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At least 433 records · Page 24Linked to original sources
PubMed2002

An automated approach for direct measurement of two-dimensional strain distributions within articular cartilage under unconfined compression.

An automated approachfor measuring in situ two-dimensional strain fields was developed and validated for its application to cartilage mechanics. This approach combines video microscopy, optimized digital image correlation (DIC), thin-plate spline smoothing (TPSS) and generalized cross-validation (GCV) techniques to achieve the desired efficiency and accuracy. Results demonstrate that sub-pixel accuracies can be achieved for measuring tissue displacements with this methodology with a measurement uncertainty ranging from 0.25 to 0.30 pixels. The deformational gradients (from which the strains are determined) can be evaluated directly using the optimized DIC, with a measurement uncertainty of 0.017 to approximately 0.032. In actual measurements of strain in cartilage, TPSS and differentiation can be used to achieve a more accurate measurement of the gradients from the displacement data. Using this automated approach, the two-dimensional strain fields inside immature bovine carpometacarpal joint cartilage specimens under unconfined compression were characterized (n=21). The depth-dependent apparent elastic modulus and Poisson's ratio were also determined and found to be smallest at the articular surface and increasing with depth. The apparent Poisson's ratio is found to decrease with increasing compressive strain, with values as low as 0.01 observed near the articular surface at 25% compression. The variation of the apparent Poisson's ratio with depth is found to be consistent with a theoretical model of cartilage which accounts for the disparity in its tensile and compressive moduli.

Animals↗
PubMed2000

Chondrocyte translocation response to direct current electric fields.

Using a custom galvanotaxis chamber and time-lapse digital video microscopy, we report the novel observation that cultured chondrocytes exhibit cathodal migration when subjected to applied direct current (DC) electric fields as low as 0.8 V/cm. The response was dose-dependent for field strengths greater than 4 V/cm. Cell migration appeared to be an active process with extension of cytoplasmic processes in the direction of movement. In some cells, field application for greater than an hour induced elongation of initially round cells accompanied by perpendicular alignment of the long axis with respect to the applied field. Antagonists of the inositol phospholipid pathway, U-73122 and neomycin, were able to inhibit cathodal migration. Cell migration toward the cathode did not require the presence of serum during field application. However, the directed velocity was nearly threefold greater in studies performed with serum. Studies performed at physiologic temperatures (approximately 37 degrees C) revealed a twofold enhancement in migration speed compared to similar studies at room temperature (approximately 25 degrees C). Findings from the present study may help to elucidate basic mechanisms that mediate chondrocyte migration and substrate attachment. Since chondrocyte migration has been implicated in cartilage healing, the ability to direct chondrocyte movement has the potential to impact strategies for addressing cartilage healing/repair and for development of cartilage substitutes.

Animals↗
PubMed2004

Requirement of Rac1 and Rac2 expression by mature dendritic cells for T cell priming.

Upon maturation, dendritic cells (DCs) acquire the unique ability to activate naïve T cells. We used time-lapse video microscopy and two-photon imaging of intact lymph nodes to show that after establishing initial contact between their dendrites and naïve T lymphocytes, mature DCs migrate toward the contacted lymphocytes. Subsequently, the DCs tightly entrap the T cells within a complex net of membrane extensions. The Rho family guanosine triphosphatases Rac1 and Rac2 but not Rho itself control the formation of dendrites in mature DCs, their polarized short-range migration toward T cells, and T cell priming.

Animals↗
PubMed2000

Single-molecule study of transcriptional pausing and arrest by E. coli RNA polymerase.

Using an optical-trap/flow-control video microscopy technique, we followed transcription by single molecules of Escherichia coli RNA polymerase in real time over long template distances. These studies reveal that RNA polymerase molecules possess different intrinsic transcription rates and different propensities to pause and stop. The data also show that reversible pausing is a kinetic intermediate between normal elongation and the arrested state. The conformational metastability of RNA polymerase revealed by this single-molecule study of transcription has direct implications for the mechanisms of gene regulation in both bacteria and eukaryotes.

DNA, Bacterial↗
PubMed2000

Differential clustering of CD4 and CD3zeta during T cell recognition.

Whereas T helper cells recognize peptide-major histocompatibility complex (MHC) class II complexes through their T cell receptors (TCRs), CD4 binds to an antigen-independent region of the MHC. Using green fluorescent protein-tagged chimeras and three-dimensional video microscopy, we show that CD4 and TCR-associated CD3zeta cluster in the interface coincident with increases in intracellular calcium. Signaling-, costimulation-, and cytoskeleton-dependent processes then stabilize CD3zeta in a single cluster at the center of the interface, while CD4 moves to the periphery. Thus, the CD4 coreceptor may serve primarily to "boost" recognition of ligand by the TCR and may not be required once activation has been initiated.

Animals↗
PubMed1995

Natural assemblages of marine bacteria exhibiting high-speed motility and large accelerations.

Natural communities of marine bacteria, an isolate (FMB-Bf3) from one marine community, and Escherichia coli were examined by video microscopy for the magnitude and uniformity of their speed. Natural communities formed tight microswarms that showed higher speeds (mean = 230 microns s-1) than did E. coli (15 microns s-1) or FMB-Bf3 (mean = 62 microns s-1). Outside the microswarms, the marine bacteria slowed to 45 microns s-1. Between turns, in mid run, and while travelling in straight lines, the natural-community bacteria accelerated up to 1,450 microns s-2 while the cultured bacteria showed maximum accelerations of 70 and 166 microns s-2. The frequency distribution of speed change for the marine bacteria was skewed towards a few large negative accelerations and a range of positive accelerations. The general pattern was one of relatively slow increases in speed followed by abrupt declines. The results indicate that the mechanical generation and energetic maintenance, as well as the environmental function, of bacterial motility need reappraisal. We conclude that the standard bacterial motility parameters of low and uniform speed, derived from culture-based studies, are not necessarily applicable to marine bacterial communities.

Escherichia coli↗
PubMed2000

Use of self-assembled monolayers of different wettabilities to study surface selection and primary adhesion processes of green algal (Enteromorpha) zoospores.

We investigated surface selection and adhesion of motile zoospores of a green, macrofouling alga (Enteromorpha) to self-assembled monolayers (SAMs) having a range of wettabilities. The SAMs were formed from alkyl thiols terminated with methyl (CH(3)) or hydroxyl (OH) groups or mixtures of CH(3)- and OH-terminated alkyl thiols and were characterized by measuring the advancing contact angles and by X-ray photoelectron spectroscopy. There was a positive correlation between the number of spores that attached to the SAMs and increasing contact angle (hydrophobicity). Moreover, the sizes of the spore groups (adjacent spores touching) were larger on the hydrophobic SAMs. Video microscopy of a patterned arrangement of SAMs showed that more zoospores were engaged in swimming and "searching" above the hydrophobic sectors than above the hydrophilic sectors, suggesting that the cells were able to "sense" that the hydrophobic surfaces were more favorable for settlement. The results are discussed in relation to the attachment of microorganisms to substrata having different wettabilities.

Cell Adhesion↗
PubMed2001

Motility of Marichromatium gracile in response to light, oxygen, and sulfide.

The motility of the purple sulfur bacterium Marichromatium gracile was investigated under different light regimes in a gradient capillary setup with opposing oxygen and sulfide gradients. The gradients were quantified with microsensors, while the behavior of swimming cells was studied by video microscopy in combination with a computerized cell tracking system. M. gracile exhibited photokinesis, photophobic responses, and phobic responses toward oxygen and sulfide. The observed migration patterns could be explained solely by the various phobic responses. In the dark, M. gracile formed an approximately 500-microm-thick band at the oxic-anoxic interface, with a sharp border toward the oxic zone always positioned at approximately 10 microM O(2). Flux calculations yielded a molar conversion ratio S(tot)/O(2) of 2.03:1 (S(tot) = [H(2)S] + [HS(-)] + [S(2-)]) for the sulfide oxidation within the band, indicating that in darkness the bacteria oxidized sulfide incompletely to sulfur stored in intracellular sulfur globules. In the light, M. gracile spread into the anoxic zone while still avoiding regions with >10 microM O(2). The cells also preferred low sulfide concentrations if the oxygen was replaced by nitrogen. A light-dark transition experiment demonstrated a dynamic interaction between the chemical gradients and the cell's metabolism. In darkness and anoxia, M. gracile lost its motility after ca. 1 h. In contrast, at oxygen concentrations of >100 microM with no sulfide present the cells remained viable and motile for ca. 3 days both in light and darkness. Oxygen was respired also in the light, but respiration rates were lower than in the dark. Observed aggregation patterns are interpreted as effective protection strategies against high oxygen concentrations and might represent first stages of biofilm formation.

Chromatiaceae↗
PubMed2004

Successful predation of filamentous bacteria by a nanoflagellate challenges current models of flagellate bacterivory.

Current models suggest that (i) filamentous bacteria are protected against predation by nanoflagellates, (ii) prey size is positively correlated with prey-predator contact probability, and (iii) contact probability is mainly responsible for size-selective predation by interception-feeding flagellates. We used five strains of filamentous bacteria and one bacterivorous nanoflagellate, Ochromonas sp. strain DS, to test these assumptions. The five strains, including one spirochete and four Betaproteobacteria strains, were isolated by the filtration-acclimatization method. All five strains possess flexible cells, but they differ in average cell length, which ranged from 4.5 to 13.7 micro m. High-resolution video microscopy was used to measure contact, capture, and ingestion rates, as well as selectivity of the flagellate feeding. Growth and feeding experiments with satiating and nonsatiating food conditions, as well as experiments including alternative well-edible prey, were performed. In contrast to predictions by current models, the flagellate successfully consumed all the tested filamentous strains. The ingestion rate was negatively correlated with bacterial length. On the other hand, the lengths of the filamentous bacteria were not positively correlated to the contact rate and capture rate but were negatively correlated to ingestion efficiency. In experiments including alternative nonfilamentous prey, the flagellates showed negative selection for filamentous bacteria, which was independent of food concentration and is interpreted as a passive selection. Our observations indicate that (i) size alone is not sufficient to define a refuge for filamentous bacteria from nanoflagellate predation and (ii) for the investigated filamentous bacteria, prey-predator contact probability could be more influenced by factors other than the prey size.

Animals↗
PubMed2001

How Giardia swim and divide.

To determine how binuclear giardia swim, we used video microscopy to observe trophozoites of Giardia intestinalis, which were labeled with an amino-specific Alexa Fluor dye that highlighted the flagella and adherence disc. Giardia swam forward by means of the synchronous beating of anterior, posterolateral, and ventral flagella in the plane of the ventral disc, while caudal flagella swam in a plane perpendicular to the disc. Giardia turned in the plane of the disc by means of a rudder-like motion of its tail, which was constant rather than beating. To determine how giardia divide, we used three-dimensional confocal microscopy, the same surface label, nuclear stains, and antitubulin antibodies. Giardia divided with mirror-image symmetry in the plane of the adherence disc, so that the right nucleus of the mother became the left nucleus of the daughter. Pairs of nuclei were tethered together by microtubules which surrounded nuclei and prevented mother or daughter giardia from receiving two copies of the same nucleus. New adherence discs formed upon a spiral backbone of microtubules, which had a clockwise rotation when viewed from the ventral surface. These dynamic observations of the parasite begin to reveal how giardia swim and divide.

Animals↗
PubMed2005

Gliding motility leads to active cellular invasion by Cryptosporidium parvum sporozoites.

We examined gliding motility and cell invasion by an early-branching apicomplexan, Cryptosporidium parvum, which causes diarrheal disease in humans and animals. Real-time video microscopy demonstrated that C. parvum sporozoites undergo circular and helical gliding, two of the three stereotypical movements exhibited by Toxoplasma gondii tachyzoites. C. parvum sporozoites moved more rapidly than T. gondii sporozoites, which showed the same rates of motility as tachyzoites. Motility by C. parvum sporozoites was prevented by latrunculin B and cytochalasin D, drugs that depolymerize the parasite actin cytoskeleton, and by the myosin inhibitor 2,3-butanedione monoxime. Imaging of the initial events in cell entry by Cryptosporidium revealed that invasion occurs rapidly; however, the parasite does not enter deep into the cytosol but rather remains at the cell surface in a membrane-bound compartment. Invasion did not stimulate rearrangement of the host cell cytoskeleton and was inhibited by cytochalasin D, even in host cells that were resistant to the drug. Our studies demonstrate that C. parvum relies on a conserved actin-myosin motor for motility and active penetration of its host cell, thus establishing that this is a widely conserved feature of the Apicomplexa.

Actins↗
PubMed1994

Substratum-induced morphological changes in a marine bacterium and their relevance to biofilm structure.

The effects of surfaces on the physiology of bacteria adhering to surfaces or immobilized within biofilms are receiving more interest. A study of the effects of hydrophobic and hydrophilic substrata on the colonization behavior of a marine bacterium, SW5, revealed major differences in the morphology of SW5 on these surfaces. Using epifluorescence, scanning confocal laser, and on-line visualization (time-lapse video) microscopy, the organisms at hydrophobic surfaces were characterized by the formation of tightly packed biofilms, consisting of single and paired cells, whereas those at hydrophilic surfaces exhibited sparse colonization and the formation of chains more than 100 microns long, anchored at the surface by the terminal (colonizing) cell. The results are discussed in terms of the possible factors inducing the observed morphological differences and the significance of these differences in terms of biofilm structure and plasmid transfer when SW5 is the recipient organism.

Bacteria↗
PubMed1995

Gliding movements in Myxococcus xanthus.

Prokaryotic gliding motility is described as the movement of a cell on a solid surface in the direction of the cell's long axis, but its mechanics are unknown. To investigate the basis of gliding, movements of individual Myxococcus xanthus cells were monitored by employing a video microscopy method by which displacements as small as 0.03 micron could be detected and speeds as low as 1 micron/min could be resolved. Single cells were observed to glide with speeds varying between 1 and 20 microns/min. We found that speed variation was due to differences in distance between the moving cell and the nearest cell. Cells separated by less than one cell diameter (0.5 micron) moved with an average speed of 5.0 micron/min, whereas cells separated by more than 0.5 micron glided with an average speed of 3.8 microns/min. The power to glide was found to be carried separately at both ends of a cell.

Cell Communication↗
PubMed2006

Palytoxin-induced cell death cascade in bovine aortic endothelial cells.

The plasmalemmal Na(+)-K(+)-ATPase (NKA) pump is the receptor for the potent marine toxin palytoxin (PTX). PTX binds to the NKA and converts the pump into a monovalent cation channel that exhibits a slight permeability to Ca(2+). However, the ability of PTX to directly increase cytosolic free Ca(2+) concentration ([Ca(2+)](i)) via Na(+) pump channels and to initiate Ca(2+) overload-induced oncotic cell death has not been examined. Thus the purpose of this study was to determine the effect of PTX on [Ca(2+)](i) and the downstream events associated with cell death in bovine aortic endothelial cells. PTX (3-100 nM) produced a graded increase in [Ca(2+)](i) that was dependent on extracellular Ca(2+). The increase in [Ca(2+)](i) initiated by 100 nM PTX was blocked by pretreatment with ouabain with an IC(50) < 1 microM. The elevation in [Ca(2+)](i) could be reversed by addition of ouabain at various times after PTX, but this required much higher concentrations of ouabain (0.5 mM). These results suggest that the PTX-induced rise in [Ca(2+)](i) occurs via the Na(+) pump. Subsequent to the rise in [Ca(2+)](i), PTX also caused a concentration-dependent increase in uptake of the vital dye ethidium bromide (EB) but not YO-PRO-1. EB uptake was also blocked by ouabain added either before or after PTX. Time-lapse video microscopy showed that PTX ultimately caused cell lysis as indicated by release of transiently expressed green fluorescent protein (molecular mass 27 kDa) and rapid uptake of propidium iodide. Cell lysis was 1) greatly delayed by removing extracellular Ca(2+) or by adding ouabain after PTX, 2) blocked by the cytoprotective amino acid glycine, and 3) accompanied by dramatic membrane blebbing. These results demonstrate that PTX initiates a cell death cascade characteristic of Ca(2+) overload.

Acrylamides↗
PubMed2004

Coverslip hypoxia: a novel method for studying cardiac myocyte hypoxia and ischemia in vitro.

In vitro experimental models designed to study the effects of hypoxia and ischemia typically employ oxygen-depleted media and/or hypoxic chambers. These approaches, however, allow for metabolites to diffuse away into a large volume and may not replicate the high local concentrations that occur in ischemic myocardium in vivo. We describe herein a novel and simple method for creating regional hypoxic and ischemic conditions in neonatal rat cardiac myocyte monolayers. This method consists of creating a localized diffusion barrier by placing a glass coverslip over a portion of the monolayer. The coverslip restricts covered myocytes to a thin film of media while leaving uncovered myocytes free to access the surrounding bulk media volume. Myocytes under the coverslip undergo marked morphology changes over time as assessed by video microscopy. Fluorescence microscopy shows that these changes are accompanied by alterations in mitochondrial membrane potential and plasma membrane dynamics and eventually result in myocyte death. We also show that the metabolic activity of myocytes drives cell necrosis under the coverslip. In addition, the intracellular pH of synchronously contracting myocytes under the coverslip drops rapidly, which further implicates metabolic activity in regulating cell death under the coverslip. In contrast with existing models of hypoxia/ischemia, this technique provides a simple and effective way to create hypoxic/ischemic conditions in vitro. Moreover, we conclude that myocyte death is hastened by the combination of hypoxia, metabolites, and acidosis and is facilitated by a reduction in media volume, which may better represent ischemic conditions in vivo.

Acidosis↗
PubMed2002

Enhanced myogenic tone in cerebral arteries from a rabbit model of subarachnoid hemorrhage.

Cerebral artery vasospasm is a major cause of death and disability in patients experiencing subarachnoid hemorrhage (SAH). Currently, little is known regarding the impact of SAH on small diameter (100-200 microm) cerebral arteries, which play an important role in the autoregulation of cerebral blood flow. With the use of a rabbit SAH model and in vitro video microscopy, cerebral artery diameter was measured in response to elevations in intravascular pressure. Cerebral arteries from SAH animals constricted more (approximately twofold) to pressure within the physiological range of 60-100 mmHg compared with control or sham-operated animals. Pressure-induced constriction (myogenic tone) was also enhanced in arteries from control animals organ cultured in the presence of oxyhemoglobin, an effect independent of the vascular endothelium or nitric oxide synthesis. Finally, arteries from both control and SAH animals dilated as intravascular pressure was elevated above 140 mmHg. This study provides evidence for a role of oxyhemoglobin in impaired autoregulation (i.e., enhanced myogenic tone) in small diameter cerebral arteries during SAH. Furthermore, therapeutic strategies that improve clinical outcome in SAH patients (e.g., supraphysiological intravascular pressure) are effective in dilating small diameter cerebral arteries isolated from SAH animals.

Animals↗
PubMed1997

Arteriolar responses to extracellular ATP in striated muscle.

Blood flow and its distribution must be appropriately regulated to ensure that perfusion is matched to local tissue demands. We investigated the role of ATP in triggering a conducted alteration in arteriolar diameter in the Saran-covered cheek pouch retractor muscle of anesthetized hamsters (n = 60). Vascular responses were observed using in vivo video microscopy upstream from the site of micropressure application of ATP (10(-8)-10(-4) M) either into the lumen or just outside the wall of first- and second-order arterioles. The role of nitric oxide (NO) in the vascular responses to ATP was determined by inhibiting NO synthase activity with N(omega)-nitro-L-arginine methyl ester (L-NAME) with and without coadministration of an excess of L-arginine. Intraluminal application of ATP led to a concentration-dependent vasodilation, which was conducted upstream along the arteriole. The dilatory response was blocked by systemic pretreatment with L-NAME and was maintained in the presence of an excess of L-arginine. In contrast, ATP introduced extraluminally resulted in a conducted vasoconstrictor response that was enhanced by pretreatment with L-NAME. The dilator response to intraluminal ATP, in the context of ATP release from erythrocytes under conditions associated with decreased supply relative to demand, supports a role for the erythrocyte in communicating local tissue needs to the vasculature, enabling the appropriate matching of oxygen supply to demand.

Adenosine↗
PubMed1997

Role of CD18-ICAM-1 in the entrapment of stimulated leukocytes in alveolar capillaries of perfused rat lungs.

This study aimed to examine the behavior of stimulated leukocytes in the pulmonary microcirculation. The leukocyte-endothelium interaction was visualized under physiological shear rates in perfused rat lungs using high-speed confocal laser video microscopy. Leukocytes labeled with carboxyfluorescein were stimulated with cytokine-induced neutrophil chemoattractant (CINC/gro), which caused L-selectin shedding and inverse upregulation of CD18. Neither unstimulated nor stimulated leukocytes exhibited rolling in either pulmonary arterioles or venules, whereas both were sequestered in capillaries. Approximately 50% of stimulated leukocytes showed a transient cessation of movement in pulmonary capillaries. The CINC/ gro stimulation, which inhibited leukocyte rolling and adhesion to mesenteric venules, reduced leukocyte velocity and increased leukocytes in pulmonary capillaries. Pretreatment with monoclonal antibodies against intercellular adhesion molecule-1 (ICAM-1) or CD18 attenuated these changes. Confocal microfluorography revealed constitutive expression of ICAM-1 not only in venules but also abundantly in capillary networks. These results suggest that selectin-independent, CD18-ICAM-1-dependent capillary sequestration is one of the major mechanisms by which activated leukocytes accumulate in the lungs.

Animals↗