

Cytoarchitectonics of some cortical areas of the feline neocortex (a quantitative study in semithin sections).

This paper presents quantitative analysis of the size of neurons in individual architectonic layers of the neocortex of the cat. In semithin sections the area, perimeter and maximal diameter of the perikarya and their nuclei were measured. Eight different cortical areas were evaluated, including the motor cortex (anterior and lateral sigmoid gyrus), the sensory cortex (coronal gyrus and anterior ectosylvian gyrus), the association cortex (lateral gyrus), the auditory cortex (medial and posterior ectosylvian gyrus) and the visual cortex (occipital gyrus). The obtained quantitative data are summarized in tables.

Animals↗
PubMed1991

Morphology of epithelizing varicose ulcers following He-Ne laser therapy.

The present study is a report on the morphological, particularly ultrastructural findings in the newly formed epidermis of varicose ulcerations after a series of irradiations with a low energy He-Ne laser. The ultrastructural alterations are interpreted as most probably conditioned by the biostimulating effect of the radiation and they are compared with the results obtained by other investigators studying these problems and with findings in the epithelium of embryonal epidermis as well as in the epidermis exposed to ultraviolet radiation.

Epithelium↗