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Latex Beads (latex + bead)
Selected AbstractsHeterogeneous modes of uptake for latex beads revealed through live cell imaging of phagocytes expressing a probe for phosphatidylinositol-(3,4,5)-trisphosphate and phosphatidylinositol-(3,4)-bisphosphateCYTOSKELETON, Issue 9 2008Jennifer Giorgione Abstract Latex beads are the preferred phagocytic substrate in biochemical studies of phagosome composition and maturation. Using living Dictyostelium cells and fluorescent probes, we compared the properties of phagosomes formed to ingest latex beads or digestible prey. Significant differences were found during the initial steps of phagocytosis. During uptake of bacteria or yeast, PHcrac-GFP, a probe that binds to membranes enriched in PI(3,4,5)P3 and PI(3,4)P2, always labeled the nascent phagosome and faded shortly after it sealed. However, labeling of bead-containing phagosomes was highly variable. Beads were engulfed by phagosomes either lacking or displaying the PHcrac-GFP label, and that label, if present, often persisted for many minutes, revealing that early trafficking steps for bead-containing phagosomes are quite heterogeneous. Later stages of the endocytic pathway appeared more similar for phagosomes containing prey and latex beads. Both types of phagosomes fused with acidic endosomes while undergoing transport along microtubules, both acquired the V-ATPase and lost it prior to exocytosis, and both bound the late endosome marker vacuolin B, which was transferred to the plasma membrane upon exocytosis. We conclude that caution is needed in extrapolating results from latex bead phagosomes to phagosomes containing physiological substances, especially in early stages of the endocytic pathway. Cell Motil. Cytoskeleton 2008. © 2008 Wiley-Liss, Inc. [source] Actin-dependent motility of melanosomes from fish retinal pigment epithelial (RPE) cells investigated using in vitro motility assaysCYTOSKELETON, Issue 2 2004E. L. McNeil Melanosomes (pigment granules) within retinal pigment epithelial (RPE) cells of fish and amphibians undergo massive migrations in response to light conditions to control light flux to the retina. Previous research has shown that melanosome motility within apical projections of dissociated fish RPE cells requires an intact actin cytoskeleton, but the mechanisms and motors involved in melanosome transport in RPE have not been identified. Two in vitro motility assays, the Nitella assay and the sliding filament assay, were used to characterize actin-dependent motor activity of RPE melanosomes. Melanosomes applied to dissected filets of the Characean alga, Nitella, moved along actin cables at a mean rate of 2 ,m/min, similar to the rate of melanosome motility in dissociated, cultured RPE cells. Path lengths of motile melanosomes ranged from 9 to 37 ,m. Melanosome motility in the sliding filament assay was much more variable, ranging from 0.4,33 ,m/min; 70% of velocities ranged from 1,15 ,m/min. Latex beads coated with skeletal muscle myosin II and added to Nitella filets moved in the same direction as RPE melanosomes, indicating that the motility is barbed-end directed. Immunoblotting using antibodies against myosin VIIa and rab27a revealed that both proteins are enriched on melanosome membranes, suggesting that they could play a role in melanosome transport within apical projections of fish RPE. Cell Motil. Cytoskeleton 58:71,82, 2004. © 2004 Wiley-Liss, Inc. [source] Heterogeneous modes of uptake for latex beads revealed through live cell imaging of phagocytes expressing a probe for phosphatidylinositol-(3,4,5)-trisphosphate and phosphatidylinositol-(3,4)-bisphosphateCYTOSKELETON, Issue 9 2008Jennifer Giorgione Abstract Latex beads are the preferred phagocytic substrate in biochemical studies of phagosome composition and maturation. Using living Dictyostelium cells and fluorescent probes, we compared the properties of phagosomes formed to ingest latex beads or digestible prey. Significant differences were found during the initial steps of phagocytosis. During uptake of bacteria or yeast, PHcrac-GFP, a probe that binds to membranes enriched in PI(3,4,5)P3 and PI(3,4)P2, always labeled the nascent phagosome and faded shortly after it sealed. However, labeling of bead-containing phagosomes was highly variable. Beads were engulfed by phagosomes either lacking or displaying the PHcrac-GFP label, and that label, if present, often persisted for many minutes, revealing that early trafficking steps for bead-containing phagosomes are quite heterogeneous. Later stages of the endocytic pathway appeared more similar for phagosomes containing prey and latex beads. Both types of phagosomes fused with acidic endosomes while undergoing transport along microtubules, both acquired the V-ATPase and lost it prior to exocytosis, and both bound the late endosome marker vacuolin B, which was transferred to the plasma membrane upon exocytosis. We conclude that caution is needed in extrapolating results from latex bead phagosomes to phagosomes containing physiological substances, especially in early stages of the endocytic pathway. Cell Motil. Cytoskeleton 2008. © 2008 Wiley-Liss, Inc. [source] HGF induction of postsynaptic specializations at the neuromuscular junctionDEVELOPMENTAL NEUROBIOLOGY, Issue 2 2006Raghavan Madhavan Abstract A critical event in the formation of vertebrate neuromuscular junctions (NMJs) is the postsynaptic clustering of acetylcholine receptors (AChRs) in muscle. AChR clustering is triggered by the activation of MuSK, a muscle-specific tyrosine kinase that is part of the functional receptor for agrin, a nerve-derived heparan sulfate proteoglycan (HSPG). At the NMJ, heparan sulfate (HS)-binding growth factors and their receptors are also localized but their involvement in postsynaptic signaling is poorly understood. In this study we found that hepatocyte growth factor (HGF), an HS-binding growth factor, surrounded muscle fibers and was localized at NMJs in rat muscle sections. In cultured Xenopus muscle cells, HGF was enriched at spontaneously occurring AChR clusters (hot spots), where HSPGs were also concentrated, and, following stimulation of muscle cells by agrin or cocultured neurons, HGF associated with newly formed AChR clusters. HGF presented locally to cultured muscle cells by latex beads induced new AChR clusters and dispersed AChR hot spots, and HGF beads also clustered phosphotyrosine, activated c-Met, and proteins of dystrophin complex; clustering of AChRs and associated proteins by HGF beads required actin polymerization. Lastly, although bath-applied HGF alone did not induce new AChR clusters, addition of HGF potentiated agrin-dependent AChR clustering in muscle. Our findings suggest that HGF promotes AChR clustering and synaptogenic signaling in muscle during NMJ development. © 2005 Wiley Periodicals, Inc. J Neurobiol, 2005 [source] Characterization in vivo of bilaterally branching pontocerebellar mossy fibre to Golgi cell inputs in the rat cerebellumEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 2 2009Tahl Holtzman Abstract Golgi cells regulate the flow of information from mossy fibres to the cerebellar cortex, through a mix of feedback and feedforward inhibitory actions on granule cells. The aim of the current study was to examine mossy fibre input to Golgi cells, in order to assess their impact on switching Golgi cells into feedforward behaviour. In urethane-anaesthetized rats, extracellular recordings were made from Golgi cells in Crus II (n = 18). Spikes were evoked in all Golgi cells by microstimulation within the contralateral hemispheral cortex, via branches of mossy fibres that terminate in both cerebellar hemispheres. The latencies of these responses were very short, consistent with a monosynaptic mossy fibre contact [average onset latency 2.3 ± 0.1 ms (SEM)]. The same stimuli had no measurable effect on spike responses of nearby Purkinje cells (n = 12). Systematic mapping in the contralateral cerebellar hemisphere (Crus Ib, IIa, IIb and the paramedian lobule) usually revealed one low-intensity stimulus ,hotspot' (12,35 ,A) from which short-latency spikes could be evoked in an individual Golgi cell. Microinjections of red and green retrograde tracers (latex beads, ,50,150 nL injection volume) made at the recording site and the stimulation hotspot resulted in double-labelled neurons within the pontine nuclei. Overall, this suggests that subsets of pontine neurons supply mossy fibres that branch to both hemispheres, some of which directly target Golgi cells. Such an arrangement may provide a common feedforward inhibitory link to temporally couple activity on both sides of the cerebellum during behaviour. [source] Heparin-binding hemagglutinin (HBHA) of Mycobacterium leprae is expressed during infection and enhances bacterial adherence to epithelial cellsFEMS MICROBIOLOGY LETTERS, Issue 2 2009Cristiana Soares De Lima Abstract A heparin-binding hemagglutinin (HBHA) expressed on the surface of Mycobacterium tuberculosis is an antigenic protein that has been implicated in bacterial adherence to epithelial cells and systemic dissemination. In this study, the potential role of the Mycobacterium leprae HBHA (ML-HBHA) homologue in leprosy was investigated. Initially, the in vivo expression of HBHA and its association with the M. leprae cell envelope was confirmed by immunoblotting and proteomic analysis. Mycobacterium leprae recombinant HBHA (rML-HBHA) bound to a heparin,Sepharose column, and its capacity to act as an adhesin was demonstrated in experiments showing that the exogenous addition of the protein to latex beads or to M. leprae cells promotes a dramatic increase in association with epithelial cells. Finally, serum anti-HBHA immunoglobulin G levels were investigated in individuals infected with M. leprae. Altogether, our data indicate that HBHA is recognized during the course of bacterial infection in humans and may play a role in leprosy pathogenesis. [source] Mechanism of H-8 inhibition of Cyclin-dependent kinase 9: study using inhibitor-immobilized matricesGENES TO CELLS, Issue 3 2003Daisuke Shima Background: Positive transcription elongation factor b (P-TEFb), which phosphorylates the carboxyl-terminal domain (CTD) of RNA polymerase II (RNAPII), is comprised of the catalytic subunit cyclin-dependent kinase 9 (CDK9) and the regulatory subunit cyclin T. The kinase activity and transcriptional activation potential of P-TEFb is sensitive to various compounds, including H-8, 5,6-dichloro-1-,-d-ribofuranosylbenzimidazole (DRB), and flavopiridol. Results: We investigated the molecular mechanism of the H-8 inhibition of CDK9 using matrices to which H-9, an amino derivative of H-8, was immobilized. CDK9 bound specifically to H-9, and this interaction was competitively inhibited by ATP and DRB, but not by flavopiridol. Mutational analyses demonstrated that the central region of CDK9, which encompasses the T-loop region, was important for its binding to H-9. Conclusions: H-9-immobilized latex beads are useful for trapping CDK9 and a subset of kinases from crude cell extracts. The flavopiridol-binding region of CDK9 is most likely different from its H-9-binding region. These biochemical data support previously reported observations which were based on crystallographic data. [source] Phagocytic capacity of leucocytes in sheep mammary secretions following weaningJOURNAL OF ANATOMY, Issue 5 2002Liliana Tatarczuch Abstract Lactating animals are particularly susceptible to mastitis during the early stages of mammary gland involution following weaning. In this study we compared the phagocytic capacity of cells collected from sheep mammary secretions at different stages of involution. The ability of neutrophils and macrophages to ingest latex beads in an in vitro phagocytosis assay was found to be dependent on how heavily the phagocytes were loaded with milk constituents. There was a decline in the phagocytic capacity of neutrophils from 1 to 2 days after weaning, while macrophages collected from fully involuted glands were more effective phagocytes compared with earlier stages (7,15 days) of involution. In addition, dendritic cells present in fully involuted mammary gland secretions (30 days after weaning) were highly phagocytic. These studies demonstrate that neutrophils and macrophages in sheep mammary secretions at early stages of involution are incapacitated, and as such may compromise the immune status of the mammary gland. [source] Zernike-type phase-contrast hard X-ray microscope with a zone plate at the Photon FactoryJOURNAL OF SYNCHROTRON RADIATION, Issue 3 2002Hiroki Yokosuka A Zernike-type phase-contrast X-ray microscope with a zone plate and a phase plate was constructed at the Photon Factory BL3C2. Parallel monochromatic X-rays of 8.97,keV were incident on a specimen and a direct beam transmitted through the specimen was focused on the back focal plane of the zone plate, where an aluminium phase plate was placed. Tantalum line patterns as fine as 0.3,µm could be imaged. Phase-contrast images of polypropylene wires and polystyrene latex beads were obtained, which showed better contrast than that of their bright field images. [source] Binding of extracellular matrix molecules by probiotic bacteriaLETTERS IN APPLIED MICROBIOLOGY, Issue 4 2003tyriak Abstract Aims: The aim of this study was to investigate extracellular matrix (ECM) and mucin binding of selected bacterial isolates with probiotic features in comparison with commercially used probiotic bacteria. Methods and Results: ECM molecules were immobilized in microtitre plates (mucin and fetuin) or on the surface of latex beads. Porcine mucin was bound by all 13 probiotic strains tested with important inter-strain differences; however, fetuin binding was similar (weak) for all 14 strains tested. Strongly positive (three) binding of bovine fibrinogen was expressed by strains from fermented food (Lactobacillus rhamnosus GG, L. casei Shirota and L. johnsonii La1) as well as by L. casei L.c., Lactobacillus sp. 2I3 and by L. plantarum LP. The other strains expressed moderate (2) or weakly positive (1) binding of bovine fibrinogen. Strongly positive (3) binding of porcine fibronectin was observed only with two strains; however, all other strains also bound this molecule. Bovine lactoferrin was bound to a higher extent than transferrins. Significance and Impact of the Study: Some animal strains (at least L. casei L.c. and Lactobacillus sp. 2I3) are comparable with the commercially used strains with respect to their ECM binding ability. As this feature is important for probiotic bacteria to be able to colonize intestine, these strains should be considered for their wider use in fermented feed (or probiotic preparations) for animals. [source] Corynebacterium diphtheriae employs specific minor pilins to target human pharyngeal epithelial cellsMOLECULAR MICROBIOLOGY, Issue 1 2007Anjali Mandlik Summary Adherence to host tissues mediated by pili is pivotal in the establishment of infection by many bacterial pathogens. Corynebacterium diphtheriae assembles on its surface three distinct pilus structures. The function and the mechanism of how various pili mediate adherence, however, have remained poorly understood. Here we show that the SpaA-type pilus is sufficient for the specific adherence of corynebacteria to human pharyngeal epithelial cells. The deletion of the spaA gene, which encodes the major pilin forming the pilus shaft, abolishes pilus assembly but not adherence to pharyngeal cells. In contrast, adherence is greatly diminished when either minor pilin SpaB or SpaC is absent. Antibodies directed against either SpaB or SpaC block bacterial adherence. Consistent with a direct role of the minor pilins, latex beads coated with SpaB or SpaC protein bind specifically to pharyngeal cells. Therefore, tissue tropism of corynebacteria for pharyngeal cells is governed by specific minor pilins. Importantly, immunoelectron microscopy and immunofluorescence studies reveal clusters of minor pilins that are anchored to cell surface in the absence of a pilus shaft. Thus, the minor pilins may also be cell wall anchored in addition to their incorporation into pilus structures that could facilitate tight binding to host cells during bacterial infection. [source] Photobleaching of Melanosomes from Retinal Pigment Epithelium: I. Effects on Protein OxidationPHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 4 2007Janice M. Burke ABSTRACT Melanin in the long-lived melanosomes of the retinal pigment epithelium (RPE) may undergo photobleaching with aging, which appears to diminish the antioxidant function of melanin and could make photobleached melanosomes less efficient in protecting biomolecules from oxidative modification. Here we analyzed whether photobleaching of melanosomes affects their ability to modify the oxidation state of nearby protein. As conventional methods developed to study soluble antioxidants are not well suited for analysis of granules such as melanosomes, we developed a new analytic method to focus on particle surfaces that involves experimentally coating granules with the cytoskeletal protein ,-actin to serve as a reporter for local protein oxidation. Isolated porcine RPE melanosomes were photobleached with visible light to simulate aging, then photobleached melanosomes, untreated melanosomes and control particles (black latex beads) were actin coated and illuminated in a photosensitized cell free system. Protein was re-stripped from particles and analyzed for carbonylation by Western blotting. Quantitative densitometry showed no reproducible differences for protein associated with untreated melanosomes when compared with control particles. Melanin has both anti- and pro-oxidant functions when light irradiated, but neither of these functions predominated in the protein oxidation assay when untreated melanosomes were used. However, protein extracted from photobleached melanosomes showed markedly increased carbonylation, both of associated actin and of endogenous melanosomal protein(s), and the effect increased with extent of granule photobleaching. Photobleaching of RPE melanosomes therefore changes the oxidation state of protein endogenous to the organelle and reduces the ability of the granule to modify the oxidation of exogenous protein near the particle surface. The results support the growing body of evidence that photobleaching of RPE melanosomes, which is believed to occur with aging, changes the physicochemical properties of the organelle and reduces the likelihood that the granules perform an antioxidant function. [source] Photobleaching of Melanosomes from Retinal Pigment Epithelium: II.PHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 4 2007Effects on the Response of Living Cells to Photic Stress Melanosomes of the retinal pigment epithelium (RPE) are long lived organelles that may undergo photobleaching with aging, which can diminish the antioxidant efficiency of melanin. Here, isolated porcine RPE melanosomes were experimentally photobleached with visible light to simulate aging and compared with untreated granules or control particles (black latex beads) for their effects on the survival of photically stressed ARPE-19 cultures. Particles were delivered to cultures for uptake by phagocytosis then cells were exposed to violet light and analyzed by a new live cell imaging method to identify the time of apoptotic blebbing as a dynamic measure of reduced cell survival. Results indicated that untreated melanosomes did not decrease photic injury to ARPE-19 cells when compared with cells lacking particles or with cells containing control particles, as might be expected if melanin performed an antioxidant function. Instead cells with untreated melanosomes showed reduced survival indicated by an earlier onset of blebbing and a lower fraction of surviving cells after photic stress. Cell survival was reduced even further in stressed cells containing melanosomes that were photobleached, and survival decreased with increasing photobleaching time. Photobleaching of RPE melanosomes therefore makes cells containing them more sensitive to light-induced cytotoxicity. This observation raises the possibility that aged melanosomes increase RPE cell photic stress in situ, perhaps contributing to reduced tissue function and to degeneration of the adjacent retina that the RPE supports. How melanosomes (photobleached or not) interact with their local subcellular environment to modify RPE cell survival is poorly understood and is likely determined by the physicochemical state of the granule and its constituent melanin. The live cell imaging method introduced here, which permitted detection of a graded effect of photobleaching, provides a sensitive bioassay for probing the effects of melanosome modifications. [source] Visual detection of IS6110 of Mycobacterium tuberculosis in sputum samples using a test based on colloidal gold and latex beadsCLINICAL MICROBIOLOGY AND INFECTION, Issue 11 2006P. Upadhyay Abstract The IS6110 sequence was detected visually in sputum samples of tuberculosis patients using a bi-probe system. One of the probes was an oligonucleotide conjugated to colloidal gold particles, complementary to one end of the target strand. The other probe was an oligonucleotide conjugated to latex beads complementary to the other end of the target strand. In a reaction mix, these two probes bind to the target strand, and the latex beads are then separated by filtration. Bound latex beads have gold colloid particles at the other end of the target strand. These gold colloid particles were made visible to the naked eye by silver autometallography on the ,invisible' colloidal gold particles. The lower detection limit was 50 ng of genomic DNA of Mycobacterium tuberculosis. This new test, together with conventional PCR, was performed on DNA extracted from sputum samples of suspected tuberculosis patients. The new test was simple to perform, the results were visible to the naked eye, and the test was highly specific, as even single point mutations in the target strand sequence could be differentiated. The test could be useful in field-level laboratories because it requires no sophisticated equipment. [source] | ||||||||||||||||||||||