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Biomedical subjects

Michael R King

Publications and source records attributed to Michael R King.

14 recordsLinked to original sources
PubMed2006

Mechanical shedding of L-selectin from the neutrophil surface during rolling on sialyl Lewis x under flow.

The interaction of L-selectin expressed on leukocytes with endothelial cells leads to capture and rolling and is critical for the recruitment of leukocytes into sites of inflammation. It is known that leukocyte activation by chemoattractants, the change of osmotic pressure in cell media, or cross-linking of L-selectin all result in rapid shedding of L-selectin. Here we present a novel mechanism for surface cleavage of L-selectin on neutrophils during rolling on a sialyl Lewis x-coated surface that involves mechanical force. Flow cytometry and rolling of neutrophils labeled with Qdot(R)-L-selectin antibodies in an in vitro flow chamber showed that the mechanical shedding of L-selectin occurs during rolling and depends on the amount of shear applied. In addition, the mechanical L-selectin shedding causes an increase in cell rolling velocity with rolling duration, suggesting a gradual loss of L-selectin and is mediated by p38 mitogen-activated protein kinase activation. Thus, these data show that mechanical force induces the cleavage of L-selectin from the neutrophil surface during rolling and therefore decreases the adhesion of cells to a ligand-presenting surface in flow.

Humans↗
PubMed2006

Adhesive dynamics simulations of the shear threshold effect for leukocytes.

Many experiments have measured the effect of force on the dissociation of single selectin bonds, but it is not yet clear how the force dependence of molecular dissociation can influence the rolling of cells expressing selectin molecules. Recent experiments using constant-force atomic force microscopy or high-resolution microscopic observations of pause-time distributions of cells in a flow chamber show that for some bonds, the dissociation rate is high at low force and initially decreases with force, indicating a catch bond. As the force continues to increase, the dissociation rate increases again, like a slip bond. It has been proposed that this catch-slip bond leads to the shear threshold effect, in which a certain level of shear rate is required to achieve rolling. We have incorporated a catch-slip dissociation rate into adhesive dynamics simulations of cell rolling. Using a relatively simple model for the shear-controlled association rate for selectin bonds, we were able to recreate characteristics of the shear threshold effect seen most prominently for rolling through L-selectin. The rolling velocity as a function of shear rate showed a minimum near 100 s-1. Furthermore, cells were observed to roll at a shear rate near the threshold, but detach and move more quickly when the shear rate was dropped below the threshold. Finally, using adhesive dynamics, we were able to determine ranges of parameters necessary to see the shear threshold effect in the rolling velocity. In summary, we found through simulation that the catch-slip behavior of selectin bonds can be responsible for the shear threshold effect.

Cell Adhesion↗
PubMed2006

Interviewing the incarcerated offender convicted of sexually assaulting the elderly.

Sexual assault is considered one of the most terrible crimes committed against the elderly, particularly as they are noted to be among the most innocent and vulnerable of populations. To better understand offender motivation and related behaviors, this article presents suggestions that are possible guidelines for forensic interviews, including an examination of potential methods for obtaining cooperation to optimize a truthful and accurate disclosure of offenders' crimes for research purposes.

Aged↗
PubMed2005

Anisotropic Brownian diffusion near a nanostructured surface.

The Brownian diffusion of sub-micrometer-sized particles (diameter 0.52 microm) in the vicinity of a nanostructured surface was experimentally characterized. The surface consisted of a repeating pattern of rectangular grooves with depth 35 nm and pitch 400 nm. It was found that the one-dimensional particle diffusivity parallel to the nanogrooves was significantly higher than the diffusivity perpendicular to the grooves (1.518+/-0.274 (S.E.) microm2/s compared to 0.704+/-0.090 microm2/s). No such anisotropy in the one-dimensional Brownian diffusivity was found for particles near a flat surface.

Anisotropy↗
PubMed2005

Rolling dynamics of a neutrophil with redistributed L-selectin.

The most common white blood cell is the neutrophil, which slowly rolls along the walls of blood vessels due to the coordinated formation and breakage of chemical selectin-carbohydrate bonds. We show that L-selectin receptors are rapidly redistributed to form a cap at one end of the cell membrane during rolling via selectins or chemotactic stimulation. This topography significantly alters the adhesive dynamics as demonstrated by computer simulations of neutrophils rolling on a carbohydrate selectin-ligand substrate under flow. It was found that neutrophils with a redistributed L-selectin cap roll on sialyl Lewis-x with a quasi-periodic motion, as characterized by relatively low velocity intervals interspersed with regular jumps in the rolling velocity. On average, neutrophils with redistributed L-selectin rolled at a lower velocity when compared with cells having a uniform L-selectin distribution of equal average density. We speculate on the possible biological implications that these differences in adhesion dynamics will have during the inflammatory response.

Cell Adhesion↗
PubMed2005

The distribution of rolling neutrophils in venular convergences.

The aim of this study was to characterize the distribution of adherent leukocytes in branched venular convergences in vivo. Intravital microscopy was used to obtain video images of leukocyte adhesion in multiple branched sites in mouse cremaster muscle, during the mild inflammatory response induced by surgical preparation. The average number of cells/vessel length was obtained over several minutes for seven venular convergences with varying geometrical configurations. Results from this study demonstrate a strong tendency of leukocytes to adhere at junctional points between converging vessels. Different vessel configurations were studied and results were shown to be insensitive to precise vessel geometry. Thus, in post-capillary venules, leukocytes are most likely to adhere at points between converging vessels, regardless of the precise geometrical properties or configuration of the vessels. Hydrodynamic mechanisms due to flow behavior through convergences likely play a significant role in determining locations of cellular adhesion. Future work should concentrate on quantifying the relative contributions of hydrodynamic and biochemical mechanisms to aid in understanding disease processes and development of treatments or therapeutics.

Animals↗
PubMed2004

Nano-to-micro scale dynamics of P-selectin detachment from leukocyte interfaces. III. Numerical simulation of tethering under flow.

Transient capture of cells or model microspheres from flow over substrates sparsely coated with adhesive ligands has provided significant insight into the unbinding kinetics of leukocyte:endothelium adhesion complexes under external force. Whenever a cell is stopped by a point attachment, the full hydrodynamic load is applied to the adhesion site within an exceptionally short time-less than the reciprocal of the hydrodynamic shear rate (e.g., typically <0.01 s). The decay in numbers of cells or beads that remain attached to a surface has been used as a measure of the kinetics of molecular bond dissociation under constant force, revealing a modest increase in detachment rate at growing applied shear stresses. On the other hand, when detached under steady ramps of force with mechanical probes (e.g., the atomic force microscope and biomembrane force probe), P-selectin:PSGL-1 adhesion bonds break at rates that increase enormously under rising force, yielding 100-fold faster off rates at force levels comparable to high shear. The comparatively weak effect of force on tether survival in flow chamber experiments could be explained by a possible partition of the load amongst several bonds. However, a comprehensive understanding of the difference in kinetic behavior requires us to also inspect other factors affecting the dynamics of attachment-force buildup, such as the interfacial compliance of all linkages supporting the adhesion complex. Here, combining the mechanical properties of the leukocyte interface measured in probe tests with single-bond kinetics and the kinetics of cytoskeletal dissociation, we show that for the leukocyte adhesion complex P-selectin:PSGL-1, a detailed adhesive dynamics simulation accurately reproduces the tethering behavior of cells observed in flow chambers. Surprisingly, a mixture of 10% single bonds and 90% dimeric bonds is sufficient to fully match the data of the P-selectin:PSGL-1 experiments, with the calculated decay in fraction of attached cells still appearing exponential.

Binding Sites↗
PubMed2004

Mechanics of transient platelet adhesion to von Willebrand factor under flow.

A primary and critical step in platelet attachment to injured vascular endothelium is the formation of reversible tether bonds between the platelet glycoprotein receptor Ibalpha and the A1 domain of surface-bound von Willebrand factor (vWF). Due to the platelet's unique ellipsoidal shape, the force mechanics involved in its tether bond formation differs significantly from that of leukocytes and other spherical cells. We have investigated the mechanics of platelet tethering to surface-immobilized vWF-A1 under hydrodynamic shear flow. A computer algorithm was used to analyze digitized images recorded during flow-chamber experiments and track the microscale motions of platelets before, during, and after contact with the surface. An analytical two-dimensional model was developed to calculate the motion of a tethered platelet on a reactive surface in linear shear flow. Through comparison of the theoretical solution with experimental observations, we show that attachment of platelets occurs only in orientations that are predicted to result in compression along the length of the platelet and therefore on the bond being formed. These results suggest that hydrodynamic compressive forces may play an important role in initiating tether bond formation.

Antigens↗
PubMed2004

Apparent 2-D diffusivity in a ruffled cell membrane.

Most biological cell membranes have a microtopology that increases their surface area, including a highly ruffled surface in the case of leukocytes. Thus, molecular membrane diffusivities as measured by fluorescence recovery after photobleaching or other methods are decreased when projected onto a plane. We use a two-dimensional crested cycloid as a parameterized surface to simulate the random-walk diffusion of a molecule within a ruffled membrane. The apparent 2-D diffusivity was then calculated when the ruffled membrane is projected onto a plane. It is shown that the apparent diffusivity decreases as a function of the membrane area, to the -1.4 power.

Animals↗
PubMed2004

The effect of hematocrit and leukocyte adherence on flow direction in the microcirculation.

We sought to characterize how adherent leukocytes at the vessel wall, and the presence of erythrocytes, alter the streamlines (paths) of blood flow in the postcapillary venules. We directly visualized blood flow and leukocyte-endothelial cell interactions in postcapillary venules located in the cremaster muscle of anesthetized mice. Fluid streamlines were visualized by perfusing the cremaster muscle tissue with 0.5-micron fluorescent beads suspended in either buffer or whole blood, to examine the effect that erythrocytes have on the directionality of flow. Acute inflammation was induced in some animals by pretreatment of the vessels with tumor necrosis factor-alpha. To quantify the flow direction, the average deflection angle was defined as a scalar metric. Tracer bead trajectories were measurably altered by the presence of systemic levels of hematocrit, determined in each animal to be about 45%. Deviation from undirectional flow was also found to: (i) decrease with increasing vessel diameter, and (ii) increase with the number of adherent leukocytes. Fluid streamlines in the presence or absence of leukocyte adhesion or red cells agreed qualitatively with those obtained from theoretical calculations of blood flow using multiparticle adhesive dynamics. The microscale characteristics of venular flow are significantly altered during inflammation or changes in local hematocrit.

Animals↗
PubMed2003

The state diagram for cell adhesion mediated by two receptors.

Leukocyte recruitment from the bloodstream to surrounding tissues is an essential component of the immune response. Capture of blood-borne leukocytes onto vascular endothelium proceeds via a two-step mechanism, with each step mediated by a distinct receptor-ligand pair. Cells first transiently adhere, or "roll" (via interactions between selectins and sialyl-Lewis-x), and then firmly adhere to the vascular wall (via interactions between integrins and ICAM-1). We have reported that a computational method called adhesive dynamics (AD) accurately reproduces the fine-scale dynamics of selectin-mediated rolling. This paper extends the use of AD simulations to model the dynamics of cell adhesion when two classes of receptors are simultaneously active: one class (selectins or selectin ligands) with weakly adhesive properties, and the other (integrins) with strongly adhesive properties. AD simulations predict synergistic functions of the two receptors in mediating adhesion. At a fixed density of surface ICAM-1, increasing selectin densities lead to greater pause times and an increased tendency toward firm adhesion; thus, selectins mechanistically facilitate firm adhesion mediated by integrins. Conversely, at a fixed density of surface selectin, increasing ICAM-1 densities lead to greater pause times and an increased tendency to firm adhesion. We present this relationship in a two-receptor state diagram, a map that relates the densities and properties of adhesion molecules to various adhesive behaviors that they code, such as rolling or firm adhesion. We also present a state diagram for neutrophil activation, which relates beta(2)-integrin density and integrin-ICAM-1 kinetic on rate to neutrophil adhesive behavior. The predictions of two-receptor adhesive dynamics are validated by the ability of the model to reproduce in vivo neutrophil rolling velocities from the literature.

Binding Sites↗
PubMed2003

Hydrodynamic interactions between rolling leukocytes in vivo.

OBJECTIVE: The aim of this study was to characterize the hydrodynamic interactions between rolling and free-stream leukocytes in an in vivo model of selectin-mediated rolling, and to identify those physical mechanisms that influence the dynamics of transient adhesion with the walls of postcapillary venules. METHODS: Postcapillary venules of diameter 22-37 microm in the cheek pouch of anesthetized hamsters were visualized using intravital microscopy, with selectin-mediated rolling occurring in response to surgical preparation. RESULTS: Rolling velocity was found to be a strong function of the center-to-center separation with the nearest cell, and also was found to correlate strongly with the number of nearby cells. These effects are shown to be beyond that attributable to variations along the length of the vessel. Adherent leukocytes were observed to provide a nucleation site, precipitating further adhesion events of free-stream cells. CONCLUSIONS: The dynamics of the transient adhesion of leukocytes to the vessel wall in postcapillary venules is strongly dependent on the local concentration of adherent leukocytes, due to the complex hydrodynamics induced by their presence. The results are shown to agree well with theoretical considerations of the flow field induced by multiple nearby cells, suggesting a need for the future exploration of multicellular effects in the microcirculation.

Animals↗
PubMed2002

Purification and characterization of a thermostable alpha-galactosidase from Thermoanaerobacterium polysaccharolyticum.

Food ingredients containing alpha-1,6-galactoside bonds elicit gastrointestinal disturbances in monogastric animals, including humans. Pretreatment of such ingredients with alpha-galactosidase (EC 3.2.1.22) has the potential to alleviate this condition. For this purpose, a thermostable alpha-galactosidase from Thermoanaerobacterium polysaccharolyticum was purified by a combination of anion exchange and size exclusion chromatographies. The enzyme has a monomeric molecular weight of approximately 80 kDa; however, it is active as a dimer. The optimum temperature for enzyme activity is 77.5 degrees C. Approximately 84 and 88% of enzyme activity remained after 36.5 h of incubation at 70 and 65 degrees C, respectively. Optimum activity was observed at pH 8.0, with a broad range of activity from pH 5.0 to 9.0. Different transition metals had weak to strong inhibitory effects on enzyme activity. The K(m) and V(max) of the enzyme are 0.29-0.345 mM and 200-232 micromol/min/mg of protein, respectively. Importantly, enzyme activity was only slightly inhibited by 75-100 mM galactose, an end product of hydrolysis. Enzyme activity was specific for the alpha-1,6-galactosyl bond, and activity was demonstrated on melibiose and soy molasses.

Amino Acid Sequence↗