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At least 775 records · Page 43Linked to original sources
PubMed1998

Simultaneous inhibition of contractile ring and central spindle formation in mammalian cells treated with cytochalasin B.

In this work we have used the inhibitor of F-actin polymerisation cytochalasin B (Cyt B) to test the hypothesis that the contractile ring and the central spindle are mutually interdependent structures in mammalian mitotic cells. Double fluorescence staining of alpha-tubulin and F-actin was employed to analyse anaphase and telophase figures from asynchronously growing cultures and prometaphase-synchronised cells. Testing for the presence of the central spindle and contractile ring in human primary fibroblasts, human hepatoma cells and Chinese hamster cells after Cyt B treatment showed that both structures were simultaneously absent in over 60% of treated anaphases and 80% of telophases. Experiments on resumption of cytokinesis in cleavage-arrested cells further showed that Cyt B-treated human fibroblasts proceeded to cleavage within minutes after removal of the drug from the medium, concomitant with the re-formation of both cellular structures in cleaving cells. These data suggest that the presence of a correctly assembled contractile ring is essential for the formation and persistence of the central spindle during ana-telophase and provide further support for the idea of a strong co-operative interaction between these two structures during cytokinesis.

Actins↗
PubMed1999

Cellular pharmacokinetics: effects of cytoplasmic diffusion and binding on organ transit time distribution.

Distribution between well-stirred compartments is the classical paradigm in pharmacokinetics. Also in capillary-issue exchange modeling a barrier-limited approach is mostly adopted. As a consequence of tissue binding, however, drug distribution cannot be regarded as instantaneous even at the cellular level and the distribution process consists of at least two components: transmembrane exchange and cytoplasmic transport. Two concepts have been proposed for the cytoplasmic distribution process of hydrophobic or amphipathic molecules, (i) slowing of diffusion due to instantaneous binding to immobile cellular structures and (ii) slow binding after instantaneous distribution throughout the cytosol. The purpose of this study was to develop a general approach for comparing both models using a stochastic model of intra- and extravascular drug distribution. Criteria for model discrimination are developed using the first three central moments (mean, variance, and skewness) of the cellular residence time and organ transit time distribution, respectively. After matching the models for the relative dispersion the remaining differences in relative skewness are predicted, discussing the relative roles of membrane permeability, cellular binding and cytoplasmic transport. It is shown under which conditions the models are indistinguishable on the basis of venous organ outflow concentration-time curves. The relative dispersion of cellular residence times is introduced as a model-independent measure of cytoplasmic equilibration kinetics, which indicates whether diffusion through the cytoplasm is rate limiting. If differences in outflow curve shapes (their relative skewness) cannot be detected, independent information on binding and/or diffusion kinetics is necessary to avoid model misspecification. The method is applied to previously published hepatic outflow data of enalaprilat, triiodothyronine, and diclofenac. It provides a general framework for the modeling of cellular pharmacokinetics.

Animals↗
PubMed1989

Tandem scanning reflected-light microscopy of cell-substratum adhesions and stress fibres in Swiss 3T3 cells.

This paper describes two applications of the tandem scanning reflected-light microscope (TSM) for the observation of the structure of individual cells growing in tissue culture. First, the TSM is used as an alternative to interference reflection microscopy (IRM) or total internal reflection aqueous fluorescence microscopy (TIRAF) to observe cell-substratum adhesions in unstained living cells growing on a glass coverslip. Second, the TSM is used to produce improved images of cellular structures in 3T3 cells stained with various protein dyes including Napthol Blue Black (NBB) and Coomassie Brilliant Blue (CBB). More specifically, close contacts and focal contacts are resolved in living 3T3 cells, and features of the nucleus, the cytoskeleton and extracellular matrix are resolved in both NBB- and CBB-stained cells. The focal contacts and associated stress fibres are clearly imaged in NBB-stained cells. The TSM is an improvement over conventional incident light microscopy because of the confocal image excludes information from out-of-focus regions of the cytoplasm, and, unlike the laser-based confocal microscope, the actual colour of the specimen is viewed directly with TSM in almost real-time.

Amido Black↗
PubMed1987

Stimulatory effect of cisplatin on production of lipid peroxidation in renal tissues.

Cisplatin (cis-diamminedichloroplatinum II), an anticancer chemotherapeutic agent with the dose-limiting side effect of nephrotoxicity, caused a statistically significant increase in lipid peroxidation, monitored by measuring the production of malondialdehyde, in rat kidney 72 hr after injection. Treatment of rats beforehand with the antioxidant alpha-tocopherol or N-N'-diphenyl-p-phenylenediamine (DPPD) effectively decreased such peroxidation. DPPD was a more effective inhibitor than alpha-tocopherol, since it is known for its ability to scavenge free radicals more powerfully. The ability of renal cortical slices to accumulate p-aminohippurate (PAH) was examined as a biochemical parameter that would change in nephrotoxicity. The ability to accumulate PAH by the incubated slices decreased 72 hr after administration of cisplatin. The pretreatment with DPPD prevented the decrease in PAH accumulation in the slices from rats treated with cisplatin. Structural changes of the renal proximal tubule caused by cisplatin, analyzed in a transmission electron microscope, were also prevented by the pretreatment with DPPD. The results suggest that cisplatin affects renal tissues in which free radicals generated by cisplatin may interact with membrane lipids to cause the production of lipid peroxidation, which affects both cellular structure and function.

Aminohippuric Acids↗
PubMedDate not supplied

[Changes in the fine structure of microbial cells induced by chaotropic salts].

The electron microscopic examination of the thin sections of cells of the yeasts Saccharomyces cerevisiae and Pichia pastoris and the gram-positive bacteria Micrococcus luteus and Bacillus subtilis showed that cell treatment with the chaotropic salts guanidine hydrochloride (6 M) and guanidine thiocyanate (4 M) at 37 degrees C for 3-5 h or at 100 degrees C for 5-6 min induced degradative processes, which affected almost all cellular structures. The cell wall, however, retained its ultrastructure, integrity, and rigidity, due to which the morphology of cells treated with the chaotropic salts did not change. High-molecular-weight DNA was localized in a new cell compartment, ectoplasm (a peripheral hydrophilic zone). The chaotropic salts destroyed the outer and inner membranes and partially degraded the outer and inner protein coats of Bacillus subtilis spores, leaving their cortex (the murein layer) unchanged. The spore core became accessible to stains and showed the presence of regions with high and low electron densities. The conditions of cell treatment with the chaotropic salts were chosen to provide for efficient in situ PCR analysis of the 16S and 18S rRNA genes with the use of oligonucleotide primers.

Bacillus subtilis↗
PubMed1992

Evaluation of cytocentrifuge apparatus with special reference to the cellular recovery rate.

Two types of commercially available cytocentrifuge apparatus (type A and type B apparatus) using disposable funnels were compared for percentage of cell recovery and degree of cell preservation. The cellularity of each cell suspension was determined using a Sysmex micro cell counter for blood analysis, and the cell recovery was obtained by counting cells in the total smeared area on the May-Grünwald Giemsa (MGG)-stained slide. Overall recovery rate by the type A apparatus was between 54.3% and 74.9% with a mean of 63.0%, whereas, the recovery rate for type B apparatus was between 30.6% and 51.8%, with a mean of 42.5%, indicating that the type A apparatus was significantly better. In the type A apparatus, a higher yield of all cells was obtained (69.7-74.9%) in the group of low cell counts (350 cells/0.5 ml), which was run for 10 minutes at 2,000 rpm. On the other hand, in the type B apparatus a higher yield of all cells was obtained (38.6-42.6%) in the group of low cell counts which was run for 10 minutes at 2,000 rpm. Cellular structure was better preserved on the slides in the type A apparatus. However, the percentage of ghost cells was somewhat higher in the type B apparatus. The cytocentrifugation of the type A apparatus consistently recovered a higher percentage of cells than with the type B apparatus. Using the type A apparatus, a high rate of cellular recovery, which is extremely important, such as for accurate morphological evaluation of cerebrospinal fluid, can be consistently obtained.

Centrifugation↗
PubMed1977

Ultrastructure of the dikaryotic form of Cyathus bulleri Brodie.

The fine structure of the dikaryotic form of Cyathus bulleri Brodie was generally found to be similar to that of other hyphal forms of the Basidiomycetes. The nuclear walls were doubled, porous and in some cases connected to the endoplasmic reticulum. The presence of typical as well as filamentous and U-shaped mitochondria was confirmed. Other cellular structures and organelles, among them vacuoles, vesicular and myelinoid-like bodies, often associated with the cell membranes, glycogen and ribosomes were also observed in the cytoplasm. The presence of the dolipore/parenthesome apparatus and clamp connections typical of the Basidiomycetes was established.

Basidiomycota↗
PubMedDate not supplied

Effects of a 5 alpha-reductase inhibitor, finasteride, on the developing prostate and testis of a marsupial.

This study examines the role of dihydrotestosterone in virilization of the developing male tammar. The onset of prostate differentiation in this marsupial species normally occurs around 25 days postpartum, long after the onset of testicular testosterone production immediately after birth and the appearance of 5 alpha-reductase in the urogenital sinus before day 10. Males treated with the 5 alpha-reductase inhibitor Finasteride had reduced prostatic growth and development, and their testicular structure was disorganized. Exogenous testosterone in males enhanced the development of prostatic buds but also caused damage to the testis structure. Treatment of female tammars with testosterone between days 20-30 postpartum stimulated prostatic tissue formation and Wolffian duct development, confirming that prostatic differentiation is initiated by androgens and occurs over a relatively narrow window of time. Testosterone had a deleterious effect on the ovary, destroying the germ cells. Although treatment with testosterone damaged gonadal cellular structure in both male and female tammar young, dihydrotestosterone is apparently necessary for stability of the seminiferous tubules in the testis. Taken together, these results suggest that dihydrotostesterone initiates prostatic development between days 20 and 25 after birth in this marsupial.

5-alpha Reductase Inhibitors↗
PubMed2001

Dynamics of alpha-actinin in focal adhesions and stress fibers visualized with alpha-actinin-green fluorescent protein.

Motile cells undergo changes in cell adhesion, behavior, and shape that are mediated by small-scale cytoskeletal rearrangements. These rearrangements have proven difficult to follow quantitatively in living cells, without disrupting the very structures and delicate protein balances under study. We have expressed a prominent cytoskeletal protein, alpha-actinin, as a fusion with green fluorescent protein (alpha AGFP), and have followed this construct's movements within transfected mouse Swiss 3T3 and BALB/c fibroblasts. alpha AGFP was expressed at low levels to avoid overexpression artifacts. alpha AGFP localized to cellular structures, including stress fibers, focal adhesions, microspikes, and lamellipodia. High-resolution video-microscopy revealed that the alpha AGFP construct could be seen relocating to focal adhesions early in their formation and shortly thereafter to stress-fiber dense bodies. By Fluorescent Recovery After Photo-bleaching (FRAP) techniques, alpha AGFP was found to have similar exchange rates and protein stability in focal adhesions and stress fibers (despite the known differences in protein composition in these two structures). This raises the possibility that the two structures share common key regulatory factors and may not be as affected by protein-protein binding interactions as previously suggested. Additionally, the exchange rates revealed by video-microscopy and FRAP analysis of alpha AGFP are more rapid than those reported previously, which were obtained using microinjection of large excesses of fluorescently-tagged protein.

3T3 Cells↗
PubMed2006

Cytoskeletal tension regulates both expression and degradation of h2-calponin in lung alveolar cells.

Calponin is an actin filament-associated regulatory protein, and its h2 isoform is expressed in lung alveolar epithelial cells under postnatal upregulation during lung development corresponding to the commencement of respiratory expansion. Consistent with this correlation to mechanical tension, the expression of h2-calponin in alveolar cells is dependent on substrate stiffness and cytoskeleton tension. The function of h2-calponin in the stability of actin cytoskeleton implicates a role in balancing the strength and compliance of alveoli. An interesting finding is a rapid degradation of h2-calponin in lung after prolonged deflation, which is prevented by inflation of the lung to the in situ expanded volume. Decreasing mechanical tension in cultured alveolar cells by reducing the dimension of culture matrix reproduced the degradation of h2-calponin. Inhibition of myosin II ATPase also resulted in the degradation of h2-calponin in alveolar cells, showing a determining role of the tension in the actin cytoskeleton. Alveolar cells statically cultured on silicon rubber membrane build high tension in the cytoskeleton corresponding to a high expression of h2-calponin. Chronic cyclic stretching of cells on the membrane did not increase but decreased the expression of h2-calponin. This finding suggests that when cellular structure adapts to the stretched dimension, cyclic relaxations periodically release cytoskeleton tension and lower the total amount of tension that the cell senses over time. Therefore, the isometric tension, other than tension dynamics, determines the expression of h2-calponin. The tension regulation of h2-calponin synthesis and degradation demonstrates a novel mechanical regulation of cellular biochemistry.

Animals↗
PubMed1999

The coronin-like protein POD-1 is required for anterior-posterior axis formation and cellular architecture in the nematode caenorhabditis elegans.

Establishment of anterior-posterior (a-p) polarity in the Caenorhabditis elegans embryo depends on filamentous (F-) actin. Previously, we isolated an F-actin-binding protein that was enriched in the anterior cortex of the one-cell embryo and was hypothesized to link developmental polarity to the actin cytoskeleton. Here, we identify this protein, POD-1, as a new member of the coronin family of actin-binding proteins. We have generated a deletion within the pod-1 gene. Elimination of POD-1 from early embryos results in a loss of physical and molecular asymmetries along the a-p axis. For example, PAR-1 and PAR-3, which themselves are polarized and required for a-p polarity, are delocalized in pod-1 mutant embryos. However, unlike loss of PAR proteins, loss of POD-1 gives rise to the formation of abnormal cellular structures, namely large vesicles of endocytic origin, membrane protrusions, unstable cell divisions, a defective eggshell, and deposition of extracellular material. We conclude that, analogous to coronin, POD-1 plays an important role in intracellular trafficking and organizing specific aspects of the actin cytoskeleton. We propose models to explain how the role of POD-1 in basic cellular processes could be linked to the generation of polarity along the embryonic a-p axis.

Actins↗
PubMedDate not supplied

[Possibilities of measurement of impedance by the use of long-range intracerebral electrodes].

An investigation of constituents in the complex (overall) resistance of deep-lying brain structures of patients with hyperkinesis treated by employing implanted electrodes helped disclose some specific features common to the impedance of conduction pathways, ventricles and some basal ganglia. The actual value of the impedance depends upon the technical peculiarities of the method. The latter does not permit it to identify the position of the electrodes amidsts various cellular structures, but is of a definite value in defining more accurately the localization of electrodes and in identifying them.

Brain↗
PubMed2006

Morphological signs of apoptosis in axotomized ganglion cells of the rabbit retina.

Optic nerve section in mammals induces apoptotic death of retinal ganglion cells (RGCs). However, a small population of RGCs survives for a relatively long time. These cells experience significant morphological changes due to the apoptotic process, but some of these changes are not clearly differentiated from those experienced in necrotic cells. In the present work, rabbit RGCs were studied 1 month after optic nerve section using light microscopy after neurobiotin injection, transmission electron microscopy (EM) and scanning electron microscopy (SEM). Apoptosis was identified by terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling and characteristic signs of apoptosis were observed in the EM images. Ultrastructural analyses showed vacuolar degeneration in the cytoplasm and normal cellular structure loss. Signs of membrane changes were observed in axotomized RGCs by SEM. Early changes seen in the cell membrane suggest that axotomy may cause important changes in the cytoskeleton. We conclude that characteristic signs of apoptosis at the cell membrane level are clearly observed in rabbit RGCs after axotomy and they may be responsible for the cellular death.

Animals↗
PubMed1993

Clusters, superclusters, and large-scale structure: a consistent picture.

Observations of the large-scale structure in the universe using different tracers and techniques, including the spatial distribution of galaxies, clusters of galaxies, narrow pencil-beam surveys, and quasars, appear to be yielding a consistent picture of the universal structure. A network of large-scale superclusters with scales up to approximately 150h-1 Mpc is suggested (where h approximately 0.5-1 is the Hubble constant in units of 100 km.s-1.Mpc-1; 1 pc = 3.09 x 10(16) m; h = 1 is used throughout this paper). The supercluster network surrounds low-density regions, suggesting a "cellular" structure of the universe. The universal dimensionless cluster correlation function, supported by new data from automated cluster surveys, is consistent with this picture. The "standard" Omega = 1 cold dark matter (CDM) model for the universe appears to be inconsistent with the details of the observed large-scale structure distribution; a low-density, Omega approximately 0.2-0.3, CDM model provides a considerably better fit to the observations.

Journal Article↗
PubMed2005

Overexpression and properties of wild-type and Tyr437His mutated myocilin in the eyes of transgenic mice.

PURPOSE: To obtain experimental in vivo information on the functional properties of myocilin for aqueous humor (AH) outflow and to study in vivo the processing of mutated Tyr437His myocilin. Myocilin is a secreted glycoprotein that is mutated in some forms of primary open-angle glaucoma (POAG), and patients with the Tyr437His mutation have severe phenotypes. METHODS: The chicken betaB1-crystallin promoter was used to overexpress wild-type human myocilin and mutated Tyr437His myocilin in the lenses of transgenic mice. Expression of transgenic mRNA was monitored by Northern blot analysis and in situ hybridization. The localization and secretion of transgenic myocilin was investigated by Western blot analysis and light and electron microscopy. Intraocular pressure (IOP) was measured by anterior chamber cannulation. RESULTS: Two independent lines were established with each of the constructs that showed a strong expression of transgenic mRNA in their lenses. Transgenic expression resulted in a 4.7 +/- 1.8-fold increase of secreted normal myocilin in mouse AH, compared with its concentration in human AH. Immunoreactivity for transgenic myocilin was observed along the surfaces of lens and corneal endothelium, and in the chamber angle. At 12 weeks of age, the ultrastructure of the trabecular meshwork in mice expressing normal myocilin was not different from that of control eyes, and IOP of transgenic animals did not significantly differ from that of control littermates. In contrast, mutated Tyr437His myocilin was not secreted from lens fibers, but accumulated in dilated cisterns of rough endoplasmic reticulum. Although no structural changes were observed in lenses of animals expressing normal myocilin, lenses with Tyr437His expression developed nuclear cataracts, completely lost transparency, and eventually ruptured. Structural changes in lenses of Tyr437His expressing mice were correlated with a significant increase in IOP. CONCLUSIONS: The results do not support the concept that increasing amounts of myocilin in the outflow tissues obstruct the system and directly cause an increase in outflow resistance. Mutated Tyr437His myocilin is not secreted in vivo and causes severe alterations of cellular structure and function. A comparable mechanism may cause POAG in patients with myocilin mutations.

Animals↗
PubMed2006

Phylogenetic and structural analysis of centromeric DNA and kinetochore proteins.

BACKGROUND: Kinetochores are large multi-protein structures that assemble on centromeric DNA (CEN DNA) and mediate the binding of chromosomes to microtubules. Comprising 125 base-pairs of CEN DNA and 70 or more protein components, Saccharomyces cerevisiae kinetochores are among the best understood. In contrast, most fungal, plant and animal cells assemble kinetochores on CENs that are longer and more complex, raising the question of whether kinetochore architecture has been conserved through evolution, despite considerable divergence in CEN sequence. RESULTS: Using computational approaches, ranging from sequence similarity searches to hidden Markov model-based modeling, we show that organisms with CENs resembling those in S. cerevisiae (point CENs) are very closely related and that all contain a set of 11 kinetochore proteins not found in organisms with complex CENs. Conversely, organisms with complex CENs (regional CENs) contain proteins seemingly absent from point-CEN organisms. However, at least three quarters of known kinetochore proteins are present in all fungi regardless of CEN organization. At least six of these proteins have previously unidentified human orthologs. When fungi and metazoa are compared, almost all have kinetochores constructed around Spc105 and three conserved multi-protein linker complexes (MIND, COMA, and the NDC80 complex). CONCLUSION: Our data suggest that critical structural features of kinetochores have been well conserved from yeast to man. Surprisingly, phylogenetic analysis reveals that human kinetochore proteins are as similar in sequence to their yeast counterparts as to presumptive Drosophila melanogaster or Caenorhabditis elegans orthologs. This finding is consistent with evidence that kinetochore proteins have evolved very rapidly relative to components of other complex cellular structures.

Centromere↗
PubMed1993

A rapid and simple staining method, using toluidine blue, for analysing mitotic figures in tissue sections.

Mitotic index is a clinically important parameter in cancer pathology. We developed a staining method using Toluidine Blue to detect efficiently and rapidly mitotic figures in sections of formalin-fixed paraffin-embedded human and rat tissues. Sections were stained at acid pH with a 0.01% Toluidine Blue solution after removal of RNA with hydrochloric acid or ribonuclease. The optimal pH of the TB staining solution was found to be 4.5 for rat tissues and 3.5 for human tissues. This procedure stained mitotic figures much more intensely than other (extra)cellular structures. A quantitative estimate of the total number of nuclei in the field where mitotic figures were counted, was obtained in an adjacent section hydrolysed in 5 N hydrochloric acid and stained by the Feulgen reaction with a Schiff-type reagent containing 0.01% Toluidine Blue. This method specifically stained interphase and mitotic nuclei and the field cellularity could be quantified by image cytometry. When these procedures were performed on two consecutive serial sections, a mitotic index could be determined accurately by relating the count of mitotic figures to the number of tumour cells.

Animals↗
PubMed1990

Contrasting of Lowicryl K4M thin sections.

A method is presented for increasing the contrast of cellular structures on ultrathin sections from tissues embedded in Lowicryl K4M. The method, designated UA/MC adsorption staining, is based on the uranyl acetate/methyl cellulose staining of thawed cryosections. Ultrathin Lowicryl K4M sections were exposed to a uranyl acetate/methyl cellulose solution and the excess solution was removed with filter paper, leaving the remainder to air dry on the section. Sections on the grids were then directly observed in the electron microscope. Parameters such as methyl cellulose and uranyl acetate concentrations, duration of staining, temperature and pH were all assessed for their effect on subsequent contrast formation. Conditions were achieved which yielded intense contrast of cellular membranes, basement membranes and extracellular matrix components usually not apparent in Lowicryl K4M thin sections routinely counter-stained with uranyl acetate and lead acetate. The enhancement of the contrast of these structures does not obscure colloidal gold particles used for immunocytochemistry or lectin labeling, thus making the UA/MC adsorption staining method useful for increasing membrane contrast in routine post-embedding immuno- and lectin cytochemistry on Lowicryl K4M thin sections.

Acrylic Resins↗