Neutrophil string formation: hydrodynamic thresholding and cellular deformation during cell collisions.
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Neutrophils unexpectedly display flow-enhanced adhesion (hydrodynamic thresholding) to L-selectin in rolling or aggregation assays. We report that the primary collision efficiency (epsilon) of flowing neutrophils with preadhered neutrophils on intercellular adhesion molecule-1 (ICAM-1) or fibrinogen also displayed a maximum of epsilon approximately 0.4-0.45 at a wall shear rate of 100 s(-1), an example of thresholding. Primary collision lifetime with no detectable bonding decreased from 130 to 10 ms as wall shear rate increased from 30 to 300 s(-1), whereas collision lifetimes with bonding decreased from 300 to 100 ms over this shear range using preadhered neutrophils on ICAM-1, with similar results for fibrinogen. Antibodies against L-selectin, but not against CD11a, CD11b, or CD18, reduced epsilon at 100 s(-1) by >85%. High resolution imaging detected large scale deformation of the flowing neutrophil during the collision at 100 s(-1) with the apparent contact area increasing up to approximately 40 microm(2). We observed the formation of long linear string assemblies of neutrophils downstream of neutrophils preadhered to ICAM-1, but not fibrinogen, with a maximum in string formation at 100 s(-1). Secondary capture events to the ICAM-1 or fibrinogen coated surfaces after primary collisions were infrequent and short lived, typically lasting from 500 to 3500 ms. Between 5 and 20% of neutrophil interactions with ICAM-1 substrate converted to firm arrest (>3500 ms) and greatly exceeded that observed for fibrinogen, thus defining the root cause of poor string formation on fibrinogen at all shear rates. Additionally, neutrophils mobilized calcium after incorporation into strings. Static adhesion also caused calcium mobilization, as did the subsequent onset of flow. To our knowledge, this is the first report of 1). hydrodynamic thresholding in neutrophil string formation; 2). string formation on ICAM-1 but not on fibrinogen; 3). large cellular deformation due to collisions at a venous shear rate; and 4), mechanosensing through neutrophil beta(2)-integrin/adhesion. The increased contact area during deformation was likely responsible for the hydrodynamic threshold observed in the primary collision efficiency since no increase in primary collision lifetime was detected as shear forces were increased (for either surface coating). (+info)
Phg2, a kinase involved in adhesion and focal site modeling in Dictyostelium.
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The amoeba Dictyostelium is a simple genetic system for analyzing substrate adhesion, motility and phagocytosis. A new adhesion-defective mutant named phg2 was isolated in this system, and PHG2 encodes a novel serine/threonine kinase with a ras-binding domain. We compared the phenotype of phg2 null cells to other previously isolated adhesion mutants to evaluate the specific role of each gene product. Phg1, Phg2, myosin VII, and talin all play similar roles in cellular adhesion. Like myosin VII and talin, Phg2 also is involved in the organization of the actin cytoskeleton. In addition, phg2 mutant cells have defects in the organization of the actin cytoskeleton at the cell-substrate interface, and in cell motility. Because these last two defects are not seen in phg1, myoVII, or talin mutants, this suggests a specific role for Phg2 in the control of local actin polymerization/depolymerization. This study establishes a functional hierarchy in the roles of Phg1, Phg2, myosinVII, and talin in cellular adhesion, actin cytoskeleton organization, and motility. (+info)
Sperm selection for ICSI: shape properties do not predict the absence or presence of numerical chromosomal aberrations.
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BACKGROUND: We hypothesize that the potential relationship between abnormal sperm morphology and increased frequency of numerical chromosomal aberrations is based on two attributes of diminished sperm maturity: (i) cytoplasmic retention and consequential sperm shape abnormalities; and (ii) meiotic errors caused by low levels of the HspA2 chaperone, a component of the synaptonemal complex. Because sperm morphology and aneuploidies were assessed in semen, but not in the same spermatozoa, previous studies addressing this relationship were inconclusive. We recently demonstrated that sperm shape is preserved following fluorescence in situ hybridization (FISH). Thus, we examined the shape and chromosomal aberrations in the same sperm. METHODS: We performed phase contrast microscopy and FISH, using centromeric probes for chromosomes X, Y, 10, 11 and 17 in 15 men. The fluorescence and respective phase contrast images were digitized using the Metamorph program. We studied 1286 sperm (256 disomic, 130 diploid and 900 haploid sperm) by three criteria: head and tail dimensions, head shape and Kruger strict morphology. Furthermore, in each analysis, we considered whether disomic or diploid sperm may be distinguished from haploid sperm. RESULTS: There was an overall, but not discriminative, relationship between abnormal sperm dimensions or shape and increased frequencies of numerical chromosomal aberrations. However, approximately 68 of the 256 disomic, and four of 130 diploid sperm showed head and tail dimensions comparable with the most normal, lowest tertile of the 900 haploid spermatozoa. Considering all 1286 sperm, among those with the most regular, symmetrical shape (n = 367), there were 63 and five with disomic and diploid nuclei, respectively. In line with these findings, among the 256 disomic sperm, 10% were Kruger normal. CONCLUSIONS: Sperm dimensions or shape are not reliable attributes in selection of haploid sperm for ICSI. (+info)
Engineering the morphology and electrophysiological parameters of cultured neurons by microfluidic surface patterning.
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The ability to control the orientation, morphology, and electrophysiological characteristics of neurons in culture allows the construction of neural circuits with defined physiological properties. Using microfluidic protein deposition onto chemically modified glass, we achieve the controlled growth of Aplysia neurons on geometrical patterns of poly-L-lysine and collagen IV, surrounded by nonadhesive regions of bovine albumin. We investigate the parameters essential for forming functional neuronal networks, the morphology, biochemistry, and electrophysiology under engineered cell culture conditions. We demonstrate that not only the orientation of neurite extension but also the number of primary neurites originating from the cell soma, their length, and branching pattern depend on the spatial constraints presented by the size and shape of the adhesion region on the patterned substrate. In addition, the physicochemical properties of the support layer influence the electrical activity of the cultured neurons. Substrate-dependent changes in the amplitude and in the dynamic parameters of the action potential cause decreased spike broadening in patterned neurons, which reflects changes in the number or functioning of active membrane ion channels. In contrast to morphology and electrophysiology, the neuropeptide content, as determined by mass spectrometry of individual patterned neurons, is not affected by the growth on patterned surfaces. Our results suggest that the morphological and electrophysiological parameters of neurons can be predictably altered/engineered by modulation of the chemical, physical, and topographical features of culture substrates. We also demonstrate that a full suite of techniques is required for functional characterization of neurons on engineered substrates. (+info)
Sphingosine 1-phosphate inhibits migration of RBL-2H3 cells via S1P2: cross-talk between platelets and mast cells.
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To analyze the involvement in allergic reactions of platelets and sphingosine 1-phosphate (Sph-1-P), a lysophospholipid mediator released from activated platelets, the effects of Sph-1-P and a supernatant prepared from activated platelets on mast cell line RBL-2H3 were examined. Sph-1-P strongly inhibited the migration of both non-stimulated and fibronectin-stimulated RBL-2H3 cells, which was reversed by JTE-013, a specific antagonist of G protein-coupled Sph-1-P receptor S1P(2); S1P(2) was confirmed to be expressed in these cells. A similar anti-motility effect of Sph-1-P was observed in a phagokinetic assay. Consistent with these results, treatment of RBL-2H3 cells with Sph-1-P resulted in a rounded cell morphology, which was blocked by JTE-013. Under the present conditions, Sph-1-P failed to induce intracellular Ca(2+) mobilization or histamine degranulation, responses postulated to be elicited by intracellular Sph-1-P. Importantly, the Sph-1-P effect, i.e., the regulation of RBL-2H3 cell motility, was mimicked by the supernatant (both with and without boiling) prepared from activated platelets, and this effect of the supernatant was also blocked by JTE-013. Our results suggest that the motility of mast cells can be regulated by Sph-1-P and also platelets (which release Sph-1-P), via cell surface receptor S1P(2) (not through intracellular Sph-1-P actions, postulated previously in the same cells). (+info)
Analysis of differentially regulated proteins in TM4 cells treated with bisphenol A.
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BPA, bisphenol A, a monomer of epoxy resins and polycarbonate plastic, is used in many consumer products including the plastic linings of cans for food and babies' bottles. BPA has been reported to cause reproductive toxicity and affects cells in rats and mice at high doses. In this study, the effect of BPA on protein expression in TM4 cells (a mouse Sertoli cell line) known to play an essential role in Spermatogenesis was investigated by two-dimensional electrophoresis (2-DE). After 16 h exposure to 50, 100, 150, 200, and 250 microM of BPA, the viability of TM4 cells decreased to about 90, 85, 78, 55, and 30% of control respectively. Approximately 800 protein spots in TM4 cells were analyzed by 2-DE with pH 4-7 linear immobilized pH gradient (IPG) Dry Strip, and 11 proteins which showed significantly different expression levels were identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). Among these, HSP 27 and placental calcium binding protein may be proteins differentially expressed by BPA exposure. (+info)
Axons in cat visual cortex are topologically self-similar.
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The axonal arbors of the different types of neocortical and thalamic neurons appear highly dissimilar when viewed in conventional 2D reconstructions. Nevertheless, we have found that their one-dimensional metrics and topologies are surprisingly similar. To discover this, we analysed the axonal branching pattern of 39 neurons (23 spiny, 13 smooth and three thalamic axons) that were filled intracellularly with horseradish peroxidase (HRP) during in vivo experiments in cat area 17. The axons were completely reconstructed and translated into dendrograms. Topological, fractal and Horton-Strahler analyses indicated that axons of smooth and spiny neurons had similar complexity, length ratios (a measure of the relative increase in the length of collateral segments as the axon branches) and bifurcation ratios (a measure of the relative increase in the number of collateral segments as the axon branches). We show that a simple random branching model (Galton-Watson process) predicts with reasonable accuracy the bifurcation ratio, length ratio and collateral length distribution of the axonal arbors. (+info)
A study of pyramidal cell structure in the cingulate cortex of the macaque monkey with comparative notes on inferotemporal and primary visual cortex.
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Recent studies have revealed a marked degree of variation in the pyramidal cell phenotype in visual, somatosensory, motor and prefrontal cortical areas in the brain of different primates, which are believed to subserve specialized cortical function. In the present study we carried out comparisons of dendritic structure of layer III pyramidal cells in the anterior and posterior cingulate cortex and compared their structure with those sampled from inferotemporal cortex (IT) and the primary visual area (V1) in macaque monkeys. Cells were injected with Lucifer Yellow in flat-mounted cortical slices, and processed for a light-stable DAB reaction product. Size, branching pattern, and spine density of basal dendritic arbors was determined, and somal areas measured. We found that pyramidal cells in anterior cingulate cortex were more branched and more spinous than those in posterior cingulate cortex, and cells in both anterior and posterior cingulate were considerably larger, more branched, and more spinous than those in area V1. These data show that pyramidal cell structure differs between posterior dysgranular and anterior granular cingulate cortex, and that pyramidal neurons in cingulate cortex have different structure to those in many other cortical areas. These results provide further evidence for a parallel between structural and functional specialization in cortex. (+info)