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PubMed2000

Defined nuclear changes accompany the reprogramming of the microspore to embryogenesis.

The switch of the gametophytic developmental program toward pollen embryogenesis to form a haploid plant represents an important alternative for plant breeding. In the present study, the switch of the gametophytic developmental program toward a sporophytic pathway, "embryogenesis," has been studied in three different plant species, Brassica, tobacco, and pepper. The switch has been induced by stress (heat shock) at the very responsive stage of the microspore, which is the vacuolate period. As a result, the cell nucleus undergoes striking structural changes with regard to late gametophytic development, including alterations of biosynthetic activities and proliferative activity. An enrichment in HSP70 heat-shock protein and in the presence of Ntf6-MAP kinase was observed after inductive treatment in the nuclei during early embryogenesis. This apparently reflected the possible roles of these proteins, specifically the protective role of HSP70 for the nuclear machinery, and signal transduction of Ntf6-MAPK for the entry of cells into proliferation. Importantly, the observed nuclear changes were similar in the three species investigated and represented convenient markers for early monitoring of embryogenesis and selection purposes for obtaining double-haploid plants in plant breeding.

Brassica↗
PubMed1995

Evolution of the cell theory.

The problem of the nature of life has a long history going back to the Greeks. There was little real progress until the 19th century and Aristotle may have been at home with many 18th century ideas about vital forces and basic units. Although Hooke described cells in 1665 it took a further 200 years for the significance and nature of cells to be appreciated. In the mid 18th century some considered the basic building blocks of living matter to be fibrous. Globular theories, the precursor to the cell theory, were quite popular at the beginning of the 19th century. Many workers, as microscopes improved, had described various cell types and structures including the nucleus but the idea that cells were the universal units is associated with in 1838 and that of Schwann in 1839. However, Schwann mistakenly thought that cells could form de novo. Cell division was established by Remak and others in the 1850s. Mitosis was first understood by Flemming in 1882. The existence of the animal cell membrane was only established by the beautiful experiments of Overton in 1895. The history of the cell theory can be used to show that progress can be based on incorrect but productive ideas. It is one of the most important ideas in all of biology.

Animals↗
PubMed1986

Altered structures in the cytoplasm of the ependymal cells next to the periventricular nucleus of the turtle Mauremys caspica.

In the apical cytoplasm of some ependymal cells of the Hypothalamic Periventricular Nucleus of the turtle Mauremys caspica large amount of cell structures of difficult diagnose are found. Their morphology is variable seeming to correspond to cell organelles in a process of degeneration, characteristic feature of the high metabolic activity in the ventricular barrier. Their possible physiological significance is discussed in the present work.

Actin Cytoskeleton↗
PubMed1997

Accessibility of nuclear DNA to triplex-forming oligonucleotides: the integrated HIV-1 provirus as a target.

The control of gene transcription by antigene oligonucleotides rests upon the specific recognition of double-helical DNA by triplex-forming oligonucleotides. The development of the antigene strategy requires access to the targeted DNA sequence within the chromatin structure of the cell nucleus. In this sudy we have used HIV-1 chronically infected cells containing the HIV provirus as endogenous genes to demonstrate that the integrated HIV-1 proviral genome is accessible to triplex-forming oligonucleotides within cell nuclei. An oligonucleotide-psoralen conjugate targeted to the polypurine tract (PPT) of the HIV-1 proviral sequence was used as a tool to convert the noncovalent triple-helical complex into a covalent lesion on genomic DNA after UV irradiation of cells. Triplex-derived adducts were analyzed using two different methods. The photo-induced psoralen cross-link prevented cleavage of the target sequence by DraI restriction endonuclease, and the sequence-specific inhibition of cleavage was revealed and quantitated by Southern blot analysis. A quantitative analysis of cross-linking efficiency was also carried out by a competitive PCR-based assay. These two approaches allowed us to demonstrate that a triplex-forming oligonucleotide can recognize and bind specifically to a 15-bp sequence within the chromatin structure of cell nuclei.

Cell Membrane Permeability↗
PubMed1999

Calcium-mediated structural changes of native nuclear pore complexes monitored by time-lapse atomic force microscopy.

Nuclear pore complexes (NPCs) are large macromolecular assemblies embedded in the double membrane nuclear envelope. They are the major gateways mediating transport of ions, small molecules, proteins, RNAs, and ribonucleoprotein particles in and out of the nucleus in interphase cells. Understanding structural changes at the level of individual pores will be a prerequisite to eventually correlate the molecular architecture of the NPC with its distinct functional states during nucleocytoplasmic transport. Toward this goal, we have employed time-lapse atomic force microscopy of native NPCs kept in buffer, and recorded calcium-mediated structural changes such as the opening (i.e. +Ca2+) and closing (i.e. -Ca2+) of individual nuclear baskets. Most likely, this structural change of the nuclear basket involves its distal ring which may act as an iris-like diaphragm. In order to directly correlate distinct structural features with corresponding functional states and dynamic aspects, we also addressed the question of whether the "central plug" or "transporter" actually represents a calcium-sensitive component of the NPC involved in mediating nucleocytoplasmic transport. Our data indicate that in the absence of ATP, cytoplasmic plugging/unplugging of the NPC is insensitive to calcium.

Animals↗
PubMed2001

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

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

Amanitins↗
PubMed2000

Anomalous diffusion of fluorescent probes inside living cell nuclei investigated by spatially-resolved fluorescence correlation spectroscopy.

We have investigated spatial variations of the diffusion behavior of the green fluorescent protein mutant EGFP (F64L/S65T) and of the EGFP-beta-galactosidase fusion protein in living cells with fluorescence correlation spectroscopy. Our fluorescence correlation spectroscopy device, in connection with a precision x-y translation stage, provides submicron spatial resolution and a detection volume smaller than a femtoliter. The fluorescence fluctuations in cell lines expressing EGFP are caused by molecular diffusion as well as a possible internal and a pH-dependent external protonation process of the EGFP chromophore. The latter processes result in two apparent nonfluorescent states that have to be taken into account when evaluating the fluorescence correlation spectroscopy data. The diffusional contribution deviates from ideal behavior and depends on the position in the cell. The fluorescence correlation spectroscopy data can either be evaluated as a two component model with one fraction of the molecules undergoing free Brownian motion with a diffusion coefficient approximately five times smaller than in aqueous solution, and another fraction diffusing one or two orders of magnitude slower. This latter component is especially noticeable in the nuclei. Alternatively, we can fit the data to an anomalous diffusion model where the time dependence of the diffusion serves as a measure for the degree of obstruction, which is large especially in nuclei. Possible mechanisms for this long tail behavior include corralling, immobile obstacles, and binding with a broad distribution of binding affinities. The results are consistent with recent numerical models of the chromosome territory structure in the cell nucleus.

Animals↗
PubMed1981

Immunocytochemical localization of intermediate filament proteins during lymphocyte capping.

Using double immuno-fluorescence techniques on frozen-thick sections, we have examined the fate of intermediate filaments during Con A receptor capping in lymphoid cells. Our results indicate that during capping intermediate filaments are preferentially aggregated between the surface receptor cap structure and the cell nucleus. It is possible, therefore, that intermediate filaments are directly involved in lymphocyte capping.

Animals↗
PubMed2004

Microchemical element imaging of yeast and human cells using synchrotron X-ray microprobe with Kirkpatrick-Baez optics.

Trace element imaging and speciation analysis in cells and subcellular compartments is a challenging and important objective for modern analytical chemistry in order to better understand the biological chemistry of essential and toxic elements. A focusing system based on Kirkpatrick-Baez design optics mounted on a synchrotron radiation scanning X-ray microscope has been developed at the ESRF and was used for trace element quantitative imaging in single cells. The focused microbeam (1.3 x 3.2 microm(2)) obtained in that way led to a photon flux as bright as 1.5 x 10(11) photons/s at 14 keV. The absolute detection limit of this analytical probe, as measured on standard reference materials, was shown to be 2 x 10(-)(17) g for most elements. Chemical maps of human carcinoma and of Saccharomyces cerevisiae cells were obtained for minor (P, S, Cl, K) and trace elements (Fe, Zn). Within human cancer cells, chemical elements are homogeneously distributed at the current spatial resolution and correlated with the sample's mass, except Fe, which shows micrometer-sized structures around the cell nucleus, and Zn, which slightly concentrates in the nucleus, while chemical maps of S. cerevisiae show homogeneous pattern distribution at the cellular level.

Cell Line, Tumor↗
PubMed1999

Ultrastructural characterization of isolated rat Kupffer cells by transmission X-ray microscopy.

The ultrastructure of primary cultured rat Kupffer cells was studied using transmission X-ray microscopy as well as transmission electron microscopy. X-ray microscopical images of intact, hydrated Kupffer cells demonstrated structures such as cell nucleus separated by a nuclear membrane and filaments concentrated in the perinuclear area. Within the cytoplasm, a number of vacuoles were visible; some of these were crescent-shaped vacuoles that were half X-ray lucent, half X-ray dense; others were uniformly dense. The number of crescent-shaped vacuoles was predominant. After phagocytosis of haematite particles, enlarged vacuoles containing the ingested material were visible within the cytoplasm of Kupffer cells while crescent-shaped vacuoles were no longer detectable. Densitometric analysis of the two types of vacuole revealed that the X-ray absorption of the uniform vacuole was approximately half that of the dense part of the crescent-shaped vacuoles. This observation led to speculation on the existence of only one type of vacuole in the cytoplasm of Kupffer cells. The different morphological aspects--crescent-shaped versus uniform vacuoles--might be due to different three-dimensional orientation with respect to the image plane. Using transmission electron microscopy, the morphology of vacuoles differed more widely in diameter, density and shape. Two main types of vacuole were identified: electron-lucent and electron-dense. Based on the observation of only one type of vacuole by transmission X-ray microscopy, the different morphological aspects of vacuoles obtained by transmission electron microscopy could be explained by imaging several different sections of a crescent-shaped vacuole. From the present data it can be concluded that transmission X-ray microscopy is a versatile technique that reveals the ultrastructure of intact, unsectioned biological specimens in their aqueous environment, thereby allowing a more comprehensive interpretation of data obtained by transmission electron microscopy.

Animals↗
PubMed2003

Nuclear interaction of the dynein light chain LC8a with the TRPS1 transcription factor suppresses the transcriptional repression activity of TRPS1.

The TRPS1 gene codes for a 1281 amino acids nuclear transcription factor with an unusual combination of different types of zinc finger motifs, including GATA-type DNA-binding and IKAROS-like zinc fingers. TRPS1 is a repressor of GATA-regulated genes and implicated in the human tricho-rhino-phalangeal syndromes. We found that two distinct regions of TRPS1 can physically interact with the dynein light chain 8 protein, LC8a, that are at least 458 amino acids apart from each other. Region A covers 89 amino acids (635-723), spanning three potential C(2)H(2) zinc finger structures, and region B covers the 100 most C-terminal amino acids (1182-1281) containing the IKAROS-like motif. LC8a is known to interact with more than 10 different molecules, both proteins and nucleic acids. In most cases, LC8a was identified as a transport molecule in the cytoplasm. Interestingly, we found that LC8a co-localizes with TRPS1 in dot-like structures in the cell nucleus. In an electrophoretic mobility shift assay we could show that the interaction of LC8a and TRPS1 lowers the binding of TRPS1 to the GATA consensus sequence. In addition, GATA-regulated reporter gene assay indicated that LC8a is able to suppress the transcriptional repression activity of TRPS1.

Amino Acid Sequence↗
PubMedDate not supplied

[Morphological changes in the structure of liver cell nuclei in rats with PP anitaminosis following injection of nicotinic acid].

The injection of the surplus quantities of nicotinic acid (150 mg/kg of body weight) to PP-deficient rats causes morphological changes in the structure of hepatocyte nuclei 3-6 h after the injection. These changes reach maximum by 9h, and the liver returns to its normal state by 24 h after administration. This time coincides with that of intensified new-formation of nicotinamide coenzymes (9h) and normalization of NAD content 24h in the rat liver after injection of the mentioned dose of nicotinic acid that evidence for a definite linkage of the biosynthesis processes with structures of the cell nucleus.

Animals↗
PubMed1979

Inhibition of thymidine incorporation into epidermal and dermal DNA of HRS/J mice after a Colcemid pulse.

The introduction of a Colcemid pulse, which is known to inhibit microtubulin assembly and thereby influence cellular events dependent on the assembly, delayed the normal passage of G1 cells to S-phase. Experimental support for this hypothesis was obtained by the pulsing of cultured inbred HRS/J mouse skin for 30 minutes with 20 micrograms Colcemid/ml and by measurement of thymidine (dThd) incorporation for a 4-hour incubation period at different intervals after the pulse. Incorporation of dThd into epidermal and dermal DNA was maximally inhibited 9.5 and 17.5 hours after the Colcemid pulse. Maximal inhibition and minimal inhibition of dThd incorporation were observed 17.5 hours after different concentrations of Colcemid (1--30 micrograms/ml) were pulsed. Maximal inhibition of dThd incorporation occurred after pulsing with 20 micrograms Colcemid/ml. The acceleration of G0 or G1 events to S-phase in proliferative cells caused by mild epidermal stripping was also inhibited after the addition of the Colcemid pulse. The data suggest a time relationship between the Colcemid pulse, the subsequent disruption of microtubulin structure, and the inhibition of dThd incorporation into epidermal and dermal DNA. The intact microtubulin structure is apparently essential for the normal intracellular dependency among the cell membrane, the cytoskeleton structure, and the cell nucleus and occurs when the cells transit from G1 to S-phase.

Animals↗
PubMed1985

[Brain tumor induced in dogs by intracerebral inoculation of SR-RSV induced cultured tumor cells--electron microscopic study].

An experimental transplantable canine brain tumor model with the advantages of rapid tumor growth within 10 days and relative safety for the investigator is presently available. The tumor is produced by intracerebral inoculation of cultured cells derived from a canine brain tumor induced by the Schmidt-Ruppin strain of the Rous-Sarcoma virus (SR-RSV). It has potential use as a model in experiments designed to evaluate the effectiveness of chemotherapy and radiotherapy with serial computerized tomography scans. However, characterization of the induced tumor is essential. Ideally, it should have features attributable to glioma and/or neuroectodermal tumors. Utilizing the technique of intracerebral inoculation of cells cultured from the original dog brain tumor induced by SR-RSV, Salcman et al identified the tumor they induced in brains of mongrel puppies as a glioma by light microscopic criteria (Reference). The purpose of our study was to further characterize this experimental tumor by electron microscopic and immunohistochemical techniques. Tumor was induced in 6 mongrel puppies. Stains of the tumor for immunohistochemical reactivity to glial fibrillary acid protein, S-100 protein and 210K neurofilament protein were all negative. With the electron microscope, the intracerebral tumor cells were mostly undifferentiated. They had a few cell processes, occasional punctate adhesions and some microvilli-like structure. The tumor cell nucleus was usually oval shaped and sometimes had nuclear indentations. The cytoplasm contained abundant free ribosomes, some rough endoplasmic reticulum and mitochondria. Collagen fibers and basal lamina were not observed in the intercellular spaces. The capillaries within the tumor were characterized by proliferation of immature endothelial cells which were non-fenestrated.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Low-LET and high-LET radiation action of 125I decays in DNA: effect of cysteamine on micronucleus formation and cell killing.

Chinese hamster ovary cells were pulse-labeled with 125I-iododeoxyuridine during early S phase, and cell samples were harvested 30 min or 5 h after labeling. The samples were frozen (with or without 25 mM cysteamine) and stored at -196 degrees C for accumulation of 125I decays. X-ray control experiments were performed at 37 degrees C and -196 degrees C. Aliquots of cells were plated for evaluating micronucleus formation and cell survival. The results demonstrated a striking shift in micronucleus formation and cell death with time after labeling. Cells frozen 30 min after labeling exhibited effects typical of low-LET radiation, but cells frozen 5 h after labeling showed a response characteristic of high-LET radiation. Cysteamine provided protection against the effects of 125I during the initial phase of effects characteristic of low-LET radiation, but no protection was seen during the phase characteristic of high-LET radiation. When cell survival was evaluated as a function of micronucleus frequency rather than dose in decays/cell, the survival curves for all treatment groups became superimposed. Previous work using the same experimental system had failed to show a direct link between 125I-induced DNA double-strand breaks and cell death. These findings are consistent with the hypothesis that DNA damage may not be the sole mechanism for cell killing and that damage to higher-order structures in the cell nucleus may contribute to (or modify) radiation-induced cell death.

Animals↗
PubMed1993

Three monoclonal antibodies differentiate human from murine epidermis.

In order to establish a set of epidermal species markers, normal human skin, murine skin and human skin transplanted to nude mice were stained with monoclonal antibodies directed to cell membrane-bound carbohydrates, a basement membrane component and a structure in the cell nucleus. Three epidermal species markers were identified. Two markers stained exclusively human epidermis: LH7.2 detects type VII collagen and stained the basement membrane of human epidermis in unfixed frozen sections, while LP4N stained cell nuclei in human epidermis in methanol-acetone-fixed frozen sections. The third marker, HH14, stained exclusively murine epidermis. HH14 defines the histo-blood group H carbohydrate antigen and stained spinous cell membranes of murine epidermis in both frozen and formalin-fixed sections.

Animals↗
PubMed2005

Nuclear lamins: building blocks of nuclear structure and function.

The cell nucleus is surrounded by a complex membranous envelope which separates the nucleoplasm from the cytoplasm. Unlike the cytoplasm, the nucleoplasm is not subdivided into membrane-bound compartments, which allows for the efficient segregation of a wide range of complex metabolic activities. In the absence of such membrane compartmentalization, the nucleus is faced with the daunting task of efficiently segregating and interconnecting an enormous array of critically important functions. These include the assembly of the large multi-component complexes or 'factories' involved in DNA replication and transcription. These structures are dynamic as they are assembled and disassembled both spatially and temporally at different times, implying the existence of an infrastructure or nucleoskeleton responsible for establishing and maintaining a complex nuclear architecture. There is increasing evidence that the nuclear lamins are essential elements of this nuclear infrastructure, and that their proper assembly and organization are required for numerous essential nuclear functions. Our goal has been to determine the roles of the nuclear lamins in vital nuclear processes including DNA replication and transcription. The hypothesis directing our investigations is that the lamins form a 3D network that courses throughout the nucleoplasm providing an infrastructure for the assembly and distribution of numerous multicomponent complexes involved in a wide range of nuclear functions.

Animals↗
PubMed1992

A behavioral study of the contributions of cells and fibers of passage in the red nucleus of the rat to postural righting, skilled movements, and learning.

Although the red nucleus consists of cells of origin for the rubro-spinal and rubro-olivary tracts, fibers of passage, including those of the superior cerebellar peduncle, which project from the cerebellum to the ventrolateral thalamus, pass through it. This study examined the relative effect of cell vs. fiber damage in the red nucleus on a number of behaviors thought to involve the red nucleus, including a skilled movement of reaching for food with a forelimb, postural righting on a surface and in the air, and learning a place response in a swimming pool test. Rats received unilateral or bilateral red nucleus lesions, using either the relatively cell-specific neurotoxins, ibotenic and quinolinic acid, or non-specific electrolytic anodal lesions. Both neurotoxic lesions effectively eliminated all red nucleus cell bodies, and in some animals they produced small cavities in the red nucleus and/or loss of cells in adjacent structures. Electrolytic lesions destroyed both cells and fibers, leaving a large cavity. The severity of the behavioral deficits were not related to the loss of red nucleus cells and there was a close relation between fiber damage and behavioral impairments on all of the tasks. The results suggest that for a number of behaviors, which have been thought to involve the red nucleus, impairments are more closely associated with fiber damage or damage to structures outside the red nucleus than they are to damage to cells of the red nucleus.

Animals↗