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H P Ting-Beall

Publications and source records attributed to H P Ting-Beall.

At least 19 recordsLinked to original sources
PubMed2001

Correlating the kinetics of cytokine-induced E-selectin adhesion and expression on endothelial cells.

Many human diseases are mediated through the immune system. In chronic inflammatory disorders, the processes ordinarily involved in tissue healing become destructive. Endothelial cells normally recruit leukocytes to inflamed tissue using cytokine-induced adhesion receptors on the surfaces of interacting cells. Leukocyte capture depends on specialized characteristics of these receptors, particularly the binding kinetics. This study is designed to clarify the relationship between cytokine-induced changes in cell properties and binding kinetics. Here, we measure the kinetics of expression and monoclonal antibody binding for E-selectin in interleukin-1alpha-stimulated microvascular endothelium in vitro and incorporate the data into kinetic models. Quantitative flow cytometry is used to determine molecular density (expression), and micropipette assays are used to find the probability of adhesion (function). Within five hours of interleukin-1alpha stimulation, E-selectin density increases from 0 to 742 sites/microm(2), and antibody-E-selectin adhesion probability increases from a baseline of 6.3% to 64%. A kinetic model is applied to find an apparent association rate constant, k(f), of 3.7 x 10(-14) cm(2)/sec for antibody-E-selectin binding. Although the model successfully predicts experimental results, the rate constant is undervalued for a diffusion-limited process, suggesting that functional adhesion may be modified through cytokine-induced changes in microtopology and receptor localization.

Animals↗
PubMed1999

Viscoelastic properties of intervertebral disc cells. Identification of two biomechanically distinct cell populations.

STUDY DESIGN: A combined experimental and theoretical biomechanical study to quantify the mechanical properties of living cells of the porcine intervertebral disc. OBJECTIVES: To quantify zonal variations in the mechanical properties and morphology of cells isolated from the intervertebral disc. SUMMARY OF BACKGROUND DATA: Cellular response to mechanical stimuli is influenced by the mechanical properties of cells and of the extracellular matrix. Significant zonal variations in intervertebral disc matrix properties have been reported. No information is currently available on the corresponding regional variations in the mechanical properties of intervertebral disc cells, despite evidence of significant differences in cellular phenotype and biologic response to loading. METHODS: The micropipette aspiration test was used in combination with a three-parameter viscoelastic solid model to measure the mechanical properties of cells isolated from the anulus fibrosus, transition zone, and nucleus pulposus. RESULTS: Intervertebral disc cells exhibited viscoelastic solid behaviors. Highly significant differences were observed in the morphology, cytoskeletal arrangement, and biomechanical properties of the nucleus pulposus cells as compared with anulus fibrosus or transition zone cells. Cells of the nucleus pulposus were approximately three times stiffer and significantly more viscous than cells of the anulus fibrosus or transition zone. CONCLUSIONS: The findings of this study provide new evidence for the existence of two biomechanically distinct cell populations in the intervertebral disc. These differences in mechanical behavior may be related to observed differences in the cytoskeletal architecture between these cells, and may further play an important role in the development, maintenance, and degeneration of the intervertebral disc.

Animals↗
PubMed1999

Myosin I contributes to the generation of resting cortical tension.

The amoeboid myosin I's are required for cellular cortical functions such as pseudopod formation and macropinocytosis, as demonstrated by the finding that Dictyostelium cells overexpressing or lacking one or more of these actin-based motors are defective in these processes. Defects in these processes are concomitant with changes in the actin-filled cortex of various Dictyostelium myosin I mutants. Given that the amoeboid myosin I's possess both actin- and membrane-binding domains, the mutant phenotypes could be due to alterations in the generation and/or regulation of cell cortical tension. This has been directly tested by analyzing mutant Dictyostelium that either lacks or overexpresses various myosin I's, using micropipette aspiration techniques. Dictyostelium cells lacking only one myosin I have normal levels of cortical tension. However, myosin I double mutants have significantly reduced (50%) cortical tension, and those that mildly overexpress an amoeboid myosin I exhibit increased cortical tension. Treatment of either type of mutant with the lectin concanavalin A (ConA) that cross-links surface receptors results in significant increases in cortical tension, suggesting that the contractile activity of these myosin I's is not controlled by this stimulus. These results demonstrate that myosin I's work cooperatively to contribute substantially to the generation of resting cortical tension that is required for efficient cell migration and macropinocytosis.

Animals↗
PubMed1999

Alterations in the Young's modulus and volumetric properties of chondrocytes isolated from normal and osteoarthritic human cartilage.

The mechanical environment of the chondrocyte is an important factor that influences the maintenance of the articular cartilage extracellular matrix. Previous studies have utilized theoretical models of chondrocytes within articular cartilage to predict the stress-strain and fluid flow environments around the cell, but little is currently known regarding the cellular properties which are required for implementation of these models. The objectives of this study were to characterize the mechanical behavior of primary human chondrocytes and to determine the Young's modulus of chondrocytes from non-osteoarthritic ('normal') and osteoarthritic cartilage. A second goal was to quantify changes in the volume of isolated chondrocytes in response to mechanical deformation. The micropipette aspiration technique was used to measure the deformation of a single chondrocyte into a glass micropipette in response to a prescribed pressure. The results of this study indicate that the human chondrocyte behaves as a viscoelastic solid. No differences were found between the Young's moduli of normal (0.65+/-0.63 kPa, n = 44) and osteoarthritic chondrocytes (0.67+/-0.86 kPa, n = 69, p = 0.93). A significant difference in cell volume was observed immediately and 600 s after complete aspiration of the cell into the pipette (p < 0.001), and the magnitude of this volume change between normal (11+/-11%, n = 40) and osteoarthritic (20+/-11%, n = 41) chondroctyes was significantly different at both time points (p < 0.002). This finding suggests that chondrocytes from osteoarthritic cartilage may have altered volume regulation capabilities in response to mechanical deformation. The mechanical and volumetric properties determined in this study will be of use in analytical and finite element models of chondrocyte-matrix interactions in order to better predict the mechanical environment of the cell in vivo.

Adult↗
PubMed1999

The deformation behavior and mechanical properties of chondrocytes in articular cartilage.

INTRODUCTION: Chondrocytes in articular cartilage utilize mechanical signals to regulate their metabolic activity. A fundamental step in determining the role of various biophysical factors in this process is to characterize the local mechanical environment of the chondrocyte under physiological loading. METHODS: A combined experimental and theoretical approach was used to quantify the in-situ mechanical environment of the chondrocyte. The mechanical properties of enzymatically-isolated chondrocytes and their pericellular matrix (PCM) were determined using micropipette aspiration. The values were used in a finite element model of the chondron (the chondrocyte and its PCM) within articular cartilage to predict the stress-strain and fluid flow microenvironment of the cell. The theoretical predictions were validated using three-dimensional confocal microscopy of chondrocyte deformation in situ. RESULTS: Chondrocytes were found to behave as a viscoelastic solid material with a Young's modulus of approximately 0.6 kPa. The elastic modulus of the PCM was significantly higher than that of the chondrocyte, but several orders of magnitude lower than that of the extracellular matrix. Theoretical modeling of cell-matrix interactions suggests the mechanical environment of the chondrocyte is highly non-uniform and is dependent on the viscoelastic properties of the PCM. Excellent agreement was observed between the theoretical predictions and the direct measurements of chondrocyte deformation, but only if the model incorporated the PCM. CONCLUSIONS: These findings imply that the PCM plays a functional biomechanical role in articular cartilage, and alterations in PCM properties with aging or disease will significantly affect the biophysical environment of the chondrocyte.

Biomechanical Phenomena↗
PubMed1998

Static and dynamic lengths of neutrophil microvilli.

Containing most of the L-selectin and P-selectin glycoprotein ligand-1 (PSGL-1) on their tips, microvilli are believed to promote the initial arrest of neutrophils on endothelium. At the rolling stage following arrest, the lifetimes of the involved molecular bonds depend on the pulling force imposed by the shear stress of blood flow. With two different methods, electron microscopy and micropipette manipulation, we have obtained two comparable neutrophil microvillus lengths, both approximately 0.3 microm in average. We have found also that, under a pulling force, a microvillus can be extended (microvillus extension) or a long thin membrane cylinder (a tether) can be formed from it (tether formation). If the force is </=34 pN (+/- 3 pN), the length of the microvillus will be extended; if the force is >61 pN (+/- 5 pN), a tether will be formed from the microvillus at a constant velocity, which depends linearly on the force. When the force is between 34 pN and 61 pN (transition zone), the degree of association between membrane and cytoskeleton in individual microvilli will dictate whether microvillus extension or tether formation occurs. When a microvillus is extended, it acts like a spring with a spring constant of approximately 43 pN/microm. In contrast to a rigid or nonextendible microvillus, both microvillus extension and tether formation can decrease the pulling force imposed on the adhesive bonds, and thus prolonging the persistence of the bonds at high physiological shear stresses.

Cell Movement↗
PubMed1998

A role for Dictyostelium racE in cortical tension and cleavage furrow progression.

The small GTPase racE is essential for cytokinesis in Dictyostelium. We found that this requirement is restricted to cells grown in suspension. When attached to a substrate, racE null cells form an actomyosin contractile ring and complete cytokinesis normally. Nonetheless, racE null cells fail completely in cytokinesis when in suspension. To understand this conditional requirement for racE, we developed a method to observe cytokinesis in suspension. Using this approach, we found that racE null cells attempt cytokinesis in suspension by forming a contractile ring and cleavage furrow. However, the cells form multiple blebs and fail in cytokinesis by regression of the cleavage furrow. We believe this phenotype is caused by the extremely low level of cortical tension found in racE null cells compared to wild-type cells. The reduced cortical tension of racE null cells is not caused by a decrease in their content of F-actin. Instead, mitotic racE null cells contain abnormal F-actin aggregates. These results suggest that racE is essential for the organization of the cortical cytoskeleton to maintain proper cortical integrity. This function of racE is independent of attachment to a substrate, but can be bypassed by other signaling pathways induced by adhesion to a substrate.

Actins↗
PubMed1997

The secretion-coupled endocytosis correlates with membrane tension changes in RBL 2H3 cells.

Stimulated secretion in endocrine cells and neuronal synapses causes a rise in endocytosis rates to recover the added membrane. The endocytic process involves the mechanical deformation of the membrane to produce an invagination. Studies of osmotic swelling effects on endocytosis indicate that the increased surface tension is tightly correlated to a significant decrease of endocytosis. When rat basophilic leukemia (RBL) cells are stimulated to secrete, there is a dramatic drop in the membrane tension and only small changes in membrane bending stiffness. Neither the shape change that normally accompanies secretion nor the binding of ligand without secretion causes a drop in tension. Further, tension decreases within 6 s, preceding shape change and measurable changes in endocytosis. After secretion stops, tension recovers. On the basis of these results we suggest that the physical parameter of membrane tension is a major regulator of endocytic rate in RBL cells. Low tensions would stimulate endocytosis and high tensions would stall the endocytic machinery.

Animals↗
PubMed1995

Comparison of different drying procedures for scanning electron microscopy using human leukocytes.

Using human leukocytes as test specimens, three different drying procedures for scanning electron microscopy: critical-point drying (CPD), Peldri II, and tetramethylsilane (TMS), were compared. All three procedures produced identical surface morphology preservation. An equal amount of volume shrinkage was observed regardless of the dehydrants and drying techniques employed. Considering the simplicity, convenience, and time saved, air-drying with TMS is by far the best choice for preparing animal cells for scanning electron microscopy.

Desiccation↗
PubMed1993

Volume and osmotic properties of human neutrophils.

Quantitative models describing the dynamics of human neutrophils in the microcirculation require accurate morphometric parameters such as volume and surface membrane area. Using both a micropipette technique and video light microscopy (LM) to measure the diameters of the spherical cells, we have accurately determined the volume of the human neutrophil. Our value, 299 +/- 32 microns 3, is in good agreement with our earlier results, but 55% larger than that reported by Schmid-Schönbein et al (Blood 56:866, 1980). However, the measurements of Schmid-Schönbein et al were based on the actual mass of the cells derived from transmission electron microscopic (TEM) images. The membrane surface area, at lysis, was calculated to be 2.6 times its initial projected area. After lysis, the cells do not reduce their size, indicative of the possibility of a F-actin network formation that would stiffen the structure. Further, we show that neutrophils behave as ideal osmometers when exposed to anisotonic solutions at 21 degrees C, as predicted by the Boyle-Van't Hoff relationship. The calculated Ponder's value, R, is 0.77, which corresponds to 77% of the cell volume being osmotically active under isotonic conditions. However, at 37 degrees C, the cells are able to regulate their volumes toward the original volumes after an osmotic stress.

Humans↗
PubMed1993

Viscosity of passive human neutrophils undergoing small deformations.

At issue is the type of constitutive equation that can be used to describe all possible types of deformation of the neutrophil. Here a neutrophil undergoing small deformations is studied by aspirating it into a glass pipet with a diameter that is only slightly smaller than the diameter of the spherically shaped cell. After being held in the pipet for at least seven seconds, the cell is rapidly expelled and allowed to recover its undeformed, spherical shape. The recovery takes approximately 15 s. An analysis of the recovery process that treats the cell as a simple Newtonian liquid drop with a constant cortical (surface) tension gives a value of 3.3 x 10(-5) cm/s for the ratio of the cortical tension to cytoplasmic viscosity. This value is about twice as large as a previously published value obtained with the same model from studies of large deformations of neutrophils. This discrepancy indicates that the cytoplasmic viscosity decreases as the amount of deformation decreases. An extrapolated value for the cytoplasmic viscosity at zero deformation is approximately 600 poise when a value for the cortical tension of 0.024 dyn/cm is assumed. Clearly the neutrophil does not behave like a simple Newtonian liquid drop in that small deformations are inherently different from large deformations. More complex models consisting either of two or more fluids or multiple shells must be developed. The complex structure inside the neutrophil is shown in scanning electron micrographs of osmotically burst cells and cells whose membrane has been dissolved away.

Biophysical Phenomena↗
PubMed1990

Identification of monoclonal antibody binding domains of Na+,K(+)-ATPase by immunoelectron microscopy.

Treatment of purified preparations of porcine Na+,K(+)-ATPase with phospholipase A2, MgCl2 and NaVO3 leads to the formation of two-dimensional crystals exclusively in a dimeric configuration. Two-dimensional computer-averaged projections of the electron microscopy images of the crystalline enzyme with bound Fab fragments of monoclonal antibody M10-P5-C11 were accomplished using image enhancement software and showed that the antibody fragments caused only a modest increase in the unit cell size, while reducing the extent of asymmetry of the two promoters in each unit cell. The digital imaging also showed that the antibody's epitope on the alpha subunit resides on the 'lobe' or 'hook' region of the intracellular portion of the enzyme. Since functional studies indicate that M10-P5-C11 binds near or between the ATP binding site and the phosphorylation site, this visualized 'lobe' region of alpha may comprise the catalytic site. In addition, the binding of another inhibitory antibody, 9-A5, has been found to prevent crystal formation and the presence of the carbohydrate sugars on the enzyme's beta subunit shown to be required for crystal formation.

Animals↗
PubMed1990

Proton and divalent cations induce synergistic but mechanistically different destabilizations of pH-sensitive liposomes composed of dioleoyl phosphatidylethanolamine and oleic acid.

Protons and divalent cations show synergistic effects on the destabilization of liposomes composed of unsaturated phosphatidylethanolamine and oleic acid (Düzgünes et al., Biochemistry (1985) 24, 3091). We have extended these observations and investigated the effects of Ca2+ and Mg2+ on the proton-induced destabilization of dioleoyl phosphatidylethanolamine/oleic acid (DOPE/OA) (4:1 molar ratio) liposomes. Temperature-induced aggregation was measured by 90 degrees light scattering. Lipid mixing was used to monitor vesicle destabilization and freeze-fracture electron microscopy was used to examine the structures formed from DOPE/OA vesicles in the presence of Ca2+ and/or protons. Both Mg2+ and Ca2+ shift the pH required for 50% lipid mixing to higher values. Temperature-induced vesicle aggregation occurs at lower temperatures in the presence of divalent cations and/or protons, indicating that intervesicular repulsions are decreased. Freeze-fracture electron micrographs show that the structures formed from DOPE/OA in the presence of Ca2+ differ significantly from those found in the presence of protons. In general, protons induce the formation of hexagonal phase, while the presence of Ca2+ leads to the formation of extensive regions of lamellar sheets with numerous lipidic particles. The synergistic effect of divalent cations and proton may be important for the maximal biological activity of DOPE/OA liposomes.

Calcium↗
PubMed1989

Digital image analysis of two-dimensional Na,K-ATPase crystals: dissimilarity between pump units.

Two-dimensional crystals of purified Na,K-ATPase were induced by treatment with phospholipase-A2 and vanadate. The negatively stained crystals were imaged by electron microscopy and analysed by digital image processing. Two-dimensional averaged projections of the crystals were calculated by the technique of correlation analysis, utilizing SPIDER (System for Processing of Image Data in Electron microscopy and Related fields) image processing software. The calculated dimensions of the unit cell were found to be 13.3 X 4.59 nm with included angle of 98 degrees, comparable to those reported by others. However, the two protomers of the unit cell were found always to be dissimilar in shape and in orientation. All protomers of one side of the dimer ribbon had a triangular outline, and all protomers of the opposing side had a comma shape. This dissimilarity could be explained by two orientations of identical protomers: one orientation for one side of the dimer ribbon, and another orientation for the protomers of the opposing side of the ribbon. An alternative explanation is that the protomers of one side of the dimer ribbon are actually in a conformation different from that of the protomers of the opposing of the ribbon.

Animals↗
PubMed1988

Kinetic and ultrastructural studies of interactions of target-sensitive immunoliposomes with herpes simplex virus.

The bilayer phase of dioleoylphosphatidylethanolamine (PE) can be stabilized with palmitoyl-IgG monoclonal antibody to the glycoprotein gD of the herpes simplex virus (HSV). Interactions of PE immunoliposomes with the target virions were characterized by analyzing the kinetics of lipid mixing, by liposomal content release, and by ultrastructural studies. As revealed by a resonance energy transfer assay, lipid mixing between PE immunoliposomes and virions was very rapid, with a second-order rate constant (kapp) of 0.173 (min)-1 (microgram/mL virus)-1. In comparison, content release from PE immunoliposomes was much slower and exhibited multiple-phase, mixed-order kinetics, indicating that liposome destabilization involved fusion of liposomes with HSV. The extent and the apparent rate of liposome destabilization were strongly dependent on liposome concentration. This was evident by the fact that only one to two liposomes were destabilized by each virus particle at low liposome concentration (0.1 microM). For higher liposome concentrations (1-10 microM), this value was 35-104. This finding implies that collision among the virus-bound liposomes is essential for the eventual collapse of PE immunoliposomes to form the hexagonal (HII) equilibrium phase which was observed using freeze-fracture electron microscopy. Studies employing soluble gD, immobilized on latex beads, indicated that a multivalent antigen source is essential for PE immunoliposome destabilization. Immediately after liposome-virus binding, fusion of liposome with the viral membrane then follows. Upon growth of the fusion complexes, which increase to 35-104 liposomes for each virus, an eventual collapse of the structure results, driving PE to its equilibrium structure of HII phase.

Antibodies, Monoclonal↗
PubMed1988

Effects of tributyltin on biomembranes: alteration of flow cytometric parameters and inhibition of Na+, K+-ATPase two-dimensional crystallization.

Carboxyfluorescein diacetate (CFDA) is a lipophilic nonfluorescent molecule that readily crosses the cell membrane. In the cytoplasm, it is hydrolyzed by nonspecific esterases to carboxyfluorescein (CF), a negatively charged fluorescent molecule, which is retained incompletely by cells with an intact plasma membrane. Exposure (4 hr) of the murine erythroleukemic cell (MELC) to micromolar quantities (0.1 to 5.0 microM) of tributyltin (TBT) results in increased cellular CF fluorescence. The increase occurs within a range below a critical value of the product (CPV) of the concentration (C) of TBT X duration (T) of exposure to TBT. Fluorescence increase is a sensitive indicator of the interaction of TBT with the cell: it is observed following exposure to 0.1 microM TBT for 4 hr at 37 degrees C. In the range above the CPV, cellular CF fluorescence is reduced apparently resulting from perturbation of membrane structure. For example, exposure of MELC to 2.5 microM TBT for 4 hr at 37 degrees C produces resistance to detergent-mediated cytolysis and inhibition of vanadate-mediated two-dimensional crystallization of Na+, K+-ATPase molecules in porcine renal microsomal membrane preparations, a process requiring molecular mobility within the membrane. Taken together, the increased cellular CF fluorescence and resistance of the MELC to cytolysis along with the inhibition of Na+, K+-ATPase crystallization in the microsomal membrane preparations suggest fixation (protein denaturation, cross-linking, etc.) at the level of the plasma membrane as a mode of toxic action of TBT.

Animals↗
PubMed1987

Electron microscopic analysis of two-dimensional crystals of the Ca2+-transport ATPase--a freeze-fracture study.

Two distinct forms of Ca2+-ATPase crystals have been analysed in sarcoplasmic reticulum (SR) membranes. The E1-type crystals, induced by Ca2+ or lanthanide ions, consist of single chains of ATPase monomers, and the E2-type crystals, induced by vanadate ions, consist of dimer chains. Using improved freeze-fracture techniques we have obtained high-resolution images of complementary surface replicas of SR membranes containing these crystal forms. In E1 crystals, the concave fracture (P) faces display obliquely oriented rows of intramembrane particles (IMPs) spaced at congruent to 6-7 nm along both crystal axes, while the convex fracture (E) faces show corresponding rows of pits. In E2 crystals, regular arrays of oblique parallel ridges with spacing of congruent to 10.5-11 nm appear on the P-faces and complementary grooves or furrows on the E-faces. In many instances the ridges break up into elongated particles repeating every 5.5 nm. When the direction of the shadow is almost parallel to the axis of the ridges, these 9.5 nm particles can be resolved into two domains, which represent intramembranous contacts between the two monomers of the two adjacent dimer chains. Complementary grooves on the E-faces can also be resolved into rows of pits complementary to the particles of the ridges on the P-faces. In the control SR membranes, randomly dispersed IMPs and corresponding pits are observed on the P- and E-faces, respectively. The data suggest that transport of Ca2+ involves significant structural changes of the enzyme molecule, reflected in the ATPase-ATPase interactions both on the cytoplasmic surface and in the lipid bilayer.

Animals↗