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| 2. |
- Lönnbro, Per, et al.
(author)
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Heat production in chemically skinned smooth muscle of guinea-pig taenia coli
- 1991
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In: Journal of Physiology. - 1469-7793. ; 440, s. 385-402
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Journal article (peer-reviewed)abstract
- 1. The rate of heat production of chemically skinned guinea-pig taenia coli smooth muscle at 25 degrees C was measured using microcalorimetric techniques. 2. Muscle strips were mounted isometrically and incubated in solutions containing MgATP (3.2 mM) and phosphocreatine (PCr, 12 mM), pH 6.9. Activation was obtained by the injection of Ca2+ into the sample compartment of the calorimeter. 3. The heat production rate of the resting preparation (pCa 9) was 0.40 +/- 0.03 mW g-1 wet weight (n = 23). During maximal activation (pCa 4.8) the heat rate increased to 1.12 +/- 0.07 mW g-1 (mean +/- S.E.M., n = 15). With stepwise increase in [Ca2+] from pCa 9 to 4.8 the energetic cost of force maintenance tended to increase at higher [Ca2+]. 4. After activation by Ca2+, the heat production rate reached its maximum while force was still increasing. 5. Changing ionic strength from 90 to 150 mM had no effect on either basal or activated heat rate. Oligomycin, amphotericin B and the adenylate kinase inhibitor Ap5A had no effect on the basal heat rate. 6. Exchanging ATP in the incubation medium for inosine triphosphate (ITP) reduced the force and heat production after injection of Ca2+. The basal heat production was not lowered when ATP was exchanged for ITP. 7. The observed enthalpy change for PCr splitting at 25 degrees C (pH 6.9, ionic strength 90 mM) was -28 +/- 3 kJ mol-1 (mean +/- S.E.M., n = 9). After correction for the phosphate equilibrium, buffer reactions, and Mg2+ binding to PCr and HPO42-, the net enthalpy change is calculated to be -39 +/- 3 kJ mol-1. 8. Heat production in the skinned smooth muscle consists of one basal component present in relaxed muscle, and one component associated with contraction. The nature of the basal heat production is unclear but does not seem to involve turnover of phosphate on the myosin light chains. The increase in the energetic tension cost with increasing activation by Ca2+ has implications for the understanding of the contractile mechanism in smooth muscle.
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| 3. |
- Lönnbro, Per, et al.
(author)
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Analysis of neutrophil membrane traffic during phagocytosis
- 2007
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In: Neutrophil Methods and Protocols (Methods in Molecular Biology). - 1588297888 - 9781588297884
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Book chapter (pop. science, debate, etc.)abstract
- In this chapter, we describe methods to study membrane traffic during phagosome formation and maturation.
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| 4. |
- Lönnbro, Per, et al.
(author)
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Isolation of bacteria-containing phagosomes by magnetic selection.
- 2008
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In: BMC Cell Biology. - : Springer Science and Business Media LLC. - 1471-2121. ; 9:June 27
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Journal article (peer-reviewed)abstract
- BACKGROUND: There is a growing awareness of the importance of intracellular events in determining the outcome of infectious disease. To improve the understanding of such events, like phagosome maturation, we set out to develop a versatile technique for phagosome isolation that is rapid and widely applicable to different pathogens. RESULTS: We developed two different protocols to isolate phagosomes containing dead or live bacteria modified with small magnetic particles, in conjunction with a synchronized phagocytosis protocol and nitrogen cavitation. For dead bacteria, we performed analysis of the phagosome samples by microscopy and immunoblot, and demonstrated the appearance of maturation markers on isolated phagosomes. CONCLUSION: We have presented detailed protocols for phagosome isolation, which can be adapted for use with different cell types and prey. The versatility and simplicity of the approach allow better control of phagosome isolation, the parameters of which are critical in studies of host-bacteria interaction and phagosome maturation.
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| 5. |
- Nordenfelt, Pontus, et al.
(author)
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Different Requirements for Early and Late Phases of Azurophilic Granule-Phagosome Fusion
- 2009
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In: TRAFFIC. - : Wiley. - 1398-9219 .- 1600-0854. ; 10:12, s. 1881-1893
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Journal article (peer-reviewed)abstract
- Phagocytosis and killing of microorganisms are complex processes that involve tightly regulated membrane traffic events. Because many signaling molecules associate with membrane rafts and because these structures can be found on azurophilic granules, we decided to investigate raft recruitment and the signaling requirements for azurophilic granule secretion during phagosome maturation. At the site of phagocytosis of immunoglobulin G-opsonized prey in human neutrophils, we found that early secretion of azurophilic granules was both raft- and calcium-dependent. Subsequently, rafts at the phagocytic site were internalized with the prey. At the fully formed phagosome, the fusion of azurophilic granules was no longer dependent on rafts or calcium. These findings were found to be true also when using Streptococcus pyogenes bacteria as prey, and depletion of calcium affected the kinetics of bacterial intracellular survival. These findings suggest that the mechanisms for delivery of azurophilic content to nascent and sealed phagosomes, respectively, differ in their dependence on calcium and membrane rafts.
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| 6. |
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| 7. |
- Nordenfelt, Pontus, et al.
(author)
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Phagocytosis of Streptococcus pyogenes by all-trans retinoic acid-differentiated HL-60 cells: roles of azurophilic granules and NADPH oxidase.
- 2009
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In: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 4:10
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Journal article (peer-reviewed)abstract
- BACKGROUND: New experimental approaches to the study of the neutrophil phagosome and bacterial killing prompted a reassessment of the usefulness of all-trans retinoic acid (ATRA)-differentiated HL-60 cells as a neutrophil model. HL-60 cells are special in that they possess azurophilic granules while lacking the specific granules with their associated oxidase components. The resulting inability to mount an effective intracellular respiratory burst makes these cells more dependent on other mechanisms when killing internalized bacteria. METHODOLOGY/PRINCIPAL FINDINGS: In this work phagocytosis and phagosome-related responses of ATRA-differentiated HL-60 cells were compared to those earlier described in human neutrophils. We show that intracellular survival of wild-type S. pyogenes bacteria in HL-60 cells is accompanied by inhibition of azurophilic granule-phagosome fusion. A mutant S. pyogenes bacterium, deficient in M-protein expression, is, on the other hand, rapidly killed in phagosomes that avidly fuse with azurophilic granules. CONCLUSIONS/SIGNIFICANCE: The current data extend our previous findings by showing that a system lacking in oxidase involvement also indicates a link between inhibition of azurophilic granule fusion and the intraphagosomal fate of S. pyogenes bacteria. We propose that differentiated HL-60 cells can be a useful tool to study certain aspects of neutrophil phagosome maturation, such as azurophilic granule fusion.
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| 8. |
- Nordenfelt, Pontus, et al.
(author)
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Phagosomal membrane rafts : azurophilic origin, Ca2+ dependence, and modulation by Streptococcus pyogenes bacteria
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Other publication (other academic/artistic)abstract
- Uptake and killing of microorganisms by neutrophils involve tightly regulated membrane traffic events that are governed by complex signals. Many of these are raft-associated, which implies that raft dynamics may be important during phagosome formation. Locally restricted, calcium-dependent, parallel upregulation of markers for membrane rafts and azurophilic granules was observed at the site of phagocytosis of IgG-opsonized prey in human neutrophils. Subsequent internalization of the prey reduced the levels of these markers in the plasma membrane. Streptococcus pyogenes bacteria, that can survive phagocytosis by neutrophils, modulated phagosomal raft acquisition by means of M proteins. Continued, but not early, delivery of rafts to the membrane of phagosomes in neutrophils and HL-60 cells was independent of calcium, as was fusion between azurophilic granules and phagosomes. Nevertheless, calcium depletion affected bacterial killing kinetics. These findings suggest that early delivery of membrane rafts is important for phagosomal maturation in neutrophils and provide new mechanistic insight into the processes required for generation of bactericidal phagosomes.
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| 9. |
- Petersson, Jessica, et al.
(author)
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The human IFN-inducible p53 target gene TRIM22 colocalizes with the centrosome independently of cell cycle phase.
- 2010
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In: Experimental Cell Research. - : Elsevier BV. - 1090-2422 .- 0014-4827. ; 316, s. 568-579
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Journal article (peer-reviewed)abstract
- TRIM22 (Staf50), a member of the TRIM protein family, is an interferon (IFN)-inducible protein as well as a p53 target gene. The function of TRIM22 is largely unknown, but TRIM22 is suggested to play a role in viral defense by restriction of viral replication. In addition, TRIM22 may function as a ubiquitin E3 ligase. In contrast to previous reports showing solely cytoplasmic localization of exogenous TRIM22, we report here that endogenous TRIM22 is localized to both nucleus and cytosol in primary human mononuclear cells, as well as in the human osteosarcoma cell line U2OS. Moreover, we demonstrate a colocalization of TRIM22 with the centrosomes in primary cells as well as in U2OS cells, and show that this colocalization is independent of cell cycle phase. Additionally, our data suggest the colocalization with centrosomes to be independent on the microtubule network. Given that some viral protein assembly takes place in the close vicinity of the centrosome, our data suggest that important functions of TRIM22 such as regulation of viral replication and protein degradation may take place in the centrosome. However, further studies are warranted to certify this notion.
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