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Various cytoplasmic and intranuclear inclusions found in macrophages and astrocytes of lead-implanted rat brains were studied with an energy dispersive x-ray microanalytical technique. Cytoplasmic inclusions contained large quantities of lead, calcium and phosphorus. The proportions of these elements were different within each inclusion. Intranuclear inclusions also contained small amounts of lead and, occasionally, calcium.
Mitochondrion is not only the major ATP manufacture center of animal cells, but also plays a key role, as a main switch, in the regulation of apoptos is. Opening of the mitochondrion permeability transition pore (MPTP), the life-or-death switch of cells, causes an increase of the permeability of mitochondrial membrane and the release of several types of apoptogenic factors from the intermembrane space, such as cytochrome c, apoptosis inducing factors (AIFs),procaspases and Ca(2+). These apoptotic factors can either activate caspases, the main member of apoptotic proteins, or destruct independently the intranuclear chromatin, or interact with other Ca(2+) -dependent proteins. These result in structural rupture and dysfunction of cells, and the cells are finally degraded into apoptotic bodies and died. Studies on the regulation mechanisms of mitochondrion in apoptosis will be of great value theoretically as well as potential practical usage in designing drugs for cancer, Parkinson's disease and so on, using mitochondria as targets. To understand the key role of the mitochondrion in the determination of life and death of the cells, recent findings in this field are reviewed in this article.
The baboon preimplantation stages were examined using light and electron microscopy. Six cases were studied at 2, 3, 4, 5, 7 and 8 days estimated fertilization age. The first 3 specimens were composed of 2, 8 and 24 blastomeres respectively. At 5 days, 30 to 40 cells were counted and more than 60 cells in later stages. Primitive "trophoblast cells" differentiate at 7 days and a crescentic blastocoele appears at 8 days. Shedding of the zona pellucida is not observed in the 7 and 8 day specimens. The preimplantation period is longer in the baboon than in man. C-type viruses are observed in the zona pellucida, in the perivitelline and interblastomeric spaces. Microvilli and caveolae cover ther periphery of the baboon conceptus. As in many other mammals, transformation of the mitochondria, changes in the ribosomes distribution, multivesicular bodies, myelin figures, nucleoli and intranuclear clusters of granules are described in the baboon. Cytoplasmic fibrous strands are not present as in the mouse. Experiments on the influence of hormones and drugs on ultrastructural changes would help to evaluate the importance of biohazards during the early development of primates.
Cationic polymer/DNA complexes are widely used for gene delivery, although the influence of the cationic polymer on the biophysical properties of the resulting complex is poorly understood. Here, several series of cationic polymers have been used to evaluate the influence of structural parameters on properties of DNA complexes. Parameters studied included the length of side chain, charge type (primary versus tertiary and quaternary), polymer molecular weight, and charge spacing along the polymer backbone. Cationic polymers with short side chains (such as polyvinylamine) formed small complexes, resistant to destabilization by polyanions, with low surface charge, limited transfection activity, and efficient intranuclear transcription. Conversely, cationic polymers with long side chains (e.g., poly[methacryloyl-Gly-Gly-NH-(CH(2))(6)-NH(2))] showed inefficient complex formation, high positive surface charge, and better transfection activity. The effects of molecular weight varied between polymers, for example, low molecular weight poly(L-lysine) produced relatively small complexes, whereas low molecular weight poly[2-(trimethylammonio)ethyl methacrylate chloride] produced large aggregates. Polymers containing quaternary ammonium groups showed efficient complex formation but poor transfection. Finally, spreading charges widely on the polymer structure inhibited their ability to condense DNA. In summary, to achieve small, stable complexes, the use of cationic polymers with short side chains bearing primary amino groups is suggested.
An ultrastructural study of pleomorphic liposarcoma with an unusual clinical presentation is described. A 69-year-old Caucasian female presented with recurrent superficial phlebitis of left leg, which responded only partially to conventional therapy. Following investigations, a mass was excised. Diagnosis of pleomorphic liposarcoma was made on microscopic examination. Numerous lipid droplets in the cytoplasm and often within intranuclear extensions of cytoplasm were seen on oil red O stain of frozen sections. Ultrastructural features are quite distinctive. The cells varied from small to often large pleomorphic cells with numerous lipid spaces without limiting membrane, large pleomorphic nuclei, and an abundance of cytoplasmic organelles. Centrioles were very prominent, in close proximity to nuclei, and collections of autophagocytic inclusions. Intercellular collagen was immature and scant. These features are compared to ultrastructural features of malignant fibrous histiocytoma, pleomorphic rhabdomyosarcoma, and cardiac myxoma, indicating that ultrastructural features are distinctive and help differentiate similar soft tissue tumors.
Homogenized lung tissue was used to experimentally reproduce lethal viral pneumonia in guinea pigs. The resultant lesions corresponded with those of the spontaneous disease. Pneumonia with necrotic bronchiolar epithelium accompanied by basophilic intranuclear inclusion bodies was the primary finding. With transmission electron microscopy, numerous viral particles were found--mainly within the nucleus of pulmonary epithelial cells but also within the cytoplasm and in extracellular space. The appearance of viral particles, in particular their paracrystalline and crystalline deposition within the nucleus, indicates that our experimentally induced pneumonia was caused by an adenovirus.
Using the pyroantimoniate technique, a study was conducted at electron microscope level on the distribution of the calcium ion in the pineal glands of normal adult Sprague-Dawley rats with initial weights of 150-200 g subjected to a 12:12 light dark cycle and others under the same conditions were subjected to modifications in the noradrenergic signal, such as continuous illumination over 7 days, blinding by bilateral enucleation (7 or 90 days) before sacrifice and bilateral superior cervical gangliectomy at 21 days before sacrifice. All the animals were sacrificed by decapitation, half of them at midday and the other half at midnight. Abundant fine precipitations of calcium were found in the intercellular spaces of the pineal glands of the normal rats. By contrast, in the gangliectomized animals subjected to constant illumination and chronic binding these precipitations were few in number. Additionally, two types of pinealocytes were observed regarding the distribution and concentration of intracytoplasmic calcium in both the normal and experimentally manipulated animals. Type I correspond to the classic light pinealocytes, with an absence of intracytoplasmic precipitations, although in the normal and gangliectomized animals sacrificed at midnight it was possible to observe fine deposits inside the mitochondrial matrix. Type II correspond to the classic dark pinealocytes, with a dense cytoplasmic matrix and numerous deposits of intracytoplasmic and intranuclear calcium; these were never seen in the type I pinealocytes.
A case of testicular plasmacytoma is described at the light and fine structure level. The patient was a 54-year-old male with a history of multiple myeloma. The testicular tumor was restricted to an interstitial space growth. Plasma cells varied in differentiation with few obtaining the cartwheel nucleus and prominent Golgi of connective tissue plasma cells. Cytoplasmic inclusions were characteristic of most cells and a large number of intranuclear inclusions were present. To our knowledge, this is the first report of such inclusions in the cells of a testicular plasmacytoma. Their significance in cases of end stage myeloma is discussed.
The runt-related transcription factors (RUNX/Cbfa/AML) are essential for cellular differentiation and fetal development. C-terminal truncations of RUNX factors that eliminate the targeting of these factors to subnuclear foci result in lethal hematopoietic and skeletal phenotypes. Here we demonstrate that in living cells the RUNX C-terminus is necessary for the dynamic association of RUNX into stable subnuclear domains. Time-lapse fluorescence microscopy shows that RUNX1 and RUNX2 localize to punctate foci that remain stationary in the nuclear space. By fluorescence recovery after photobleaching assays, both proteins are shown to dynamically associate at these subnuclear foci, with a 10 second half-time of recovery. A truncation of RUNX2, removing its intranuclear targeting signal (NMTS), increases its mobility by an order of magnitude, resulting in a half-time of recovery equivalent to that of EGFP alone. We propose that the dynamic shuttling of RUNX factors in living cells to positionally stabilized foci, which is dependent on the C-terminus, is a component of the mechanism for gene regulation in vivo.
A 46-year-old female, with an 11 year history of malignant lymphoreticular disease, developed a neurological illness clinically manifested by a focal mass lesion in the left frontal lobe. In biopsied tissue, immunofluorescence study revealed the presence of JC antigen in the glial cells. Histologically, the lesion was characteristic of PML consisting of PML consisting of focal necrosis in the subcortical white matter, numerous fat laden macrophages and marked hypertrophy of oligodendrocytes and astrocytes. By electron microscopy, hypertrophic astrocytes contained intranuclear viral particles consistent with papova virions and aggregates of intracytoplasmic viral particles consisting of a single to several virions tightly surrounded by a single membrane. The membrane appeared to have been derived from that of the cellular vesicles. Fusion of the virus-associated membrane to the astroglial plasmalemma occurred when the virions appeared to shift towards extracellular space. The virioncontaining astrocytes showed cytoplasmic "fibrillar hypertrophy" similar to the characteristic gigantic astroglias of PML. This fact would provide an additional evidence that these gigantic cells, although lacking identifiable viral structures, were the result of anaplastic transformation by JC virus. Many virus-bearing astroglias were noted to be in the early stage of cellular necrosis, of "edematous degeneration". This further indicates that the JC virus is capable of inducing both lytic and abortive astroglial infections. Many oligodendroglias were hypertrophic due to the presence of intranuclear viral particles and markedly increased numbers of microtubules and free ribosomes in the cytoplasm. Membrane-bound intracytoplasmic viral particles were also noted in the oligodendroglias. Some fat laden macrophages contained large intracytoplasmic viral bodies, presumably originating from phagocytized virus-bearing cells.
A female bearded dragon (Amphibolurus barbatus) died following intermittent periods of inappetance. No significant gross lesions were found at autopsy, but histological examination revealed disordered liver architecture with numerous foci of coagulative necrosis. Eosinophilic intranuclear inclusions were present in many hepatocytes, some epithelial cells of the bile ductules and occasional epithelial cells of renal tubes and glomeruli. Large numbers of viral particles within many nuclei, associated with the intranuclear inclusion were demonstrated by electronmicroscopy. Similar particles, sometimes in paracrystalline arrays, were also seen within membrane-bound vesicles located next to the nuclei and to a lesser degree within the cytoplasm and extracellular spaces. The virus was considered to be an adenovirus on the basis of its size, morphology, site of formation and lack of envelopment. It was considered to he the cause of the hepatitis.
A number of structural and functional subnuclear compartments have been described, including regions exclusive of chromosomes previously hypothesized to form a reactive nuclear space. We have now explored this accessible nuclear space and interchromosomal nucleoplasmic domains experimentally using Xenopus vimentin engineered to contain a nuclear localization signal (NLS-vimentin). In stably transfected human cells incubated at 37 degrees C, the NLS-vimentin formed a restricted number of intranuclear speckles. At 28 degrees C, the optimal temperature for assembly of the amphibian protein, NLS-vimentin progressively extended with time out from the speckles into strictly orientated intranuclear filamentous arrays. This enabled us to observe the development of a system of interconnecting channel-like areas. Quantitative analysis based on 3-D imaging microscopy revealed that these arrays were localized almost exclusively outside of chromosome territories. During mitosis the filaments disassembled and dispersed throughout the cytoplasm, while in anaphase-telophase the vimentin was recruited back into the nucleus and reassembled into filaments at the chromosome surfaces, in distributions virtually identical to those observed in the previous interphase. The filaments also colocalized with specific nuclear RNAs, coiled bodies and PML bodies, all situated outside of chromosome territories, thereby interlinking these structures. This strongly implies that these nuclear entities coexist in the same interconnected nuclear compartment. The assembling NLS-vimentin is restricted to and can be used to delineate, at least in part, the formerly proposed reticular interchromosomal domain compartment (ICD). The properties of NLS-vimentin make it an excellent tool for performing structural and functional studies on this compartment.
In 1974, Berezney and Coffey described what they called the nuclear matrix (NM), thus ignoring our priority, since we had isolated and characterized virtually the same skeletal structure 25 years before this discovery. The presence of NM in the live cell was doubted, because of unsuccessful attempts to recognize it in vivo. NM comprises the lamina, extracted nucleoli and an intranuclear fibrogranular network. The internal matrix is very labile, its presence and abundance depending on methods of isolation, whereas the isolated NM can be revealed as granules 25-30 nm in diameter. As the state of the interchromatin space changes with varying in vivo conditions, temperature and methods of isolation, doubts cast upon the very existence of NM are to be regarded as hardly valid, and new progress in its study may be expected in the XXI century.
Lamin A is synthesized in the cytoplasm as a precursor bearing a carboxyl-terminal CaaX box or isoprenylation signal. This precursor is post-translationally processed through multiple steps: isoprenylation with a farnesyl residue on the cysteine of the CaaX box, proteolytic removal of the last three amino acids, carboxymethylation of the cysteine residue and, finally, proteolytic removal of 15 amino acids from the carboxyl terminus. This last step gives rise to mature lamin A from which the isoprenylated terminus has been removed. Isoprenylation is a prerequisite for all other steps of processing. The subcellular location of these processing steps for lamin A is still a matter of debate. We have produced an antibody specific to the 18 amino acid carboxyl terminus of the lamin A precursor that does not recognize mature lamin A. This antibody detects intranuclear foci by immunofluorescence. Larger amounts of lamin A precursor were accumulated by treating cells with mevinolin (MVN), an inhibitor of isoprenoid synthesis. In MVN-treated cells, the lamin A precursor accumulated most strikingly in the peripheral nuclear lamina where it was assembled, while intranuclear foci were maintained. The addition of an excess of mevalonate (MVA), which restores isoprenylation activity, to MVN-treated cells led to a progressive disappearance of the lamin A precursor from the peripheral lamina. This process was completed after 4 hours of MVA treatment, after which the lamin A precursor was restricted to intranuclear foci. We conclude from these results that the non-isoprenylated lamin A precursor appears competent for assembly into the peripheral nuclear lamina, and that all the processing steps leading to mature lamin A can occur within the nuclear space.
Liver biopsies from 4 baboons taken during 15 months of iron-polymaltose injections, were compared with specimens from 2 controls. A morphometric method was used to assess ferritin concentration in various cells. Initially, ferritin and siderosomes were conspicuous in reticuloendothelial cells but rare in hepatocytes. Unusual findings included intranuclear ferritin and coalesced ferritin within bile canaliculi. With advancing overload, ferritin and hemosiderin increased not only in sinusoidal cells, but also in hepatocytes, with concomitant elevation of transaminases. The hepatocytes now showed evidence of damage and excessive collagen was present mainly around portal spaces. A year after cessation of iron injections, hepatocyte ultrastructure was near normal while sinusoidal cells were still heavily overloaded. The baboon appeared to be a useful model for the study of iron overload. Although in this study most of the damage was reversible, it is suspected that more prolonged overload, a different route of administration or other, more toxic iron compounds, may lead to cirrhosis similar to that of the iron-loading anemias.
Hepatoma cells, though sharing many similarities with non-neoplastic hepatocytes, showed conspicuous alterations--increased nuclear/cytoplasmic ratio, intranuclear invapinations of cytoplasm, lipoid material, high mitotic activity, and the presence of abundant small fat droplets and glycogen in the cytoplasm. "Capillarization" of sinusoids was a constant feature with the appearance of junctions between endothelial cells, deposition of fibrillary material in Disse's spaces, flattening of hepatocyte microvilli and deposition of basement-membrane-like material. Abundant microtubules and the presence of annulate lamellae were other features noted in the tumor cells.
Globular inclusions are present in the nuclei of the columnar epithelial cells lining the ductus epididymidis in the garden dormouse, Eliomys quercinus L. Such inclusions have been reported in the dog where they are very abundant, in the stallion and in man. They have been found to be absent from sheep, cat, rabbit, guinea pig, rat and mouse. We have now found them in the garden dormouse, an ascrotal hibernator, but have been unable to detect them in another hibernator, the thirteen-lined ground squirrel Citellus tridecemlineatus, this time a scrotal mammal. The globular inclusions are first seen in the perinuclear space formed by the two membranes of the nuclear envelope; they are then invested by the inner membrane of the nuclear envelope and transferred into the nucleus. They later disintegrate and the vacuole is finally left with an electron-transparent content. The number of inclusions varies with the hormonal status of the animal: the granules are always present as soon as there is a rise in plasma testosterone. Electron energy-loss spectroscopy in the electron microscope (EELS) shows that inclusions contain C, N, O, Fe, Mg, P and S. This suggests that the intranuclear granules are made of protein cross-linked by disulphur bonds and contain iron, magnesium and phosphorus.
There is much interest in recent years in the possible role of different nuclear compartments and subnuclear domains in the regulation of gene expression, signalling, and cellular functions. The nucleus contains inositol phosphates, actin and actin-binding proteins and myosin isoforms, multiple protein kinases and phosphatases targeting Cdk-1 and Cdk-2, MAPK/SAPK, and Src-related kinases and their substrates, suggesting the implication of several signalling pathways in the intranuclear organization and function of nuclear bodies (NBs). NBs include the well-characterized Cajal bodies (CBs; or coiled bodies), the nucleolus, perinucleolar and perichromatin regions, additional NBs best illustrated by the promyelocytic leukemia nuclear bodies [PML-NBs, also named PML oncogenic dots (PODs), ND10, Kr-bodies] and similar intranuclear foci containing multi-molecular complexes with major role in DNA replication, surveillance, and repair, as well as messenger RNA and ribosomal RNA synthesis and assembly. Chromatin modifying proteins, such as the CBP acetyltransferase and type I histone deacetylase, accumulate at PML-NBs. PML-NBs and Cajal bodies are very dynamic and mobile within the nuclear space and are regulated by cellular stress (heat shock, apoptosis, senescence, heavy metal exposure, viral infection, and DNA damage responses). NBs strongly interact, using signalling mechanisms for the directional and ordered traffic of essential molecular components. NBs organize the delivery and storage of essential RNAs and proteins that play a role in transcription, pre-mRNA biosynthesis and splicing, and the sequestration and/or degradation of regulatory proteins, such as heterogenous nuclear ribonuclear proteins (hnRNPs), p53, Rb1, CBP, STAT3, and others. The objective of this review is to summarize some aspects of these nuclear structures/bodies/domains, including their proposed roles in cellular signalling and in human diseases, mainly neurodegenerative disorders and cancer.