scholarly journals Ribonucleoprotein components in liver cell nuclei as visualized by cryoultramicrotomy.

1975 ◽  
Vol 67 (1) ◽  
pp. 200-214 ◽  
Author(s):  
E Puvion ◽  
W Bernhard
Keyword(s):  
Cell Nuclei ◽  
Glutaraldehyde Fixation ◽  
Rat Hepatocyte ◽  
Colloidal Iron ◽  
Dna Structures ◽  
Normal Rat ◽  
Ultrathin Frozen Sections ◽  
Interchromatin Granules ◽  
Perichromatin Granules ◽  
Perichromatin Fibrils

The interphase nucleus of the normal rat hepatocyte has been studied in ultrathin frozen sections after glutaraldehyde fixation and the modification of various staining procedures known to be specific for DNA structures (Moyne's thallium stain, Gautier's osmium-ammine) or preferential for RNP carriers and basic proteins (regressive stains based on the use of EDTA or citrate, negatively charged colloidal iron). The results are comparable to those obtained after classical dehydration and embedding. Particular attention has been paid to the nucleolus and extranucleolar RNP components, such as perichromatin fibrils and granules, as well as interchromatin granules. A striking observation was the uneven size and the strongly increased number of perichromatin granules, and the appearance of a contiguous interchromatin net, containing nucleoproteins. Cryoultramicrotomy without embedding appears to be very useful for the exploration of the nucleus in thick sections which remain sufficiently transparent even with the usual accelerating voltages.

Use of human autoantibodies and immunoelectron microscopy to study structure and function of the nucleolus and nuclear bodies

1992 ◽  
Vol 50 (1) ◽  
pp. 506-507
Author(s):  
Robert L. Ochs
Keyword(s):  
Electron Microscopy ◽  
Structure And Function ◽  
Nuclear Bodies ◽  
Nuclear Interior ◽  
Coiled Bodies ◽  
Interchromatin Granules ◽  
And Function ◽  
Conventional Electron Microscopy ◽  
Perichromatin Granules ◽  
Perichromatin Fibrils

By conventional electron microscopy, the formed elements of the nuclear interior include the nucleolus, chromatin, interchromatin granules, perichromatin granules, perichromatin fibrils, and various types of nuclear bodies (Figs. 1a-c). Of these structures, all have been reasonably well characterized structurally and functionally except for nuclear bodies. The most common types of nuclear bodies are simple nuclear bodies and coiled bodies (Figs. 1a,c). Since nuclear bodies are small in size (0.2-1.0 μm in diameter) and infrequent in number, they are often overlooked or simply not observed in any random thin section. The rat liver hepatocyte in Fig. 1b is a case in point. Historically, nuclear bodies are more prominent in hyperactive cells, they often occur in proximity to nucleoli (Fig. 1c), and sometimes they are observed to “bud off” from the nucleolar surface.

scholarly journals Nuclear RNA Is Extruded from Apoptotic Cells

1998 ◽  
Vol 46 (9) ◽  
pp. 999-1005 ◽  
Author(s):  
Marco Biggiogera ◽  
Maria Grazia Bottone ◽  
Carlo Pellicciari
Keyword(s):  
Appreciable Amount ◽  
Apoptotic Cells ◽  
Spontaneous Apoptosis ◽  
Systemic Autoimmune Diseases ◽  
Nucleolar Material ◽  
Nuclear Rna ◽  
Interchromatin Granules ◽  
Possible Cause ◽  
Perichromatin Granules ◽  
Perichromatin Fibrils

During spontaneous apoptosis of thymocytes there is extrusion of ribonucleoproteins (RNPs) from the cell. The aim of this investigation was to elucidate whether the RNP aggregates in apoptotic cells and bodies still contain RNA in an appreciable amount. We demonstrated by specific cytochemical techniques that the aggregates of nuclear RNPs extruded in the cytoplasm of spontaneously apoptotic thymocytes contain RNA in a sufficient amount to be detected cytochemically. These heterogeneous ectopic RNP-derived structures (HERDS) are formed by perichromatin fibrils, interchromatin granules, perichromatin granules, and nucleolar material. The RNA detected inside these clusters should therefore correspond to both mRNA and snRNA as well as to rRNA. We never observed DNA-contaning aggregates in the cytoplasm of apoptotic thymocytes. The presence of RNA in the HERDS that may be released from apoptotic cells suggests that the decrease in the amount of total RNA during apoptosis may be mostly linked to cellular extrusion rather than to degradation of RNA by RNase activities. Another interesting aspect of these results lies in the hypothesis of apoptosis as a possible cause for the presence of autoantibodies in the serum of patients with systemic autoimmune diseases.

scholarly journals Negative charge distribution and density on the surface of oxygenated normal and sickle red cells

Blood ◽  
1981 ◽  
Vol 57 (4) ◽  
pp. 675-678
Author(s):  
LJ Clark ◽  
LS Chan ◽  
DR Powars ◽  
RF Baker
Keyword(s):  
Charge Distribution ◽  
Electron Micrographs ◽  
Negative Charge ◽  
External Surface ◽  
Iron Hydroxide ◽  
Red Cells ◽  
Glutaraldehyde Fixation ◽  
Colloidal Iron ◽  
Membrane Area ◽  
Significant Difference

Negative charges on the external surface of red cells were visualized by colloidal iron hydroxide labelling of 50% of the membrane area after osmotic hemolysis and glutaraldehyde fixation. Counts were made over randomly selected areas on electron micrographs at 350,000 x magnification. Statistical analyses showed that at the 95% level of confidence there was no significant difference between oxygenated normal (AA) and sickle (SS) cells in either the distribution or the density of negative charges.

scholarly journals Ultrastructural distribution of nuclear ribonucleoproteins as visualized by immunocytochemistry on thin sections.

1984 ◽  
Vol 98 (1) ◽  
pp. 358-363 ◽  
Author(s):  
S Fakan ◽  
G Leser ◽  
T E Martin
Keyword(s):  
Protein A ◽  
Nuclear Architecture ◽  
Gold Complex ◽  
Thin Sections ◽  
Border Regions ◽  
Core Proteins ◽  
Interchromatin Granules ◽  
Lowicryl K4m ◽  
Perichromatin Fibrils

The ultrastructural distribution of nuclear ribonucleoproteins (RNP) has been investigated by incubation of thin sections of mouse or rat liver, embedded in Lowicryl K4M or prepared by cryoultramicrotomy, with antibodies specific for RNP. The antibodies were localized by means of a protein A-colloidal gold complex. Anti-small nuclear (sn)RNP antibodies, specific for determinants of the nucleoplasmic snRNP species containing U1, U2, U4, U5, and U6 RNAs, were found associated preferentially with perichromatin fibrils, interchromatin granules, and coiled bodies. This indicates an early association of snRNP with structural constituents containing newly synthesized heterogeneous nuclear RNA. It also suggests a possible structural role of some snRNPs in nuclear architecture. Antibodies against the core proteins of heterogeneous nuclear RNP particles associate preferentially with the border regions of condensed chromatin, and in particular with perichromatin fibrils and some perichromatin granules. These results are discussed in view of recent knowledge about the possible role of nucleoplasmic RNP-containing components in the functions of the cell nucleus.

scholarly journals Biosynthesis, surface expression and function of the fibronectin receptor after rat liver cell transformation to tumorigenicity

1993 ◽  
Vol 291 (1) ◽  
pp. 247-255 ◽  
Author(s):  
M Decastel ◽  
M A Doyennette-Moyne ◽  
E Gouet ◽  
M Aubery ◽  
P Codogno
Keyword(s):  
Molecular Mass ◽  
Hepatoma Cell ◽  
Rat Hepatocytes ◽  
Hepatoma Cells ◽  
Rat Hepatocyte ◽  
Fibronectin Receptor ◽  
Zajdela Hepatoma ◽  
Sds Page ◽  
Normal Rat ◽  
And Function

Zajdela hepatoma cells are poorly-adherent cells derived from an undifferentiated tumour and transplanted into rat. We compared the biosynthesis, structure and function of the fibronectin receptor in normal rat hepatocytes with that in Zajdela hepatoma cells. The rat hepatocyte fibronectin receptor has been isolated. It is composed of two subunits: alpha 5 (molecular mass 155 kDa) and beta 1 (molecular mass 115 kDa). However, its biosynthesis has not yet been described. Using polyclonal antibodies raised against each of the subunits of the receptor, we observed that the alpha 5-subunit was synthesized as a 155-kDa polypeptide in normal rat hepatocytes and Zajdela hepatoma cells. In contrast, the molecular mass of the beta 1-subunit was 130 kDa in Zajdela hepatoma cells versus 115 kDa in normal rat hepatocytes. Pulse-chase experiments showed that the apparent transition time from the 100-kDa beta 1-precursor to the 130-kDa mature form was abnormally prolonged in Zajdela hepatoma cells since the latter was not detected until 24 h, while the transition from the 100-kDa precursor to the 115-kDa mature form began within 3 h in normal rat hepatocytes. Digestion of both the normal rat hepatocytes and Zajdela hepatoma cells 100-kDa beta 1-precursors with endo-beta-N-acetylglucosaminidase H and peptide N-glycosidase yielded products from 100 kDa to 84 kDa and 82 kDa, respectively, as judged by SDS/PAGE, suggesting that the same polypeptide chain is synthesized in normal rat hepatocytes and in Zajdela hepatoma cells. Incubation of the mature normal rat hepatocyte beta 1-subunit with peptide N-glycosidase reduced its molecular mass from 115 kDa to 82 kDa, as judged by SDS/PAGE, while the molecular mass of the abnormal mature Zajdela hepatoma cell beta 1-subunit decreased from 130 to 110 kDa. Thus, in addition to alterations in the Asn-linked oligosaccharide processing, ‘ascitic growth’ induced other post-translational modifications in the Zajdela hepatoma cell beta 1-subunit. Furthermore, both the abnormal mature 130-kDa and precursor 100-kDa beta 1-subunits were detected on the surface of Zajdela hepatoma cells, associated with the alpha 5-subunit. The relationship between these structural alterations in the fibronectin receptor and the impaired Zajdela hepatoma cell binding to soluble fibronectin or to a coated fibronectin matrix that was observed in this study is discussed.

scholarly journals ANIONIC SITES OF HUMAN ERYTHROCYTE MEMBRANES

1973 ◽  
Vol 57 (2) ◽  
pp. 373-387 ◽  
Author(s):  
Garth L. Nicolson
Keyword(s):  
Phospholipase C ◽  
Human Erythrocyte ◽  
Colloidal Particles ◽  
Iron Hydroxide ◽  
Glutaraldehyde Fixation ◽  
Anionic Sites ◽  
Colloidal Iron ◽  
Acetylneuraminic Acid ◽  
Induced Aggregation ◽  
Distributed Clusters

The effects of pH, trypsin, and phospholipase C on the topographic distribution of acidic anionic residues on human erythrocytes was investigated using colloidal iron hydroxide labeling of mounted, fixed ghost membranes. After glutaraldehyde fixation at pH 6.5–7.5, the positively charged colloidal particles were bound to the membranes in small randomly distributed clusters. The clusters of anionic sites were reversibly aggregated by incubation at pH 5.5 before fixation at the same pH. These results correlate with the distribution of intramembranous particles found by Pinto da Silva (J. Cell Biol. 53:777), with the exception that the distribution of anionic sites on a majority of the fixed ghosts at pH 4.5 was aggregated instead of dispersed. The randomly distributed clusters could be nonreversibly aggregated by trypsin or phospholipase C treatment of intact ghosts before glutaraldehyde fixation. Previous glutaraldehyde fixation prevented trypsin and pH induced aggregation of the colloidal iron sites. Evidence that N-acetylneuraminic acid groups are the principal acidic residues binding colloidal iron was the elimination of greater than 85% of the colloidal iron labeling to neuraminidase-treated cell membranes. Colloidal iron binding N-acetylneuraminic acid residues may reside on membrane molecules such as glycophorin, a sialoglycoprotein which contains the majority of the N-acetylneuraminic acid found on the human erythrocyte membrane.

scholarly journals Organization of Highly Acetylated Chromatin around Sites of Heterogeneous Nuclear RNA Accumulation

1998 ◽  
Vol 9 (9) ◽  
pp. 2491-2507 ◽  
Author(s):  
Michael J. Hendzel ◽  
Michael J. Kruhlak ◽  
David P. Bazett-Jones
Keyword(s):  
Histone Deacetylases ◽  
Active Regions ◽  
Fibroblast Cell ◽  
Cell Nuclei ◽  
Transmission Electron ◽  
Interchromatin Granule ◽  
Nuclear Rna ◽  
Interchromatin Granules ◽  
The Individual ◽  
Rna Accumulation

Histones found within transcriptionally competent and active regions of the genome are highly acetylated. Moreover, these highly acetylated histones have very short half-lives. Thus, both histone acetyltransferases and histone deacetylases must enrich within or near these euchromatic regions of the interphase chromatids. Using an antibody specific for highly acetylated histone H3, we have investigated the organization of transcriptionally active and competent chromatin as well as nuclear histone acetyltransferase and deacetylase activities. We observe an exclusion of highly acetylated chromatin around the periphery of the nucleus and an enrichment near interchromatin granule clusters (IGCs). The highly acetylated chromatin is found in foci that may reflect the organization of highly acetylated chromatin into “chromonema” fibers. Transmission electron microscopy of Indian muntjac fibroblast cell nuclei indicates that the chromatin associated with the periphery of IGCs remains relatively condensed, most commonly found in domains containing chromatin folded beyond 30 nm. Using electron spectroscopic imaging, we demonstrate that IGCs are clusters of ribonucleoprotein particles. The individual granules comprise RNA-rich fibrils or globular regions that fold into individual granules. Quantitative analysis of individual granules indicates that they contain variable amounts of RNA estimated between 1.5 and >10 kb. We propose that interchromatin granules are heterogeneous nuclear RNA-containing particles, some of which may be pre-mRNA generated by nearby transcribed chromatin. An intermediary zone between the IGC and surrounding chromatin is described that contains factors with the potential to provide specificity to the localization of sequences near IGCs.

Cryo-Ultramicrotomy of Islets of Langerhans

1974 ◽  
Vol 15 (3) ◽  
pp. 591-603
Author(s):  
S. L. HOWELL ◽  
MARGARET TYHURST
Keyword(s):  
Polyethylene Glycol ◽  
B Cells ◽  
Islets Of Langerhans ◽  
Structural Features ◽  
Silicotungstic Acid ◽  
Frozen Sections ◽  
Glutaraldehyde Fixation ◽  
Ultrathin Frozen Sections ◽  
Direct Immersion ◽  
A And B Cells

A procedure is described for the preparation of ultrathin frozen sections of glutaraldehyde-fixed or unfixed islets of Langerhans by cryo-ultramicrotomy. Freezing of the tissue was accomplished by direct immersion of isolated islets in liquid nitrogen. Sectioning was performed at a specimen temperature of -80 °C and a knife temperature of -40 °C, the ribbon of sections being collected on a trough containing 60 % dimethyl sulphoxide. Staining was accomplished with 4 % silicotungstic acid and sections were protected from drying artifacts by rinsing with 0.5% polyethylene glycol. Even in tissue not subjected to prior glutaraldehyde fixation, most of the structural features of A and B cells were well preserved in frozen sections, which were obtained in a number and quality which should render them suitable for ultrastructural, cytochemical or radioautographic studies.

scholarly journals Subnuclear Localization and Dynamics of the Pre-mRNA 3′ End Processing Factor Mammalian Cleavage Factor I 68-kDa Subunit

2007 ◽  
Vol 18 (4) ◽  
pp. 1282-1292 ◽  
Author(s):  
Stefano Cardinale ◽  
Barbara Cisterna ◽  
Paolo Bonetti ◽  
Chiara Aringhieri ◽  
Marco Biggiogera ◽  
...  
Keyword(s):  
Rna Polymerase Ii ◽  
Ultrastructural Level ◽  
Essential Factor ◽  
Processing Factor ◽  
Nuclear Speckles ◽  
Subnuclear Localization ◽  
Factor I ◽  
Active Transcription ◽  
Interchromatin Granules ◽  
Perichromatin Fibrils

Mammalian cleavage factor I (CF Im) is an essential factor that is required for the first step in pre-mRNA 3′ end processing. Here, we characterize CF Im68 subnuclear distribution and mobility. Fluorescence microscopy reveals that in addition to paraspeckles CF Im68 accumulates in structures that partially overlap with nuclear speckles. Analysis of synchronized cells shows that CF Im68 distribution in speckles and paraspeckles varies during the cell cycle. At an ultrastructural level, CF Im68 is associated with perichromatin fibrils, the sites of active transcription, and concentrates in interchromatin granules-associated zones. We show that CFIm68 colocalizes with bromouridine, RNA polymerase II, and the splicing factor SC35. On inhibition of transcription, endogenous CF Im68 no longer associates with perichromatin fibrils, but it can still be detected in interchromatin granules-associated zones. These observations support the idea that not only splicing but also 3′ end processing occurs cotranscriptionally. Finally, fluorescence recovery after photobleaching analysis reveals that the CF Im68 fraction associated with paraspeckles moves at a rate similar to the more dispersed molecules in the nucleoplasm, demonstrating the dynamic nature of this compartment. These findings suggest that paraspeckles are a functional compartment involved in RNA metabolism in the cell nucleus.

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