Biomedical subjects

P Hozák

Publications and source records attributed to P Hozák.

At least 19 recordsLinked to original sources

PubMed2006

Nuclear myosin is ubiquitously expressed and evolutionary conserved in vertebrates.

Nuclear myosin I (NMI) is a single-headed member of myosin superfamily localized in the cell nucleus which participates along with nuclear actin in transcription and chromatin remodeling. We demonstrate that NMI is present in cell nuclei of all mouse tissues examined except for cells in terminal stages of spermiogenesis. Quantitative PCR and western blots demonstrate that the expression of NMI in tissues varies with the highest levels in the lungs. The expression of NMI is lower in serum-starved cells and it increases after serum stimulation. The lifespan of NMI is longer than 16 h as determined by cycloheximide translation block. A homologous protein is expressed in human, chicken, Xenopus, and zebrafish as shown by RACE analysis. The analysis of genomic sequences indicates that almost identical homologous NMI genes are expressed in mammals, and similar NMI genes in vertebrates.

Amino Acid Sequence↗

PubMed2005

Distinct localization of a beta-tubulin epitope in the Tetrahymena thermophila and Paramecium caudatum cortex.

Many of the highly organized microtubular arrangements in ciliates are located in the cortical area containing membrane vesicles and vacuoles. In Tetrahymena thermophila and Paramecium caudatum, immunofluorescence microscopy with the monoclonal antibody TU-06, directed against beta-tubulin, revealed distinct staining of this cortical region alone, while the cilia and other microtubular structures were unstained. The specificity of the antibody was confirmed by immunoblotting and by preabsorption of the antibody with purified tubulin. Double-label immunofluorescence with antibodies against gamma-tubulin, detyrosinated alpha-tubulin, and centrin showed that the TU-06 epitope is localized outside the basal body region. This was also confirmed by immunogold electron microscopy of thin sections. Proteolytic digestion of porcine brain beta-tubulin combined with a peptide scan of immobilized, overlapping peptides disclosed that the epitope was in the beta-tubulin region beta81-95, a region which is phylogenetically highly conserved. As known posttranslational modifications of beta-tubulin are located outside this area, the observed staining pattern cannot be interpreted as evidence of subcellular sequestration of modified tubulin. The limited distribution of the epitope could rather reflect the dependence of TU-06 epitope exposition on conformations of tubulin molecules in microtubule arrangements or on differential masking by interacting proteins.

3T3 Cells↗

PubMed2005

Post-treatment plasma transforming growth factor beta 1 (TGF-beta1) level predicts for late morbidity in patients with advanced head and neck cancer.

Between 2001 and 2002, 29 patients with advanced inoperable squamous head and neck cancer treated with radiotherapy with or without simultaneous chemotherapy were evaluated for their plasma TGF-beta1 levels prior to the treatment, in the middle of the radiotherapy course and at the end of the treatment. Patients were assessed for treatment response and late morbidity. Predictive value of TGF-beta1 level on either of the assessed parameters was tested. From 29 eligible patients (pts), 18 achieved complete response, 8 partial response and three pts progressed primarily. After a median follow-up of 16 months we recorded 16 cases of grade >1 late morbidity. We found that post-treatment elevated plasma TGF-beta1 level predicts late morbidity grade >1 (p=0.05) rather than pre-treatment level (p=0.062). Neither pre-treatment nor post-treatment plasma TGF-beta1 level has a predictive value to the treatment response (CR vs. no CR, p=0.125 and 0.252, respectively). The post-treatment plasma TGF-beta 1 level can predict late morbidity grade >1 in advanced head and neck cancer treated with radio(chemo)therapy. This could make a basis for dose escalation in selected patients.

Adult↗

PubMed2003

Different patterns of chromogranin A and Leu-7 (CD57) expression in gastrointestinal carcinoids: immunohistochemical and confocal laser scanning microscopy study.

Thirty-seven carcinoids of the gastrointestinal tract were studied with immunohistochemical staining for chromogranin A (CgA) and Leu-7 (CD57). The aim of this study was to distinguish and describe the differences in patterns of distribution of immunostaining of these two non-specific neuroendocrine markers in neuroendocrine tumors of different degree of differentiation (typical, vs. atypical carcinoids) at different gastrointestinal sites. Selected 5 tumors from this group were studied in detail using confocal laser scanning microscopy (CLSM) and double immunofluorescence staining to disclose the patterns of distribution of CgA and CD57 positive granules within the individual tumor cells. Prominent differences in the patterns of immunohistochemical staining for both studied markers related to the degree of differentiation of the tumors were observed in studied neoplasms. Regular (diffuse) strongly positive immunoreaction for CgA predominated in typical carcinoids, whereas atypical tumors were characterized by irregular patchy staining. Both typical and atypical tumors displayed predominantly irregular patchy staining for CD57. The results of CLSM study indicate that different modes of CgA and CD57 expression and/or co-expression can occur in neuroendocrine tumors. Neoplastic cells that contained either CgA positive neuroendocrine granules (NEG), or Leu-7 positive NEG, were frequently observed in different areas of the tumor samples, especially in atypical carcinoids. Varying number of cells revealed co-localisation of both CgA and Leu-7 within the NEG. Similar co-localisation of CgA and CD57 was found in non-neoplastic Kultschitski cells of the mucosa of small intestine. In conclusion, our results suggest that the differences in CgA and CD57 expression in human neuroendocrine tumors are related to the degree of differentiation of the neoplasms and probably reflect the degree of maturation (functional state) of neuroendocrine granules within the neoplastic cells.

Biomarkers, Tumor↗

PubMed2002

The localization of nuclear DNA helicase II in different nuclear compartments is linked to transcription.

Nuclear DNA helicase II (NDH II) is a member of the DEAH superfamily of helicases and functions as a pre-mRNA- and mRNA-binding protein in human cells. Here we report for the first time that human NDH II is associated with the nucleolus of transformed and nontransformed cells as shown by immunofluorescence and by ultrastructural studies. When RNA polymerase II (POL II) transcription is inhibited, NDH II highly accumulates in the nucleolus and shows predominant association with subdomains in DFC and in a portion of GC attached to DFC. Furthermore, these subdomains completely co-localize with mRNA-binding protein TLS. In addition, we show that nucleolar accumulation of NDH II is closely related to G(0)-phase growth arrest in human fibroblasts. Thus, the nucleolar localization of NDH II depends upon the metabolic state of the cell. Based on the data we propose that NDH II operates in both nucleoplasmic and nucleolar mode, and that its redistribution reflects accumulations indicating a possible cycling of NDH II between nucleoplasm and the nucleolus. The nucleolus can serve as a temporary storage or recycling center for NDH II. Possible functions of NDH II in pre-rRNA biogenesis, or in nucleolar mRNA metabolism, are also discussed.

Adenosine Triphosphatases↗

PubMed2002

LR White is preferable to Unicryl for immunogold detection of fixation-sensitive nuclear antigens.

The purpose of this study was to compare two electron microscopy embedding media - LR White and Unicryl - with regard to cell morphologyical and immunohistochemical preservation properties for the study of fixation-sensitive nuclear antigens. Human cervical carcinoma (HeLa) cells were fixed with 2% paraformaldehyde and 0.1% glutaraldehyde, and embedded in parallel in the two resins: LR White and Unicryl using; two different polymerization protocols were used for each resin. Preservation of fine nuclear structure was good after LR White and poor after Unicryl embedding. Immunogold labeling of Sm antigen was significantly stronger on LR White sections. Polymerization by UV light resulted in stronger and more specific labeling than heat polymerization. These results show that LR White is advantageous over Unicryl for the study of nuclear antigens requiring delicate aldehyde fixation.

Acrylic Resins↗

PubMed2001

Arrangement of ribosomal genes in nucleolar domains revealed by detection of "Christmas tree" components.

We investigated how the transcribing ribosomal genes ("Christmas trees") of HeLa cells are arranged in the nucleolus. Hypotonic conditions let the granular component disperse, while fibrillar centres and parts of the dense fibrillar component were resistant to low ionic strength conditions. Both remained within the former nucleolar territory. We used immunocytochemistry and in situ hybridisation at the light microscopic and ultrastructural level for the analysis of the internal nucleolar structures. The 5' ends of ribosomal RNA and ribosomal DNA sequences were found associated with the periphery of fibrillar centres. The hypotony-resistant parts of the dense fibrillar component did not contain the 5' end of the transcript or the gene. The downstream ribosomal DNA sequences were found in the nucleolar territory but not associated with any hypotony-resistant structures. The downstream ribosomal RNA revealed a similar distribution. We show that transcription initiation and transcript elongation occur in different molecular and structural environments. Transcription initiation is located at the periphery of fibrillar centres. Evidently the dense fibrillar component is non-homogeneous in molecular composition. Transcript elongation is continued in a part of the dense fibrillar component which is dissolved under intermediate hypotonic conditions. A structural model of nucleolar transcription is suggested.

3' Flanking Region↗

PubMed2001

The nucleoskeleton: a permanent structure of cell nuclei regardless of their transcriptional activity.

Nuclear matrix or nucleoskeleton is thought to provide structural basis for intranuclear order. However, the nature of this structure is still uncertain because of numerous technical difficulties in its visualization. To reveal the "real" morphology of the nucleoskeleton, and to identify possible sources of structural artifacts, three methods of nucleoskeleton preparations were compared. The nucleoskeleton visualized by all these techniques consists of identical elements: nuclear lamina and an inner network comprising core filaments and the "diffuse" nucleoskeleton. We then tested if the nucleoskeleton is a stable structure or a transient transcription-dependent structure. Incubation with transcription inhibitors (alpha-amanitin, actinomycin D, and DRB) for various periods of time had no obvious effect on the morphology of the nucleoskeleton. A typical nucleoskeleton structure was observed also in a physiological model-in transcriptionally inactive mouse 2-cell embryos and in active 8- to 16-cell embryos. Our data suggest that the nucleoskeleton is a permanent structure of the cell nucleus regardless of the nuclear transcriptional state, and the principal architecture of the nucleoskeleton is identical throughout the interphase.

Amanitins↗

PubMed2001

Differential subcellular distribution of tubulin epitopes in boar spermatozoa: recognition of class III beta-tubulin epitope in sperm tail.

The exposure of tubulin epitopes was studied in ejaculated boar spermatozoa using a panel of four monoclonal antibodies specific to the N-terminal or C-terminal structural domains of tubulin and three monoclonal antibodies against class III beta-tubulin. The specificity of the antibodies was confirmed by immunoblotting. Immunocytochemical staining showed that antibodies discriminated between various parts of a spermatozoon, and that epitopes of class III beta-tubulin were present in the flagellum. A tubulin epitope from the C-terminal domain of beta-tubulin was detected in the triangular segment of the postacrosomal part of the sperm head. Its distribution changed after an A23187 ionophore-induced acrosome reaction, indicating that tubulin participates in the early stages of fertilization. Three monoclonal antibodies, TU-20, SDL.3D10, and TUJ1 directed against epitopes on the C-terminal end of neuron-specific class III beta-tubulin that is widely used as a neuronal marker, stained the flagella. The reactivity of TU-20 was further confirmed by absorbing the antibody with the immunizing peptide and by immunoelectron microscopy. Immunoblotting after two-dimensional electrophoresis revealed that the corresponding epitope was not present on all beta-tubulin isoforms. These results suggest that various tubulins are involved in the functional organization of the mammalian sperm flagellum and head.

Acrosome Reaction↗

PubMed2001

Caveolae are involved in the trafficking of mouse polyomavirus virions and artificial VP1 pseudocapsids toward cell nuclei.

Electron and confocal microscopy were used to observe the entry and the movement of polyomavirus virions and artificial virus-like particles (VP1 pseudocapsids) in mouse fibroblasts and epithelial cells. No visible differences in adsorption and internalization of virions and VP1 pseudocapsids ("empty" or containing DNA) were observed. Viral particles entered cells internalized in smooth monopinocytic vesicles, often in the proximity of larger, caveola-like invaginations. Both "empty" vesicles derived from caveolae and vesicles containing viral particles were stained with the anti-caveolin-1 antibody, and the two types of vesicles often fused in the cytoplasm. Colocalization of VP1 with caveolin-1 was observed during viral particle movement from the plasma membrane throughout the cytoplasm to the perinuclear area. Empty vesicles and vesicles with viral particles moved predominantly along microfilaments. Particle movement was accompanied by transient disorganization of actin stress fibers. Microfilaments decorated by the VP1 immunofluorescent signal could be seen as concentric curves, apparently along membrane structures that probably represent endoplasmic reticulum. Colocalization of VP1 with tubulin was mostly observed in areas close to the cell nuclei and on mitotic tubulin structures. By 3 h postinfection, a strong signal of the VP1 (but no viral particles) had accumulated in the proximity of nuclei, around the outer nuclear membrane. However, the vast majority of VP1 pseudocapsids did not enter the nuclei.

Adsorption↗

PubMed2000

Statistical evaluation of colocalization patterns in immunogold labeling experiments.

The ultrastructural localization of various antigens in a cell using antibodies conjugated to gold particles is a powerful instrument in biological research. However, statistical or stereological tools for testing the observed patterns for significant clustering or colocalization are missing. The paper presents a method for the quantitative analysis of single or multiple immunogold labeling patterns using interpoint distances and tests the method using experimental data. The clustering or colocalization of gold particles was detected using various characteristics of the distribution of distances between them. Pair correlation and cross-correlation functions were used for exploratory analysis; second order reduced K (or cross-K) functions were used for testing the statistical significance of observed events. Confidence intervals of function values were estimated by Monte Carlo simulations of the Poisson process for independent particles, and results were visualized in histograms. Furthermore, a suitability of K functions modified by censoring or weighting was tested. The reliability of the method was assessed by evaluating the labeling patterns of nascent DNA and several nuclear proteins with known functions in replication foci of HeLa cells. The results demonstrate that the method is a powerful tool in biological investigations for testing the statistical significance of observed clustering or colocalization patterns in immunogold labeling experiments.

Cell Nucleus↗

PubMed1999

Intranuclear anchoring of repetitive DNA sequences: centromeres, telomeres, and ribosomal DNA.

Centromeres, telomeres, and ribosomal gene clusters consist of repetitive DNA sequences. To assess their contributions to the spatial organization of the interphase genome, their interactions with the nucleoskeleton were examined in quiescent and activated human lymphocytes. The nucleoskeletons were prepared using "physiological" conditions. The resulting structures were probed for specific DNA sequences of centromeres, telomeres, and ribosomal genes by in situ hybridization; the electroeluted DNA fractions were examined by blot hybridization. In both nonstimulated and stimulated lymphocytes, centromeric alpha-satellite repeats were almost exclusively found in the eluted fraction, while telomeric sequences remained attached to the nucleoskeleton. Ribosomal genes showed a transcription-dependent attachment pattern: in unstimulated lymphocytes, transcriptionally inactive ribosomal genes located outside the nucleolus were eluted completely. When comparing transcription unit and intergenic spacer, significantly more of the intergenic spacer was removed. In activated lymphocytes, considerable but similar amounts of both rDNA fragments were eluted. The results demonstrate that: (a) the various repetitive DNA sequences differ significantly in their intranuclear anchoring, (b) telomeric rather than centromeric DNA sequences form stable attachments to the nucleoskeleton, and (c) different attachment mechanisms might be responsible for the interaction of ribosomal genes with the nucleoskeleton.

Cell Nucleus↗

PubMed1998

Association of protein kinase A type I with detergent-resistant structures of mammalian sperm cells.

The finding that flagellar movement in detergent-permeabilized sperm cells is restored when Mg ATP and cAMP are added implicated detergent-resistant protein kinase A (PKA) in the regulation of sperm motility. It is widely believed that only the PKA regulatory subunit RII can associate with the cytoskeleton and/or organelles. In this paper we used monoclonal antibodies against the PKA catalytic subunit and RI subunit and demonstrated that PKA type I is also associated with the sperm cytoskeleton. To our knowledge, this is the first report showing anchored PKA type I. This association was found in sperm of nonrodent mammalian species and, to a lesser extent, also in mouse sperm. The PKA catalytic subunit is bound to the cytoskeleton secondarily via its complex with the regulatory subunit. The detergent-resistant complexes of RI and catalytic subunits localize predominantly to the flagellum. Ultrastructural immunogold labeling revealed the association of detergent-resistant PKA type I with outer dense fibers (ODF) and the fibrous sheath (FS) but not with microtubules. This location is consistent with a proposed role of PKA in regulation of FS sliding on underlying ODF.

Animals↗

PubMed1998

Preferential silver reaction of nucleolar regions adjacent to fibrillar centers in ring shaped nucleoli of leukemic lymphocytes.

Silver stained proteins (SSPs) characteristic for interphasic nucleolus organizer regions (NORs) associated with fibrillar centers (FCs) and adjacent nucleolar regions of ring shaped nucleoli in leukemic lymphocytes exhibit a different sensitivity to the mild acid extraction including that with HCl. Such extractions permit a preferential visualization of fibrillar centers adjacent regions (FCARs) which are believed to represent sites of the ribosomal RNA (rRNA) transcription. The resistance of SSPs in FCARs to the extraction with HCl seems to be due to their binding to other components present in these regions. The extractibility of SSPs with HCl was influenced by the fixatives used. The largest resistance of SSPs to the extraction with HCl was noted after fixation with glutaraldehyde. In contrast, the largest extractibility of these proteins was observed after fixation with unbuffered formaldehyde.

B-Lymphocytes↗

PubMed1997

Evidence for the presence of myosin I in the nucleus.

We produced and affinity-purified polyclonal antibodies to adrenal myosin I. These antibodies recognize adrenal myosin I by Western blot analysis (116 kDa) and inhibit the actin-activated ATPase activity of purified adrenal myosin I. They also recognize a 120-kDa protein in extracts prepared from many different cell lines. Fluorescence microscopy demonstrated the presence of immunoreactive material in the perinuclear region, the leading edges, and the nuclei of 3T3 cells. Fluorescence microscopy also demonstrated nuclear staining in mouse oocytes at the germinal vesicle stage and in the pronuclei during fertilization. Confocal and immunoelectron microscopy confirmed the intranuclear localization. Electron microscopy also demonstrated staining of structures in nucleoli that are thought to be associated with rDNA transcription. Western blot analyses revealed the presence of the 120-kDa protein in extracts prepared from nuclei that are apparently free of cytosolic contamination. The same nuclear protein binds 125I-calmodulin and is photoaffinity labeled with [alpha-32P]ATP. The 120-kDa protein was partially purified from twice washed nuclei using ammonium sulfate fractionation and gel filtration chromatography. Column fractions containing 120-kDa protein as revealed by Western blot analysis also contain K+-EDTA ATPase activity. The 120-kDa protein was also shown to bind actin in the absence, but not the presence, of ATP. Since K+-EDTA ATPase activity, actin, and ATP binding are defining features of the members of the myosin superfamily of proteins, we propose that the 120-kDa protein is a previously undescribed myosin I isoform that is an intranuclear actin-based molecular motor.

3T3 Cells↗

PubMed1996

The nucleoskeleton and attached activities.

This review briefly summarizes recent results obtained on the nucleoskeleton of HeLa cells. When the nucleoskeleton is observed in HeLa cells after removal of most chromatin using "physiological" conditions, four structural elements can be recognized: nuclear lamina, core filaments, diffuse skeleton, and nuclear bodies. After dispersing the nucleolar granular component, two additional structures can be visualized: the intranucleolar skeleton, and the fibrillar centers. The relationship between the nucleoskeleton and nuclear synthetic activities-DNA replication and ribosomal transcription-is discussed. The nucleoskeleton is an important nuclear structure, involved in nuclear compartmentalization and probably critical for replication and transcription.

Binding Sites↗

PubMed1996

The transcription unit of ribosomal genes is attached to the nuclear skeleton.

The relationship between various loci of the ribosomal gene repeat and the nucleoskeleton was examined in agarose-embedded HeLa cells. The accessibility of intranucleolar structures to molecular probes was improved by dispersing the granular component of nucleoli, and unattached DNA was removed from permeabilized nuclei under "physiological" conditions by enzymatic digestion and subsequent electroelution. The cells were then hybridized in situ with various human rDNA probes for the transcription unit or for the intergenic spacer. A strong signal was detected with probes for the transcription unit but no signal was seen with probes for the intergenic spacer. These results show that only the transcription unit is strongly attached to the nucle(ol)ar skeleton and imply that rDNA is probably attached to the skeleton primarily via RNA polymerase complexes rather than via sequence-specific attachment sites. Nucleolar fibrillar centers, embedded into the nucle(ol)ar skeleton, provide structural support for these attachments.

Cell Nucleolus↗

PubMed1996

Transcriptional activity and nucleolar ultrastructure of embryonic rabbit nuclei after transplantation to enucleated oocytes.

Changes in the level of transcriptional activity in 32-cell stage morula nuclei were studied after blastomere electrofusion to enucleated oocytes. Nuclear transplant recipients were pulse labelled with 3H-uridine during cultivation in vitro, embryos were then fixed and processed for autoradiography and electron microscopy. Transcriptional activity substantially decreased after 4.5 hr and was completely inhibited at last 15 hr after fusion. Transcription resumed thereafter in two-cell stage embryos and could be detected in both nuclei from 70% of the embryos analyzed. Transcription activity rapidly increased at the eight 16-cell stages, reaching the level typical for 32-cell stage nuclei used for the transfer. Changes in nucleolar ultrastructure after the nuclear transfer reflected the inhibition and subsequent reactivation of rRNA transcription. Nucleoli of 32-cell embryos had a typical structure of active nucleoli; many fibrillar centers surrounded and interconnected by threads of the dense fibrillar component and embedded in the granular component. Six hours following nuclear transplantation, these nucleoli underwent drastic changes including loss of granular material, collapse of nucleolar structure, and segregation of nucleolar components. Following the first cleavage, segregated fibrillar components of nucleoli manifested a complete inhibition of nucleolar transcription. Ribosomal RNA transcription was restored at the eight-cell stage and the sequence of ultrastructural changes was similar to that of the normal development. However, at the 32-cell stage, excessive extrusion of pre-ribosomal particles in the cytoplasm occurred, suggesting a possible alteration in regulating mechanisms of ribosome delivery. These results show that after fusion with enucleated metaphase II cytoplasm and subsequent activation, transcription is inhibited in donor embryonic nuclei and progressively increases again during cleavage; almost as in normal embryos. Migration of ribosomes into cytoplasm appears more intense in 32-cell stage reconstituted embryos but this does not seem to inhibit blastocyst building.

Animals↗