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At least 433 records · Page 24Linked to original sources
PubMed1991

Cell shape and organization of the contractile apparatus in cultured adult cardiac myocytes.

The isolation and culture of adult cardiac myocytes has proved to be an ideal model system to explore myocardial biology at the cellular level. A major criticism of this model, however, has been that organ-specific characteristics such as cell shape and subcellular structural organization cannot be retained in vitro for prolonged periods of time. Encasing freshly isolated myocytes in a matrix of calcium alginate enables one to maintain the rod-like, three-dimensional (3D) shape of the cultured myocyte. Such preparations more closely resemble their in vivo counterparts with respect to the organization of the contractile apparatus, the transverse tubular system and the sarcoplasmic reticulum than do heart cells cultured on a two-dimensional (2D) plastic surface. Stereologic measurements reveal that myofibrillar volume density (VvMYF) decreases in both non-beating preparations over a 2-week interval, but VvMYF is conserved in cells cultured in an alginate matrix when compared to those myocytes maintained on a laminin-coated substratum. The present observations suggest that in the absence of contractile function myofibrillar atrophy appears responsible for the decline in VvMYF in alginate (3D) preparations, whereas atrophy and subcellular remodelling probably mediate the myofibrillar loss and reorganization that develops when adult heart cells are cultured on a 2D surface.

Alginates↗
PubMed1985

Changes in DNA and in nuclear and nucleolar dry mass and area in senescing parenchyma cells of corn cob and stalk tissues.

Corn (Zea mays L.) cob and stalk parenchyma tissues were used for a study of senescence using quantitative interference microscopy and quantitative Feulgen cytophotometry. In both parenchyma tissues, nuclear dry mass (NDM), nucleolar dry mass (NoDM), nuclear area (NA), and nucleolar area (NoA) increased during cell elongation and then decreased prior to cell death. These changes were more distinct in corn cob parenchyma tissue than in stalk parenchyma tissue. DNA content (determined for cob parenchyma, center section, only) decreased over the entire study period, slowly at first, them more rapidly. Because NoDM and NoA trends in cob parenchyma tissue were similar to those of NDM and NA but with earlier decreases, we conclude that nucleolar degeneration is an early event in corn parenchyma cell senescence. Loss of DNA also occurs earlier than loss of NDM in cob parenchyma tissue. It is further suggested that the increases in NDM as DNA content decreased represents nuclear protein and RNA content increases prior to senescence. However, no indication of nucleoli degeneration was found in stalk parenchyma cells even though NDM and NA decreased prior to cell death.

Cell Nucleolus↗
PubMed1979

Aggregation of microtubule initiation sites preceding neurite outgrowth in mouse neuroblastoma cells.

By examining microtubule regrowth using immunofluorescence with antibody to tubulin, we have studied the structure and intracellular localization of microtubule initiation sites in undifferentiated and differentiated mouse neuroblastoma cells. The undifferentiated cells are round and lack cell processes. They contain an average of 12 initiation sites per cell. Each of these sites, which are located near the cell nucleus, initiates the growth of several microtubules in a radial formation. In contrast to the undifferentiated cells, neuroblastoma cells stimulated to differentiate by serum deprivation are asymmetrical, containing one or two very long neurites. These cells have a single, large microtubule initiation center which can be visualized not only by immunofluorescence but by phase-contrast and differential interference microscopy as well. The initiation site measures 3-4 mu in diameter and is located in the cell body along a line defined by the neurite. During cell differentiation, the large initiation, the large initiation center seems to be formed by the aggregation of many smaller sites. This process procedes neurite extension by about 24 hr. The growth of microtubules from this center appears to be highly oriented, since most microtubules initially grow into the neurite processes rather than into the cell interior. Thus major changes in the structure and location of microtubule initiation sites occur during the differentiation of neuroblastoma cells. Similar changes are likely to be involved in alterations in the morphology of other cell types.

Animals↗
PubMed1988

The microtubule-dependent formation of a tubulovesicular network with characteristics of the ER from cultured cell extracts.

The formation of a dynamic tubulovesicular membrane network that resembles the endoplasmic reticulum (ER) has been observed in extracts of cultured chick embryo fibroblasts (CEF cells) using video-enhanced differential interference contrast microscopy. Initially, membranes in the CEF extracts appeared amorphous and aggregated, but with time, membrane tubules moved out along stationary microtubules. The membrane tubules formed new branches on intersecting microtubules and fused with other branches to form a network of interconnected polygons. The tubulovesicular network was solubilized by detergent and took on a beaded morphology in a hypotonic buffer. Formation of the tubulovesicular network required ATP and microtubules. The network did not contain elements of the plasma membrane, Golgi apparatus, or mitochondria but could be labeled with ER markers. We suggest that the tubulovesicular network contains components from the ER and is formed by membrane associated motors moving upon microtubules in a process we call microtubule-dependent tethering.

Adenosine Triphosphate↗
PubMed1992

Relationship between fertility of bovine semen and in vitro induction of acrosome reactions by heparin.

This study was designed to relate nonreturn rates of bulls in a commercial artificial insemination programme to the in vitro induction of acrosome reactions by the glycosaminoglycan, heparin. Semen was collected from twelve 1 to 2 year-old Holstein bulls. Washed spermatozoa from 4 to 6 ejaculates were incubated for 6 hours with 10 mug/ml heparin. Acrosome reactions were determined by differential interference microscopy. The percentage increase in acrosome reaction in heparin-treated compared to control samples was significantly correlated to the 90-day nonreturn rate of the bulls (r = 0.86; P<0.001). A significant correlation was obtained between the fertility of bulls predicted on the basis of acrosome reaction induction and achieved 90-day nonreturn rate (r=0.81; P<0.01).

Journal Article↗
PubMed1995

Purification and characterization of cytoplasmic dynein of rabbit liver.

Cytoplasmic dynein is a microtubule-dependent motor protein, which plays a role in intracellular transport. However, there have been few studies regarding the role of cytoplasmic dynein in the liver. Purification of cytoplasmic dynein from rabbit liver took advantage of the affinity of microtubule-dependent motor proteins for microtubules. Purified dynein contained heavy chain (450 kDa), intermediate chain (75 kDa), light chains (45-58 kDa) and dynactin (150 kDa). The subunit composition was consistent with previously reported data on brain cytoplasmic dynein. Microtubules prepared from bovine brain were driven by purified cytoplasmic dynein from rabbit liver, and movements of microtubules were visualized by video-enhanced differential interference contrast microscopy. The mean velocity of the motile microtubules was 1.09 +/- 0.13 microns/s. Our study provides evidence of rapid intracellular transport in hepatocytes controlled by cytoplasmic dynein.

Animals↗
PubMed1989

Flow cytometric comparison of the effects of trialkyltins on the murine erythroleukemic cell.

Cellular effects of exposure to tributyltin (TBT), triethyltin (TET), or trimethyltin (TMT) were investigated by flow cytometry employing the murine erythroleukemic cell (MELC) as a model cellular system. Cell viability was investigated by the carboxyfluorescein diacetate (CFDA) uptake/propidium iodide (PI) exclusion method: above a critical concentration (exposure for 4 h), which was specific for each of the trialkyltin compounds, the cell becomes permeable to PI, indicating loss of viability. Cellular CF fluorescence (derived from intracellular hydrolysis of CFDA) increased as a function of alkyltin concentration below the critical concentration and decreased as viability decreased above the critical concentration. Relative membrane potential, monitored with a cyanine dye (DiOC6), correlated with viability (PI exclusion), remaining essentially unaltered below the critical concentration and decreasing above it. At/above 1 microM TBT, 5 microM TET, or 100 microM TMT, the cell cycle was blocked in the G2/M phase. The 90 degrees light scatter (a measure of refractive index), axial light loss (a measure of volume), and fluorescein isothiocyanate (FITC) fluorescence (a measure of protein content) of nuclei isolated from trialkyltin-treated MELC by detergent treatment, increased as a function of organotin dose. Fluorescence and interference microscopy revealed increased quantities of residual cytoplasmic tags adherent to the nuclei as a function of organotin dose, apparently resulting from increased cytoplasmic resistance to detergent-mediated solubilization. The effects of the trialkyltins correlated with their lipophilicity (octanol/water coefficient). These data support the hypothesis that fixation (protein denaturation, cross-linking, etc.) is an important mode of organotin cytotoxicity.

Animals↗
PubMed1980

Visualization of cell-substratum adhesion plaques by antibody exclusion.

We report on an antibody exclusion method for detecting cell adhesion plaques. Substrata coated by plasma fibronectin )pFN) appeared uniformly fluorescent when analyzed by indirect immunofluorescence using a specific anti-pFN antibody. When cells that had spread on the substrata were permeabilized and analyzed by indirect immunofluorescence, areas of non-fluorescence were apparent beneath the cells and in focal streaks along the cell margins. The nonfluorescent focal streaks were coincident with adhesion plaques visualized by interference reflection microscopy.

Cell Adhesion↗
PubMed1994

How do oligodendrocytes ensheath and myelinate nerve fibers?

Oligodendrocyte precursor cells were cultured from newborn rat brain and studied their differentiation and proliferation. They have identified type-1, type-2, and type-3 oligodendrocytes based on the expression of characteristic marker molecules that frequently used to stage oligodendrocyte development. The type-3 oligodendrocytes were observed to send but tentative that locate axons prior to myelination. These processes terminate in lamellipodia, which eventually enwrap the axon and begin the myelination process with several steps. At the first stage, ruffling is immediately induced at the lamellipodia with filopodia made of oligodendrocyte processes, and the axon is contacted several times; then process retraction occurs to reform the filopodial and lamellipodial parts prior to the onset of the myelination. Second, after filopodial movements and lamellipodial ruffling occur again, their morphology is dramatically changed to become three thick filopodia that anchor to the axon. Finally, lamellipodial ruffling parts ripple, the angle between the position of the resting filopodium and the axon change, depending on the start of axonal movement, and the lamellipodia turn around the axon like a transverse wave with one stroke of the brush, as observed on the video screen, and their rolling membrane changes to the bursting form within minutes in real time.

Animals↗
PubMed2006

Characterisation of membrane mimetics on a dual polarisation interferometer.

Dual polarisation interferometry (DPI) has been used to characterise the formation of hybrid bilayer membranes (HBM) on a silicon-oxynitride surface. This technique allows the simultaneous determination of multiple physical properties of an HBM, as the HBM is being formed in a single experiment: mass, thickness in the z-direction (normal to the surface), tilt angle of the first layer and refractive index. Decanoic acid was covalently attached to an amine modified silicon-oxynitride sensor chip surface via 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride condensation reaction. The decanoic acid layer was 0.92+/-0.12 nm thick, indicating a tilt angle of 57 degrees from surface normal, and possessed a mass of 1.05+/-0.10 ng mm(-2) and a refractive index (RI) of 1.450+/-0.020. Phospholipid vesicles made from 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) or 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) were injected over the fatty acid surface to form an HBM. The DPPC HBM was 4.32+/-0.68 nm thick, with a total mass of 3.18+/-0.60 ng mm(-2) and a RI of 1.404+/-0.007. The DMPC HBM was 2.12+/-0.34 nm thick, with a total mass of 2.25+/-0.51 ng mm(-2), and a RI of 1.435+/-0.007. DPI thus provides an insight into HBM formation and differences between the structural organisation of HBMs of different composition.

Biomimetic Materials↗
PubMed2006

Homopolyvalent antibody-antigen interaction kinetic studies with use of a dual-polarization interferometric biosensor.

We used dual-polarization interferometry (DPI) to study the interaction kinetics between a 'homopolyvalent' antigen (Ag) and a monoclonal antibody (Ab). A model system, which uses a monoclonal Ab against a homopentameric Ag, C-reactive protein (CRP), is presented with principle and experiments for the study of the interactions between an Ab and an Ag that has multiple identical epitopes. This allows evaluation of the dissociation constant (K(D)) and of the binding stoichiometry by DPI based on measurements of phase changes of Ab-Ag complexes in the transverse magnetic (TM) and transverse electric (TE) polarization modes. The average experimental value of K(D) found by the DPI technique for anti-CRP Ab was shown to be in close agreement with the value obtained by an indirect competition-enzyme-linked immunosorbent assay (ELISA). Moreover, the total number of Ab combining sites on the DPI sensor chip was calculated, and the binding stoichiometry of the surface Ag-Ab complex was obtained. This study illustrates the advantages of the DPI method in biosensing in its capacity for simultaneous evaluation of the thickness and refractive index (density, mass) of adsorbed layers. This allowed a comprehensive analysis of affinity reactions between an Ab having two binding sites and a multi-sited Ag.

Antibodies, Monoclonal↗
PubMed2006

In vitro effects of micafungin against Candida biofilms on polystyrene and central venous catheter sections.

Long-term inserted and surgically implanted catheters can be colonised by Candida spp. Candida biofilms in vitro are often resistant to antifungal agents. The aim of this study was to investigate the in vitro activity of micafungin (MFG) against six Candida spp. biofilms on polystyrene (PS) and central venous catheter (CVC) sections. Safranin staining and differential interference contrast microscopy were used to demonstrate biofilm production. MFG activity was determined by the reduction in metabolic activity (%RMA) by tetrazolium reduction assay on both substrates. In vitro, Candida albicans, Candida parapsilosis, Candida glabrata, Candida tropicalis, Candida dubliniensis and Candida kefyr produced mature biofilms on PS and CVC sections. MFG was active against C. kefyr (0.5 microg/mL) and C. glabrata (<0.5 microg/mL) on PS. However, MFG displayed resistance (>16 microg/mL) against C. albicans, C. dubliniensis,C. tropicalis and C. parapsilosis. On CVC disks, MFG was active against C. glabrata (1 microg/mL) as well as C. parapsilosis and C. albicans (<0.5 microg/mL). MFG was resistant (>16 microg/mL) against C. dubliniensis, C. tropicalis and C. kefyr. MFG was active in vitro against all six Candida spp. on both substrates. However, MFG could not reduce the metabolic activity completely even at the highest concentration.

AIDS-Related Opportunistic Infections↗