Biomedical subjects

I Raska

Publications and source records attributed to I Raska.

At least 37 records · Page 2Linked to original sources

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

Human autoantibodies identify a protein in dense fibrillar and granular components of the nucleolus.

A novel nucleolar protein of an approximate molecular weight of 60 kDa was identified by immunoprecipitation in human cells with an autoimmune sclerodermic serum. It maps at the ultrastructural level to nucleolar granular and dense fibrillar components. This 60 kDa protein could not be demonstrated in Western blots suggesting that the epitope structure is complex and/or is sensitive to the treatment of cells. The immunoprecipitation results indicate that the 60 kDa protein is not a phosphoprotein and is not associated with a nucleolar RNA containing particle. The identified protein represents a new autoimmune marker in the field of systemic connective tissue diseases.

Autoantibodies↗

PubMed1998

Histones and DNA ultrastructural distribution in plant cell nucleus: a combination of immunogold and cytochemical methods.

In this work we report for the first time the ultrastructural distribution of histones and DNA in the nuclear compartments in two different plant cell types: Allium cepa L. root meristems and Capsicum annuum L. microspores and pollen grains, by using antibodies against histones H2B and H4 and anti-DNA. Immunolocalizations were combined with ultrastructural cytochemistry for nucleic acids (methylation-acetylation method), DNA (NAMA-Ur) and RNPs (EDTA), to relate the subcellular location of histones and DNA with the chemical subcompartmentalization of the cell nucleus. This is particularly interesting concerning the presence of histones or not on fibers of the interchromatin region and on the fibrillar components of the nucleolus, nuclear subcompartments where transcription has been shown to take place at some regions. Our methodological approach permitted to define precisely the structures where histones were detected in relation to the ultrastructural localization of chromatin in various structural condensation levels. Concerning the localization of DNA and histones on the different components of the nucleolus, the combination of immunogold labeling with the methylation-acetylation cytochemical method, developed in our laboratory, was very useful, thus permitting a clear recognition of the nucleolar components and a correct assignment of labeling, which is not always evident on uranyl-lead-stained Lowicryl sections. Double immunogold assays were also done for a simultaneous visualization of histones and DNA. Our results show a coincident distribution of histones and DNA on the same nuclear compartments revealing the presence of both antigens on condensed chromatin, fibers of the interchromatin region, principally located at the periphery of the condensed chromatin, and in the fibrillar components of the nucleolus.

Acetic Anhydrides↗

PubMed1998

Long-term action of potassium bromide on the rat thyroid gland.

Male rats fed by a standard diet with determined of bromine and iodine content were exposed to a 133-day oral administration of KBr (100, 200, 400 mg Br-/l drinking water). Their thyroid glands showed increased growth of the epithelial cells reflected by a microfollicular rearrangement of the parenchyma due to proliferation of very small follicles with a low or zero content of colloid. Morphometric analysis of thyroids of Br(-)-exposed animals revealed a significant decrease in the volume of intrafollicular colloid and marked increase in the number of the smallest follicles (areas up to 100 and 100-300 micron 2). In addition, the nuclei of thyrocytes showed an increased number of mitoses. The vascularization was increased as well. In the blood plasma of the Br(-)-exposed animals the T4 concentration was significantly decreased in dependence on the bromine concentrations. Thyroglobulin immunoreactivity in the colloid of Br(-)-exposed animals decreased after administration of 400 mg Br-/l drinking water. Increasing concentrations of Br- in the drinking water caused an increased bromine concentration in the thyroid, a decreased iodine content and a decreased I/Br molar ratio. The changes in the rat thyroid caused by long-term administration of 100 mg Br-/l were similar to hyperplastic parenchymal goitre and were comparable to those induced in previous experiments by the same bromine concentration administered over a 16- and 66-day period respectively.

Administration, Oral↗

PubMed1998

Processing of free cells for electron microscopy using a fibrin clot.

A method of fibrin clot preembedding permitting the simple and gentle handling of free cells to be processed for electron microscopy is described. This technique is particularly useful for immunocytochemical techniques such as Lowicryl and thawed croysection approaches and represents a convenient alternative to procedures such as gelatine or agar preembeddings.

Antibodies, Monoclonal↗

PubMed1997

Heterogenous nuclear RNP C1 and C2 core proteins are targets for an autoantibody found in the serum of a patient with systemic sclerosis and psoriatic arthritis.

OBJECTIVE: To determine a target recognized by anti-Bh autoantibody, found in the serum of a patient with the unusual coexistence of systemic sclerosis (SSc) and psoriatic arthritis (PsA). METHODS: Antigens recognized by the anti-Bh serum were characterized by indirect immunofluorescence on HeLa cells, by conventional immunoblotting using nuclear extract or partially purified preparation of heterogenous nuclear RNP (hnRNP) proteins, and by 2-dimensional immunoblotting. For the analysis of cross-reactivity and immunofluorescence patterns, autoantibodies were affinity-purified by blot elution and then retested. RESULTS: Comparison of the reactivity of the anti-Bh antibody with the monoclonal antibody 4F4 against both the hnRNP C proteins, together with the determination of biochemical properties of the autoantigens, led to the identification of C1 and C2 core proteins as the targets for the anti-Bh autoantibody. CONCLUSION: Several essential components of the spliceosome are targeted by autoantibodies that are present in the sera of patients with systemic rheumatic diseases. We also found that the hnRNP core proteins C1 and C2 are recognized by the autoantibody present in the serum of a patient with SSc and PsA. C1 and C2 hnRNP proteins should be added to the several intracellular autoantigens recently shown to be cleaved by interleukin-1beta-converting enzyme-like enzymes during apoptosis.

Aged↗

PubMed1997

Expression of the proliferating cell nuclear antigen (PCNA) in the rat thyroid gland after exposure to bromide.

Analysis of expression of the proliferating cell nuclear antigen (PCNA) was used to determine the presumed hyperplastic character of morphological changes in the rat thyroid evoked by bromide administration. Male rats fed by a standard diet with determined iodine and bromine content were given potassium bromide. Control animals received no bromide. Experimental animals were given 10, 50 or 100 mg Br- per 11 drinking water for 16 and 66 days, or 100, 200, 400 mg Br-/l drinking water for 133 days. The thyroids of treated animals showed activation of growth of the epithelial follicular component as well as diffuse and focal microfollicular rearrangement of the parenchyma with higher follicular cells accompanied by a decrease of the amount of colloid even at low bromine concentrations (10-100 mg Br-/l drinking water). Using the PCNA-LI index (PCNA-positive nuclei.100/total number of follicular cell nuclei in the section), immunohistochemical analysis of PCNA in the nuclei of the follicular cells was carried out in parrafin sections. The index was significantly higher in bromide exposed animals (P < 0.01) and correlated well with the histological changes, with bromide concentration and with a increased mitotic activity of the follicular cells. PCNA analysis showed that morphological changes resembling a parenchymatic goitre reflect a microfollicular rearrangement of the thyroid of rats exposed to bromide and have the character of hyperplasia owing to the increased mitotic activity of the follicular epithelium.

Animals↗

PubMed1997

Potassium bromide and the thyroid gland of the rat: morphology and immunohistochemistry, RIA and INAA analysis.

The increasing environmental concentration of bromine has resulted in attempts to obtain information on its possibly deleterious effect on humans, particularly on a major target organ of this halogen i.e. the thyroid gland. In order to establish the morphological and functional effects of bromine on the thyroid, we have performed experiments on male rats which, in addition to a standard diet with an estimated iodine/bromine content, were fed for periods of 16 and 66 days with the small quantities of bromide expected to be encountered in the environment (10, 50 and 100 mg of Br-/l in drinking water). This treatment induced growth of the follicular epithelial component and microfollicular tissue rearrangement, a reduction of intrafollicular colloid, an increase in the height of the follicular cells and the number of mitoses, and it enhanced vascularization. Image analysis revealed a significant reduction in the volume of colloid, despite the accompanying rise in the number of minute follicles. The immunohistochemical positivity of the thyroglobulin fell in the microfollicular colloid of the exposed animals, although this was affected to a lesser extent in the larger follicles. The concentration of bromine in the thyroid increased with the amount of bromine intake, while at the same time the molar ratio of iodine/bromine decreased. The plasma level of T4 was lowered after both 16 and 66 days of treatment, but the T3 level only after 66 days treatment. The level of TSH did not exhibit any significant change. The observed changes, which have a parenchymatous goitre-like character, may have a direct relevance for human medicine, since the concentrations of bromide chosen in these experiments are readily encountered in the environment.

Animals↗

PubMed1996

The RNA 3' cleavage factors CstF 64 kDa and CPSF 100 kDa are concentrated in nuclear domains closely associated with coiled bodies and newly synthesized RNA.

The cleavage stimulation factor (CstF), and the cleavage and polyadenylation specificity factor (CPSF) are necessary for 3'-terminal processing of polyadenylated mRNAs. To study the distribution of 3' cleavage factors in the nuclei of human T24 cells, monoclonal antibodies against the CstF 64 kDa subunit and against the CPSF 100 kDa subunit were used for immunofluorescent labelling. CstF 64 kDa and CPSF 100 kDa were distributed in a fibrogranular pattern in the nucleoplasm and, in addition, were concentrated in 1-4 bright foci. Double immunofluorescence labelling experiments revealed that the foci either overlapped with, or resided next to, a coiled body. Inhibition of transcription with alpha-amanitin or 5,6-dichloro-beta-D-ribofuranosyl-benzimidazole (DRB) resulted in the complete co-localization of coiled bodies and foci containing 3' cleavage factors. Electron microscopy on immunogold double-labelled cells revealed that the foci represent compact spherical fibrous structures, we named 'cleavage bodies', intimately associated with coiled bodies. We found that approximately 20% of the cleavage bodies contained a high concentration of newly synthesized RNA, whereas coiled bodies were devoid of nascent RNA. Our results suggest that the cleavage bodies that contain RNA are those that are adjacent to a coiled body. These findings reveal a dynamic and transcription-dependent interaction between different subnuclear domains, and suggest a relationship between coiled bodies and specific transcripts.

Amanitins↗

PubMed1995

Nuclear ultrastructures associated with the RNA synthesis and processing.

Relatively little is known about the spatial organization of RNA synthesis, processing, and transport in (mammalian) cell nuclei. This review summarizes results of electron microscopic mapping of RNA synthetic sites and macromolecules involved directly, or indirectly, in the metabolism of RNAs in somatic cell mammalian nuclei. Significance of these results will be discussed in the context of the molecular mechanisms underlying spatial arrangements of RNA metabolism.

Cell Nucleolus↗

PubMed1994

The Drosophila lethal(2)giant larvae tumor suppressor protein is a component of the cytoskeleton.

Tumor suppressor genes act as recessive determinants of cancer. In Drosophila these genes play a role in normal development and are essential for regulating cell growth and differentiation. Mutations in the gene, lethal(2)giant larvae, l(2)gl, besides causing malignant tumors in the brain and imaginal discs, generate developmental defects in a number of other tissues. Much of the uncertainty regarding the function of the l(2)gl gene product, p127, results from a lack of knowledge as to the precise location of this protein in the cell. We have investigated the cellular and subcellular localization of p127, using confocal and electron microscopy as well as biochemical and cell fractionation procedures. Our analyses indicate that p127 is located entirely within the cell in both the cytoplasm and bound to the inner face of lateral cell membranes in regions of cell junctions. On the membrane, p127 can form large aggregates which are resistant to solubilization by nonionic detergents, indicating that p127 is participating in a cytoskeletal matrix. These findings suggest that the changes in cell shape and the loss of apical-basal polarity observed in tumorous tissues are a direct result of alterations in the cytoskeleton organization caused by l(2)gl inactivation and also suggest that p127 is involved in a cytoskeletal-based intercellular communication system directing cell differentiation.

Amino Acid Sequence↗

PubMed1993

Nonisotopic ultrastructural mapping of transcription sites within the nucleolus.

A nonradioactive ultrastructural method based on the incorporation of 5-bromouridine-5'-triphosphate into the RNA of streptolysin O-permeabilized cultured HeLa cells is described and used for the visualization of rRNA transcription sites. Even though the method provides much better resolution than ultrastructural autoradiography, the results obtained do not allow the assignment of rRNA transcription to a single nucleolar structural component. We locate the rRNA transcription sites at the border region of fibrillar centers with dense fibrillar components. In addition, the method represents a convenient tool for the in situ immunodetection of extranucleolar RNA synthesis.

Cell Nucleolus↗

PubMed1993

Experimental thyroiditis in guinea pigs and rabbits. Immunization with thyroglobulin and bovine thyroid gland suspension.

Morphological changes in the thyroid glands of the guinea pigs with autoimmune thyroiditis (EAT) experimentally induced by thyroglobulin (TGL) or immunization by the suspension of thyroid gland cells with CFA manifested mainly by atrophy and alterations of follicular cells, fibrotic tissue changes, formation of inflammatory lymphoplasmocytic infiltrations, multiplication of C-cells and by the increase in the proportion of lymphocytes with activated nucleoli in the tissue. The antigenic effects of TGL differed from those of the cell suspension; the effects of TGL participated especially in the formation of the infiltrates, the effects of cell suspension participated in the diapedesis of mononuclears and in the multiplication of C-cells. The findings correspond to the principal findings in human autoimmune lymphocytic thyroiditis. In an electron microscope, strongly dilated cisterns of endoplasmic reticulum (ER) and multiplied mitochondria in the cytoplasm of altered follicular cells were found. The wall of the follicles exhibited fully intact or altered C-cells. The latter had a large number of granules in cytoplasm with an unusually clear medullary substance. In the rabbit thyroid glands no morphological changes were observed following the immunization with both antigens. TGL antibodies examined immunohistochemically in the sera were present in all the sera of guinea pigs immunized with TGL and CFA. The antibodies determined by dot immunodetection were present in the sera of all guinea pigs immunized with TGL+CFA, the titres reached the level of 1:81 to 1:729; the highest titres were observed in the guinea pigs following the immunization by dose of 7.7 mg/kg after 12 weeks of immunization.

Animals↗

PubMed1992

Structure-function subcompartments of the mammalian cell nucleus as revealed by the electron microscopic affinity cytochemistry.

This electron microscopy review documents the in situ cytochemical localization of important nuclear structures and relates this to the important nuclear functions of RNA transcription and processing. With the help of specific probes (antibodies, nucleic acid probes), a comprehensive picture of nuclear subcompartmentalization is beginning to emerge.

3T3 Cells↗

PubMed1991

Human autoantibody to a novel protein of the nuclear coiled body: immunological characterization and cDNA cloning of p80-coilin.

Antibodies producing an unusual immunofluorescent pattern were identified in the sera of patients with diverse autoimmune features. This pattern was characterized by the presence of up to six round discrete nuclear bodies in interphase cell nuclei. Immunoblotting analysis showed that these sera recognized an 80-kD nuclear protein, and affinity-purified anti-p80 antibody from the protein band reproduced the fluorescent staining of nuclear bodies. Colloidal gold immunoelectron microscopy showed that the affinity-purified anti-p80 antibody recognized the coiled body, an ultramicroscopic nuclear structure probably first described by the Spanish cytologist Ramon y Cajal. Five cDNA clones were isolated from a MOLT-4 cell lambda gt-11 expression library using human antibody and oligonucleotide probes. The longest cDNA insert was 2.1 kb and had an open reading frame of 405 amino acids. A clone encoding a 14-kD COOH-terminal region of the protein was used for expression of a beta-galactosidase fusion protein. An epitope was present in this COOH-terminal 14-kD region, which was recognized by 18 of 20 sera with anti-p80 reactivity, and affinity-purified antibody from the recombinant protein also reacted in immunofluorescence to show specific staining of the coiled body. This is the first demonstration and molecular cloning of a protein that appears to have particular identification with the coiled body, and it was designated p80-coilin. Autoantibody to p80-coilin may be useful for the elucidation of the structure and function of the coiled body, and the availability of a cDNA sequence could be helpful in further studies to clarify the clinical significance of this autoantibody response.

Amino Acid Sequence↗

PubMed1991

Ultrastructural cryoimmunocytochemistry is a convenient tool for the study of DNA replication in cultured cells.

In the present study, we have optimized an immunocytochemical ultrastructural approach for in situ localization of newly synthesized DNA in unsynchronized as well as in synchronized human HeLa cells and in exponentially growing mouse P815 cells, which had incorporated bromodeoxyuridine (BrdU) during short pulses varying from 1 to 20 minutes. The incorporated BrdU was detected in hydrolyzed ultrathin cryosections or Lowicryl sections by means of a monoclonal antibody, revealed by secondary colloidal gold-labeled probes. The results demonstrate our ability to study, with high resolution and reproducibility, DNA replication during consecutive periods of the S-phase, which is monitored by the incorporation of tritiated thymidine. In addition, this approach allows one to perform a concomitant mapping of replicated DNA and various enzymes of the replisome.

Animals↗

PubMed1991

Immunological and ultrastructural studies of the nuclear coiled body with autoimmune antibodies.

Studies with human autoimmune sera identified auto-antibodies reacting with a novel antigen of 80 kDa. In interphase mammalian cells, the 80-kDa antigen was enriched in nuclear coiled bodies and was used as a marker for this nuclear structure. This antigen was subsequently named p80-coilin. By light and electron microscopic immunocytochemistry, a number of other antigens were also localized to the coiled body, including components of small nuclear ribonucleoproteins which are involved in the processing of nucleolar and extranucleolar RNA. Although the function of the coiled body is unknown, the presence of these subcellular particles might indicate an involvement in RNA metabolism. The identification of a protein highly enriched in this structure and the availability of specific antibodies might help in its isolation and the study of its function.

Animals↗