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Permeability of dechorionated one-cell and six-somite stage zebrafish (Brachydanio rerio) embryos to water and methanol.

The permeability of dechorionated one-cell and six-somite stage zebrafish (Brachydanio rerio) embryos to water and the cryoprotectant methanol at 22 degreesC was studied, using real-time video microscopy to determine the volumetric changes of the embryos during cryoprotectant exposure. The equilibrium volumetric behavior of the embryos and the Boyle-van't Hoff relationships were determined using sucrose as a nonpermeating compound. These showed the embryos to behave as nearly ideal osmometers over the range of 253-1724 mOsm, with osmotically inactive volumes of 72.9 and 82.6% for one-cell and six-somite stage embryos, respectively. The Boyle-van't Hoff relationship of the ovary eggs was also determined for comparison and their osmotically inactive volume was 63.9%. The Kedem-Katchalsky parameters of water permeability (Lp), cryoprotectant permeability (Ps), and reflection coefficient (sigma) were determined using DIFFCHAM software. The parameters reported in this study are phenomenological parameters referring to the overall embryo response. The mean values of these parameters were Lp = 0.34 and 0.35 (micrometer/min*atm), Ps = 0.45 and 0.04 (micrometer/s), and sigma = 0.88 and 0.93 for one-cell and six-somite stage embryos, respectively. While the water permeability of the dechorionated zebrafish embryos at different developmental stages remained relatively stable, the permeability to the cryoprotectant methanol (Ps) appeared to decrease during embryo development. The Ps and sigma values for methanol are the first reported for dechorionated fish embryos at these stages.

Animals↗

Neuron-muscle contact changes presynaptic resting calcium set-point.

The role of retrograde signaling between target cells and their presynaptic partners during early cell-cell interactions was examined in cell culture. Using time-lapse video microscopy and fura-2 calcium analysis, we followed contacts between presynaptic neurites of Helisoma motoneurons and muscle fibers dissociated from identified partners in chemical synaptogenesis. Cytosolic calcium rose dramatically at the region of cell-cell contact and subsequently increased throughout the entire neuron. Changes in the calcium set-point of presynaptic neurons were maintained even when connections with muscle targets were severed. Changes in presynaptic calcium concentration were not detected following contact with partners in electrical synapse formation. These data, taken together with the fact that calcium changes coincide with the time course of chemical synaptogenesis in these neuron-muscle cultures, suggest that calcium changes may be involved in early stages of synapse formation.

Animals↗

Tracing the incorporation of the sperm tail in the mouse zygote and early embryo using an anti-testicular alpha-tubulin antibody.

The mechanism of sperm tail incorporation and the fate of the tail during mouse fertilization and early embryogenesis were examined. Time-lapse video microscopy and anti-tubulin immunofluorescence show that the incorporation of the sperm tail, but not the sperm head, is sensitive to cytochalasin B (a microfilament inhibitor). Colcemid, a microtubule inhibitor, does not affect tail incorporation. High-resolution, low-voltage scanning electron microscopy demonstrates that the plasma membrane covering the sperm tail does not appear to fuse with the oocyte membrane during in vitro fertilization in the presence of cytochalasin. In control and colcemid-treated oocytes, the plasma membrane along the sperm tail, which is oriented tangential to the egg surfaces, appears to fuse with the oocyte membrane at multiple sites. An antibody to testicular alpha-tubulin detects sperm-derived, but not egg, microtubules and this has permitted us to trace the behavior and disappearance of the sperm tail during embryogenesis. Conventional and confocal microscopy show that following sperm incorporation, the tail often splays into multiple fibers. At the two-cell stage, the axoneme may be localized in either blastomere or it may be found to run through the midbody between both blastomeres. The tail appears to shorten by the 8-cell stage and is undetectable after the 16-32 cell stage. In morulae, tail fragments have been found in outer cells but not in inner ones, and fragments have not be found in blastocysts. These data suggest that microtubules of sperm and oocytes contain different isotypes of alpha-tubulin, nongenomic sperm-derived components survive at least to the morula stage of mouse development, and egg microfilaments are involved in the incorporation of the sperm tail but not the sperm head, which demonstrates that motility during sperm incorporation is different in mammals when compared to lower vertebrates and invertebrates.

Actin Cytoskeleton↗

Caffeine-induced calcium release in sea urchin eggs and the effect of continuous versus pulsed application on the mitotic apparatus.

Unfertilized and fertilized eggs of the sea urchins Strongylocentrotus purpuratus and Lytechinus pictus, injected with the fluorescent probe calcium green dextran (Molecular Probes, Inc.) and monitored with single cell fluorimetry, rarely responded to 10 mM caffeine, but with 20 mM caffeine they produced a transient rise in intracellular calcium. Unfertilized eggs of both species produced a sharp peak approximately 2/3 the height of the normal fertilization peak, with the L. pictus response usually slightly lower and more variable. Image-intensified video microscopy of S. purpuratus eggs showed that this release originated in the center of the egg as well as in the cortex. When caffeine was applied to fertilized eggs within 10-15 min after the fertilization peak, during the period of elevated calcium, the size of the resulting calcium release increased with the elapsed time between the peak and the time of caffeine application. For example, there was no release when caffeine was applied at the height of the peak. When caffeine was washed out after the calcium level returned to baseline, there was a secondary peak (in the absence of caffeine) whose size decreased in proportion to the elapsed time. The source of this secondary calcium release is not known. Caffeine applied after the fertilization peak had returned to baseline produced a transient peak that was not followed by a secondary peak. The caffeine response gradually increased during the cell cycle. Although 10 mM caffeine did not produce a measurable calcium release, light microscopy of fixed and sectioned material showed that fertilized eggs incubated throughout the cell cycle in 7-10 mM caffeine-sea water had greatly reduced numbers of microtubules. Eggs treated at prometaphase with 10 mM caffeine rapidly lost a formed mitotic apparatus by shortening of the spindle and movement of the two centrosomes to the metaphase plate. Within 15 min all the mitotic apparatus-associated vesicles, which are known to sequester calcium, aggregated into two dense spheres of packed membranes located at the sites of the former asters. Microtubules and asters rapidly recovered when caffeine was removed. These observations suggest: (1) that the caffeine-sensitive store contributes to the fertilization calcium release and is an important sink for calcium after the fertilization peak, (2) that the caffeine-sensitive store in unfertilized eggs is located in the central part of the egg as well as in the cortex, and (3) that the caffeine-sensitive store is associated with the membrane system of the mitotic apparatus and may serve as a regulator of microtubule polymerization.

Animals↗

Migration of oligodendrocyte precursors on astrocytes and meningeal cells.

Oligodendrocytes populate developing white matter and repopulate demyelinated regions of the CNS by migration. Although little is known about their migratory routes, the environment through which these cells migrate, whether during development, disease, or injury, is packed with astrocytes infiltrated with or bounded by meningeal cells. In the present study, the migration of oligodendrocyte precursors from primary cultures and of the precursor cell lines (CG4 and Oli-neu) on astrocytes and meningeal cells was investigated using tissue culture migration assays and time lapse video microscopy. Oligodendrocyte precursors and the cell lines were found to migrate poorly on astrocytes and meningeal cells compared to migration on laminin even though both astrocytes and meningeal cells express cell surface laminin. The migration-inhibitory activity was not detected in conditioned media derived from either astrocytes or meningeal cells, nor was it detected from matrix deposited by these cells. Analyses of the events immediately following cell-cell contacts revealed that oligodendrocyte precursor-astrocyte contacts were typically long-lasting and appeared to be adhesive, whereas precursor-meningeal cell contacts usually resulted in rapid withdrawal of the precursor cell process. No correlation was found, however, between general adhesiveness and the rate of migration. Our results suggest that both astrocytes and meningeal cells retard migration of oligodendrocyte precursors, consistent with the view that they may impede the movement of oligodendrocyte precursors into CNS lesion sites.

Animals↗

Cell motility, invasion, and malignancy induced by overexpression of K-FGF or bFGF.

We have tested the hypothesis that fibroblast growth factors contribute to cell locomotion and invasion, by investigating the properties of NIH-3T3 fibroblasts that secrete K-FGF following transfection with the K-fgf proto-oncogene and NIH-3T3 fibroblasts transfected with either the normal basic fibroblast growth factor (bFGF) coding sequence that lacks a known secretory signal sequence, or a chimeric bFGF sequence fused to an immunoglobulin signal sequence that targets the growth factor to the secretory pathway. The cell lines were tested for altered invasive characteristics on a physiologically relevant collagen substratum, and their motility rates were measured in cell locomotion assays using time-lapse video microscopy in the absence or presence of suramin, an inhibitor of growth factor-receptor interaction. The data were analyzed by comparing the motility rates of all K-fgf- and bFGF-transfected cell lines as a function of malignant potential. These studies show for the first time that K-fgf gene expression potently increases cell motility characteristics. Further, the bFGF transfectants generally exhibited increased rates of cell locomotion which were not dependent upon the presence of a signal sequence for secretion. These results support models of cell locomotion in which stimulation of this process can occur through both autocrine and intracrine pathways. In addition, a significant correlation was found to exist between cell locomotion and malignant potential, supporting the view that growth factor-induced motility, although not sufficient on its own to induce metastasis, is important in the promotion of tumor cell dissemination.

3T3 Cells↗

Functional characterization of the spontaneously transformed human umbilical vein endothelial cell line ECV304: use in an in vitro model of angiogenesis.

To gain insight into the role of the endothelial cell during the pathophysiology of the angiogenic response, investigators have isolated micro- and macrovascular endothelial cells from a wide range of both animal and human vessels, including the umbilical vein. Human umbilical vein endothelial cells (HUVECs) isolated from umbilical cords remain a readily available and popular source of endothelial cell. However, the isolation and culture of these cells have several disadvantages, including the risk of infection, exogenous growth factor requirement, and low proliferative capacity. The heterogeneity of endothelial cells from different vascular beds as well as the heterogeneity between HUVEC isolates from different cords can make the critical interpretation of results difficult. ECV304 is a unique spontaneously transformed human umbilical vein endothelial cell line. In this report, the novel use of ECV304 cells as an alternative to HUVECs in an in vitro model of angiogenesis using the reconstituted basement membrane extract (Matrigel) was investigated. ECV304 cells were characterized immunohistochemically and their angiogenic behavior on Matrigel was analyzed functionally by phase-contrast, electron, and time-lapse video microscopy. ECV304 cells had several practical advantages over HUVEC culture and in contrast to HUVECs, ECV304 cells exhibited an enhanced and highly reproducible capacity for in vitro angiogenesis. However, several differentiated functions were lost or reduced in the ECV304 cell line which also exhibited anomalous cytokeratin expression. ECV304 cells may provide novel insights into the mechanisms governing angiogenesis under both normal physiological and pathological conditions.

Animals↗

A fibrin or collagen gel assay for tissue cell chemotaxis: assessment of fibroblast chemotaxis to GRGDSP.

Fibroblast chemotaxis is implicated in many physiological processes, including wound healing and morphogenesis. We present a novel assay for chemotaxis of fibroblasts (and other slow-moving tissue cells) in a direct-viewing chamber containing a physiologically relevant three-dimensional fibrin or collagen gel in which long-lasting, spatially continuous gradients have been sustained for at least 24 h, long enough for significant fibroblast migration. This combination of features is not available in any alternative assay of comparable setup simplicity. Using a putative fibroblast chemotactic factor, the fibronectin peptide GRGDSP, we measured human foreskin fibroblast alignment in the direction along the gradient, which followed a biphasic dependence on GRGDSP concentration with an optimal concentration of about 10 nM. Time-lapse video microscopy revealed that cell migration was up the soluble GRGDSP gradient, confirming positive chemotaxis to GRGDSP and rejecting the possibility of dominant haptotaxis down the soluble GRGDSP gradient, that is, up a putative gradient of integrin-mediated adhesion induced by the soluble GRGDSP gradient.

Cell Line↗

Nuclear envelope fission is linked to cytokinesis in budding yeast.

We have investigated the relationship between nuclear envelope fission and cytokinesis during mitotic cell division in budding yeast. By carrying out time-lapse and optical sectioning video microscopy analysis of cells that express green fluorescent protein (GFP)-tagged nuclear envelope and actomyosin ring components, we found that nuclear division is temporally coupled to cytokinesis. Light and electron microscopy analysis also showed that nuclear envelope fission and the division of the nucleoplasm are severely delayed in cytokinesis mutants, resulting in discoupling between the nuclear division cycle and the budding cycle. These results suggest that homotypic membrane fusion may be activated by components or the mechanical action of cytokinetic structures and presents a mechanism for the equal partitioning of the nucleus and the temporal coordination of this event with chromosome segregation during mitosis.

Cell Division↗

Effects of K+Channel blockers on acetylcholine-induced vasodilation in guinea-pig choroid.

The purpose of this study is to clarify which K+channels contribute to the acetylcholine (ACh)-induced vasodilation from the diameter changes in arterioles of the guinea-pig choroid. The choroid was isolated from the guinea-pig eyeball, pinned flat on a silicone rubber plate and superfused with warmed oxygenated (35 degrees C) Krebs solution. Diameters of choroidal arterioles were measured using video microscopy and a computer program for analysis. The effects of K+channel inhibitors (glibenclamide, tetraethylammonium [TEA], apamin and charybdotoxin [ChTX]) on the ACh-induced vasodilation were examined in arterioles which had been constricted by either norepinephrine (NE) or high K+solution. In NE (10(-5)m)-constricted arterioles, the combination of nitroarginine (10(-4)m) and indomethacin (10(-5)m) reduced ACh (10(-6)m)-induced vasodilatation by 24%. When high K+solution was used to constrict the arterioles, ACh-induced vasodilation was abolished by nitroarginine and indomethacin. In the presence of nitroarginine and indomethacin, the ACh-induced dilatation of NE-constricted arterioles was attenuated by TEA (10(-3)m), apamin (10(-7)m), and ChTX (10(-7)m) but not by glibenclamide (2x10(-5)m). Simultaneous application of apamin and ChTX inhibited the ACh (10(-6)m)-induced dilatation by 85%. In arterioles of guinea pig-choroid, nitric oxide and prostacyclin are not main mediators in ACh-induced vasodilation. Simultaneous activation of a set of Ca2+-sensitive K+channels may take most part of ACh-induced vasodilation.

Acetylcholine↗

Quantitative effects of peripheral monocytes and nerve growth factor on CNS neural morphometric outgrowth parameters in vitro.

Would healing of the central nervous system (CNS) is a complex process involving interactions between cells from both the vascular and the neural environments, extracellular matrix proteins, and a cocktail of agonistic and antagonistic bioactive molecules. Vascular cells, particularly peripheral monocytes and macrophages, are believed to play an important role in organizing and mediating CNS tissue reactions subsequent to penetrating injuries that compromise the blood-brain barrier. Although many investigators have studied the effect of macrophages and microglia (resident brain macrophages) on neural outgrowth, little is known regarding monocyte effects. We have combined tissue culture, video microscopy, and digital image processing and analysis to quantify morphometric parameters of neurons exposed to monocyte secretory products in vitro. The experimental system developed is simple in design but provides a quantitative understanding of cellular function and molecular mechanisms and has the ability to both study processes of graded complexity and relate cellular function to overall systems behavior. We evaluate the efficacy of the experimental model developed by measuring morphometric parameters of human neural cells (hNT cell line) in the presence of nerve growth factor (NGF). Results suggest that monocyte-conditioned media (MCM) increases neuron outgrowth parameters, such as neuritic output, mean arbor output, neurite branching, and effective cell diameter. Moreover, we show that the bioactive factor present in MCM is not IL-1 and the activity of the factor with respect to neural outgrowth is between that of 10 and 100 ng/ml NGF.

Cell Line↗

Lysis of Large Unilamellar Vesicles Induced by Analogs of the Fusion Peptide of Influenza Virus Hemagglutinin

We have developed a micropipette aspiration assay to observe the lysis of large (20-30 &mgr;m diameter) vesicles aspirated using micropipettes. Single membrane lysis events can be seen with the light microscope and are followed using fluorescence assays and video microscopy. In this study, we have examined the ability of two analogs of the fusion peptide from influenza virus hemagglutinin to induce the lysis of large unilamellar egg phosphatidylcholine vesicles, as a function of peptide concentration and pH. X31 is a wild-type peptide from one strain of Influenza A, and E5 is an analogue which has several residues replaced by glutamate residues. Both peptides were found to induce lysis of large vesicles in a pH-dependent manner. Both peptides exhibited maximal activity at pH 5, measured in terms of both rate and extent of lysis. E5 was active at much lower concentrations than X31. Our results with both peptides are compared to results published from other laboratories.

Journal Article↗

Colloidal Particles at Solid-Liquid Interfaces: Mechanisms of Desorption Kinetics.

We study the sorption of colloids on equally charged surfaces. Our focus is on the time scale from hours to weeks, where adsorption is not an irreversible process but interplays with (spontaneous) desorption. Using model calculations, we show how the desorption kinetics is influenced by readsorption, a potential barrier, a secondary potential minimum, local variation of the potential, and bond aging. In the experimental part we present results of in situ observation of the sorption kinetics of polystyrene latex particles onto a glass surface. Combining the evanescent field method with video microscopy, we were able to identify the particle arrival and departure times individually and therefrom determine the adhesion time distribution function. The nonexponentiality of this function can be explained by a gamma distribution of the potential depth at the binding sites as well as by logarithmic bond aging. Copyright 1998 Academic Press.

Journal Article↗

Dewetting of Thin Block Copolymer Films.

The dewetting of thin films of low molecular weight diblock and triblock copolymers of poly(oxyethylene)/poly(oxybutylene) on silicon has been studied using video microscopy and X-ray reflectivity. Dewetted films were observed to comprise polygonal domains of polymer droplets, with a domain size of several millimeters. The dewetted films were studied using X-ray reflectivity, which showed that a film of polymer <50 Å thick remains on the substrate at the same time as the macroscopic droplets. This suggests that autophobic dewetting occurred in this system; i.e., a microscopically thin film was in equilibrium with macroscopic droplets. The growth velocity of holes in the polymer film was found to be constant at a fixed temperature in the stage of hole growth that could be studied using optical microscopy. The velocity was found to be an exponential function of temperature. Copyright 1999 Academic Press.

Journal Article↗

Spreading Dynamics of Polydimethylsiloxane Drops: Crossover from Laplace to Van der Waals Spreading.

The spreading dynamics of small polydimethylsiloxane (PDMS) drops was studied on substrates with varying surface energies. For experimental parameters near the wetting transition, we observed small PDMS drops of different drop volumes as a function of time using interference video microscopy. While for large drops the contact angle &theta; decreases with the well-established power-law relation &theta; approximately t(-0.3) (Tanner's law), the effect of dispersive van der Waals (VW) interactions must be taken into account when interpreting the evolution of small drops. Two signatures of the VW forces are observed. For a positive Hamaker constant, the disjoining pressure acts as an additional driving force, leading to an acceleration of droplet spreading as soon as the drop height becomes comparable to the range of the VW interactions. In addition, a precursor film forms ahead of the contact line, leading to an apparent volume loss, particularly noticeable for very small drops. Contact line pinning may be a problem and we describe its effect on our experimental results. We present a theory that discusses the interplay of surface tension and VW forces in the case of a spreading drop. This model predicts a new spreading regime for very thin drops, in agreement with our experimental results. Copyright 2001 Academic Press.

Journal Article↗

Dinitrophenol pretreatment of rat ventricular myocytes protects against damage by metabolic inhibition and reperfusion.

We have investigated the protective effects of pretreatment with the mitochondrial uncoupler 2,4-dinitrophenol on the cellular damage induced by metabolic inhibition (with cyanide and iodoacetic acid) and reperfusion in freshly isolated adult rat ventricular myocytes. Damage was assessed from changes in cell length and morphology measured using video microscopy. Intracellular Ca(2+), mitochondrial membrane potential, and NADH were measured using fura-2, tetramethylrhodamine ethyl ester and autofluorescence, respectively. During metabolic inhibition myocytes developed rigor, and on reperfusion 73.6+/-8.1% hypercontracted and 10.8+/-6.7% recovered contractile function in response to electrical stimulation. Intracellular Ca(2+) increased substantially, indicated by a rise in the fura-2 ratio (340/380 nm) on reperfusion from 0.86+/-0.04 to 1.93+/-0.18. Myocytes pretreated with substrate-free Tyrode containing 50 microm dinitrophenol showed reduced reperfusion injury: 29.0+/-7.4% of cells hypercontracted and 65.3+/-7.3% recovered contractile function (P<0.001 vs control). The fura-2 ratio on reperfusion was also lower at 1.01+/-0.08. Fluorescence measurements showed that dinitrophenol caused mitochondrial depolarisation, and decreased NADH. The presence of the substrates glucose and pyruvate reduced these effects, and abolished the protection against damage by metabolic inhibition and reperfusion. However protection was unaffected by block of ATP-sensitive potassium channels. Thus the protective effects of pretreatment with dinitrophenol may result from a reduction in NADH in response to mitochondrial depolarisation.

Animals↗

Beta-adrenergic modulation of the collateral-dependent coronary microcirculation.

The effect of chronic, collateral-dependent perfusion on beta-adrenergic coronary microvascular responses was examined. Ameroid constrictors were placed on the proximal left circumflex (LCx) coronary artery in 16 pigs. In 8 pigs, heparinized saline containing vascular endothelial growth factor (VEGF) was administered into the perivascular space of the proximal LCx artery using an implanted osmotic pump. After 5-7 weeks, coronary arterial microvessels (70-150 microns) were studied in a pressurized (40 mm Hg) no-flow state with video-microscopy. beta-Adrenoceptor-mediated relaxations of isolated microvessels from the collateral-dependent LCx region to isoproterenol (P < 0.01) were markedly reduced, as were those to the adenylate cyclase activator forskolin (P < 0.01), compared to the respective response of vessels from the normally perfused left anterior descending artery region. Responses to the Gs-protein activator NaF showed a similar trend, but the differences were not significant. Chronic treatment with VEGF normalized responses to isoproterenol, NaF, and forskolin in the collateral-dependent LCx region. Blood flow in the LCx region increased in both control (P < 0.01) and VEGF-treated (P < 0.05) groups during rapid atrial pacing. The absolute increase in LCx blood flow was greater in the VEGF group than in the control group at rest (P < 0.05), but not during rapid pacing. Thus, beta-adrenergic microvascular relaxation is impaired in the collateral-dependent coronary microcirculation. The periadventitial delivery of VEGF improves myocardial perfusion to the collateral-dependent area and preserves beta-adrenergic-mediated relaxation of microvessels in the collateral-dependent myocardium.

Animals↗

Extracorporeal plasma perfusion of cultured hepatocytes: effect of intermittent perfusion on hepatocyte function and morphology.

The most promising approaches to developing a temporary bioartificial liver support system involve incorporating cultured primary hepatocytes into an extracorporeal perfusion device. As a result, it is important to characterize both the phenotypic response of these cells during extracorporeal perfusion and the critical factors involved in maintaining differentiated cell function over extended periods of perfusion. In this study, hepatocytes cultured in a collagen sandwich configuration were connected to a rat via a hollow fiber plasma separator and perfused with plasma on line. Perfusions were either continuous for 48 hr or intermittent for up to 174 hr with 6 hr per day of extracorporeal plasma perfusion alternating with 18 hr of culture medium perfusion. During perfusion cell morphology was continuously monitored by time-lapse video microscopy. After the procedure, hepatocytes were returned to static culture and function was evaluated by measuring the rates of urea synthesis daily for 7 days. During plasma perfusion all hepatocytes accumulated cytoplasmic lipid droplets in a time dependent manner. Urea synthesis was maintained at initial levels for up to 20 hr of continuous plasma perfusion. However, urea synthesis rates were reduced by 31 and 52% after 30 and 48 hr of continuous plasma exposure, respectively. With intermittent perfusions, as well as with control cells perfused with culture medium only, urea synthesis rates did not decrease for at least 78 hr of total perfusion. There was no difference between the urea synthesis rates after 48 hr of cumulative plasma exposure time between cells subjected to continuous and intermittent plasma perfusion. These results suggest that cultured hepatocytes may be exposed to plasma for at least 20 hr with no significant reduction in liver-specific function. Furthermore, an intermittent plasma perfusion schedule can be used to divide the useful plasma perfusion time over several days with no adverse effects on cell function.

Animals↗