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At least 1,153 records · Page 64Linked to original sources
PubMed1989

Stabilization of microtubules by tubulin-GDP-Pi subunits.

Microtubule dynamic instability has been accounted for by assuming that tubulin subunits at microtubule ends differ from the tubulin-GDP subunits that constitute the bulk of the microtubule. It has been suggested that this heterogeneity results because ends contain tubulin subunits that have not yet hydrolyzed an associated GTP molecule. Alternatively, in a recent model it was proposed that ends contain tubulin-GDP-Pi subunits from which Pi has not yet dissociated. The models differ in their predicted response to added ligands: because GDP in subunits in microtubules does not exchange with nucleotide in solution, the heterogeneity from a tubulin-GTP cap will not be eliminated by added GTP; however, the dissociability of Pi in tubulin-GDP-Pi subunits will allow a heterogeneity resulting from a tubulin-GDP-Pi cap to be eliminated by added excess Pi. Elimination of the heterogeneity is expected to be manifested by an elimination of dynamic instability behavior. Using video microscopy to study the kinetic behavior of individual microtubules under reaction conditions where dynamic instability is the dominant mechanism for microtubule length changes, we have determined the effects of 0.167 M Pi on the rate of subunit addition in the elongation phase, the rate of subunit dissociation in the rapid shortening phase, and the rates of the phase transitions from elongation to rapid shortening and from rapid shortening to growing. Since 0.167 M Pi did not decrease the subunit dissociation rate in the rapid shortening phase or the rate of the phase transition from growing to rapid shortening, our results provide no support for the hypothesis that tubulin-GDP-Pi subunits are responsible for dynamic instability behavior of microtubules.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2004

Antimitotic antifungal compound benomyl inhibits brain microtubule polymerization and dynamics and cancer cell proliferation at mitosis, by binding to a novel site in tubulin.

The antifungal agent benomyl [methyl-1-(butylcarbamoyl)-2-benzimidazolecarbamate] is used throughout the world against a wide range of agricultural fungal diseases. In this paper, we investigated the interaction of benomyl with mammalian brain tubulin and microtubules. Using the hydrophobic fluorescent probe 1-anilinonaphthalene-8-sulfonic acid, benomyl was found to bind to brain tubulin with a dissociation constant of 11.9 +/- 1.2 microM. Further, benomyl bound to at a novel site, distinct from the well-characterized colchicine and vinblastine binding sites. Benomyl altered the far-UV circular dichroism spectrum of tubulin and reduced the accessibility of its cysteine residues to modification by 5,5'-dithiobis-2-nitrobenzoic acid, indicating that benomyl binding to tubulin induces a conformational change in the tubulin. Benomyl inhibited the polymerization of brain tubulin into microtubules, with 50% inhibition occurring at a concentration of 70-75 microM. Furthermore, it strongly suppressed the dynamic instability behavior of individual brain microtubules in vitro as determined by video microscopy. It reduced the growing and shortening rates of the microtubules but did not alter the catastrophe or rescue frequencies. The unexpected potency of benomyl against mammalian microtubule polymerization and dynamics prompted us to investigate the effects of benomyl on HeLa cell proliferation and mitosis. Benomyl inhibited proliferation of the cells with an IC(50) of 5 microM, and it blocked mitotic spindle function by perturbing microtubule and chromosome organization. The greater than expected actions of benomyl on mammalian microtubules and mitosis together with its relatively low toxicity suggest that it might be useful as an adjuvant in cancer chemotherapy.

Animals↗
PubMed2005

Conformational changes in HIV-1 gp41 in the course of HIV-1 envelope glycoprotein-mediated fusion and inactivation.

HIV-1 envelope glycoprotein-mediated fusion is driven by the concerted coalescence of the HIV-1 gp41 N- and C-helical regions, which results in the formation of 6-helix bundles. These two regions are considered prime targets for peptides and antibodies that inhibit HIV-1 entry. However, the parameters that govern this inhibition have yet to be elucidated. We address this issue by monitoring the temporal sequence of conformational states of HIV-1 gp41 during the course of HIV-1-mediated cell-cell fusion by quantitative video microscopy using reagents that bind to N- and C-helical regions, respectively. Env-expressing cells were primed by incubation with target cells at different times at 37 degrees C followed by washing. The reactivity of triggered gp41 to the NC-1 monoclonal antibody, which we demonstrate here to bind to N-helical gp41 trimers, increased rapidly to a maximal level in the primed state but decreased once stable fusion junctions had formed. In contrast, reactivity with 5-helix, which binds to the C-helical region of gp41, increased continuously as a function of time following the priming. The peptide N36(Mut(e,g)) reduced NC-1 monoclonal antibody binding and enhanced 5-helix binding, consistent with the notion that this molecule promotes dissociation of gp41 trimers. This inactivation pathway may be important for the design of entry inhibitors and vaccine candidates.

AIDS Vaccines↗
PubMed1998

One-headed kinesin derivatives move by a nonprocessive, low-duty ratio mechanism unlike that of two-headed kinesin.

A single molecule of the "two-headed" motor enzyme kinesin can move along a microtubule continuously for many enzymatic turnovers (processive movement), and the velocity produced by one kinesin molecule is the same as that produced by many kinesin molecules (high duty ratio). We studied the microtubule movement driven at 1 mM ATP by biotinated N-terminal fragments of Drosophila kinesin heavy chain attached to streptavidin-coated coverslips at various surface densities. K448-BIO has velocity at a high density of vmax = 750 nm s-1 and is dimeric (hence two-headed); K365-BIO (vmax = 200 nm s-1) and K340-BIO (vmax = 90 nm s-1) are monomeric. Escape of microtubules from the surface was prevented by methylcellulose so that continuous trajectories of microtubules not continuously attached to motor molecules could be recorded by video microscopy. The component of instantaneous velocity parallel to the microtubule axis (v) was analyzed in trajectories with a mean velocity 0.4-0.7 times vmax. In K448-BIO trajectories, the distribution of v was bimodal with peaks near 0 and 750 nm s-1. Temporal autocorrelation analysis of v detected lengthy episodes of high-velocity movement consistent with isolated processive microtubule runs driven at vmax by single K448-BIO dimers. K365-BIO and K340-BIO trajectories had unimodal distributions of v and autocorrelation times much shorter than those for K448-BIO. Therefore the monomeric motors have duty ratio < 55% (i.e., no forward movement is generated for at least 45% of the enzymatic cycle time) or processivity below the detection limit of approximately 300 turnovers even in methylcellulose. Continuous movement at maximal velocity thus requires more than one kinesin head.

Adenosine Triphosphate↗
PubMed1998

Suppression of microtubule dynamics by binding of cemadotin to tubulin: possible mechanism for its antitumor action.

Cemadotin (LU103793) (NSC D-669356) is a water-soluble synthetic analogue of dolastatin 15 that inhibits cell proliferation in vitro and the growth of human tumor xenografts. Cemadotin is in phase II clinical trials as a promising cancer chemotherapeutic agent. The drug blocks cells at mitosis. Its primary mode of action has been unclear but is believed to involve an action on microtubules. We have found that cemadotin binds to tubulin and strongly suppresses microtubule dynamics. Scatchard analysis of cemadotin binding to tubulin indicated that there are two affinity classes of cemadotin-binding sites with Kd values of 19.4 microM and 136 microM. Cemadotin did not inhibit the binding of vinblastine to tubulin, and, conversely, vinblastine did not inhibit the binding of cemadotin to tubulin. By quantitative video microscopy of individual microtubules, we found that cemadotin strongly suppressed dynamic instability of microtubules assembled to steady state using bovine brain tubulin devoid of microtubule-associated proteins. It reduced the rate and extent of growing and shortening, increased the rescue frequency, and increased the percentage of time the microtubules spent in an attenuated or paused state, neither growing nor shortening detectably. At the lowest effective cemadotin concentrations, dynamics were suppressed in the absence of significant microtubule depolymerization. The results suggest that cemadotin exerts its antitumor activity by suppressing spindle microtubule dynamics through a distinct molecular mechanism by binding at a novel site in tubulin.

Animals↗
PubMed2000

Suppression of microtubule dynamic instability and treadmilling by deuterium oxide.

Deuterium oxide (D(2)O) is known to promote the assembly of tubulin into microtubules in vitro, to increase the volume of mitotic spindles and the number and length of spindle microtubules, and to inhibit mitosis. Reasoning that its actions on cellular microtubules could be due to modulation of microtubule dynamics, we examined the effects of replacing H(2)O with D(2)O on microtubule dynamic instability, treadmilling, and steady-state GTPase activity. We found that replacing 50% or more of the H(2)O with D(2)O promoted microtubule polymerization and stabilized microtubules against dilution-induced disassembly. Using steady-state axoneme-seeded microtubules composed of pure tubulin and video microscopy, we found that 84% D(2)O decreased the catastrophe frequency by 89%, the shortening rate by 80%, the growing rate by 50%, and the dynamicity by 93%. Sixty percent D(2)O decreased the treadmilling rate of microtubules composed of tubulin and microtubule-associated proteins by 42%, and 89% D(2)O decreased the steady-state GTP hydrolysis rate by 90%. The mechanism responsible for the ability of D(2)O to stabilize microtubule dynamics may involve enhancement of hydrophobic interactions in the microtubule lattice and/or the substitution of deuterium bonds for hydrogen bonds.

Animals↗
PubMed2002

Phospholipid/protein cones.

The presence of protein in tubule-forming solutions of the diacetylenic phospholipid 1,2-bis(10,12-tricosadiynoyl)-sn-glycero-3-phosphocholine results in the formation of hollow cones rather than the expected hollow cylinders. Differential phase-contrast video microscopy reveals that cones grow from proteinaceous nodules in a fashion similar to cylindrical tubule growth from spherical vesicles. Spatially resolved electron-beam energy-dispersive X-ray fluorescence spectroscopy shows the protein to be associated with the cone wall. Small-angle X-ray scattering shows that, like the protein-free cylinders, the cones are multilamellar with essentially identical interlamellar spacing.

Diynes↗
PubMed2003

Arrays of mobile tethered vesicles on supported lipid bilayers.

We report a new system of laterally mobile, arrayed vesicles that are encoded with DNA to control tethering to fluid-supported phospholipid bilayers. The motion of individual fluorescently labeled vesicles, specifically bound, are easily visualized by fluorescence video microscopy and observed to collide reversibly on the surface. This system is an ideal model for studying interactions involving membranes, in particular integral membrane proteins.

Fluorescent Dyes↗
PubMed2006

Swelling kinetics of poly(N-isopropylacrylamide) minigels.

We synthesize poly(N-isopropylacrylamide) (PNIPAM) gels with different sizes in the micrometer scale by a slight variation of a recent emulsion polymerization method (ref 1). The procedure is different than that typically used for obtaining macroscopic PNIPAM hydrogels. The resultant minigel suspension is polydisperse thus allowing the swelling kinetics for different gel sizes to be studied; we do so at temperatures below the volume-transition temperature by wetting with water previously dried particles. The resultant swelling is followed by optical video microscopy. We find that the characteristic swelling time scales with the inverse of the particle dimension squared, in agreement with theoretical predictions (ref 2). The proportionality constant is the network diffusion coefficient D, which for the minigels under consideration appears to be in between that of PNIPAM macrogels and the self-diffusion coefficient of water.

Acrylic Resins↗
PubMed2005

Direct measurement of single and ensemble average particle-surface potential energy profiles.

This work involves the development of a novel technique that integrates total internal reflection and video microscopy methods to simultaneously measure single particle and ensemble average particle-surface interactions. For the 2 mum silica colloids and glass coverslip used in this study, particle size polydispersity is found to be a dominant factor in determining the distribution of single particle profiles about ensemble average profiles. In conjunction with this observation, chemical and physical nonuniformity are not evident in any of our measurements even with sensitivity to interactions on the order of kT. One advantage of using ensemble averaging in conjunction with time averaging is the ability to dramatically decrease the time required to measure average particle-wall interactions which scales inversely with interfacial particle concentration. A number of experimental issues are addressed in the development of this technique including (1) combining single particle distribution functions, (2) statistical sampling of distribution functions using both time and ensemble averaging, and (3) correcting overlapping scattering signals between adjacent particles. The capabilities of the ensemble averaging technique are also demonstrated to provide unique measurements of particle-surface interactions in metastable systems by selecting only height excursions of levitated particles when calculating potentials. Ultimately, this new technique provides several important advantages over single particle measurements, which provides a foundation for measuring interactions in increasingly complex interfacial systems.

Journal Article↗
PubMed2005

Capillary forces between spherical particles floating at a liquid-liquid interface.

We study the capillary forces acting on sub-millimeter particles (0.02-0.6 mm) trapped at a liquid-liquid interface due to gravity-induced interface deformations. An analytical procedure is developed to solve the linearized capillary (Young-Laplace) equation and calculate the forces for an arbitrary number of particles, allowing also for a background curvature of the interface. The full solution is expressed in a series of Bessel functions with coefficients determined by the contact angle at the particle surface. For sub-millimeter spherical particles, it is shown that the forces calculated using the lowest order term of the full solution (linear superposition approximation; LSA) are accurate to within a few percents. Consequently the many particle capillary force is simply the sum of the isolated pair interactions. To test these theoretical results, we use video microscopy to follow the motion of individual particles and pairs of interacting particles at a liquid-liquid interface with a slight macroscopic background curvature. Particle velocities are determined by the balance of capillary forces and viscous drag. The measured velocities (and thus the capillary forces) are well described by the LSA solution with a single fitting parameter.

Journal Article↗
PubMed2006

Control over colloidal aggregation in monolayers of latex particles at the oil-water interface.

The controlled generation of 2D aggregate networks is studied experimentally using micrometer-sized polystyrene latex particles attached to the oil-water interface. Starting from an initially crystalline monolayer, appropriate combinations of carefully added electrolyte and surfactant enable control over both the fractal dimension and the kinetics of aggregation. Remarkably, the colloidal crystals formed upon first spreading remain stable, even for days, when substantial amounts of electrolyte are added to the aqueous phase. Pressure-area isotherms reveal a slow time evolution of the electrostatic dipole-dipole interaction. When the electrostatic interaction has been sufficiently weakened, aggregation proceeds in well-defined, reproducible manner. The aggregation process is analyzed using quantitative video microscopy. The evolution of the cluster size distribution and its moments is characterized, and static and dynamic scaling exponents are derived to identify the nature of the aggregation process. In the range of concentrations studied here, the kinetics all agree with a "fast", diffusion-limited cluster type of aggregation. However, the fractal dimension strongly depends on the composition of the aqueous subphase. Rather dense structures are found when only electrolyte is used, whereas more open structures are obtained when even small amounts of surfactant are added. It is suggested that this structural dependency is related to the effect of surfactant on the contact angle and its consequences for the anisotropic nature of the capillary interactions.

Journal Article↗
PubMed2006

Mapping patterned potential energy landscapes with diffusing colloidal probes.

We report a new method for mapping patterned surfaces based on monitoring the interactions of freely diffusing colloidal probes with pattern features to generate measured potential energy landscapes. Evanescent wave scattering and video microscopy are used to track 3D center positions of nominal 2 microm silica colloids as they diffuse over 5-20-nm-thick patterned gold films. An analysis of ensemble-averaged particle height histograms on different pattern features using Boltzmann's equation produces local electrostatic and van der Waals potentials in good agreement with independent measurements and predictions. Absolute separation is obtained from theoretical fits to measured potential-energy profiles and direct measurement by depositing silica colloids onto gold surfaces via electrophoretic deposition. As colloidal probe and pattern feature dimensions become comparable, potential energy profiles suffer some distortion due to the increased probability of probes sampling pattern feature edges. An analysis of interfacial colloidal probe diffusion in conjunction with potential energy measurements demonstrates a consistent interpretation of dissipative and conservative forces in these measurements. Future extensions of this work should produce similar approaches for interrogating physical, chemical, and biomolecular heterogeneous/patterned surfaces and structures with diffusing colloidal probes.

Colloids↗
PubMed1997

Mitogens as motogens.

Numerous in vivo methodologies have documented the invasive behavior of glioma cells through normal brain parenchyma. Glioma cell locomotion has also been assessed with a number of in vitro assays including the Boyden chamber and other chemotaxis assays, colloidal gold cell tracking, analysis of migration of cells tumor cells from spheroids, confrontation cultures of glioma cells with aggregates of non-neoplastic tissue, time-lapse video microscopy, electron microscopic examination of the cytomorphologic correlates of cell motility, the radial dish assay, and quantitative enzyme immunoassay of proteins associated with invasion (e.g. laminin). Several of these techniques have been specifically modified to assess the effects of cytokines on glioma cell motility in vitro. Cytokines studied utilizing these methods include: epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), the bb dimer of platelet-derived growth factor (PDGFbb), nerve growth factor (NGF), interleukin 2 (IL-2), transforming growth factors alpha and beta 1 (TGF alpha and TGFstraat1), and tumor necrosis factor alpha (TNF alpha). This review summarizes the investigational methods used to evaluate random and directional glioma cell motility and invasion in vivo and in vitro. The roles of specific mitogens as motogens, as evaluated with these methods are then presented.

Animals↗
PubMed1998

Induction of apoptosis by gemcitabine in BG-1 human ovarian cancer cells compared with induction by staurosporine, paclitaxel and cisplatin.

A variety of chemotherapeutic agents induce cell death via apoptosis. We had shown previously that gemcitabine (2',2'-difluorodeoxycytidine) induced an atypical apoptosis in BG-1 human ovarian cancer cells; therefore, further studies were conducted to characterize more precisely gemcitabine-induced apoptosis in BG-1 cells compared to a general inducer of apoptosis, staurosporine. BG-1 cells exposed to 0.5, 1.0 and 10 microM gemcitabine for 8 h, or staurosporine (1.0 microM) for 6 h, exhibited high molecular weight DNA fragmentation (50 kbp); however, only staurosporine treatment produced internucleosomal DNA fragments (200 bp) in a laddered pattern on the agarose gel. Staurosporine (1.0 microM) rapidly induced phosphatidylserine plasma membrane translocation that increased linearly with time as measured by annexin V-FITC binding, and similar kinetics were observed for caspase activation by staurosporine in BG-1 cells. In contrast, 10 microM gemcitabine increased phosphatidylserine expression in a small fraction of cells (5-10%) vs. untreated controls over the course of 48 h and significant caspase activity was detected within 12 h of drug exposure. Time-lapse video microscopy of BG-1 cells exposed to 1.0 microM staurosporine or 10 microM gemcitabine for up to 72 h showed that the morphologic changes and kinetics of cell death induced by these agents differed significantly. We also evaluated the apoptosis induced by paclitaxel (a mitotic poison) and cisplatin (an agent not dependent on cell cycle functions) in BG-1 cells by these methods because these drugs are used clinically to treat ovarian cancer. Our findings demonstrate that the kinetics of apoptotic cell death induced by gemcitabine and other chemotherapeutic agents should be taken into account when designing treatment strategies for ovarian cancer.

Journal Article↗
PubMed2001

Platelet-activating factor evokes Ca2+ transients after the blockade of ryanodine receptor by dantrolene in RAW 264.7 macrophages.

In the present study we studied platelet-activating factor (PAF)-, and ATP-induced increases in intracellular Ca2+ concentration ([Ca2+]i) using RAW 264.7 macrophages filled with fura-2/AM and imaged with fluorescence video microscopy. We found that the prevalence of detectable [Ca2+]i responses to PAF application was significantly higher in the presence of dantrolene. Dantrolene itself significantly decreased basal [Ca2+]i of macrophages compared to control cases after a 20-min incubation period. In the dantrolene-treated cells even the peak [Ca2+]i in response to PAF (as an average of all cells) was below the baseline of control suggesting that decreased [Ca2+]i plays a permissive role in the Ca2+ rise induced by PAF in macrophages. In contrast to the effect of PAF, neither the amplitude of response to ATP nor the frequency of responding cells changed significantly during dantrolene treatment in our experiments. These cells were able to respond to a standard immune stimulus as well: lipopolysaccharide (LPS) was able to increase [Ca2+]i. Our data indicate that the effectiveness of PAF to increase [Ca2+]i in RAW 264.7 macrophages depends on the resting [Ca2+]i. It has also been shown in this study that PAF and ATP differently regulate Ca2+ homeostasis in macrophages during inflammatory response and therefore they possibly differently modulate cytokine production by macrophages.

Adenosine Triphosphate↗
PubMed1993

Transport of ionic species in skin: contribution of pores to the overall skin conductance.

Two methods are reported that allow visualization of high conductance paths in skin at current densities typically used during clinical iontophoretic drug delivery (10-200 microA/cm2). In the first method, the counter-directional iontophoretic transport of Fe(CN)6(4-) and Fe3+ across skin results in the precipitation of colloidal prussian blue, Fe4[Fe(CN)6]3, at sites of high iontophoretic flux. The appearance of localized deposits of Fe4[Fe(CN)6]3 is recorded by video microscopy and used to document the activation of low-resistance paths. In the second method, the ionic flux of Fe(CN)6(4-) through pores is directly imaged by scanning electrochemical microscopy (SECM). Both methods demonstrate that the iontophoretic flux across skin is highly localized. Activation of low-resistance pores in hairless mouse skin is shown to occur during iontophoresis. The spatial density of current carrying pores increases from 0 to 100-600 pores/cm2 during the first 30-60 min of iontophoresis. At longer times, the active pore density approaches a quasi-steady-state value that is proportional to the applied current density. The total conductance of the skin is proportional to the number of pores, consistent with a model of conduction in skin that is comprised of low-resistivity pores in parallel with a high-resistivity bulk phase. The contribution of pores to the total skin conductance during iontophoresis increases from an initial value of 0-5% to a quasi-steady-state value of 50-95%.

Animals↗
PubMed2002

The significance of inter- and intraspecific variation in bacterivorous and herbivorous protists.

This paper reviews the emerging evidence on the significance of inter- and intraspecific variation in the feeding behaviour of aquatic protists. Small heterotrophic nanoflagellates (HNF) have been identified as the primary bacterial consumers in most aquatic environments. Recent research using novel techniques such as flow cytometry and high resolution video microscopy revealed that their feeding strategies and grazing rates are diverse. There is an important conceptual difference between uptake rates measured in short-term (min to h) experiments and grazing rates averaged over a longer-term (d). This is because the latter are strongly affected by digestion rates which are species-specific, i.e. the same bacterial prey may be digested differently by various grazers, and the same predator may selectively digest variable prey. Planktonic ciliates are the most important algal consumers in many lakes and marine systems. Large species-specific differences in their feeding behaviour and growth rates have been documented for closely related species. Intraspecific variation, which is, most likely, caused by varying clonal composition may be as important as interspecific variation. Finally, there is some evidence that the individual variability within a given population is generally large, both among bacterivorous HNF and among herbivorous ciliates. The consequences of this diversity becoming apparent at the levels of the species, population, clone and individual need to be considered by aquatic ecologists in their conceptual models.

Animals↗