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Results for “Intranuclear Space”

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PubMed2004

Human topoisomerase IIalpha: targeting to subchromosomal sites of activity during interphase and mitosis.

Mammalian topoisomerase IIalpha (topo IIalpha) plays a vital role in the removal of topological complexities left on DNA during S phase. Here, we developed a new assay to selectively identify sites of catalytic activity of topo IIalpha with subcellular resolution. We show that topo IIalpha activity concentrates at replicating heterochromatin in late S in a replication-dependent manner and at centric heterochromatin during G2 and M phases. Inhibitor studies indicate that this cell cycle-dependent concentration over heterochromatin is sensitive to chromatin structure. We further show that catalytically active topo IIalpha concentrates along the longitudinal axis of mitotic chromosomes. Finally, we found that catalytically inert forms of the enzyme localize predominantly to splicing speckles in a dynamic manner and that this pool is differentially sensitive to changes in the activities of topo IIalpha itself and RNA polymerase II. Together, our data implicate several previously unsuspected activities in the partitioning of the enzyme between sites of activity and putative depots.

Antigens, Neoplasm↗

PubMed2004

Identification and characterization of two novel proteins affecting fission yeast gamma-tubulin complex function.

The gamma-tubulin complex, via its ability to organize microtubules, is critical for accurate chromosome segregation and cytokinesis in the fission yeast, Schizosaccharomyces pombe. To better understand its roles, we have purified the S. pombe gamma-tubulin complex. Mass spectrometric analyses of the purified complex revealed known components and identified two novel proteins (i.e., Mbo1p and Gfh1p) with homology to gamma-tubulin-associated proteins from other organisms. We show that both Mbo1p and Gfh1p localize to microtubule organizing centers. Although cells deleted for either mbo1(+) or gfh1(+) are viable, they exhibit a number of defects associated with altered microtubule function such as defects in cell polarity, nuclear positioning, spindle orientation, and cleavage site specification. In addition, mbo1Delta and gfh1Delta cells exhibit defects in astral microtubule formation and anchoring, suggesting that these proteins have specific roles in astral microtubule function. This study expands the known roles of gamma-tubulin complex components in organizing different types of microtubule structures in S. pombe.

Binding Sites↗

PubMed2005

The slow Wallerian degeneration protein, WldS, binds directly to VCP/p97 and partially redistributes it within the nucleus.

Slow Wallerian degeneration (Wld(S)) mutant mice express a chimeric nuclear protein that protects sick or injured axons from degeneration. The C-terminal region, derived from NAD(+) synthesizing enzyme Nmnat1, is reported to confer neuroprotection in vitro. However, an additional role for the N-terminal 70 amino acids (N70), derived from multiubiquitination factor Ube4b, has not been excluded. In wild-type Ube4b, N70 is part of a sequence essential for ubiquitination activity but its role is not understood. We report direct binding of N70 to valosin-containing protein (VCP; p97/Cdc48), a protein with diverse cellular roles including a pivotal role in the ubiquitin proteasome system. Interaction with Wld(S) targets VCP to discrete intranuclear foci where ubiquitin epitopes can also accumulate. Wld(S) lacking its N-terminal 16 amino acids (N16) neither binds nor redistributes VCP, but continues to accumulate in intranuclear foci, targeting its intrinsic NAD(+) synthesis activity to these same foci. Wild-type Ube4b also requires N16 to bind VCP, despite a more C-terminal binding site in invertebrate orthologues. We conclude that N-terminal sequences of Wld(S) protein influence the intranuclear location of both ubiquitin proteasome and NAD(+) synthesis machinery and that an evolutionary recent sequence mediates binding of mammalian Ube4b to VCP.

Adenosine Triphosphatases↗

PubMed2006

Role of mitogen-activated protein kinase kinase in Porphyromonas gingivalis-induced myocardial cell hypertrophy and apoptosis.

Secreted factors present in the medium following growth of the periodontal pathogen Porphyromonas gingivalis cause increased cardiomyocyte hypertrophy and apoptosis, whereas secreted factors from Actinobacillus actinomycetemcomitans and Prevotella intermedia have no such effects. The purpose of this study was to clarify the role of mitogen-activated protein kinase (MAPK)/extracellular-regulated protein kinase (ERK) pathways in P. gingivalis medium-induced H9c2 myocardial cell hypertrophy and apoptosis. Cellular morphology, DNA fragmentation, nuclear condensation, total mitogen-activated protein kinase/extracellular-regulated protein kinase-1 (ERK-1), total ERK-1 protein, and phosphorylated ERK-1 protein products in cultured H9c2 myocardial cells were measured by actin immunofluorescence, agarose gel electrophoresis, nuclear condensation, and western blotting following stimulation with P. gingivalis spent growth medium or pre-administration of U0126, a potent MEK-1/2 inhibitor. Components of P. gingivalis spent culture medium not only resulted in increased total MEK-1 and ERK-1 protein products, but also caused increased cellular size, DNA fragmentation, and nuclear condensation in H9c2 cells. These three parameters, and the phosphorylated ERK-1 protein products of H9c2 cells treated with P. gingivalis medium, were all significantly reduced after pre-administration of U0126. The results suggest that P. gingivalis-secreted factors may initiate MEK/ERK signal pathways and lead to myocardial cell hypertrophy and apoptosis.

Animals↗

PubMedDate not supplied

Quantitative methods to analyze subnuclear protein organization in cell populations with varying degrees of protein expression.

The control of gene transcription is dependent on DNA-binding and coregulatory proteins that assemble in distinct regions of the cell nucleus. We use multispectral wide-field microscopy of cells expressing transcriptional coregulators labeled with fluorescent proteins (FP) to study the subnuclear localization and function of these factors in living cells. In coexpression studies, the glucocorticoid receptor interacting protein (GRIP) coactivator protein and the silencing mediator of retinoid and thyroid (SMRT) corepressor protein form spherical subnuclear focal bodies that are spatially distinct, suggesting that specific protein interactions concentrate these divergent proteins in separate subnuclear regions. However, the variability of these subnuclear bodies between cells within the population makes analysis based on "representative images" difficult, if not impossible. To address this issue, we develop a protocol for unbiased selection of cells from the population, followed by the automated quantification of the subnuclear organization of the labeled proteins. Statistical methods identify a significant linear correlation between the FP-coregulator expression level and subnuclear focal body formation for both FP-GRIP and FP-SMRT. Importantly, we confirm that these changes in subnuclear organization could be statistically normalized for differences in coregulator expression level. This integrated quantitative image analysis method will allow the rigorous comparison of different experimental cell populations that express variable levels of FP fusion proteins.

Animals↗

PubMed2006

Identification of a dynein interacting domain in the papillomavirus minor capsid protein l2.

Papillomaviruses enter cells via endocytosis (H. C. Selinka et al., Virology 299:279-287, 2002). After egress from endosomes, the minor capsid protein L2 accompanies the viral DNA to the nucleus and subsequently to the subnuclear promyelocytic leukemia protein bodies (P. M. Day et al., Proc. Natl. Acad. Sci. USA 101:14252-14257, 2004), suggesting that this protein may be involved in the intracytoplasmic transport of the viral genome. We now demonstrate that the L2 protein is able to interact with the microtubule network via the motor protein dynein. L2 protein was found attached to microtubules after uncoating of incoming human papillomavirus pseudovirions. Based on immunofluorescence and coimmunoprecipitation analyses, the L2 region interacting with dynein is mapped to the C-terminal 40 amino acids. Mutations within this region abrogating the L2/dynein interaction strongly reduce the infectivity of pseudoviruses, indicating that this interaction mediates the minus-end-directed transport of the viral genome along microtubules towards the nucleus.

Active Transport, Cell Nucleus↗

PubMed2006

Genotoxic stress and cellular stress alter the subcellular distribution of human T-cell leukemia virus type 1 tax through a CRM1-dependent mechanism.

Human T-cell leukemia virus type 1 Tax is a predominantly nuclear viral oncoprotein that colocalizes with cellular proteins in nuclear foci known as Tax speckled structures (TSS). Tax is also diffusely distributed throughout the cytoplasm, where it interacts with and affects the functions of cytoplasmic cellular proteins. Mechanisms that regulate the distribution of Tax between the cytoplasm and nucleus remain to be identified. Since Tax has been shown to promote genome instability by perturbing cell cycle progression and DNA repair mechanisms following DNA damage, we examined the effect of genotoxic stress on the subcellular distribution and interacting partners of Tax. Tax localization was altered in response to various forms of cellular stress, resulting in an increase in cytoplasmic Tax and a decrease in Tax speckled structures. Concomitantly, colocalization of Tax with sc35 (a TSS protein) decreased following stress. Tax translocation required the CRM1 nuclear export pathway, and a transient interaction between Tax and CRM1 was observed following stress. These results suggest that the subcellular distribution of Tax and the interactions between Tax and cellular proteins respond dynamically to cellular stress. Changes in Tax distribution and interacting partners are likely to affect cellular processes that regulate cellular transformation.

Active Transport, Cell Nucleus↗

PubMed2006

RNA-templated replication of hepatitis delta virus: genomic and antigenomic RNAs associate with different nuclear bodies.

Lacking an RNA-dependent RNA polymerase, hepatitis delta virus (HDV), which contains a circular RNA of 1.7 kilobases, is nonetheless able to replicate its RNA by use of cellular transcription machineries. Previously, we have shown that the replications of genomic- and antigenomic-strand HDV RNAs have different sensitivities to alpha-amanitin, suggesting that these two strands are synthesized in different transcription machineries in the cells, but the nature of these transcription machineries is not clear. In this study, we performed metabolic labeling and immunofluorescence staining of newly synthesized HDV RNA with bromouridine after HDV RNA transfection into hepatocytes and confirmed that HDV RNA synthesis had both alpha-amanitin-sensitive and -resistant components. The antigenomic RNA labeling was alpha-amanitin resistant and localized to the nucleolus. The genomic RNA labeling was alpha-amanitin sensitive and more diffusely localized in the nucleoplasm. Most of the genomic RNA labeling appeared to colocalize with the PML nuclear bodies. Furthermore, promyelocytic leukemia protein, RNA polymerase II (Pol II), and the Pol I-associated transcription factor SL1 could be precipitated together with hepatitis delta antigen, suggesting the association of HDV replication complex with the Pol I and Pol II transcription machineries. This conclusion was further confirmed by an in vitro replication assay. These findings provide additional evidence that HDV RNA synthesis occurs in the Pol I and Pol II transcription machineries, thus extending the capability of the cellular DNA-dependent RNA polymerases to utilizing RNA as templates.

Amanitins↗

PubMed2006

Multiple controls regulate nucleostemin partitioning between nucleolus and nucleoplasm.

Nucleostemin plays an essential role in maintaining the continuous proliferation of stem cells and cancer cells. The movement of nucleostemin between the nucleolus and the nucleoplasm provides a dynamic way to partition the nucleostemin protein between these two compartments. Here, we show that nucleostemin contains two nucleolus-targeting regions, the basic and the GTP-binding domains, that exhibit a short and a long nucleolar retention time, respectively. In a GTP-unbound state, the nucleolus-targeting activity of nucleostemin is blocked by a mechanism that traps its intermediate domain in the nucleoplasm. A nucleostemin-interacting protein, RSL1D1, was identified that contains a ribosomal L1-domain. RSL1D1 co-resides with nucleostemin in the same subnucleolar compartment, unlike the B23 and fibrillarin, and displays a longer nucleolar residence time than nucleostemin. It interacts with both the basic and the GTP-binding domains of nucleostemin through a non-nucleolus-targeting region. Overexpression of the nucleolus-targeting domain of RSL1D1 alone disperses nucleolar nucleostemin. Loss of RSL1D1 expression reduces the compartmental size and amount of nucleostemin in the nucleolus. Our work reveals that the partitioning of nucleostemin employs complex mechanisms involving both nucleolar and nucleoplasmic components, and provides insight into the post-translational regulation of its activity.

Amino Acid Sequence↗

PubMed2006

Intranuclear coccidiosis in tortoises: nine cases.

Chelonian intranuclear coccidiosis has been reported once, in two radiated tortoises (Geochelone radiata), and is apparently rare. We describe intranuclear coccidiosis diagnosed histologically in two radiated tortoises, three Travancore tortoises (Indotestudo forstenii), two leopard tortoises (Geochelone pardalis), one bowsprit tortoise (Chersina angulata), and one impressed tortoise (Manouria impressa). Infection was systemic and involved alimentary, urogenital, respiratory, lymphoid, endocrine, and integumentary systems. Trophozoites, meronts, merozoites, macrogametocytes, microgametocytes, and nonsporulated oocysts were seen histologically or by electron microscopy. Intracytoplasmic and extracellular stages of parasite development also were identified histologically. Sequencing of a coccidial 18S rRNA consensus polymerase chain reaction (PCR) product revealed a novel sequence that provided phylogenetic information and may be useful for further diagnostic test design. Intranuclear coccidiosis was associated with variable degrees of inflammation in all cases, was considered the cause of death in six tortoises, and was a substantial contributing factor to the cause of death in two tortoises.

Animals↗

PubMed2004

Computer-assisted image analysis protocol that quantitatively measures subnuclear protein organization in cell populations.

Many nuclear proteins, including the nuclear receptor co-repressor (NCoR) protein are localized to specific regions of the cell nucleus, and this subnuclear positioning is preserved when NCoR is expressed in cells as a fusion to a fluorescent protein (FP). To determine how specific factors may influence the subnuclear organization of NCoR requires an unbiased approach to the selection of cells for image analysis. Here, we use the co-expression of the monomeric red FP (mRFP) to select cells that also express NCoR labeled with yellow FP (YFP). The transfected cells are selected for imaging based on the diffuse cellular mRFP signal without prior knowledge of the subnuclear organization of the co-expressed YFP-NCoR. The images acquired of the expressed FPs are then analyzed using an automated image analysis protocol that identifies regions of interest (ROIs) using a set of empirically determined rules. The relative expression levels of both fluorescent proteins are estimated, and YFP-NCoR subnuclear organization is quantified based on the mean focal body size and relative intensity. The selected ROIs are tagged with an identifier and annotated with the acquired data. This integrated image analysis protocol is an unbiased method for the precise and consistent measurement of thousands of ROIs from hundreds of individual cells in the population.

Animals↗

PubMedDate not supplied

Ultrastructural arguments for cytoplasmic engulfment phenomenon in nucleus of Entamoeba histolytica.

The electron optic study of a pathogenic E. histolytica strain revealed particular aspects concerning the shape, structure and distribution in the nuclear space of the intranuclear structures known as button-like bodies. These aspects are ultrastructural arguments useful in interpreting the intranuclear inclusions as sections through the cytoplasmic material sequestered in the nuclear space by karyophagocytosis.

Animals↗

PubMed1975

[Alpha-1-antitrypsin deficiency in children: liver ultrastructure and speculations (author's transl)].

Fourteen liver biopsies from twelve young patients with liver diseases associated with homozygous, PiZZ phenotype, alpha-1-antitrypsin deficiency in their sera were examined by electron microscopy. In all these biopsies characteristic homogeneous material was found in some hepatocytes and corresponded, when observed on adjacent semithin sections by light microscopy, to the deposit stained by periodic acid Schiff reaction. The accumulation in perinuclear spaces resulted in intranuclear invaginations, but the major deposit was located in lumens of the endoplasmic reticulum. The limiting membranes were rough and smooth but the extent of the latter was so large that only this type of reticulum seemed peculiarly involved in the accumulating process. On the contrary, Golgi complexes did not seen obligatorily involved by this process because, when observed, they appeared almost normal even in heavily overloaded liver cells. At least for the PiZZ phenotype, the abnormal substance would be an asialo form of normal alpha-1-antitrypsin. Thus the subject of this study is the morphologic translation of an impairment in the synthesis of a glycoprotein. In the light of data concerning the synthesis of such proteins our findings lead us to suggest: The ultrastructural patterns observed in alpha-1-antitrypsin deficiency cannot give the expected morphologic evidence of the biochemical data which locate the first binding steps of monosaccharide residues in the rough endoplasmic reticulum. The absence of sialic acid could not result from an enzymatic defect primarily located in Golgi complexes but could be secondary to an impairment in the binding of one monosaccharide residue which improves subsequent fixation of sialic acid, in the smooth endoplasmic reticulum. Finally it seems necessary to emphasize that the relationship between the abnormal substance and various important non specific lesions is largely unknown and that we don't know the significance of polymorphous dense bodies observed in ductular cells during the cholestatic period.

Cell Nucleus↗

PubMed1993

[Characterization of a fluorouracil-resistant human gastric carcinoma cell line and its morphological behavior].

A fluorouracil (FU)-resistant human carcinoma cell line (SGC-7901/R) was established in culture by progressively increasing the concentration of FU from 50 ng.ml-1 to 2.5 mg.ml-1. The cell line has been successfully subcultured for more than 150 passages during more than 2 years. Its degree of FU resistance was 139-fold vs that of FU sensitive cell line (SGC-7901/S), and the resistant phenotype was stable when cells were cultured for 23 passages in FU-free medium. The doubling time was 17.3 and 25.6 h for resistant cells and parental cells, respectively. Swiss (nu/nu) nude mice were used for the in vivo experiment, the FU-resistant cell line also exhibited resistance to FU and cross-resistance to mitomycin C. FU inhibited markedly the incorporation of [3H]UR into sensitive cells and only showed a 31.6% inhibition with FU 100 micrograms.ml-1 in resistant cells. For the incorporation of [3H]TdR into DNA, inhibitory rates were seen with different concentrations of FU in resistant cells. By morphologic observation, SGC-7901/R cells showed little secretion but without any tendency to glandular pattern. Their nuclei were allotype with enlarged perinuclear space and a few intranuclear pseudoinclusions. The mitotic phase of cells was found frequently. The phenotype of resistant cell line can be deduced more malignant than that of parental cell line.

Animals↗

PubMed2000

Fine-needle aspiration diagnosis of psammomatous melanotic schwannoma.

A 51-yr-old male presented with an 8-mo history of lower back pain. Computerized axial tomography (CT) and magnetic resonance imaging (MRI) studies showed a 3.5 X 3.0 cm sacral mass within the spinal canal in the region of the left S2 nerve root. A fine-needle aspirate biopsy (FNAB) was performed under CT guidance. The cytologic findings included a spindle-cell population with a fibrillary background arranged in a vaguely streaming pattern, wavy nuclei with mild atypia, wispy cytoplasm, rare intranuclear inclusions, and dilated vascular spaces. Some cells contained a nonrefractile granular brown pigment consistent with melanin. Also identified were calcified concentric laminations typical of psammoma bodies. Immunohistochemically, the neoplastic cells were strongly immunoreactive for S-100 protein and HMB-45. A diagnosis of psammomatous melanotic schwannoma was rendered. No stigmata of Carney's complex were identified on physical examination. The patient has declined the recommendation of surgical excision of the mass. We herein describe a case unequivocally diagnosed as psammomatous melanotic schwannoma by FNAB.

Antigens, Neoplasm↗

PubMed1956

Electron microscope observations on intracellular virus-like particles associated with the cells of the Lucké renal adenocarcinoma.

The common renal adenocarcinoma of the leopard frog was studied in thin sections with the electron microscope. Approximately a third of the tumors examined were found to contain spheroidal bodies of uniform size and distinctive morphology that are believed to be virus particles. These consist of hollow spheres (90 to 100 mmicro) having a thick capsule and a dense inner body (35 to 40 mmicro) that is eccentrically placed within the central cavity (70 to 80 mmicro). Virus particles of this kind occur principally in the cytoplasm but occasionally they are also found in the nucleus and in the extracellular spaces of the tumor. The intranuclear inclusion bodies that are visible with the light microscope are largely comprised of hollow, spherical vesicles with thin limiting membranes. These are embedded in a finely granular matrix. A few of the thin walled vesicles contain a dense inner body like that of the cytoplasmic virus particles. This suggests that they may be immature virus particles. The inclusion bodies are believed to be formed in the course of virus multiplication but they usually contain very few mature virus particles. Bundles of dense filaments and peculiar vacuolar inclusions also occur in the cytoplasm of the tumor cells. These seem to be related in some way to the presence of virus but their origin and significance remain obscure. These findings are discussed in relation to previous work suggesting that the Lucké adenocarcinoma is caused by an organ-specific filtrable agent. It is concluded that the "virus particles" found in electron micrographs of the tumor cells may be the postulated tumor agent. On the other hand, the possibility remains that the particles described here are not those that are causally related to the tumors.

Carcinoma, Renal Cell↗

PubMed1980

Changes of the central nervous system in herpes zoster.

Postmortem examination was conducted in eight cases of neurocutaneous herpes zoster. One generalized case with diffuse and focal meningoencephalomyelitis showed numerous infiltrates in the subarachnoid space and along the cerebral ventricles. Intranuclear inclusion bodies could be demonstrated for the first time in ependymal and in arachnoidal cells, and viral capsids were found in the latter. The parenchymal lesions, lying predominantly in the white matter, were characterized by inflammation, hemorrhage, axonal swellings, demyelination, and intranuclear inclusions in oligodendrocytes. In the second generalized case there was a granulomatous giant cell angiitis with intranuclear inclusions bodies in the vessel walls. Out of four cases with localized damage of the central nervous system (CNS), three showed demyelination as the main alteration. In another case there was a hitherto undescribed calcifying coagulation necrosis of a spinal ganglion.

Adult↗

PubMed1987

Intranuclear rod-shaped actin filament bundles in poorly differentiated axillary adenosquamous cell carcinoma.

An axillary cystic poorly differentiated adenosquamous cell carcinoma was studied by electron microscopy and immunohistochemistry for keratin, vimentin, desmin, and actin. Many tumor cells had rod-shaped intranuclear fibrillary inclusions which consisted of 60 A microfilaments. These intranuclear inclusions reacted with antiactin antibody and were considered to be F-actin bundles. The cells having intranuclear inclusions were frequently found in areas close to the cystic space, and inclusions of tumor cells close to the cyst were thicker. However, mitotic figures were predominantly found in the periphery of the tumor. Intranuclear actin is known to be mainly soluble G-actin, and one of its important functions is reported to be the chromosome condensation in mitosis. It is speculated that the cells having intranuclear fibrillary inclusions of actin have impairment of mitosis.

Actins↗