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PubMedDate not supplied

Confocal spectral imaging analysis of intracellular interactions of mitoxantrone at different phases of the cell cycle.

It is suggested that the cytotoxicity of anticancer agent mitoxantrone (MITOX) is related to a complex combination of molecular interactions which lead to slowing of S phase traverse and arresting of cells in G2 phase of the cell cycle or even to an apoptosis at high concentration of MITOX. Here intracellular molecular interactions of MITOX were visualised and studied using the confocal spectral imaging technique in synchronised K562 cells. Localisation, quantitative distributions of MITOX in the polar environment, MITOX bound to hydrophobic cellular structures (MITOXphob), nucleic acid-related complexes of MITOX (MITOXNA) and relative distributions of naphthoquinoxaline (NQX) metabolite and intrinsic cellular fluorescence of porphyrins were measured within cytoplasmic and nuclear compartments (chromosomes) of the G2, S, and M cells treated with 10 or 2 microM of MITOX for 1 hour. Colocalisation of MITOX, NQX metabolite and sites of intrinsic cellular fluorescence indicates an accumulation of MITOX within or near mitochondria. One may suppose that due to high concentration MITOX can compete with natural substrates for binding to the enzymes thus affecting the normal functioning of a mitochondria. A remarkable redistribution of MITOX and its complexes occurs in the M cells. In particular, a prominent amount of MITOX is associated with the surface of chromatids but not with the cytoplasmic structures in M cells. At the present time the exact location of the sites of MITOX accumulation in the M cells is not known. It is thought to be some cytoskeleton/microtubule structures associated directly with the chromosomes. Selective labelling of particular cytoskeleton structures and/or proteins in MITOX treated cells is in the progress now and the question will be addressed using the CSI technique.

Antineoplastic Agents↗
PubMed1989

Association of 7 SL RNA and an SRP-like particle with polysomes and endoplasmic reticulum in the developing sea urchin embryo.

We have identified the sea urchin cognate of the mammalian signal recognition particle (SRP). This particle contains the diagnostic 7 SL small RNA, sediments at a similar velocity to that reported for the mammalian particle, and is found associated with the ER and polysomes. We have examined its subcellular localization during embryogenesis in order to determine whether it could serve in a translational regulatory capacity for a subset of the stored maternal mRNAs. In these studies the 7 SL RNA was used as a marker for the particle, since we determined that the 7 SL RNA exists exclusively within the SRP-like particle at all developmental stages. The relative distribution of the SRP among cytoplasmic structures changes dramatically during development. This represents an actual change in subcellular localization because the 7 SL RNA level remains nearly constant per embryo until the pluteus stage, when it increases slightly. In eggs, the SRP exists almost entirely free in the cytoplasm as an 11 S particle. Very soon after fertilization and throughout development there is an increase in the association of the particle with rapidly sedimenting structures, until by the pluteus stage greater than 90% of the SRP exists in a bound state. The nature of the associations is complex, and the bound structures include, at least in part, ribosomes, polysomes, and microsomes. The SRP is associated with microsomal membranes in gastrula (36 hr) but not in blastula (12 hr) or earlier embryos. Using the criteria of sensitivity to Triton X-100, we determined that 16% of the SRP in a 10,000g cytoplasmic fraction was bound to membranes in a microsomal (endoplasmic reticulum)-containing fraction in the gastrula. In contrast, less than 1% was membrane associated in the blastula. The SRP was also found in a ribosome-polysome fraction in 12-, 36-, and 48-hr embryos, but not in eggs. Finally, a small but significant portion of the SRP was found associated with monosomes in cleavage stage embryos. The possible role the SRP could play in the elongation arrest of stored maternal messages for secreted proteins is discussed.

Animals↗
PubMed1996

Morphology of ricin and abrin exposed endothelial cells is consistent with apoptotic cell death.

Cultures of bovine pulmonary endothelial (BPE) cells were exposed to LC70 doses of ricin or abrin (15.5 and 4.5 pM respectively) over a period of up to 40 h. The viability of the cultures (as determined by the neutral red (NR) dye retention assay) declined after 6 h exposure to the toxins. From 15 h onwards, cellular material in toxin exposed cultures became detached from the substratum of the culture vessels. Hoffman modulation contrast photomicrography showed that this process was due to ricin and abrin exposed cells collapsing into membrane bound vesicles which retained the NR dye, became detached and floated into the medium. These apoptotic-like structural changes were further investigated by transmission electron microscopy (TEM) and by agarose gel electrophoresis of DNA from control and exposed cultures. Many of the characteristic changes associated with apoptotic cell death were seen using TEM, including heterochromatin condensation at the nuclear periphery, crenulation of the nuclear membrane and progressive degeneration of residual nuclear and cytoplasmic structures. The plasma membrane of many cells remained intact, and contained nuclear and cytoplasmic debris. Agarose gel electrophoresis of DNA extracted from toxin-treated cells revealed oligonucleosome sized DNA fragments, characteristic of apoptosis, from adherent cells at 7 h and both adherent and floating populations when harvested from 15 h; DNA from unexposed control cells did not show this fragmentation. The identification of apoptosis as being a significant additional mechanism of toxicity following exposure to ricin and abrin holotoxins raises the possibility of developing new therapeutic strategies against poisoning by these phytotoxins.

Abrin↗
PubMed1975

The use of fluorescein isothiocyanate labelled lectins for immunohistochemical demonstration of saccharides.

Cryostat sections of various tissues from man, rat, mice, hamster, rabbit, guinea pig, sparrow and carp were examined by fluorescence microscopy after incubation with fluorescein isothiocyanate labelled Concanavalin A and Lens culinaris lectin. In the same tissue of various species no basic differences in the pattern of fluorescence were observed. In general, connective tissue structures were stained by both lectins in the same degree, whereas by fluorescein isothiocyanate labelled Concanavalin A additionally a varying staining of the cell surface and of cytoplasmic structures occurred. The results show the general presence of Concanavalin A and Lens culinaris lectin receptors in normal animal tissues.

Animals↗
PubMed2001

Anthracycline-induced cardiomyopathy is manifested in decreased protein synthesis, impaired intracellular regeneration, and non-necrotic death of cardiomyocytes.

The cytostatic anthracycline antibiotic daunomycin hydrochloride led to the development of plastic myocardial insufficiency characterized by impaired intracellular regeneration of cardiomyocytes and progressive involution of cytoplasmic structures. Morphological signs of plastic myocardial insufficiency included fragmentation, annulation, or collapse of nucleoli in cardiomyocyte nuclei, lysis of myofilaments, sarcomeres, or myofibrils, focal degradation of the cytoplasm, and intensive autophagy. Fatal anthracycline-induced cardiac insufficiency was associated with massive cardiomyocyte loss due to their non-necrotic death and elimination. Our findings indicate that anthracycline-induced cardiomyopathy in laboratory animals is a convenient model for studying general mechanisms underlying the pathogenesis of regenerative and plastic cardiac insufficiency in humans.

Animals↗
PubMed1993

Multinucleated fibroblastic cells in the periodontal ligaments of aged rats.

Using 12- to 18-month-old rats, we examined the ultrastructural and cytochemical features of multinucleated fibroblastic cells (MFCs) in the periodontal ligament (PDL) of molars. In aged rats, the MFCs were distributed randomly in the PDL and exhibited cytoplasmic structural variations which were not dependent on the number of nuclei. There was a tendency for the MFCs to cluster in the PDL. The MFCs, rich in cytoplasmic organelles involved with procollagen synthesis such as rough endoplasmic reticulum and the Golgi apparatus, incorporated and secreted 3H-proline-labelled products. The MFCs also possessed many phagosomes containing intact collagen fibrils. These MFCs were apparently involved in phagocytosis and intracellular degradation of incorporated collagen fibrils. Phagosome-rich MFCs contain acid phosphatase activity in primary and secondary lysosomes, similar or stronger in intensity to that which can be demonstrated in mononuclear fibroblasts. However, unlike mononuclear fibroblasts, the MFCs did not exhibit alkaline phosphatase activity along their plasma membranes. These results suggest that MFCs demonstrate a range of fibroblastic cellular activity, including collagen phagocytosis, and that they may lack certain plasma membrane glycoproteins, which might explain the occurrence of multinucleation in these cells.

Acid Phosphatase↗
PubMed1980

Serotonin and seasonal variation in the pancreatic structure of bats: possible presence of serotonergic axons in the gland.

The serotonin concentration in the bat pancreas was determined at nine times during the year. Pancreatic ultrastructure was also examined. The pancreatic level of serotonin was uniform throughout the year except in late May, when it fell dramatically from a normal level of 3.14 +/- .19 mugm/gm to 0.40 +/- .05 mugm/gm. No seasonal changes in islet fine structure were found; however, the ultrastructure of exocrine cells was seen to be changed radically only in bats captured in late May. At this time, bats develop unusual cytoplasmic structures in the exocrine cells. These structures consist of membrane-limited bodies containing long, straight, and parallel paracrystalline arrays of stacked membranes and round or pleomorphic osmiophilic granules. Depletion of serotonin with reserpine or parachlorophenylalanine, or incubation of pancreatic tissue with different concentrations of serotonin, failed to provoke the formation of these structures in exocrine cells of active bats. Administration of 3H-5-hydroxytryptophan to bats, or incubation of bat pancreas with 3H-serotonin, failed to demonstrate a labeling of pancreatic exocrine cells. Incubation of the gland with 3H-serotonin, however, led to intense labeling of a subset of pancreatic terminal axons. It is concluded that the natural fall in the concentration of serotonin in late May is not the cause of the formation of the paracrystalline structures in pancreatic exocrine cells. The specific pancreatic uptake mechanism for serotonin found by Kovary et al. ('80) is probably a property of pancreatic neurites; these neurites might be serotonergic.

Animals↗
PubMed1985

Investigation of a test system for the rapid differentiation of nuclear and cytoplasmic damage in eucaryotes.

The effect of various agents which cause cell inactivation on the growth curves and RNA synthesis rates of yeast cells has been studied. On the basis of these investigations it was concluded that such studies can be used as a rapid test system for drawing preliminary conclusions as to whether a particular agent primarily damages the DNA of the cell nucleus or cytoplasmic structures.

Acrylonitrile↗
PubMed1984

Arachnoid mater of the bullfrog, Rana catesbeiana. A potential model for the study of intermediate filaments.

In the bullfrog, the meninges surrounding the central nervous system include an arachnoid mater that contains layers of cells with abundant intermediate filaments (IFs) having unique organizational characteristics. This membrane contains an inner lamina of cells that resemble fibroblasts and an outer lamina of flattened cells that are almost filled with IFs. The IFs of the outer arachnoid are arranged in compact, arching bundles that lie parallel to the outer surface of the central nervous system. Thus, sections cut tangentially to the membrane reveal bending of filament bundles, whereas transverse sections do not. In some cells bordering the subdural space, bundles of filaments are organized into highly-ordered spiral arrays. Attachments to the numerous desmosomes and, apparently, to the nuclear envelope suggest anchoring of cytoplasmic structures by the IF system. Microtubules occur primarily near the plasma membrane and the nucleus. Numerous caveolae also are associated with the plasma membrane. The unusual abundance, organization, and cytoplasmic relations of IFs in the bullfrog arachnoid suggest that this membrane may serve as an important model for study of fundamental cytoskeletal relations and function.

Animals↗
PubMed2004

Ubiquitin-mediated fluorescence complementation reveals that Jun ubiquitinated by Itch/AIP4 is localized to lysosomes.

Ubiquitin family peptide modifications regulate the functions and stabilities of many proteins. We have developed an approach for the visualization of ubiquitinated proteins in living cells designated ubiquitin-mediated fluorescence complementation (UbFC). This approach is based on complementation among fragments of fluorescent proteins when they are brought together by the covalent conjugation of ubiquitin fused to one fragment to a substrate protein fused to a complementary fragment. The UbFC strategy enables simultaneous visualization of proteins modified by different ubiquitin family peptides and comparison of their effects on protein localization. Visualization of ubiquitinated Jun revealed that it was localized predominantly to cytoplasmic structures. In contrast, Jun conjugated to small ubiquitin-related modifier 1 (SUMO1) was localized to subnuclear foci. Comparison of the distribution of ubiquitinated Jun with markers for various cytoplasmic compartments revealed that ubiquitinated Jun was localized to lysosomal vesicles. Fractionation of cell lysates confirmed that the majority of ubiquitinated Jun partitioned to the cytoplasmic fraction, and density gradient centrifugation analysis demonstrated that it cosedimented with lysosomal beta-hexosaminidase activity. Mutation of a recognition sequence for the E3 ligase Itch/AIP4 prevented Jun ubiquitination and stabilized it in cells. Inhibition of lysosomal protein degradation by bafilomycin or chloroquine stabilized Jun but had no effect on the stability of mutated Jun that was not ubiquitinated by Itch/AIP4. The visualization of ubiquitinated Jun in living cells has uncovered a lysosomal pathway for Jun degradation that involves ubiquitination by Itch/AIP4.

3T3 Cells↗
PubMed2002

Differential localization of chicken FIP2 homologue, Ag-9C5, in secretory epithelial cells.

When hepatocytes polarize, a subset of cellular proteins specifically localizes to the apical cell surface forming the boundary of the bile canaliculus. We have isolated a cDNA encoding a protein recognized by a monoclonal antibody (9C5) that specifically stains the bile canaliculus. The encoded protein (Ag-9C5) is a cytoplasmic protein with three leucine zippers and a zinc finger at the C-terminus. Extensive amino acid sequence similarity indicates that Ag-9C5 is likely the chicken homologue of a human protein, FIP2, which interacts with huntingtin and Rab8. Epitope-tagged Ag-9C5 colocalizes with endogenous Ag-9C5 and other canaliculus marker antigens in transfected organ cultures. In Cos7 cells and MDCK cells Ag-9C5 forms punctate cytoplasmic structures. In intact tissues Ag-9C5 is highly concentrated at the apical surfaces of cells that secrete protein from the apical surfaces, but is found in a fine punctate cytoplasmic pattern in other polarized epithelia. Because this protein has a number of characteristics of proteins that act as scaffolds for assembly of protein complexes (e.g., the cytoplasmic domain of classical cadherins and the FERM superfamily of proteins), it appears that FIP2/Ag-9C5 may act as a scaffold for assembling a complex of proteins that are involved in targeting of some secretory vesicles to defined regions of the cell surface.

Amino Acid Sequence↗
PubMed1992

The perinuclear matrix as a structural element of the mouse sperm nucleus.

The mouse sperm nucleus, after the removal of protamines and DNA, consisted of a skeletal structure that conformed to the original nuclear shape. Sperm were extracted with 1% SDS, and the isolated nuclei, along with the enveloping perinuclear theca, were incubated in 25 mM dithiothreitol, and exposed to different reagents in an effort to displace the protamines, P1 and P2. Protamines, labeled with [3H]arginine, were displaced from the nucleus by CaCl2.MgCl2, but only partially by anionic detergents, monovalent cations, and polyvalent anions. Displacement of P1 and P2 was achieved by digesting the nuclei with DNase I and simultaneously extracting with CaCl2.MgCl2 (3:2; mol:mol) in stepwise increments of 125, 150, 175, 200, and 250 mM. Protamine displacement was concentration-dependent, occurring with an EC50 of approximately 205 mM and with maximal displacement at approximately 250 mM CaCl2.MgCl2. The nucleus was reduced to a skeletal structure consisting of the perinuclear theca and an internal network of transverse fibers. The evidence was consistent with the former being derived from the perforatorium and postacrosomal nuclear sheath (both cytoplasmic structures), whereas the fibers were most likely of nuclear origin. By SDS-PAGE and isoelectric focusing (IEF), perinuclear matrices consisted of greater than or equal to 230 protein spots, with M(r)s in the range of 70,000 to 8000 and pIs of greater than or equal to 7.5 to approximately 4.7, respectively. Monoclonal antibodies prepared against perinuclear matrices bound to specific proteins on IEF immunoblots and, based on light and electron microscopic observations, to discrete domains of the sperm perinuclear theca and nucleus.

Animals↗
PubMed1976

[Quantitative ultrastructural study of the perikaryon of the Purkinje cell and of the adjacent area in normal and hypothyroid rats aged 21 days].

The effects of neonatal hypothyroidism on synaptic organization, glial surrounding and cytoplasmic structures of the Purkinje cell perikaryon of the 21 days old rat were studied by electron microscopy. Hypothyroidism decreases the size of the perikaryon but does not change the nucleo-cytoplasmic ratio of the cell. At 21 days the axonal endings that make synapse on the Purkinje cell perikaryon, already show the adult morphological features and hypothyroidism does not change these. The general synaptic density on the Purkinje perikaryon is not significantly altered, but the cells of hypothyroid animals still recieve 1/3 of their axo-somatic synapses on somatic spines, whereas euthyroid animals have practically no synapses on somatic spines. Hypothyroidism leads the disappearance of the synchronism which normally exists between the translocation or resorption of the Purkinje cell somatic spines and the establishment of the basket cell synapses. The basket cell axons of the normal animals form synapses only on the smooth surfaces of the Purkinje cell perikaryon while they also establish it on the somatic spines of the hypothyroid animals. The density of the axon terminals of basket cells is decreased, while that of the endings of Purkinje cell axon collaterals and of climbing fibers are increased. Hypothyroidism produces an increase in the size of the glial sheath around the Purkinje perikaryon. It does not alter the proportion of the cytoplasmic area occupied by mitochondria, but in thyroid deficiency there is an increase in the numbers of mitochondria which are reduced in average size. The Golgi apparatus is only slightly affected. Thyroid defiency does not alter the density of the cytoplasmic agreggates of free ribosomes but there is a decrease in density of the ergastoplasm and a disorganization of the Nissl bodies.

Animals↗
PubMed1983

An alternative fixation-processing method for preembedding ultrastructural immunocytochemistry of cytoplasmic antigens: the GBS (glutaraldehyde-borohydride-saponin) procedure.

The use and properties of an alternative fixation procedure for ultrastructural cytochemistry is described that utilized primary fixation of cells with glutaraldehyde with considerable latitude in concentration and time of fixation. This was followed by treatment with sodium borohydride (as described by Weber, Rathke and Osborn (Proc Natl Acad Sci USA 75:1820, 1978)), which increases the accessibility of some cytoplasmic compartments to antibodies, apparently through the reduction of the Schiff bases induced by glutaraldehyde. Through the use of saponin membrane permeabilization, this primary fixation method allows good ultrastructural preservation with accessibility of most, but not all, cytoplasmic structures. Localization of tubulin, clathrin, alpha2-macroglobulin present in lysosomes, vesicular stomatitis virus (VSV) G protein, and myosin are demonstrated. This technique failed to expose the antigenic structure of actin in microfilament bundles or intranuclear antigens. This method should be useful for good preservation of ultrastructure and antibody localization of some, but not all, protein antigens in the cytoplasm of cultured cells, as well as in intact tissue. This technique is especially useful for cytosolic antigens in cultured cells.

Aldehydes↗
PubMed2001

Regenerative and plastic insufficiency of cardiomyocytes during impairment of protein synthesis.

Regenerative and plastic insufficiency of cardiomyocytes comprises cell changes resulting from impaired DNA-dependent RNA synthesis and biosynthetic processes. It is accompanied by progressive involution of cytoplasmic structures and causes atrophy and partial elimination of cells without signs of coagulation and colliquative necroses. Segregation and fragmentation of nucleoli in cardiomyocyte nuclei, lysis of myofibrils, and focal degradation of the cytoplasm are ultrastructural signs of impaired protein synthesis. Intracellular cardiomyocyte regeneration is characterized by disorientation of newly formed myofibrils and their excessive elongation, which are related to DNA damages followed by changes in transcription and translation in cardiomyocytes. Calculation of the absolute number of cardiomyocytes is a new approach to evaluation of contractile myocardial insufficiency.

Animals↗
PubMed1998

Green fluorescent protein labeling of cytoskeletal structures--novel targeting approach based on leucine zippers.

Green fluorescent protein (GFP) is a valuable marker for intracellular protein localization. However the fusion of GFP with structural proteins can alter their properties, resulting in a loss of fusion protein localization, decreased GFP fluorescence or both. We describe a novel targeting approach based on noncovalent heterodimerization of GFP and cytoplasmic structural proteins. The formation of structural protein/GFP complexes was mediated by modified leucine zipper protein spacers designed to form high-affinity heterodimers. The complexes localized accurately to specific sites within cells, providing selective fluorescence labeling of subcellular structures such as microfilaments or focal contacts.

Actins↗
PubMed1991

Morphometry of cytoplasmic components of mammalian articular chondrocytes and corneal keratocytes: species and zonal variations of mitochondria in relation to nutrition.

A morphometric analysis was made of nuclei and cytoplasmic structures in electron micrographs of chondrocytes in the non-calcified layer of articular cartilage of the femoral condyles in adult mouse and dog and of the human incus. Mitochondrial volume density (% cytoplasm) was lower in dog than in mouse cells or cells of the incus. It was also lower in the cells of deep zone cartilage than in superficial cells. Analysis of keratocytes of the corneal stroma in mouse and sheep gave similar findings to that in cartilage. Mitochondrial volume density was higher in mouse than in sheep keratocytes and, in sheep, higher in subepithelial (anterior) cells than in cells of the intermediate and subendothelial strata. Values in human stromal cells were similar to those in the sheep. Endoplasmic reticulum volume density was higher in mouse than in sheep keratocytes. Lipid and filaments were more abundant in dog than in mouse chondrocytes but keratocytes contained little or none. Mitochondrial volume densities correlate with diffusion distances (tissue thickness) from the sources of nutrition, for example, for oxygen, in the two tissues. The relationship to certain aspects of chondroitin sulphate and keratansulphate synthesis and topographical distribution in cartilage and cornea is discussed.

Animals↗
PubMed1961

The normal fine structure of opossum testicular interstitial cells.

The interstitial tissue of the opossum testis includes interstitial or Leydig cells, macrophages, and small cells which morphologically resemble mesenchymal cells. The latter are thought to give rise to mature interstitial cells. The most prominent feature of the interstitial cell cytoplasm is an exceedingly abundant agranular endoplasmic reticulum. This reticulum is generally in the form of a meshwork of interconnected tubules about 300 to 450 A in diameter, but occasionally it assumes the form of flattened, fenestrated cisternae resembling those of pancreatic acinar cells, except for the lack of ribonucleoprotein particles on the surface of the membranes. The interstitial cells vary considerably in their cytoplasmic density. The majority are quite light, but some appear extremely dense, and in addition usually have a more irregular cell surface, with numerous small pseudopodia. These differences may well reflect variations in physiological state. Cytoplasmic structures previously interpreted as "crystalloids" consist of long bundles of minute parallel tubules, each about 180 A in diameter, which seem to be local differentiations of the endoplasmic reticulum. The mitochondria are rod-shaped, and contain a moderately complex internal membrane structure, and also occasional large inclusions that are spherical and homogeneous. The prominent juxtanuclear Golgi complex contains closely packed flattened sacs and small vesicles. The results of the present study, coupled with biochemical evidence from other laboratories, make it seem highly probable that the agranular endoplasmic reticulum is involved in the synthesis of the steroid hormones produced by the interstitial cell. This finding therefore constitutes one of the first functions of the agranular reticulum for which there is good morphological and biochemical evidence.

Animals↗