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At least 1,405 records · Page 78Linked to original sources
PubMed2002

Differential activation of ion channels by inositol 1,4,5-trisphosphate (IP3)- and ryanodine-sensitive calcium stores in rat basilar artery vasomotion.

Spontaneous, rhythmical contractions, or vasomotion, can be recorded from cerebral vessels under both normal physiological and pathophysiological conditions. Using electrophysiology to study changes in membrane potential, the ratiometric calcium indicator Fura-2 AM to study changes in [Ca(2+)](i) in both the arterial wall and in individual smooth muscle cells (SMCs), and video microscopy to study changes in vessel diameter, we have investigated the cellular mechanisms underlying vasomotion in the juvenile rat basilar artery. During vasomotion, rhythmical oscillations in both membrane potential and [Ca(2+)](i) were found to precede rhythmical contractions. Nifedipine depolarized SMCs and abolished rhythmical contractions and depolarizations. [Ca(2+)](i) oscillations in the arterial wall became reduced and irregular, while [Ca(2+)](i) oscillations in adjacent SMCs were no longer synchronized. BAPTA-AM, thapsigargin and U73122 hyperpolarized SMCs, relaxed the vessel, decreased basal calcium levels and abolished vasomotion. Chloride substitution abolished rhythmical activity, depolarized SMCs, increased basal calcium levels and constricted the vessel, while niflumic acid and DIDS abolished vasomotion. Ryanodine, charybdotoxin and TRAM-34, but not iberiotoxin, 4-aminopyridine or apamin, each depolarized SMCs and increased the frequency of rhythmical depolarizations and [Ca(2+)](i) oscillations. We conclude that vasomotion in the basilar artery depends on the release of intracellular calcium from IP(3) (inositol 1,4,5,-trisphosphate)-sensitive stores which activates calcium-dependent chloride channels to depolarize SMCs. Depolarization in turn activates voltage-dependent calcium channels, synchronizing contractions of adjacent cells through influx of extracellular calcium. Subsequent calcium-induced calcium release from ryanodine-sensitive stores activates an intermediate conductance potassium channel, hyperpolarizing the SMCs and providing a negative feedback pathway for regeneration of the contractile cycle.

Animals↗
PubMed2004

Basolateral anion transport mechanisms underlying fluid secretion by mouse, rat and guinea-pig pancreatic ducts.

Fluid secretion by interlobular pancreatic ducts was determined by using video microscopy to measure the rate of swelling of isolated duct segments that had sealed following overnight culture. The aim was to compare the HCO(3)(-) requirement for secretin-evoked secretion in mouse, rat and guinea-pig pancreas. In mouse and rat ducts, fluid secretion could be evoked by 10 nm secretin and 5 microm forskolin in the absence of extracellular HCO(3)(-). In guinea-pig ducts, however, fluid secretion was totally dependent on HCO(3)(-). Forskolin-stimulated fluid secretion by mouse and rat ducts in the absence of HCO(3)(-) was dependent on extracellular Cl(-) and was completely inhibited by bumetanide (30 microm). It was therefore probably mediated by a basolateral Na(+)-K(+)-2Cl(-) cotransporter. In the presence of HCO(3)(-), forskolin-stimulated fluid secretion was reduced approximately 40% by bumetanide, approximately 50% by inhibitors of basolateral HCO(3)(-) uptake (3 microm EIPA and 500 microm H(2)DIDS), and was totally abolished by simultaneous application of all three inhibitors. We conclude that the driving force for secretin-evoked fluid secretion by mouse and rat ducts is provided by parallel basolateral mechanisms: Na(+)-H(+) exchange and Na(+)-HCO(3)(-) cotransport mediating HCO(3)(-) uptake, and Na(+)-K(+)-2Cl(-) cotransport mediating Cl(-) uptake. The absence or inactivity of the Cl(-) uptake pathway in the guinea-pig pancreatic ducts may help to account for the much higher concentrations of HCO(3)(-) secreted in this species.

Animals↗
PubMed2004

Regulation of human airway ciliary beat frequency by intracellular pH.

pHi affects a number of cellular functions, but the influence of pHi on mammalian ciliary beat frequency (CBF) is not known. CBF and pHi of single human tracheobronchial epithelial cells in submerged culture were measured simultaneously using video microscopy (for CBF) and epifluorescence microscopy with the pH-sensitive dye BCECF. Baseline CBF and pHi values in bicarbonate-free medium were 7.2 +/- 0.2 Hz and 7.49 +/- 0.02, respectively (n = 63). Alkalization by ammonium pre-pulse to pHi 7.78 +/- 0.02 resulted in a 2.2 +/- 0.1 Hz CBF increase (P < 0.05). Following removal of NH4Cl, pHi decreased to 7.24 +/- 0.02 and CBF to 5.8 +/- 0.1 Hz (P < 0.05). Removal of extracellular CO2 to change pHi resulted in similar CBF changes. Pre-activation of cAMP-dependent protein kinase (10 microM forskolin), broad inhibition of protein kinases (100 microM H-7), inhibition of PKA (10 microM H-89), nor inhibition of phosphatases (10 microM cyclosporin + 1.5 microM okadaic acid) changed pHi-mediated changes in CBF, nor were they due to [Ca2+]i changes. CBF of basolaterally permeabilized human tracheobronchial cells, re-differentiated at the air-liquid interface, was 3.9 +/- 0.3, 5.7 +/- 0.4, 7.0 +/- 0.3 and 7.3 +/- 0.3 Hz at basolateral i.e., intracellular pH of 6.8, 7.2, 7.6 and 8.0, respectively (n = 18). Thus, intracellular alkalization stimulates, while intracellular acidification attenuates human airway CBF. Since phosphorylation and [Ca2+]i changes did not seem to mediate pHi-induced CBF changes, pHi may directly act on the ciliary motile machinery.

Calcium↗
PubMed2005

Functional role of Na+-HCO3- cotransport in migration of transformed renal epithelial cells.

Cell migration is crucial for immune defence, wound healing or formation of tumour metastases. It has been shown that the activity of the Na(+)-H(+) exchanger (NHE1) plays an important role in cell migration. However, so far it is unknown whether Na(+)- HCO(3)(-) cotransport (NBC), which has similar functions in the regulation of intracellular pH (pH(i)) as NHE1, is also involved in cell migration. We therefore isolated NHE-deficient Madin-Darby canine kidney (MDCK-F) cells and tested whether NBC compensates for NHE in pH(i) and cell volume regulation as well as in migration. Intracellular pH was measured with the fluorescent pH indicator 2'7'-bis(carboxyethyl)-5-carboxyfluorescein (BCECF). The expression of NBC isoforms was determined with semiquantitative PCR. Migration was monitored with time-lapse video microscopy and quantified as the displacement of the cell centre. We found that MDCK-F cells express the isoform NBC1 (SLCA4A gene product) at a much higher level than the isoform kNBC3 (SLCA4A8 gene product). This difference is even more pronounced in NHE-deficient cells so that NBC1 is likely to be the major acid extruder in these cells and the major mediator of propionate-induced cell volume increase. NHE-deficient MDCK-F cells migrate more slowly than normal MDCK-F cells. NBC activity promotes migration during an acute intracellular acid load and increases migratory speed and displacement on a short timescale (< 30 min) whereas it has no effect on the long-term behaviour of migrating MDCK-F cells. Taken together, our results show that NBC actvity, despite many functional similarities, does not have the same importance for cell migration as NHE1 activity.

Animals↗
PubMedDate not supplied

Flow-driven diameter response in rat femoral arteries perfused in vitro.

The effects of flow and flow changes on arterial diameter were investigated in vitro on isolated rat femoral arteries. Segments of femoral arteries were excised, mounted on microcannulas, and perfused with Tyrode's solution (37 degrees C). Perfusion pressure was kept constant at 90 mm Hg. The mean external diameter after equilibration at a transmural pressure of 90 mm Hg was 720 +/- 50 microm (n = 12). Vessels were then constricted with norepinephrine (1 microM in the superfusion solution) to 77% +/- 13% of the resting diameter; acetylcholine was used to check endothelial function. The external diameter was measured continuously using video microscopy. The arteries were subjected to two different types of flow variations: (a) step changes in flow (increase and decrease, n = 6) and (b) low-frequency sinusoidal flow variations (frequencies ranging from 0.002 to 0.1 Hz, n = 11). Flow ranged from 0 to 800 microl/min (shear stress ranging from 0 to 15 dyn/cm2). All measured vessels constricted as flow increased. Flow steps induced exponential-like contractions (flow increase) or relaxations (flow decrease) with mean characteristic time constants 31 +/- 4 and 22 +/- 2 s, respectively. Sinusoidal flow oscillations induced sinusoidal diameter oscillations with a time delay. An increase in the frequency of the flow led to a decrease of both the amplitude of the flow-induced diameter oscillations and the phase shift between flow and diameter. The dynamic diameter response to flow changes could be characterized by a first-order low-pass filter with a time constant of 22 s.

Animals↗
PubMed1991

Optimization of human endothelial cell attachment to vascular graft polymers.

Endothelial cells (EC) covering the blood-contacting surface of a prosthetic material could potentially enhance the subsequent nonthrombogenicity of the surface. In order to create such a surface, the EC must become attached to the surface, spread and ultimately form a monolayer. In this study we examined several factors that influence these processes. On ePTFE surfaces, surface pretreatment with human serum for 30 minutes at a concentration of 1.4 gm percent protein resulted in significantly more attached EC when compared to other concentrations or when compared to fetal calf serum or human serum albumin. The rate of EC spreading was strongly influenced by temperature, with a maximum occurring at 37 degrees C. During real-time video microscopy, it was noted that the rate of EC attachment and spreading was primarily dependent on arrival of the EC to the surface rather than attachment and spreading. Thus as a method of increasing EC delivery, the concept of filtering EC onto the graft lumenal surface was tested by pressurizing the graft lumen to speed EC delivery to the surface. This technique produced a 2 to 5-fold increase in EC attachment when compared to gravity forced cell deposition. We conclude that an ePTFE graft can be rapidly endothelialized using these simple measures.

Biocompatible Materials↗
PubMed1991

Microscopic instrumentation and analysis of laser-tissue interaction in a skin flap model.

A dorsal skin flap model for microcirculatory studies has been modified for "in vivo" studies of laser-tissue interaction with microcirculation. An experimental apparatus has been built implementing a laser delivery system, video microscopy during irradiation, and thermal recordings. This model has been used to study irradiation effects on microcirculation using the argon laser (488 and 514.5 nm) and the argon pumped dye laser at 577 nm. The results include: measurements of the optical properties of the model; dosimetry measurements for the production of embolized and stationary coaguli in arterioles and venules; and focal vessel disappearance of venules irradiated with the argon or the argon pumped dye laser at 577 nm; a method to determine light attenuation in the model; a unique method for measurements of blood flow velocity in arterioles and venules and measurements obtained with this method; measurements of transient and steady state temperatures during irradiation and a study of laser induced photorelaxation phenomena in venules.

Animals↗
PubMed2004

Stokes drag on a sphere in a nematic liquid crystal.

The Stokes-Einstein relation relates the diffusion coefficient of a spherical Brownian particle in a viscous fluid to its friction coefficient. For a particle suspended in anisotropic liquid, theory predicts that the drag coefficient should also be anisotropic. Using video microscopy coupled with particle tracking routines, the Brownian fluctuations of micrometer-sized particles were analyzed to yield a quantitative measurement of the diffusion coefficients parallel and perpendicular to the nematic director. The experimental values agree quite well with recent numerical calculations that take into account the distortions of the director field in the vicinity of the particles.

Journal Article↗
PubMed2005

Premelting at defects within bulk colloidal crystals.

Premelting is the localized loss of crystalline order at surfaces and defects at temperatures below the bulk melting transition. It can be thought of as the nucleation of the melting process. Premelting has been observed at the surfaces of crystals but not within. We report observations of premelting at grain boundaries and dislocations within bulk colloidal crystals using real-time video microscopy. The crystals are equilibrium close-packed, three-dimensional colloidal structures made from thermally responsive microgel spheres. Particle tracking reveals increased disorder in crystalline regions bordering defects, the amount of which depends on the type of defect, distance from the defect, and particle volume fraction. Our observations suggest that interfacial free energy is the crucial parameter for premelting in colloidal and atomic-scale crystals.

Journal Article↗
PubMed2006

Brownian motion of an ellipsoid.

We studied the Brownian motion of isolated ellipsoidal particles in water confined to two dimensions and elucidated the effects of coupling between rotational and translational motion. By using digital video microscopy, we quantified the crossover from short-time anisotropic to long-time isotropic diffusion and directly measured probability distributions functions for displacements. We confirmed and interpreted our measurements by using Langevin theory and numerical simulations. Our theory and observations provide insights into fundamental diffusive processes, which are potentially useful for understanding transport in membranes and for understanding the motions of anisotropic macromolecules.

Journal Article↗
PubMed1990

Target-dependent structural changes accompanying long-term synaptic facilitation in Aplysia neurons.

The mechanisms underlying structural changes that accompany learning and memory have been difficult to investigate in the intact nervous system. In order to make these changes more accessible for experimental analysis, dissociated cell culture and low-light-level video microscopy were used to examine Aplysia sensory neurons in the presence or absence of their target cells. Repeated applications of serotonin, a facilitating transmitter important in behavioral dishabituation and sensitization, produced growth of the sensory neurons that paralleled the long-term enhancement of synaptic strength. This growth required the presence of the postsynaptic motor neuron. Thus, both the structural changes and the synaptic facilitation of Aplysia sensorimotor synapses accompanying long-term behavioral sensitization can be produced in vitro by applying a single facilitating transmitter repeatedly. These structural changes depend on an interaction of the presynaptic neuron with an appropriate postsynaptic target.

Animals↗
PubMed1999

Single-polymer dynamics in steady shear flow.

The conformational dynamics of individual, flexible polymers in steady shear flow were directly observed by the use of video fluorescence microscopy. The probability distribution for the molecular extension was determined as a function of shear rate, gamma;, for two different polymer relaxation times, tau. In contrast to the behavior in pure elongational flow, the average polymer extension in shear flow does not display a sharp coil-stretch transition. Large, aperiodic temporal fluctuations were observed, consistent with end-over-end tumbling of the molecule. The rate of these fluctuations (relative to the relaxation rate) increased as the Weissenberg number, gamma;tau, was increased.

Bacteriophage lambda↗
PubMed2000

Single-file diffusion of colloids in one-dimensional channels

Single-file diffusion, prevalent in many processes, refers to the restricted motion of interacting particles in narrow micropores with the mutual passage excluded. A single-filing system was developed by confining colloidal spheres in one-dimensional circular channels of micrometer scale. Optical video microscopy study shows evidence that the particle self-diffusion is non-Fickian for long periods of time. In particular, the distribution of particle displacement is a Gaussian function.

Journal Article↗
PubMed1987

Corkscrew-like shortening in single smooth muscle cells.

The slower and more economical contraction of smooth muscle as compared to that of skeletal muscle may relate to the arrangement of its contractile apparatus. Because the arrangement of the contractile apparatus determines the manner in which a single smooth muscle cell shortens, shortening of a contracting cell was examined by tracking of marker bead movements on the cell surface by means of digital video microscopy. Smooth muscle cells were observed to freely shorten in a unique corkscrew-like fashion with a pitch of 1.4 cell lengths (that is, the length change required for one complete rotation of cell) at a rate of 27 degrees per second. Corkscrew-like shortening was interpreted in terms of a structural model in which the contractile apparatus or cytoskeleton (or both) are helically oriented within the cell. Such an arrangement of these cytoarchitectural elements may help to explain in part the contractile capabilities of smooth muscle.

Animals↗
PubMed1994

Aminoglycoside-induced increase of intracellular calcium in LLC-PK1 cells due to an artifact caused by trypsin and EDTA.

Dietary calcium supplements attenuate experimental aminoglycoside nephrotoxicity. In cultured renal tubular cells, intracellular calcium levels have been reported to rise with aminoglycoside addition to the culture medium. In experiments designed to verify the in vitro influence of calcium on cultured kidney cells, we detected an unexpected artifact. When we resuspended cultured LLC-PK1 cells with trypsin and EDTA to measure intracellular calcium levels, our results correlated well with previously reported values. However, we saw no increase in intracellular calcium levels when we measured them by digital imaging video microscopy unless trypsin-EDTA exposure preceded aminoglycoside exposure. This apparent artifact should be considered in any study of the effects of various agents on intracellular calcium levels.

Animals↗
PubMed1995

Effects of macro- and microcirculatory functions on ceftriaxone concentrations in tissues of patients with stage IV peripheral arterial occlusive disease.

The objective of the study was to determine the ceftriaxone levels achievable within lesions (toe or forefoot area) in patients with septic gangrene and to investigate the relationship between macro- and microcirculatory parameters and antibiotic concentration. Fifteen patients with severe chronic peripheral occlusive disease received an intravenous injection of 2 g of ceftriaxone. Antibiotic levels in venous and capillary blood and in an exudative part of the lesion were measured. Macrocirculatory functions were assessed by Doppler sonography, plethysmography, and angiography; microcirculatory functions were assessed by quantitative capillaroscopy and fluorescence video microscopy. The mean antibiotic concentrations measured between 4 and 8 h after injection were 92 +/- 26 micrograms/ml in venous blood and 84 +/- 46 micrograms/ml in capillary blood. The concentration in tissue reached its maximum 4 h after injection; the average concentration at between 2 and 8 h was 95 +/- 55 micrograms/ml. Only dynamic capillary parameters showed significant (P < 0.01) correlations to antibiotic levels in tissue. Significantly (P < 0.01) higher levels in tissue were observed in patients with adequate microcirculatory functions (138 +/- 48 micrograms/ml) than in patients with poor microcirculatory function (51 +/- 26 micrograms/ml). Microcirculatory dysfunction appears to be the limiting factor for tissue antibiotic levels. However, even those patients with poor microcirculatory function showed tissue antibiotic levels that were above the MICs for the pathogens most frequently isolated from gangrenous lesions. Therefore, intravenous application was found to be adequate and additional measures such as intra-arterial therapy or Bier's occlusion are basically unnecessary. Our finding that microcirculatory function is the limiting factor for the tissue antibiotic concentration is corroborated by computations based on a three-compartment model.

Aged↗
PubMed1997

Increase in Fluorescence Intensity of 16S rRNA In Situ Hybridization in Natural Samples Treated with Chloramphenicol.

Despite the numerous advantages of fluorescent in situ hybridization for the identification of single prokaryotic cells with 16S rRNA probes, use of the technique with natural samples, especially those from the marine environment, is still problematic. The low percentage of fluorescently labeled cells constitutes the primary problem for in situ hybridization of natural samples, probably due to low cellular rRNA content. This study represents an attempt to improve detection of marine prokaryotes by increasing cellular rRNA content without changing the species composition. Cells from three California coastal sites were treated with chloramphenicol, an inhibitor of protein synthesis and rRNA degradation, at 100 (mu)g/ml and then were probed with a "universal" 16S rRNA fluorescent probe and viewed by image-intensified video microscopy. Counts of fluorescent cells increased from ca. 75% for untreated samples to ca. 93 to 99% for chloramphenicol-treated samples, compared to counts produced by DAPI (4(prm1),6-diamidino-2-phenylindole) staining, after at least 45 min of exposure to the drug (these percentages include autofluorescent cells, which averaged 6%). This suggests that most cells in these samples were active. We hypothesize that the low fluorescent-cell counts previously reported were probably often due to the fluorescence intensity of labeled cells being below the detection level rather than to high levels of dead cells in marine environments. This method may aid in the characterization of bacterioplankton with fluorescent probes.

Journal Article↗
PubMed2004

Impact of violacein-producing bacteria on survival and feeding of bacterivorous nanoflagellates.

We studied the role of bacterial secondary metabolites in the context of grazing protection against protozoans. A model system was used to examine the impact of violacein-producing bacteria on feeding rates, growth, and survival of three common bacterivorous nanoflagellates. Freshwater isolates of Janthinobacterium lividum and Chromobacterium violaceum produced the purple pigment violacein and exhibited acute toxicity to the nanoflagellates tested. High-resolution video microscopy revealed that these bacteria were ingested by the flagellates at high rates. The uptake of less than three bacteria resulted in rapid flagellate cell death after about 20 min and cell lysis within 1 to 2 h. In selectivity experiments with nontoxic Pseudomonas putida MM1, flagellates did not discriminate against pigmented strains. Purified violacein from cell extracts of C. violaceum showed high toxicity to nanoflagellates. In addition, antiprotozoal activity was found to positively correlate with the violacein content of the bacterial strains. Pigment synthesis in C. violaceum is regulated by an N-acylhomoserine lactone (AHL)-dependent quorum-sensing system. An AHL-deficient, nonpigmented mutant provided high flagellate growth rates, while the addition of the natural C. violaceum AHL could restore toxicity. Moreover, it was shown that the presence of violacein-producing bacteria in an otherwise nontoxic bacterial diet considerably inhibited flagellate population growth. Our results suggest that violacein-producing bacteria possess a highly effective survival mechanism which may exemplify the potential of some bacterial secondary metabolites to undermine protozoan grazing pressure and population dynamics.

Animals↗