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At least 1,063 records · Page 59Linked to original sources
PubMed1979

The grasshopper X chromosome. I. States of condensation and the nuclear envelope at G1, S and G2 of premeiotic interphase and at early meiotic prophase.

The sub-stages of spermatocyte interphase (G1, S and G2) have been identified in the grasshopper Brachystola magna using E.M. autoradiography and serial thin sectioning techniques. The X chromosome occupies a nuclear envelope bound compartment separate from an autosome compartment during G1 and S. At G2 the X compartment is resolved by coalescence of the membranes enveloping the X chromosome and autosome compartments.--At G1 and S, the compartmentalized X chromatin is laced with nuclear membrane material. This X chromatin associated membrane decreases in amount as the cell passes through G2 and enters early meiotic prophase. There are at least 2 and possibly 3 states of condensation of the heterochromatic X during premeiotic interphase and early meiotic prophase correlated with the presence or absence of membrane material associated with the chromatin.--The X chromatin replicates asynchronously with autosomal euchromatin and synchronously with autosomal heterochromatin associated with nucleoli. The X chromatin replication appears to be associated with the nuclear membrane.--The observations indicate that the nuclear membrane is involved with X chromosome condensation and may be implicated in asynchronous X chromosome replication as well.

Animals↗
PubMed1994

Temperature-sensitive steps in the transport of Semliki Forest virus envelope proteins in mosquito C6/36 cells.

We have analysed the temperature dependence of the transport of Semliki Forest virus (SFV) envelope proteins in mosquito cells, the natural host cells of alphaviruses. These cells are cultivated at a lower temperature (28 degrees C) and have a different lipid composition as compared to mammalian cells. When the incubation temperature was reduced at early times after infection, the onset of virus shedding was delayed and the maximal titers decreased correspondingly to the temperature. No virus was shed at 12 degrees C. No evidence was observed for a block of virus release due to a shift of the sites of virus maturation. When the incubation temperature was reduced at later times after infection a critical temperature of 12 degrees C was again observed. At this temperature no transport of viral proteins took place, p62 remained uncleaved, the glycan processing of E1 did not occur and the envelope proteins accumulated in a pre-Golgi compartment. We suggest a mathematical formula which allows the extrapolation of transport data to the temperature at which intracellular protein transport becomes blocked.

Aedes↗
PubMed2000

MxA GTPase blocks reporter gene expression of reconstituted Thogoto virus ribonucleoprotein complexes.

Human MxA protein accumulates in the cytoplasm of interferon-treated cells and inhibits the multiplication of several RNA viruses, including Thogoto virus (THOV), a tick-borne orthomyxovirus that transcribes and replicates its genome in the cell nucleus. The antiviral mechanism of MxA was investigated by using two alternative minireplicon systems in which recombinant viral ribonucleoprotein complexes (vRNPs) of THOV were reconstituted from cloned cDNAs. A chloramphenicol acetyltransferase reporter minigenome RNA was expressed either by T7 RNA polymerase in the cytoplasm of transfected cells or, alternatively, by RNA polymerase I in the nucleus. The inhibitory effect of MxA was studied in both cellular compartments by coexpressing wild-type MxA or TMxA, an artificial nuclear form of MxA. Our results indicate that both MxA proteins recognize the assembled vRNP rather than the newly synthesized unassembled components. The present findings are consistent with previous data which indicated that cytoplasmic MxA prevents transport of vRNPs into the nucleus, whereas nuclear MxA directly inhibits the viral polymerase activity in the nucleus.

Animals↗
PubMed1990

Monoclonal antibody analysis of the proliferating cell nuclear antigen (PCNA). Structural conservation and the detection of a nucleolar form.

The proliferating cell nuclear antigen, PCNA, has recently been identified as the polymerase delta accessory protein. PCNA is essential for cellular DNA synthesis and is also required for the in vitro replication of simian virus 40 (SV40) DNA where it acts to coordinate leading and lagging strand synthesis at the replication fork. The cDNA for rat PCNA was cloned into a series of bacterial expression vectors and the resulting protein used to immunize mice. Eleven new monoclonal antibodies to PCNA have been isolated and characterized. Some of the antibodies recognize epitopes conserved from man to fission yeast. Immunocytochemical analysis of primate epithelial cell lines showed that the antibodies recognized antigenically distinct forms of PCNA and that these forms were localized to different compartments of the nucleus. One antibody reacted exclusively with PCNA in the nucleolus. These results suggest that the PCNA protein may fulfil several separate roles in the cell nucleus associated with changes in its antigenic structure.

Animals↗
PubMed2003

A non-canonical transferable signal mediates nuclear import of simian immunodeficiency virus Vpx protein.

Protein transport into the nucleus is generally considered to involve specific nuclear localization signals (NLS) though it is becoming increasingly evident that efficient and well controlled import of proteins which lack a canonical NLS also occurs in cells. Vpx, a 112 amino acid protein from human immunodeficiency virus type 2 (HIV-2) and the closely related simian immunodeficiency virus (SIV) is one such protein, which does not have an identifiable canonical NLS and is yet efficiently imported to the nuclear compartment. Here we report that Vpx protein is imported to the nucleus independently of virus-encoded cofactors. When fusions of truncated versions of Vpx with full-length beta-galactosidase (beta-Gal) were tested, the region from Vpx 61 to 80 was found to be sufficient to mediate the import of the heterologous cytoplasmic protein to the nucleus. Inactivation of Vpx NLS precluded nuclear import of Vpx and reduced virus replication in non-dividing macrophage cultures, even when functional integrase and Gag matrix proteins implicated in viral nuclear import were present. Importantly, we identified and characterized a novel type of 20 amino acid transferable nuclear import signal in Vpx that is distinct from other import signals described. In addition, we show that the minimal nuclear targeting domain identified here overlaps with helical domain III (amino acid (aa) 64-82) and the structural integrity of this helical motif is critical for the nuclear import of Vpx. Taken together, these data suggest that Vpx is imported to the nucleus via a novel import pathway that is dependent on its 20 amino acid unique nuclear targeting signal, and that the nuclear import property of Vpx is critical for the optimal virus replication in non-dividing cells such as macrophages.

Active Transport, Cell Nucleus↗
PubMed2006

p27(SJ), a novel protein in St John's Wort, that suppresses expression of HIV-1 genome.

Transcription of the HIV-1 genome is controlled by the cooperation of viral regulatory proteins and several host factors which bind to specific DNA sequences within the viral promoter spanning the long terminal repeat, (LTR). Here, we describe the identification of a novel protein, p27(SJ), present in a laboratory callus culture of Hypericum perforatum (St John's Wort) that suppresses transcription of the HIV-1 genome in several human cell types including primary culture of microglia and astrocytes. p27(SJ) associates with C/EBPbeta, a transcription factor that regulates expression of the HIV-1 genome in macrophages and monocytic cells, and the viral transactivator, Tat. The association of p27(SJ) with C/EBPbeta and Tat alters their subcellular localization, causing their accumulation in the perinuclear cytoplasmic compartment of the cells. Fusion of a nuclear localization signal to p27(SJ) forces its entry into the nucleus and diminishes the capacity of p27(SJ) to suppress Tat activity, but does not alter its ability to suppress C/EBPbeta activation of the LTR. Results from binding assays showed the inhibitory effect of p27(SJ) on C/EBPbeta interaction with DNA. Finally, our results demonstrate that expression of p27(SJ) decreases the level of viral replication in HIV-1-infected cells. These observations suggest the potential for the development of a therapeutic advance based on p27(SJ) protein to control HIV-1 transcription and replication in cells associated with HIV-1 infection in the brain.

Astrocytes↗
PubMed1999

The human DNA ligase III gene encodes nuclear and mitochondrial proteins.

We provide evidence that the human DNA ligase III gene encodes a mitochondrial form of this enzyme. First, the DNA ligase III cDNA contains an in-frame ATG located upstream from the putative translation initiation start site. The DNA sequence between these two ATG sites encodes an amphipathic helix similar to previously identified mitochondrial targeting peptides. Second, recombinant green fluorescent protein harboring this sequence at its amino terminus was efficiently targeted to the mitochondria of Cos-1 monkey kidney cells. In contrast, native green fluorescent protein distributed to the cytosol. Third, a series of hemagglutinin-DNA ligase III minigene constructs were introduced into Cos-1 cells, and immunocytochemistry was used to determine subcellular localization of the epitope-tagged DNA ligase III protein. These experiments revealed that inactivation of the upstream ATG resulted in nuclear accumulation of the DNA ligase III protein, whereas inactivation of the downstream ATG abolished nuclear localization and led to accumulation within the mitochondrial compartment. Fourth, mitochondrial protein extracts prepared from human cells overexpressing antisense DNA ligase III mRNA possessed substantially less DNA ligase activity than did mitochondrial extracts prepared from control cells. DNA end-joining activity was also substantially reduced in extracts prepared from antisense mRNA-expressing cells. From these results, we conclude that the human DNA ligase III gene encodes both nuclear and mitochondrial enzymes. DNA ligase plays a central role in DNA replication, recombination, and DNA repair. Thus, identification of a mitochondrial form of this enzyme provides a tool with which to dissect mammalian mitochondrial genome dynamics.

Amino Acid Sequence↗
PubMedDate not supplied

[Complexes of nuclear DNA-polymerases with 3'----5'-exonucleases from the rat liver].

"Editing" 3'----5' exonuclease activity of DNA polymerases corrects replication errors. This activity associated with procaryotic DNA polymerases is not intrinsic to purified mammalian DNA polymerases. By means of extraction and subsequent gel filtration, several subspecies of complexes of 3'----5' exonuclease (E.C. 3.1.4.26) with DNA polymerases alpha, beta (E.C. 2.7.7.7) and some other proteins were isolated from chromatin, nucleoplasm, nuclear membrane, and cytosol. Complexes containing 3'----5' exonuclease manifest from 40 to 70% of total DNA polymerase activity revealed in different compartments of a hepatocyte. Molecular masses of the complexes amount from 250 to 1500 kDa They dissociate as a result of solution hydrophobization. DNA polymerase alpha activity enhances 5--8 folds during cell transition from G0 to S-period. The value of the ratio of 3'----5' exonuclease activity of different complexes to their DNA polymerase activity varies from 0.5 to 12. Other cases of discovery of the complexes of DNA polymerases with 3'----5' exonucleases are discussed. It is suggested that the absence of 3'----5' exonuclease active site in the DNA polymerase polypeptide is compensated by the complex formation of the corresponding enzymes.

Animals↗
PubMed1999

Interferon-induced human MxA GTPase blocks nuclear import of Thogoto virus nucleocapsids.

Interferon-induced human MxA protein belongs to the dynamin superfamily of large GTPases. It exhibits antiviral activity against a variety of RNA viruses, including Thogoto virus, an influenza virus-like orthomyxovirus transmitted by ticks. Here, we report that MxA blocks the transport of Thogoto virus nucleocapsids into the nucleus, thereby preventing transcription of the viral genome. This interaction can be abolished by a mAb that neutralizes the antiviral activity of MxA. Our results reveal an antiviral mechanism whereby an interferon-induced protein traps the incoming virus and interferes with proper transport of the viral genome to its ultimate target compartment within the infected cell.

3T3 Cells↗
PubMed1992

A freeze-fracture study of the perineurium in galactose neuropathy: morphological changes associated with endoneurial oedema.

Feeding rats with galactose as 40% of their diet results in peripheral nerve oedema related to the intrafascicular accumulation of galactitol and sodium. In this study, associated changes in the perineurium were examined by the freeze-fracture replication technique. Perineurial cells are linked by tight junctions (zonulae occludentes). In normal animals these are made up of anastomosing strands organized in a belt-like arrangement along the margins of continuous cells. The majority of the tight junctions in the galactose-fed animals displayed structural abnormalities. These ranged from slight separation of the strands to fragmentation and dispersal, with looping of isolated strands. Some of the tight junctions contained large dilated compartments within the junctional network. Short lengths of intramembranous particles, probably representing assembly or disassembly of tight junctional strands, were also observed. The membranes of perineurial cells normally possess numerous openings of caveolae. A quantitative assessment showed that the mean density of these caveolae openings was increased in the galactose-fed rats as compared with controls. The alterations in the tight junctions resemble those that have been produced experimentally in epithelia by subjecting them to abnormal osmotic gradients. They also resemble those seen in human diabetic neuropathy in which osmotic disturbances involving the perineurium have been considered to occur. If the alterations involve the inner layers of the perineurium, they are likely to impair its barrier function. The increased number of caveolae openings in galactose neuropathy may represent a reaction to the endoneurial oedema and indicate that the pinocytotic-like vesicles have a transport function.

Animals↗
PubMed1993

The Jülich Environmental Specimen Bank.

The environmental specimen bank of the Federal Republic of Germany represents the modern form of a systematically designed archive for the preservative storage of selected environmental specimens. Major aims of environmental specimen banks are: (i) retrospective identification and quantification of environmental chemicals including those not known at the time of storage; (ii) retrospective replicate analysis of samples stored under preservative conditions for the determination of inorganic elements or organic compounds already examined; and (iii) retrospective control of the success of legislative environmental protection measures in handling chemicals regarded as hazardous for man and the environment. Results of the environmental specimen bank at Jülich by the Institute of Applied Physical Chemistry, Research Centre Jülich, KFA, obtaining the initiated legislative regulations providing environmental pollution, are already proved for various compartments.

Animals↗
PubMed2004

Recognition of bacteria in the cytosol of Mammalian cells by the ubiquitin system.

Recent studies have suggested the existence of innate host surveillance systems for the detection of bacteria in the cytosol of mammalian cells. The molecular details of how bacteria are recognized in the cytosol, however, remain unclear. Here we examined the fate of Salmonella typhimurium, a gram-negative bacterial pathogen that can infect a variety of hosts, in the cytosol of mammalian cells. These bacteria typically occupy a membrane bound compartment, the Salmonella-containing vacuole (SCV), in host cells. We show that some wild-type bacteria escape invasion vacuoles and are released into the cytosol. Subsequently, polyubiquitinated proteins accumulate on the bacterial surface, a response that was witnessed in several cell types. In macrophages but not epithelial cells, the proteasome was observed to undergo a dramatic subcellular relocalization and become associated with the surface of bacteria in the cytosol. Proteasome inhibition promoted replication of S. typhimurium in the cytosol of both cell types, in part through destabilization of the SCV. Surprisingly, the cytosol-adapted pathogen Listeria monocytogenes avoided recognition by the ubiquitin system by using actin-based motility. Our findings indicate that the ubiquitin system plays a major role in the recognition of bacterial pathogens in the cytosol of mammalian cells.

Actins↗
PubMed2004

Mitochondrial poly(ADP-ribosylation): from old data to new perspectives.

Poly(ADP-ribosylation) is involved in DNA repair and replication, transcription, and cell death. For a long time, only one poly(ADP-ribosylating) enzyme was known, named ADPRT/PARP (EC 2.4.2.30). The recent discovery of a family of PARPs has provided a high degree of complexity in the field. Moreover, the finding that poly(ADP-ribosylation) is not confined to the nucleus but is also carried out by cytoplasmic enzymes supports the idea that it could regulate proteins localized in different cellular compartments. In this respect, a reappraisal of the literature on mitochondrial poly(ADP-ribosylation) could be useful, as well as a discussion of its relevance regarding the current "hot" view of poly(ADP-ribosylation) as a mediator of cell death.

Animals↗
PubMed2026

Fetal signatures in the 3D genome of iPSC-derived neurons and their implications for disease modeling.

Induced pluripotent stem cells (iPSCs) have revolutionized neuroscience, providing an approach to generate patient-specific neurons for modeling of neurological diseases. However, it remains unclear how closely iPSC-derived neurons replicate the chromatin architecture of authentic brain neurons. Here, we uniformly processed newly generated Hi-C data from iPSC-derived neurons and neurons isolated from the human postmortem brain, together with previously published data sets comprising 228 human and 89 mouse Hi-C and snm3C-seq samples from different cell subtypes. These data were merged into 96 high-coverage contact maps used to examine chromatin features ranging from chromatin compartments and topologically associating domains (TADs) to chromatin loops, Polycomb-mediated contacts, and frequently interacting regions (FIREs). We find that iPSC-derived neurons largely retain the chromatin state of undifferentiated cells and resemble fetal rather than mature neurons. iPSC-derived neurons exhibit unusually strong compartmentalization, an enrichment of developmental genes at TAD borders, and a marked reduction of long-range repressive Polycomb-mediated contacts that typically silence early fetal programs. Although immature, iPSC-derived neurons offer advantages for modeling interactions between disease-associated SNPs and target genes, as many psychiatric disorders have neurodevelopmental origins. Integrating iPSC-derived and postmortem neuronal data sets therefore provides complementary insights into the chromatin landscape underlying disease-associated interactions. Our study offers a valuable Hi-C resource for the community and provides a detailed comparison of chromatin architecture throughout neuronal maturation, underscoring its importance for validating neuronal models and providing a robust framework for future studies.

Journal Article↗
PubMed2000

Intracellular growth of Legionella pneumophila in Dictyostelium discoideum, a system for genetic analysis of host-pathogen interactions.

Conditions were established in which Legionella pneumophila, an intracellular bacterial pathogen, could replicate within the unicellular organism Dictyostelium discoideum. By several criteria, L. pneumophila grew by the same mechanism within D. discoideum as it does in amoebae and macrophages. Bacteria grew within membrane-bound vesicles associated with rough endoplasmic reticulum, and L. pneumophila dot/icm mutants, blocked for growth in macrophages and amoebae, also did not grow in D. discoideum. Internalized L. pneumophila avoided degradation by D. discoideum and showed evidence of reduced fusion with endocytic compartments. The ability of L. pneumophila to grow within D. discoideum depended on the growth state of the cells. D. discoideum grown as adherent monolayers was susceptible to L. pneumophila infection and to contact-dependent cytotoxicity during high-multiplicity infections, whereas D. discoideum grown in suspension was relatively resistant to cytotoxicity and did not support intracellular growth. Some known D. discoideum mutants were examined for their effect on growth of L. pneumophila. The coronin mutant and the myoA/B double myosin I mutant were more permissive than wild-type strains for intracellular growth. Growth of L. pneumophila in a G(beta) mutant was slightly reduced compared to the parent strain. This work demonstrates the usefulness of the L. pneumophila-D. discoideum system for genetic analysis of host-pathogen interactions.

Animals↗
PubMed2001

Intracellular trafficking of adeno-associated virus vectors: routing to the late endosomal compartment and proteasome degradation.

The early steps of adeno-associated virus (AAV) infection involve attachment to a variety of cell surface receptors (heparan sulfate, integrins, and fibroblast growth factor receptor 1) followed by clathrin-dependent or independent internalization. Here we have studied the subsequent intracellular trafficking of AAV particles from the endosomal compartment to the nucleus. Human cell lines were transduced with a recombinant AAV (rAAV) carrying a reporter gene (luciferase or green fluorescent protein) in the presence of agents that affect trafficking. The effects of bafilomycin A(1), brefeldin A, and MG-132 were measured. These drugs act at the level of endosome acidification, early-to-late endosome transition, and proteasome activity, respectively. We observed that the transducing virions needed to be routed as far as the late endosomal compartment. This behavior was markedly different from that observed with adenovirus particles. Antiproteasome treatments with MG-132 led to a 50-fold enhancement in transduction efficiency. This effect was accompanied by a 10-fold intracellular accumulation of single-stranded DNA AAV genomes, suggesting that the mechanism of transduction enhancement was different from the one mediated by a helper adenovirus, which facilitates the conversion of the rAAV single-stranded DNA genome into its replicative form. MG-132, a drug currently in clinical use, could be of practical use for potentializing rAAV-mediated delivery of therapeutic genes.

Cell Nucleus↗
PubMed2003

Smooth and rough lipopolysaccharide phenotypes of Brucella induce different intracellular trafficking and cytokine/chemokine release in human monocytes.

Virulence of the intracellular pathogen Brucella for humans is mainly associated with its lipopolysaccharide (LPS) phenotype, with smooth LPS phenotypes generally being virulent and rough ones not. The reason for this association is not quite understood. We now demonstrate by flow cytometry, electron microscopy, and ELISA that human peripheral blood monocytes interact both quantitatively and qualitatively different with smooth and rough Brucella organisms in vitro. We confirm that considerably higher numbers of rough than smooth brucellae attach to and enter the monocytes in nonopsonic conditions; but only smooth brucellae replicate in the host cells. We show for the first time that rough brucellae induce higher amounts than smooth brucellae of several CXC (GRO-alpha, IL-8) and CC (MIP-1alpha, MIP-1beta, MCP-1, RANTES) chemokines, as well as pro- (IL-6, TNF-alpha) and anti-inflammatory (IL-10) cytokines released by challenged monocytes. Upon uptake, phagosomes containing rough brucellae develop selective fusion competence to form spacious communal compartments, whereas phagosomes containing smooth brucellae are nonfusiogenic. Collectively, our data suggest that rough brucellae attract and infect monocytes more effectively than smooth brucellae, but only smooth LPS phenotypes establish a specific host cell compartment permitting successful parasitism. These novel findings link the LPS phenotype of Brucella and its virulence for humans at the level of the infected host cells. Whether this is due to a direct effect of the LPS molecules or to upstream bacterial mechanisms remains to be established.

Brucella↗
PubMed2005

Individualised cancer therapeutics: dream or reality? Therapeutics construction.

The analysis of DNA microarray and proteomic data, and the subsequent integration into functional expression sets, provides a circuit map of the hierarchical cellular networks responsible for sustaining the viability and environmental competitiveness of cancer cells, that is, their robust systematics. These technologies can be used to 'snapshot' the unique patterns of molecular derangements and modified interactions in cancer, and allow for strategic selection of therapeutics that best match the individual profile of the tumour. This review highlights technology that can be used to selectively disrupt critical molecular targets and describes possible vehicles to deliver the synthesised molecular therapeutics to the relevant cellular compartments of the malignant cells. RNA interference (RNAi) involves a group of evolutionarily conserved gene silencing mechanisms in which small sequences of double-stranded RNA or intrinsic antisense RNA trigger mRNA cleavage or translational repression, respectively. Although RNAi molecules can be synthesised to 'silence' virtually any gene, even if upregulated, a mechanism for selective delivery of RNAi effectors to sites of malignant disease remains challenging. The authors will discuss gene-modified conditionally replicating viruses as candidate vehicles for the delivery of RNAi.

Animals↗