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At least 91 records · Page 5Linked to original sources
PubMed1989

Mapping replicational sites in the eucaryotic cell nucleus.

We have used fluorescent microscopy to map DNA replication sites in the interphase cell nucleus after incorporation of biotinylated dUTP into permeabilized PtK-1 kangaroo kidney or 3T3 mouse fibroblast cells. Discrete replication granules were found distributed throughout the nuclear interior and along the periphery. Three distinct patterns of replication sites in relationship to chromatin domains in the cell nucleus and the period of S phase were detected and termed type I (early to mid S), type II (mid to late S) and type III (late S). Similar patterns were seen with in vivo replicated DNA using antibodies to 5-bromodeoxyuridine. Extraction of the permeabilized cells with DNase I and 0.2 M ammonium sulfate revealed a striking maintenance of these replication granules and their distinct intranuclear arrangements with the remaining nuclear matrix structures despite the removal of greater than 90% of the total nuclear DNA. The in situ prepared nuclear matrix structures also incorporated biotinylated dUTP into replication granules that were indistinguishable from those detected within the intact nucleus.

Animals↗
PubMed2000

The role of protein kinase C in the regulation of cell growth and in signalling to the cell nucleus.

The protein kinase C (PKC) family of serine/threonine kinases consists of at least 11 mammalian isoforms, which show slight differences in their molecular structure and enzymatic properties. PKC isoforms are involved in a wide variety of intracellular signalling events and play an important role in tumour promotion and cell growth control in general. Studies of expression levels in cancer cells and studies using overexpression of single isoforms or expression of dominant negative isoforms reveal that, depending on the cellular background, PKC isoforms can either promote or inhibit cell growth. To understand the role of PKC isoforms in growth control, it is essential to understand how PKC functions in the intracellular signalling cascades towards the cell nucleus. Recent work has shown that PKC isoforms can act either in the cytoplasm, and cause nuclear effects indirectly by triggering signalling pathways directed towards the cell nucleus, or, after translocation and activation, can themselves act in the cell nucleus.

Animals↗
PubMed1982

Definition of a series of stages in the association of two herpesviral proteins with the cell nucleus.

We examined the kinetics and the nature of the association of two herpes simplex virus proteins, the major DNA-binding protein (ICP8) and the major capsid protein (ICP5), with the nuclei of infected cells. We defined a series of stages in the association of the ICP8 protein with the cell nucleus. (i) Immediately after synthesis, the protein was found in the cytoplasmic fraction but associated rapidly with the crude nuclear fraction. (ii) The initial association of ICP8 with the crude nuclear fraction was detergent sensitive but DNase resistant, and, thus, the protein was either bound to structures attached to the outside of the nucleus and had not penetrated the nuclear envelope or was loosely bound in the nucleus, (iii) At intermediate times, a low level of an intermediate form was observed in which the association of ICP8 with the nuclear fraction was resistant to both detergent and DNase treatment. The protein may be bound to the nuclear matrix at this stage. Inhibition of viral DNA synthesis caused the DNA-binding protein to accumulate in this form. (iv) At late times during the chase period, the association of ICP8 with the cell nucleus was resistant to detergent treatment but sensitive to DNase treatment. our results argue that at this stage ICP8 was bound to viral DNA. Thus, nuclear association of the DNA-binding protein did not require viral DNA replication. More important is the observation that there is a series of stages in the nuclear association of this protein, and, thus, there may be a succession of binding sites for this protein in the cell during its movement to its final site of action in the nucleus. The major capsid protein showed some similar stages of association with the cell nucleus but the initial association with the nucleus followed a lag period. Its early association with the crude nuclear fraction was also detergent sensitive but was resistant to detergent treatment at later times. Its association with the cell nucleus was almost completely resistant to DNase treatment at all times. Inhibition of viral DNA replication blocked the nuclear transport of this protein. Thus, these two viral proteins share some stages in nuclear transport, although their requirements for nuclear association are different.

Animals↗
PubMed2005

Imaging in situ protein-DNA interactions in the cell nucleus using FRET-FLIM.

Although the distribution of DNA-binding proteins inside the cell nucleus can be analyzed by immunolabeling or by tagging proteins with GFP, we cannot establish whether the protein is bound to DNA or not. Here, we describe a novel approach that allows imaging of the in situ interaction between a GFP-fusion protein and DNA in the cell nucleus, using fluorescence resonance energy transfer (FRET). We used fluorescence lifetime imaging microscopy (FLIM) as a reliable tool to detect protein in contact with DNA. The method was successfully applied to the DNA-binding proteins histone H2B and the glucocorticoid receptor and to the heterochromatin-associated proteins HP1alpha and HP1beta.

Cell Line, Tumor↗
PubMed1999

Stress and the cell nucleus: dynamics of gene expression and structural reorganization.

A growing number of experimental observations reveal that the cell nucleus is functionally compartmentalized yet organized to ensure a dynamic response to events that influence nuclear activities. The cellular and molecular response to physiological and environmental stress induces a rapid and transient change in gene expression associated with major changes in nuclear architecture that impacts on signals involved in cell growth. In this review, we will address the effects of stress on the functional compartmentation of the cell nucleus and the dynamic reorganization of nuclear structures and function.

Animals↗
PubMed1992

Stimulation of the hamster ventral lateral geniculate nucleus induces Fos-like immunoreactivity in suprachiasmatic nucleus cells.

The suprachiasmatic nucleus (SCN) functions as a pacemaker for circadian rhythms in rodents. Its activity and, therefore, circadian rhythms, are synchronized by light information from both a direct retinal projection and an indirect projection from the thalamic intergeniculate leaflet (IGL) and ventral lateral geniculate nucleus (vLGN) (geniculohypothalamic tract; GHT). The GHT also appears to be important for synchronizing circadian rhythms to non-photic cues. Light can activate expression of several immediate-early genes in the SCN, including c-fos. We examined whether electrical stimulation of the IGL/vLGN region would induce Fos-like immunoreactivity (lir) in the hamster SCN. Electrical stimulation was given for one hour during the dark period of the lighting cycle. Stimulation at night of the IGL and adjacent LGN regions induced Fos-lir in cells in the SCN. Labeled cells were found in the dorsolateral part of the caudal SCN, and not in rostral or central regions of the SCN, nor in the ventral part of the caudal SCN. These results indicate that activation of cells contributing to the hamster GHT can induce Fos-lir in a restricted region of the caudal SCN.

Animals↗
PubMed1991

Role of luteal cell nucleus in the expression of gonadotropin action.

Gonadotropin receptors are not only present in cell membranes, but also in nuclei of bovine and human luteal cells. hCG/hLH can directly regulate several nuclear functions. To further investigate the role of luteal cell nucleus in the expression of gonadotropin action, the effect of enucleation of luteal cells on gonadotropin receptors and gonadotropin response was studied. Luteal cytoplasts were prepared by colchicine treatment of purified whole luteal cells followed by centrifugation at 37 C in a Percoll gradient. The cytoplasts were 85 to 90% pure with a recovery of about 57%. Cytoplasts were viable as determined by trypan blue exclusion (87%) and metabolically competent as determined by 3H-leucine incorporation into proteins. On the day of preparation, the viability and metabolic competency of cytoplasts were similar to control cells, i.e. untreated and colchicine treated whole luteal cells. In addition, cytoplasts and control cells showed a similar decline in number and viability during storage at 4 C. While control cells continue to be metabolically competent, cytoplasts showed a dramatic decline by 48 h of storage at 4 C. Neither the cytoplasts nor control cells degraded 125I-hCG. The kinetics of 125I-hCG association and dissociation, specificity and affinity of binding to cytoplasts were similar to control cells. However, the number of available gonadotropin receptors in cytoplasts was significantly lower than in control cells. Cytoplasts contained lower progesterone levels and more importantly, they could not be stimulated by 10 nM hCG or 10 mM dibutyryl cyclic AMP to produce more progesterone. Controls cells, on the other hand, contained higher progesterone levels and responded to hCG and dibutyryl cyclic AMP stimulation. In summary, removal of nuclei from luteal cells results in a partial loss of gonadotropin receptors and complete loss of steroidogenic response to hCG and dibutyryl cyclic AMP.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2006

The Nuclear Mitotic Apparatus (NuMA) protein is contributed by the donor cell nucleus in cloned porcine embryos.

The Nuclear Mitotic Apparatus (NuMA) protein is a multifunctional protein that is localized to the nucleus in interphase and to the poles of the mitotic apparatus during mitosis. In unfertilized porcine oocytes, NuMA is localized to the meiotic spindle. NuMA is removed along with the meiotic spindle during the enucleation process before reconstructing the egg by introducing the donor cell nucleus to produce cloned embryos. Questions have been raised regarding the source for NuMA in cloned embryos, as the enucleated oocyte does not contain detectable NuMA in the cytoplasm. To determine the source of NuMA in porcine nuclear transfer (NT) embryos, we conducted an immunofluorescence microscopy study with antibodies against NuMA to investigate the appearance and distribution of NuMA before and after reconstructing NT embryos with porcine skin fibroblasts. We used donor cells from a confluent culture with all cells in interphase. For comparative studies, we also determined the immunofluorescence pattern of NuMA, gamma-tubulin, and alpha-tubulin in porcine fibroblasts, parthenogenetic embryos and in vitro fertilized (IVF) embryos. Results show that NuMA was localized in nuclei of 33.5% (163/456) of the serum-deprived fibroblasts used as donor cells. No NuMA staining was detected in enucleated pig oocytes. Immediately after nuclear transfer, NuMA staining was absent in all donor cell fibroblast nuclei (0 h) but staining was detected by 6 h within the reconstructed eggs, at which time the transferred somatic cell nucleus swelled in most cells (19/27) and became a pronucleus-like structure. NuMA was localized exclusively within the pronucleus-like structures (15/27). At 25 h, NuMA was detected inside the nucleus (16/25) either in one-cell or in 2-cell stage embryos. Interestingly, in parthenogenetic embryos, NuMA staining was not detected in all 42 eggs examined at 1 h, and evident NuMA staining was only detected inside a few (4/51 at 6 h; 6/48 at 25 h) of the nuclei. In IVF embryos, NuMA was detected within the nucleus at 6 h (5/20) and 25 h (13/16). These results show that the donor cell nucleus contains NuMA that is contributed to the reconstructed embryo and possibly activated by mechanisms in the oocyte's cytoplast.

Active Transport, Cell Nucleus↗
PubMed1995

The paradox of Ferrata and the fate of the extruded red cell nucleus: two problems concerning erythropoiesis in the human.

A critical analysis of the present erythropoietic pathway reveals two problems which require resolving. One is the paradox of the basophilic erythroblast, and the other is the fate of the extruded red cell nucleus. The problems can be overcome if erythropoiesis is looked at differently, and the orthochromatic normoblast considered to arise directly from a denuded stem cell nucleus as has been previously suggested. The orthochromatic normoblast extrudes its nucleus leaving behind a reticulocyte. The extruded but functionally impaired nucleus of the orthochromatic normoblast then gives rise to a polychromatic normoblast, a defective cell. The poorly made cytoplasm of the polychromatic normoblast is shed and its nucleus, now non-functional, undergoes complete dissolution into an aggregate of ultrafine particles. The theory has the advantage that the fate of the extruded red cell nucleus can be explained without having to introduce phagocytosis by macrophages and all the immunological difficulties which this entails. The new pathway does away with the basophilic erythroblast as a haemoglobin-producing cell, and it is argued that the cell instead is a ferritin storage cell, and that erythropoiesis is the result of two separate but interdependent pathways, a ferritin storage pathway and a haemoglobin production pathway. Evidence is put forward to support the new pathways.

Biology↗
PubMed1976

Synaptic events and discharge patterns of cochlear nucleus cells. I. Steady-frequency tone bursts.

Unitary discharge patterns (peristimulus time histograms or PSTH) and synaptic events were studies with intracellular recording techniques in 164 cat cochlear nucleus cells to steady-frequency tone bursts 250 ms in duration. There were four response types defined on the basis of the shape of the discharge patterns to tones at the characteristic or best frequency. Primarylike units resemble eighth nerve fibres and have a maximum discharge at tone onset, followed by a smooth decline to a steady level of activity. Buildup units have a transient response at tone onset, followed a period of little or not activity before gradually increasing their discharge rate for the remainder of the tone burst. Onset units have an initial burst of spikes at the onset, with little or no activity for the remainder of the tone burst. Pause units have a long latency (10-30 ms) between tone onset and the appearance of low levels of unit activity, which then gradually increase in rate for the remainder of the tone burst. Changes in signal frequency or intensity within the excitatory response area did not modify response patterns of primarylike and onset units, but could evoke primarylike patterns in buildup and pause units. Inhibition manifested by suppression of spontaneous activity and membrane hyperpolarization were of three kinds: 1) in response to signals at the edges of the excitatory response area (i.e., the inhibitory surround) and detected in onset buildup, and pause units but not in primarylike units; 2) occurring at the offset of tones in the excitatory response area and detected in all four types of cochlear nucleus cells; 3) during excitatory tone bursts in onset and buildup units associated with the periods of suppressed unit activity. Membrane hyperpolarization did not accompany the delay in unit activity after tone onset in pause units. Inhibitory events in cochlear nucleus cells provide mechanisms for producing diversity in the temporal pattern of discharges to acoustic signals which may underly the encoding of complex features of sounds.

Acoustic Stimulation↗
PubMed2005

Proteasomes degrade proteins in focal subdomains of the human cell nucleus.

The ubiquitin proteasome system plays a fundamental role in the regulation of cellular processes by degradation of endogenous proteins. Proteasomes are localized in both, the cytoplasm and the cell nucleus, however, little is known about nuclear proteolysis. Here, fluorogenic precursor substrates enabled detection of proteasomal activity in nucleoplasmic cell fractions (turnover 0.0541 microM/minute) and nuclei of living cells (turnover 0.0472 microM/minute). By contrast, cell fractions of nucleoli or nuclear envelopes did not contain proteasomal activity. Microinjection of ectopic fluorogenic protein DQ-ovalbumin revealed that proteasomal protein degradation occurs in distinct nucleoplasmic foci, which partially overlap with signature proteins of subnuclear domains, such as splicing speckles or promyelocytic leukemia bodies, ubiquitin, nucleoplasmic proteasomes and RNA polymerase II. Our results establish proteasomal proteolysis as an intrinsic function of the cell nucleus.

Animals↗
PubMed2002

Genomes, proteomes, and dynamic networks in the cell nucleus.

Post-genome era experimental strategies seek to understand cellular pathways globally rather than through examination of individual components. Genomics and proteomics provide the experimental tools to establish the framework of gene and protein pathways present in a cell. These methods are complemented by emerging in vivo microscopy approaches, which permit placement of pathways within the architectural context of the cell. Analysis of dynamic live cell microscopy data combined with computational analyses finally allows the quantitative, mechanistic description of protein properties and pathways operating in living cells. Here we discuss how genomics and proteomics are changing the study of the cell nucleus and how in vivo microscopy methods have contributed to our changing conceptual and mechanistic understanding of nuclear architecture and function. Mapping of gene loci and genome regions are beginning to reveal organizational principles of the genome within the cell nucleus, proteomic analysis of subnuclear compartments and the gene expression machinery is providing insights into the molecular nature of nuclear events, and in vivo microscopy is illuminating the dynamic nature of nuclear organization. Initial findings from these efforts make clear that nuclear and cellular behavior can not be described by linear pathways. New tools such as computational modeling are providing evidence that emerging concepts such as network organizations and stochastic interactions are crucially important for cellular function and organization. These insights are changing our view of the nucleus and the cell as a whole.

Animals↗