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U De Boni

Publications and source records attributed to U De Boni.

At least 37 records · Page 2Linked to original sources
PubMed1992

Rearrangement of centromeric satellite DNA in hippocampal neurons exhibiting long-term potentiation.

In situ hybridization in conjunction with three-dimensional reconstruction was used to examine the topology of satellite DNA (sDNA) sequences in hippocampal CA1 neurons. In slices fixed immediately after preparation, 4-5 signals/nucleus were detected in CA1, CA3 and dentate neurons. 70-80% of 154 neurons examined in these 3 areas displayed all signals at the nuclear periphery. In the remaining fraction of neurons, sDNA signals were divided between the nucleolus and the nuclear periphery. sDNA signals were consistently localized to the nuclear midplane. Slices left to equilibrate in artificial cerebral spinal fluid for 1 h, in the absence of potentiation, exhibited a significant increase in the total number of signals/nucleus in CA1 and dentate neurons. This increase in the number of signals occurred in both nucleolar and peripheral compartments, with the number of the nucleolar compartment nearly doubling. The total number of signals/nucleus was found to be consistently reduced in tetanized CA1 neurons (4.89 +/- 0.09 signals/nucleus, n = 195, P less than 0.05) as compared to neurons from unpotentiated slices (5.27 +/- 0.10 signals/nucleus, n = 81). A similar decrease in the total number of signals/nucleus was also observed in CA1 neurons exposed to N-methyl-D-aspartate (NMDA), from 5.27 +/- 0.10 signals/nucleus (n = 81) to 5.00 +/- 0.08 signals/nucleus (n = 215, P less than 0.05). In contrast, dentate neurons, employed as internal controls, did not exhibit any change in number and compartmentalization of sDNA signals.(ABSTRACT TRUNCATED AT 250 WORDS)

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1991

Chromatin motion in neuronal interphase nuclei: changes induced by disruption of intermediate filaments.

Motion of nucleoli within interphase nuclei, known as nuclear rotation, may be used as a measure of motion of chromatin domains within the global confines of the nucleus. Mechanisms by which chromatin domains are transposed remain enigmatic. It has been established that nuclei are anchored by a network of intermediate filaments, structural proteins which share epitopes with nuclear lamins and possibly representing a constraint on nuclear rotation. It is postulated that selective removal of this constraint, by acrylamide, would result in increased chromatin motion. Mean rates of nucleolar displacement were quantified in neurons, in vitro. Nuclear rotation increased from a mean control rate of 0.102 +/- 0.002 micron/min (n = 52) to a maximum mean rate of 0.207 +/- 0.026 micron/min (n = 11), after 23 hr of exposure to 4 mM acrylamide. Despite this significant increase in motion of intranuclear domains, cytoplasmic structures in the immediate juxtanuclear area did not exhibit increases in rates of motion. Immunocytochemistry was used to visualize cytoskeletal structures and to assay selective disruption of neurofilaments by acrylamide. Increased rates of chromatin motion coincided with breakdown of the intermediate filament network. Ultrastructural analyses showed that the increase in chromatin motion induced by acrylamide was also associated with a significant (P less than 0.005) change in the thickness of the nuclear lamina, decreasing from 20.9 +/- 5.10 nm (n = 159) in controls to 18.9 +/- 3.1 nm (n = 148), to 19.5 +/- 3.6 nm (n = 240) and to 16.1 +/- 4.4 nm (n = 103) at 4, 8 and 22 hr exposure, respectively. Moreover, the number of mitochondria per unit area changed significantly (P less than 0.0001) with exposure to acrylamide, increasing from 9.1 +/- 2.2 mitochondrial profiles in controls to 16.5 +/- 5.3 profiles after 22 hr exposure to acrylamide. Distribution of other cytoskeletal components, actin and microtubules, was not altered and does not appear to play a significant role in the observed increase in rates of nuclear rotation. We conclude that the removal of the damping effects on chromatin motion normally imposed by the nuclear lamina and by intermediate filaments results in increased chromatin motion.

Acrylamide↗
PubMed1991

Enhanced neuronal regeneration by retinoic acid of murine dorsal root ganglia and of fetal murine and human spinal cord in vitro.

This study demonstrates that retinoic acid (RA), an active metabolite of vitamin A, can act to enhance regeneration of neurites, at physiologic concentrations, in vitro. Explanted fragments of mouse dorsal root ganglia (DRG) and mouse and human spinal cord (SC) were maintained, in vitro, for periods up to 11 d. Murine DRG neurons were exposed to RA concentrations ranging from 100 microM to 1 nM, whereas neurons within murine and human SC explants were exposed to 10 microM to 10 nM RA. Results show that RA significantly (P less than 0.001) increases mean neurite length but not neurite number. Specifically, murine DRG neurons showed increases in mean neurite length of 30.7% with individual explants showing increases of up to 133.5%. Murine and human SC showed mean enhancements of 43.4 and 58.1%, respectively, but did so at lower concentrations of RA. The results indicate that RA may play a potentially critical role in neuronal regeneration.

Animals↗
PubMed1991

Dynamics of nucleolar fusion in neuronal interphase nuclei in vitro: association with nuclear rotation.

Nuclear rotation (NR) refers to the motion of chromatin domains in interphase nuclei of several cell types, including neurons, in vitro. It has been proposed that NR may function, during cellular differentiation, in the transposition of specific chromatin domains into the cytotypic chromosome pattern known to exist in interphase nuclei. It is controversial whether NR represents motion of nuclei in toto, including the nuclear envelope, or whether NR represents independent motion of subnuclear structures, relative to each other. Using nucleoli as markers of chromatin motion in dorsal root ganglion neurons in vitro, we now show that trajectories of individual nucleoli are spatially restricted to subnuclear domains. Nucleoli move at mean rates of 2.153 +/- 0.037 deg/min and exhibit periodic fluctuations in rate. Fast Fourier transform analyses show dominant frequencies ranging from 0.47 c/h to 2.91 c/h. The power spectra of periodic motion of 15 of 25 nucleoli monitored exhibit resonance which suggests that NR represents forced harmonic motion. Quantification of motion of nucleoli in differentiating, multinucleolate neurons showed that internucleolar distances may rapidly decrease, culminating in nucleolar fusion, and showed that nucleolar fusion was invariably associated with a transient increase in the rate of NR. These results indicate that nucleoli may move independently; that an association exists between rearrangement of chromatin domains and NR; and that NR, nucleolar fusion, and differentiation are linked.

Animals↗
PubMed1991

Arrangement of kinetochore proteins and satellite DNA in neuronal interphase nuclei: changes induced by gamma-aminobutyric acid (GABA).

Discrete chromatin domains occupy specific nuclear compartments in neuronal interphase nuclei. Nuclear rotation, defined as the motion of interphase chromatin domains, has been proposed to function in the placement of specific chromatin domains to nuclear compartments which are appropriate to the physiological state or the state of differentiation of the cell. Rates of this chromatin motion may be increased by agents, including gamma-amino butyric acid (GABA), which may cause changes in gene expression. To test whether GABA also causes a spatial rearrangement of specific chromatin domains, the three-dimensional distribution of kinetochores in nuclei of mouse dorsal root ganglion neurons was determined by immunofluorescence. In addition, centromeric satellite DNA sequences were localized by in situ hybridization using a biotinylated mouse satellite DNA probe followed by immunofluorescence. Kinetochores occurred in clusters, associated with nucleoli or in intermediate nucleoplasmic regions, between the nucleolus and the nuclear membrane. Clusters of satellite DNA sequences were found either associated with nucleoli or throughout the nucleoplasm. Strikingly, nucleoplasmic kinetochores consistently occupied a 5-microns distance from the nuclear center, representing 70% of the spherical nuclear radius. Exposure of neurons to GABA induced a significant reorganization of kinetochores which may represent movement of chromosomes to alternate nuclear compartments to accommodate a new transcriptional state.

Animals↗
PubMed1991

Dislocation of chromatin elements in prophase induced by diethylstilbestrol: a novel mechanism by which micronuclei can arise.

The in vitro micronucleus test with Syrian hamster embryo (SHE) cells assays the induction of micronuclei by chemical agents. Both chromosome fragments and lagging chromosomes can give rise to micronuclei. Nevertheless, only limited information is available on the ultrastructure of micronuclei and the mechanisms of their formation. Diethylstilbestrol (DES), a non-mutagenic carcinogen, as well as its analogue 3.3'-DES induce micronuclei in SHE cells. A comparison of the dose response of DES-induced micronucleus formation with the previously published ones for aneuploidy and transformation shows that all 3 run in parallel. Thus, a functional relationship between these endpoints, in the SHE system, may be implied. The present study is designed to address the formation of micronuclei using supravital UV microscopy, to test for the presence of defined chromosome domains within micronuclei using immunocytochemistry, and to define aspects of their ultrastructure by electron microscopy. Supravital UV microscopy showed that 3.3'-DES induces displacement of chromosomes/chromatids during prophase/anaphase and formation of micronuclei during cytokinesis. Immunocytochemistry revealed that micronuclei contain, at high frequencies, CREST antibody-reactive kinetochores, indicating the presence of whole chromosomes or centric fragments in these structures. Moreover, transmission electron microscopy showed that micronuclei exhibit ultrastructural details typical of interphase nuclei. Specifically, micronuclei exhibited morphological evidence of a nuclear lamina and segregation of karyoplasm into euchromatic and heterochromatic regions. All micronuclei examined were enclosed by a nuclear envelope of normal morphology and showed nuclear pore complexes. Together the findings provide evidence that DES interferes with the mitotic apparatus as early as prophase, resulting in the formation of micronuclei and, as a consequence, in the loss of chromatids or chromosomes.

4-Nitroquinoline-1-oxide↗
PubMed1990

Colloidal gold labeling of intracellular ligands in dorsal root sensory neurons, visualized by scanning electron microscopy.

We report a novel technique that combines high-resolution scanning electron microscopy (SEM) of intracellular structures with backscattered electron imaging (BEI) of colloidal gold-labeled intracellular ligands. Murine dorsal root ganglia were immersion-fixed, freeze-cleaved, labeled with gold complexes, and critical point-dried. Specimens were carbon-coated and viewed by BEI. They were then minimally sputter-coated with gold and previously identified cells relocated by secondary electron imaging (SEI). This permitted increased resolution of intracellular detail while gold particles remained detectable by BEI. Incubation with RNAse-gold and DNAse-gold complexes resulted in specific labeling of cytoplasm and nucleus, respectively. Immunolabeling of neurofilament (NF) and small nuclear ribonucleoproteins (snRNP) resulted in selective labeling of intracellular antigens. Nonspecific binding was abolished by use of 1% skin milk. Specifically, incubation with monoclonal anti-NF68 resulted in labeling of cytoplasm in 66% of neurons, notably of the large cells known to contain large amounts of NF. Satellite cells, which lack NF, showed low levels of background label. Human autoimmune anti-Sm serum recognizes snRNP particles, with the exception of the nucleolar U3 snRNP. Labeling with this serum resulted in specific labeling of 92% of nuclei, with only background labeling over nucleoli and cytoplasm. The results show that it is feasible to employ high-resolution SEM in conjunction with colloidal gold labeling to localize intracellular ligands in situ.

Animals↗
PubMed1989

Reversible toroidal compaction of DNA by aluminum.

Electron microscopy has been used to characterize the products of the reactions of aluminum with DNA under three different conditions, one of which, pH 5 and Al (III)/DNA(P) ratio of 0.4, has been previously shown to produce reversible interstrand crosslinking in double-stranded DNA molecules. Under this condition, aluminum produced macromolecular aggregates of DNA upon heating, with a distinctive ultrastructure reversible to double-stranded DNA after removal of the aluminum. These structures were toroidal in configuration and exhibited mean widths of 4.9 +/- 1.8 nm and were 18.6 +/- .4 nm in diameter with a toroidal internal diameter of 7.4 +/- 4.7 nm. Previous results have shown that these structures contain Al(III) - crosslinked DNA, the present data suggests that this intermolecular crosslinking is associated with the production of compacted structures.

Aluminum↗
PubMed1988

Modulation of nuclear rotation in neuronal interphase nuclei by nerve growth factor, by gamma-aminobutyric acid, and by changes in intracellular calcium.

Nuclear rotation (NR) is typically measured as motion of nucleoli within nuclei of cells in vitro. This occurs in cycling cells. However, its observation in neurons arrested in interphase indicates that mechanisms related to mitosis are not a prerequisite. We have recently shown that NR occurs in three dimensions within the nuclear space, that it occurs within the space delineated by the outer nuclear membrane and that it includes chromatin domains in addition to nucleoli and have postulated that this motion of chromatin domains is related to changes in gene expression. We now show that exposure of dorsal root, sensory neurons in vitro to nerve growth factor (NGF) or to gamma-aminobutyric acid (GABA), agents which alter gene expression, and to agents causing redistribution of calcium, such as EGTA and the calcium ionophore A23187, significantly alters NR. The NGF increased the mean rate of NR and did so at a time after exposure when activity of RNA polymerases have been shown to rise. Exposure to GABA resulted, within minutes, in shifts of the nucleolus within the three-dimensional space of the nucleus, associated in some neurons with significant, sigmoidal increases in the rate of NR. The calcium ionophore A23187 as well as chelation of extracellular calcium with EGTA similarly increased rates. Importantly, excess calcium, with EGTA remaining present, returned NR of all nucleoli to rates not different from controls. This indicates that the increase in NR seen with EGTA is specific to the chelation of calcium and not an nonspecific response to EGTA. It is difficult to link the action of agents which alter gene expression or transmembrane ion balance with changes in NR. Nevertheless, in support of our hypothesis, the results presented here show that agents known to alter gene expression, alter NR in a temporally coincident manner and that they do so, possibly, by calcium-dependent mechanisms.

Animals↗
PubMed1988

Chromatin and nuclear envelope of freeze-fractured, neuronal interphase nuclei, resolved by scanning electron microscopy.

High-resolution scanning electron microscopy (SEM) has previously been used to study intracellular detail, including chromatin. It has, however, been commonly carried out either on cellular subfractions or following extraction methods to visualize detail. In the work presented here, intracellular detail of neurons of the dorsal root was visualized in situ by viewing freeze-fracture faces obtained after hypotonic expansion. This procedure permits the detailed resolution, by SEM, of juxtanuclear and intranuclear detail to a degree impossible without hypotonic dispersal. In agreement with work previously reported, nuclear chromatin of these interphase cells presents largely as 30-nm fibers, with a next higher hierarchical structure imparted by swelling in magnesium chloride. Detailed analyses showed that particles as small as 10-nm nucleosomes comprising the 30-nm chromatin fiber could be resolved, with "end-on" views of such fibers showing 5 nucleosomes per helical turn of the fiber. Chromatin fibers positioned subjacent to nuclear pores, or associated with "nuclear spaces" communicating with nuclear pores, were frequently found to be resolved as clusters, in an apparently more decondensed conformation, rather than tightly coiled into the 30-nm fiber. In addition, details of the nuclear envelope, including nuclear pores and perinuclear filaments as well as membranes of the endoplasmic reticulum, decorated with ribosomes, were clearly resolved.

Animals↗
PubMed1986

Curvilinear, three-dimensional motion of chromatin domains and nucleoli in neuronal interphase nuclei.

The term "nuclear rotation" refers to a motion of nucleoli within interphase nuclei of several cell types. No mechanism or function has been ascribed to this phenomenon, and it was unknown whether nuclear structures in addition to nucleoli participate in this motion. Moreover, it was unclear whether nuclear rotation occurs independent of concurrent motion of juxtanuclear cytoplasm. The work reported here presents quantitative evidence, for three-dimensional intranuclear, tandem motion of fluorescently labeled chromatin domains associated with nucleoli and those remote from nucleoli. The results show that such motion is curvilinear, that it is not restricted to nucleoli, and, moreover, that it occurs independently of motion of juxtanuclear, cytoplasmic structures. These results suggest that this motion represents karyoplasmic streaming and its function is to transpose to nuclear pores those chromatin domains actively transcribed.

Cell Nucleolus↗
PubMed1985

Calcium metabolism in aluminum encephalopathy.

Electrophysiologic deficits occurred in the in vitro hippocampal slice preparation removed from rabbits at various stages of an aluminum-induced encephalopathy. These deficits were associated with a progressive increase in total tissue calcium content. The deficits were reversed, in part, by increasing extracellular calcium concentration. Whereas calmodulin and calcium-binding protein, as measured by radioimmune assay, did not change in amount, the activity of calmodulin, as a calcium-calmodulin activator of the enzyme 3',5'-cyclic nucleotide phosphodiesterase, declined progressively as the aluminum encephalopathy developed. Cholinergic neurochemical transmission, measured by choline acetyltransferase activity and muscarinic binding, did not change in the encephalopathy. We postulate that aluminum alters brain calcium homeostasis, perhaps through an effect on calmodulin.

Aluminum↗
PubMed1985

Controlled induction of paired helical filaments of the Alzheimer type in cultured human neurons, by glutamate and aspartate.

One characteristic histopathological feature of brains of patients with Senile Dementia of the Alzheimer Type (SDAT) is a neuronal change known as neurofibrillary degeneration. Ultrastructurally, this degeneration presents as aggregates of Paired Helical Filaments (PHFs). We have reported (De Boni and Crapper 1978) that brain affected with SDAT contains a factor which induces PHFs similar to those occurring in Alzheimer's disease, in human CNS neurons in vitro. We now report that PHFs may be induced in cultured, fetal human spinal cord lesions by factors other than extracts from brain affected by SDAT. Specifically, ultrastructural analyses have shown that PHFs closely similar to those found in SDAT are induced by the excitotoxic aminoacids, glutamate (Glu) and aspartate (Asp). PHFs were found with Glu and Asp in combination at 1.1 and 0.45 mM, respectively and by Glu alone at 2.2 mM, when added to the culture medium. Cultures grown and maintained in MEM Eagle medium without Glu and Asp were invariably composed of tightly packed neuropil with abundant synaptic contacts, were free of vacuolated somata or processes and did not exhibit the presence of PHFs. In contrast, cultures exposed to Glu and Asp invariably responded with the presence of vacuolated neuronal somata and degenerating neuronal processes containing intermediate filaments, frequently paired into PHFs. These induced PHFs are composed of intermediate filaments morphologically identical (diameter 10 +/- 0.9 nm, n = 55) to neurofilaments. While the induced PHFs exhibit a range of periodicities (50-540 nm) with an overall mean period of 140 +/- 68 nm (n = 245), approximately 4% are morphologically closely similar (mean period 76 +/- 10 nm, n = 10) to those in SDAT.

Alzheimer Disease↗
PubMed1984

Chromatin structure in dementia.

Nuclei extracted from neocortex of patients with Alzheimer's disease and treated with micrococcal nuclease release a population of dinucleosomes that contain an increase in the linker histones H1o and H1oo . Five other degenerative brain diseases that clinically resemble Alzheimer's disease do not result in these changes, although Pick's disease is associated with an increase in H1 on dinucleosomes. Histones from nuclei of patients with Alzheimer's disease are also more resistant to salt-induced release from chromatin than are those from age-matched control subjects. These results support the hypothesis that an alteration in chromatin structure is a marker for Alzheimer's disease.

Aged↗
PubMed1984

Organotypic development of neonate rabbit hippocampus in roller tube culture.

Maintenance of organotypic cultures of hippocampus derived from neonate rabbit has not been previously reported. The study described here was undertaken to define the conditions most suitable for organotypic development, in vitro, of this structure. Slices of hippocampus, on flying cover-slips, were maintained on plasma clots in roller tubes, for periods of up to 6 weeks. The results showed that the explanted, immature hippocampus developed in a manner which parallels the in vivo development, previously described. Specifically, pronounced neuronal differentiation was noted as the cultures matured. There is evidence that the hippocampus of rabbit, in vivo, at 3 weeks of age has assumed the mature pattern of neuronal and synaptic differentiation. Such differentiation similarly occurred in the cultured hippocampus described in this study. This system would serve as an ideal tool in applications in experimental neuropathology, where the use of a model of a phylogenetically advanced central nervous system is preferred.

Animals↗