Aromatic amino acids and their relevance in the specificity of the PH domain.

Phosphoinositides are phosphatidylinositol derived, well known to be second messengers in various cell signaling pathways as well as in processes such as cell differentiation, cellular stress response, gene transcription, and chromatin remodeling. The pleckstrin homology domain of phospholipase C-delta 1 is responsible for recognizing and binding to PI(4,5)P2 and for this reason has been widely used to study this phosphoinositide as a biosensor when it is conjugated to a fluorescent tag. In this work, we modified the primary structure of pleckstrin homology domain by site-specific mutagenesis to change the specificity for phosphoinositides. We obtained 3 mutants: K30A, W36F, and W36Y with different specificity to phosphoinositides. Mutant domain K30A recognized PI(4,5)P2 , PI(3,4,5)P3 , phosphatidic acid (PA), and weakly PI(3,5)P2 . Mutant domain W36F recognized all the phosphoinositides studied and the PA. Finally, mutant domain W36Y seemed to interact with PA and all the other phosphoinositides studied, except PI(3)P. The changes in recognition argue against a simple charge and nonpolar region model for these interactions and more in favor of a specific docking region with a specific recognition site. We conducted in silico modeling that explains the mechanisms behind the observed changes and showed that aromatic amino acids appear to play more important role, than previously thought, in the specificity of phospholipids' binding domains.

[Tumour-stroma interactions in urothelial cancer]. / Tumor-Stroma-Interaktionen im Harnblasenkarzinom.

BACKGROUND: The histopathological structure of malignant tumours involves two essential compartments - the tumour parenchyma with the actual transformed cells, and the supportive tumour stroma. The latter consists of specialized mesenchymal cells, such as fibroblasts, macrophages, lymphocytes and vascular cells, as well as of their secreted products, including components of the extracellular matrix, matrix modifying enzymes and numerous regulatory growth factors and cytokines. In consequence, the tumour stroma has the ability to influence virtually all aspects of tumour development and progression, including therapeutic response. AIM: In this article we review the current knowledge of tumor stroma interactions in urothelial carcinoma and present various experimental systems that are currently in use to unravel the biological basis of these heterotypic cell interactions. RESULTS: For urothelial carcinoma, an extensive tumour stroma is quite typical and markers of activated fibroblasts correlate significantly with clinical parameters of advanced disease. Another clinically important variable is provided by the stromal expression of syndecan-1. CONCLUSION: Integration of markers of activated stroma into clinical risk evaluation could aid to better stratification of urothelial bladder carcinoma patients. Elucidation of biological mechanisms underlying tumour-stroma interactions could provide new therapeutical targets.

Simultaneous detection of multiple targets for ultrastructural immunocytochemistry.

Simultaneous detection of biological molecules by means of indirect immunolabeling provides valuable information about their localization in cellular compartments and their possible interactions in macromolecular complexes. While fluorescent microscopy allows for simultaneous detection of multiple antigens, the sensitive electron microscopy immunodetection is limited to only two antigens. In order to overcome this limitation, we prepared a set of novel, shape-coded metal nanoparticles readily discernible in transmission electron microscopy which can be conjugated to antibodies or other bioreactive molecules. With the use of novel nanoparticles, various combinations with commercial gold nanoparticles can be made to obtain a set for simultaneous labeling. For the first time in ultrastructural histochemistry, up to five molecular targets can be identified simultaneously. We demonstrate the usefulness of the method by mapping of the localization of nuclear lipid phosphatidylinositol-4,5-bisphosphate together with four other molecules crucial for genome function, which proves its suitability for a wide range of biomedical applications.

Transcription-dependent rearrangements of actin and nuclear myosin I in the nucleolus.

Nuclear actin and nuclear myosin I (NMI) are important players in transcription of ribosomal genes. Transcription of rDNA takes place in highly organized intranuclear compartment, the nucleolus. In this study, we characterized the localization of these two proteins within the nucleolus of HeLa cells with high structural resolution by means of electron microscopy and gold-immunolabeling. We demonstrate that both actin and NMI are localized in specific compartments within the nucleolus, and the distribution of NMI is transcription-dependent. Moreover, a pool of NMI is present in the foci containing nascent rRNA transcripts. Actin, in turn, is present both in transcriptionally active and inactive regions of the nucleolus and colocalizes with RNA polymerase I and UBF. Our data support the involvement of actin and NMI in rDNA transcription and point out to other functions of these proteins in the nucleolus, such as rRNA maturation and maintenance of nucleolar architecture.

Actin complexes in the cell nucleus: new stones in an old field.

Actin is a well-known protein that has shown a myriad of activities in the cytoplasm. However, recent findings of actin involvement in nuclear processes are overwhelming. Actin complexes in the nucleus range from very dynamic chromatin-remodeling complexes to structural elements of the matrix with single partners known as actin-binding proteins (ABPs). This review summarizes the recent findings of actin-containing complexes in the nucleus. Particular attention is given to key processes like chromatin remodeling, transcription, DNA replication, nucleocytoplasmic transport and to actin roles in nuclear architecture. Understanding the mechanisms involving ABPs will definitely lead us to the principles of the regulation of gene expression performed via concerting nuclear and cytoplasmic processes.

Cytokine expression and signaling in drug-induced cellular senescence.

Cellular senescence guards against cancer and modulates aging; however, the underlying mechanisms remain poorly understood. Here, we show that genotoxic drugs capable of inducing premature senescence in normal and cancer cells, such as 5-bromo-2'-deoxyuridine (BrdU), distamycin A (DMA), aphidicolin and hydroxyurea, persistently activate Janus kinase-signal transducer and activator of transcription (JAK/STAT) signaling and expression of interferon-stimulated genes (ISGs), such as MX1, OAS, ISG15, STAT1, PML, IRF1 and IRF7, in several human cancer cell lines. JAK1/STAT-activating ligands, interleukin 10 (IL10), IL20, IL24, interferon gamma (IFNgamma), IFNbeta and IL6, were also expressed by senescent cells, supporting autocrine/paracrine activation of JAK1/STAT. Furthermore, cytokine genes, including proinflammatory IL1, tumor necrosis factor and transforming growth factor families, were highly expressed. The strongest inducer of JAK/STAT signaling, cytokine production and senescence was BrdU combined with DMA. RNA interference-mediated knockdown of JAK1 abolished expression of ISGs, but not DNA damage signaling or senescence. Thus, although DNA damage signaling, p53 and RB activation, and the cytokine/chemokine secretory phenotype are apparently shared by all types of senescence, our data reveal so far unprecedented activation of the IFNbeta-STAT1-ISGs axis, and indicate a less prominent causative role of IL6-JAK/STAT signaling in genotoxic drug-induced senescence compared with reports on oncogene-induced or replicative senescence. These results highlight shared and unique features of drug-induced cellular senescence, and implicate induction of cancer secretory phenotype in chemotherapy.

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.

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.

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.

Detection of cell type and marker specificity of nuclear binding sites for anionic carbohydrate ligands.

The emerging functionality of glycosaminoglycan chains engenders interest in localizing specific binding sites using cytochemical tools. We investigated nuclear binding of labeled heparin, heparan sulfate, a sulfated fucan, chondroitin sulfate, and hyaluronic acid in epidermal keratinocytes, bone marrow stromal cells, 3T3 fibroblasts and glioma cells using chemically prepared biotinylated probes. Binding of the markers was cell-type specific and influenced by extraction of histones, but was not markedly affected by degree of proliferation, differentiation or malignancy. Cell uptake of labeled heparin and other selected probes and their transport into the nucleus also was monitored. Differences between keratinocytes and bone marrow stromal cells were found. Preincubation of permeabilized bone marrow stromal cells with label-free heparin reduced the binding of carrier-immobilized hydrocortisone to its nuclear receptors. Thus, these tools enabled binding sites for glycosaminoglycans to be monitored in routine assays.

[Induction of nuclear envelope formation around individual chromosomes under impact of hypotonic shock]. / Induktsiia formirovaniia iadernoi obolochki vokrug individual'nykh khromosom pod deistviem gipotonicheskogo shoka.

In the present work we have studied the distribution of some proteins participating in the nuclear envelope assembly (lamins A/C, B and LAP2 alpha) in mitotic cells and after hypotonic treatment with 15% Hank's solution. In untreated cells, these proteins are localized in the nuclei of interphase cells migrate to the cytoplasm during mitosis. Hypotonic treatment of interphase, prophase and telophase cells does not lead to considerable relocalization of lamins A/C and B. However, unlike normal mitosis, in prometaphase and metaphase cells their chromosomes acquire affinity to lamins and LAP2 alpha. Comparative analysis of lamins and LAP2 alpha distribution have revealed that chromosomes have special sites for binding with different proteins.

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.

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.

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.

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.

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.

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.

Accumulation of the proteolytic marker peptide ubiquitin in the trophoblast of mammalian blastocysts.

Ubiquitination is a universal protein degradation pathway in which the molecules of 8.5-kDa proteolytic peptide ubiquitin are covalently attached to the epsilon-amino group of the substrate's lysine residues. Little is known about the importance of this highly conserved mechanism for protein recycling in mammalian gametogenesis and fertilization. The data obtained by the students and faculty of the international training course Window to the Zygote 2000 demonstrate the accumulation of ubiquitin-cross-reactive structures in the trophoblast, but not in the inner cell mass of the expanding bovine and mouse blastocysts. This observation suggests that a major burst of ubiquitin-dependent proteolysis occurs in the trophoblast of mammalian peri-implantation embryos. This event may be important for the success of blastocyst hatching, differentiation of embryonic stem cells into soma and germ line, and/or implantation in both naturally conceived and reconstructed mammalian embryos.

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.

A myosin I isoform in the nucleus.

A nuclear isoform of myosin I beta that contains a unique 16-amino acid amino-terminal extension has been identified. An affinity-purified antibody to the 16-amino acid peptide demonstrated nuclear staining. Confocal and electron microscopy revealed that nuclear myosin I beta colocalized with RNA polymerase II in an alpha-amanitin- and actinomycin D-sensitive manner. The antibody coimmunoprecipitated RNA polymerase II and blocked in vitro RNA synthesis. This isoform of myosin I beta appears to be in a complex with RNA polymerase II and may affect transcription.