| 1. |
- Bengtsson, T., et al.
(författare)
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Actin dynamics in human neutrophils during adhesion and phagocytosis is controlled by changes in intracellular free calcium
- 1993
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Ingår i: European Journal of Cell Biology. - Jena, Germany : Urban und Fischer Verlag. - 0171-9335 .- 1618-1298. ; 62:1, s. 19-58
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Tidskriftsartikel (refereegranskat)abstract
- The role of changes in cytosolic free calcium concentration ([Ca2+]i) in the assembly and disassembly of actin during adhesion and phagocytosis was evaluated. Rhodamine-phalloidin staining combined with quantitative fluorescence and confocal laser scanning microscopy was used to measure local F-actin changes in single adherent human neutrophils phagocytosing yeast particles on different surfaces and under different calcium conditions. Cells were suspended in a) calcium-containing medium (CCM) or b) calcium-free medium (CFM) or c) were first depleted of calcium (i.e., MAPT/AM-loaded in CFM) and then suspended in CFM (MAPT). In parallel, local [Ca2+]i changes were monitored using a fura-2 ratio imaging system. In CCM or CFM, attachment to the substrate and formation of pseudopods around a yeast particle generated, within a few seconds, rises in [Ca2+]i, both around the phagosome and in the cell body. During continued phagocytosis, [Ca2+]i was more elevated around the phagosome compared to the rest of the cell. No [Ca2+]i fluctuations were observed in MAPT cells. Adhesion and phagocytosis led to a several-fold increase in F-actin. The increase was transient in cells in CCM and CFM, but remained high in Ca-depleted neutrophils. A distinct ring of F-actin was formed around a phagosome with a yeast particle. Twenty min after ingestion the amount of this actin decreased more than 50% in CCM and CFM cells but increased by 40 to 100% in MAPT cells. The accumulation of F-actin in MAPT cells was reduced to resting levels by adding Ca2+ and ionomycin after ingestion. This treatment reestablished the periphagosomal [Ca2+]i rises, as observed in CCM cells. In conclusion, the present study shows that the actin polymerization, occurring in human neutrophils during adhesion and phagocytosis, is not influenced by changes in [Ca2+]i, whereas the subsequent depolymerization is. The accumulation of actin filaments around the phagosome in calcium-depleted cells could be involved in the inhibition of phagolysosome fusion seen in the absence of [Ca2+]i changes (Jaconi et al., J. Cell Biol. 110, 1555-1564 (1990)). This suggests that the actin network, controlled by [Ca2+]i, regulates the movement of granules during phagocytosis.
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| 2. |
- Franzén, Lars, et al.
(författare)
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Fractionated irradiation and early changes in noradrenaline induced potassium efflux(86Rb+) in rat parotid gland
- 1992
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Ingår i: Acta Oncologica. - 0284-186X .- 1651-226X. ; 31:3, s. 359-364
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Tidskriftsartikel (refereegranskat)abstract
- The effects of fractionated irradiation on the electrolyte fluid secretion from rat parotid gland were studied. Secretion was measured as noradrenaline stimulated potassium efflux in vitro with Rb-86+ as tracer for potassium. The irradiation was delivered either as a five-day schedule (total dose 20, 25, 30, 35, 40, 45 Gy) or a two-day schedule (total dose 24, 32 Gy). The noradrenaline stimulated efflux was decreased in comparison with contralateral controls 10 days after the last irradiation. The effect was dose-dependent. Based on the data available, alpha/beta ratio of the used system was calculated to about 20 Gy, which corresponds to other results regarding early radiation effects.
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| 3. |
- Franzén, Lars, et al.
(författare)
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Fractionated-irradiation and late changes in rat parotid-gland : effects on the number of acinar-cells, potassium efflux, and amylase secretion
- 1993
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Ingår i: International Journal of Radiation Biology. - : Informa UK Limited. - 0955-3002 .- 1362-3095. ; 64:1, s. 93-101
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Tidskriftsartikel (refereegranskat)abstract
- Irradiation of head- and neck cancer commonly results in oral dryness and discomfort for the patients due to salivary gland damage. The exact mechanisms behind the inherent radiosensitivity of salivary glands remain to be elucidated. In the present study, we used different in vitro secretory models and quantitative morphological characterization of rat parotid gland following fractionated unilateral irradiation to one gland on a 5-day fraction schedule (Monday-Friday) with 6 MV photons (total dose 30, 35, 40 and 45 Gy) or a two-fractions regimen in 5 days (Monday and Friday) with total dose of 24 and 32 Gy. The contralateral shielded gland served as control, and parallel analyses of irradiated and control glands were performed 180 days following the last irradiation treatment. The relative noradrenaline stimulated electrolyte secretion (rubidium-86 tracer for potassium) was decreased in the irradiated compared with control glands. The noradrenaline-stimulated exocytotic amylase release was not significantly affected by irradiation, but the gland content of amylase was decreased dose-dependently. The quantitative morphological analysis revealed a dose-dependent decline in the number of acinar cells, whereas the other parenchymal cells (intercalated, striated- and excretory duct cells) were un-, affected by the irradiation compared with control glands.
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| 4. |
- Gustafsson, Mikael, et al.
(författare)
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A distributed image-processing system for measurements of intracellular calcium in living cells
- 1991
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Ingår i: Computer Methods and Programs in Biomedicine. - : Elsevier. - 0169-2607 .- 1872-7565. ; 36:4, s. 199-221
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Tidskriftsartikel (refereegranskat)abstract
- During the last decade, image-processing techniques have been introduced as a valuable tool in biologically oriented research. In combination with novel fluorescent probes, these techniques permit assessment of subcellular distributions of several intracellularly important cations, such as free calcium ions and protons. Typically, systems used for image processing are located centrally around the experimental setup. This configuration has drawbacks, mainly because the laborious extraction and processing of data that generally follow an experimental session limits the access to the system for other investigators. We describe here the principles of a distributed image processing system, based on IBM-compatible personal computers (PCs), that without extra hardware can cope with all the necessary image processing involved in imaging of intracellular cations. The potential of the PC as an image processor, however, reaches beyond this specific application and many image processing tasks can be carried out successfully on a standard PC. Thus, the centrally located dedicated image processor is used only for image acquisition in the experimental situation. This in turn optimizes the utilization of expensive resources and increases efficiency. The mouse-operated software is described in detail, so that interested investigators can extract useful parts for integration into their own applications and experimental environment.
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| 5. |
- Gustafsson, Mikael, et al.
(författare)
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A novel principle for quantitation of fast intracellular calcium changes using Fura-2 and a modified image processing system : applications in studies of neutrophil motility and phagocytosis
- 1992
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Ingår i: Cell Calcium. - : Churchill Livingstone. - 0143-4160 .- 1532-1991. ; 13:8, s. 473-486
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Tidskriftsartikel (refereegranskat)abstract
- A new principle is described for imaging intracellular free calcium [Ca2+]i changes in single, living cells utilizing the fluorescent probe Fura-2. It is based upon video color mixing in real time and allows high-speed visualization, at maximum image resolution, of [Ca2+]i changes without digital image ratioing. The epifluorescence images produced by 340 and 380 nm excitations are stored in two memory buffers of a personal computer-based image processing system. Two video signals are generated independently from each buffer and connected to the red and green inputs of a video display. An image is this way created, in which [Ca2+]i shows up as a specific hue, whereas changes in dye concentration, light intensity, cell thickness show up as variations in brightness of the imaged cells. The method has advantages over conventional ratio imaging, notably simplicity and speed, since no calculations are made. Yet it can be combined with traditional digital image processing. The imaging technique allows monitoring of [Ca2+]i changes in rapidly moving cells, like neutrophils. It is demonstrated that during random locomotion on serum-coated glass surfaces, [Ca2+]i levels appeared to oscillate and that the frequency of the oscillations are related to locomotive activity. Furthermore, in Ca2+ free medium, the cells continue to move and phagocytose in the presence of Ca2+ ionophore (ionomycin) and 2 mM EGTA. In the presence of 1 mM extracellular Ca2+, ionomycin-treated cells were not able to move or phagocytose.
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| 6. |
- Gustafsson, Mikael
(författare)
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Motility of human polymorphonuclear leukocytes : An image processing approach
- 1993
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Cell motility is essential for polymorphonuclear leukocyte (PMN) function in the defense against invading microorganisms and in inflammatory processes. Using digital video microscopy and image processing, individual cells were studied during random locomotion, chemotactic locomotion and phagocytosis. When PMN moved in a specific direction, the lipid membrane was found to flow in the same direction, contradicting the rearward lipid flow model of cell locomotion. This was assessed with video photobleaching of a fluorescent lipid probe, Dii. The motility of PMN could not be blocked by ca2+ depletionand /or inhibition of myosin light chain kinase, suggesting that ea2+ and myosin are not necessary for locomotion. Temporal and spatial characteristics of intracellular free ea2+ and intracellular pH were assessed by loading the cells with esters of Fura-2 and the fluorcscein derivative BCECF, respectively. There was a distinct correlation between the direction of PMN locomotion and the slope of the Ca2+ gradient of the cells. PMN moving randomly on a surface often exhibited a rearward gradient with higher ca2+ concentration in the rear of the cell. Experimental reversal of the gradient, however, did not change the direction of motility. During phagocytosis of yeast particles, Saccharomyces cerevisiae, intracellular free calcium rose within seconds after contact with the prey. Intracellular pH varied between 7.1 and 7.3 and was uniform across the cells. After phagocytosis, phagosomal pH first decreased and then returned to neutraL A role for pH in phagosome-lysosome formation is proposed. PMN loaded with self-quenching concentrations of BCECF exhibited a strict correlation between pseudopod protrusions and increase in fluorescence, indicating water influx and dilution of the probe. This finding may be essential for the understanding of actin cytoskeleton dynamics during cell locomotion.
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| 7. |
- Holgersson, Jan, et al.
(författare)
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The blood group B type-4 heptaglycosylceramide is a minor blood group B structure in human B kidneys in contrast to the corresponding A type-4 compound in A kidneys. Structural and in vitro biosynthetic studies.
- 1992
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Ingår i: Biochimica et biophysica acta. - 0006-3002. ; 1180:1, s. 33-43
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Tidskriftsartikel (refereegranskat)abstract
- Blood group A glycolipid antigens have been found based upon at least four different core saccharides (types 1 to 4). The biological significance of this structural polymorphism is not known, although the successful outcome of transplantations of blood group A2 kidneys to blood group O individuals have been partly explained by the low expression of A type-3 and -4 chain glycolipid antigens in A2 kidneys. If graft rejection due to ABO incompatibility is, in any way, correlated to the expression of type-3 and -4 chain blood group glycolipids, it is of interest to identify possible blood group B structures based on these core saccharides. In a non-acid glycosphingolipid fraction isolated from human blood group B kidneys, mass spectrometry, high-temperature gas chromatography-mass spectrometry and probing of thin-layer chromatograms with Gal alpha 1-4Gal-specific Escherichia coli and monoclonal anti-B antibodies provided evidence for minute amounts of a Gal alpha 1-3(Fuc alpha 1-2)Gal beta-HexNAc-Gal alpha 1-4Gal beta-Hex-Ceramide structure consistent with a B type-4 chain heptaglycosylceramide. In contrast, blood group A kidneys have the corresponding A type-4 chain heptaglycosylceramide as the predominant blood group A glycolipid. No, or very low activity of the blood group B gene enzyme on the type-4 chain blood group H hexaglycosylceramide precursor was found by biosynthetic experiments in vitro, which might explain the low expression of type-4 chain blood group B heptaglycosylceramides in human blood group B kidneys.
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| 8. |
- Jacobson, Ken, et al.
(författare)
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Lipid flow in locomoting cells : Response
- 1991
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Ingår i: Science. - : American Association for the Advancement of Science. - 0036-8075 .- 1095-9203. ; 251:4991, s. 318-318
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Tidskriftsartikel (refereegranskat)abstract
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| 9. |
- Lee, Juliet, et al.
(författare)
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The direction of membrane lipid flow in locomoting polymorphonuclear leukocytes
- 1990
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Ingår i: Science. - : American Association for the Advancement of Science. - 0036-8075 .- 1095-9203. ; 247:4947, s. 1229-1233
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Tidskriftsartikel (refereegranskat)abstract
- The objective of this study was to determine the direction of membrane lipid flow in locomoting cells. The plasma membrane of human polymorphonuclear leukocytes was stained with a fluorescent lipid analog dihexadecanoyl indocarbocyanine. A line was photobleached on the cell surface perpendicular to the direction of cell motion. Low-light-level fluorescence microscopy and digital image-processing techniques were used to analyze a series of images taken at short intervals after photobleaching. The bleached line remained visible for about 5 seconds before being erased by diffusional recovery. Examination of fluorescence intensity profiles allowed a comparison to be made between the velocities of line and cell movement. Results indicate that the bleached line moves forward with the same velocity as the cell during locomotion, refuting the retrograde lipid flow model of locomotion. Instead, the plasma membrane lipid appears to move forward according to either the unit movement of membrane or the tank track model of locomotion.
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| 10. |
- Lundqvist, Helen, et al.
(författare)
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Neutrophil control of formylmethionyl-leucyl-phenylalanine induced mobilization of secretory vesicles and NADPH-oxidase activation: Effect of an association of the ligand-receptor complex to the cytoskeleton
- 1994
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Ingår i: Biochimica et Biophysica Acta. Molecular Cell Research. - : Elsevier. - 0167-4889 .- 1879-2596. ; 1224:1, s. 43-50
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Tidskriftsartikel (refereegranskat)abstract
- The stimulus formylmethionyl-leucyl-phenylalanine (FMLP) interacts with neutrophils and generates signal(s) in the cells that induces mobilization of the secretory vesicles as well as activation of the superoxide anion/hydrogen peroxide generating NADPH-oxidase. Binding, at 15°C, of FMLP to its neutrophil surface receptor is followed by an association of the ligand-receptor complex to the cell cytoskeleton, and this association occurs concomitant with a desensitization of the cells with respect to activation of the NADPH-oxidase. Other stimuli can still activate the oxidase (in fact even induce a primed response), indicating that the observed phenomenon is stimulus specific and could not be accounted for by an effect on the oxidase itself, but rather that the association of the ligand-receptor complex to the cytoskeleton eliminates the capacity of the complex to generate the signal(s) that activates the NADPH-oxidase. The cytoskeleton associated ligand-receptor complex generates, however, the signal(s) responsible for mobilization of the secretory vesicles, to the plasma membrane, and this mobilization occurs without any increase in the intracellular concentration of free Ca2+.
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