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1.
Scand J Immunol ; 86(3): 143-155, 2017 Sep.
Article in English | MEDLINE | ID: mdl-28671713

ABSTRACT

The complement and neutrophil defence systems, as major components of innate immunity, are activated during inflammation and infection. For neutrophil migration to the inflamed region, we hypothesized that the complement activation product C5a induces significant changes in cellular morphology before chemotaxis. Exposure of human neutrophils to C5a dose- and time-dependently resulted in a rapid C5a receptor-1 (C5aR1)-dependent shape change, indicated by enhanced flow cytometric forward-scatter area values. Similar changes were observed after incubation with zymosan-activated serum and in blood neutrophils during murine sepsis, but not in mice lacking the C5aR1. In human neutrophils, Amnis high-resolution digital imaging revealed a C5a-induced decrease in circularity and increase in the cellular length/width ratio. Biomechanically, microfluidic optical stretching experiments indicated significantly increased neutrophil deformability early after C5a stimulation. The C5a-induced shape changes were inhibited by pharmacological blockade of either the Cl-/HCO3--exchanger or the Cl- -channel. Furthermore, actin polymerization assays revealed that C5a exposure resulted in a significant polarization of the neutrophils. The functional polarization process triggered by ATP-P2X/Y-purinoceptor interaction was also involved in the C5a-induced shape changes, because pretreatment with suramin blocked not only the shape changes but also the subsequent C5a-dependent chemotactic activity. In conclusion, the data suggest that the anaphylatoxin C5a regulates basic neutrophil cell processes by increasing the membrane elasticity and cell size as a consequence of actin-cytoskeleton polymerization and reorganization, transforming the neutrophil into a migratory cell able to invade the inflammatory site and subsequently clear pathogens and molecular debris.


Subject(s)
Actin Cytoskeleton/immunology , Cell Shape/immunology , Complement C5a/metabolism , Inflammation/immunology , Neutrophils/immunology , Actins/metabolism , Adenosine Triphosphate/metabolism , Cells, Cultured , Chemotaxis , Chloride-Bicarbonate Antiporters/metabolism , Complement C5a/immunology , Humans , Neutrophil Activation , Neutrophils/pathology , Receptor, Anaphylatoxin C5a/metabolism , Receptors, Purinergic P2X/metabolism , Signal Transduction
2.
Biochemistry ; 39(46): 14279-91, 2000 Nov 21.
Article in English | MEDLINE | ID: mdl-11087377

ABSTRACT

DNA strand transfer reactions occur twice during retroviral reverse transcription catalyzed by HIV-1 reverse transcriptase. The 4-chlorophenylhydrazone of mesoxalic acid (CPHM) was found to be an inhibitor of DNA strand transfer reactions catalyzed by HIV-1 reverse transcriptase. Using a model strand transfer assay system described previously [Davis, W. R., et al. (1998) Biochemistry 37, 14213-14221], the mechanism of CPHM inhibition of DNA strand transfer has been characterized. CPHM was found to target the RNase H activity of HIV-1 reverse transcriptase. DNA polymerase activity was not significantly affected by CPHM; however, it did inhibit the polymerase-independent RNase H activity with an IC(50) of 2.2 microM. In the absence of DNA synthesis, CPHM appears to interfere with the translocation, or repositioning, of RT on the RNA.DNA template duplex, a step required for efficient RNA hydrolysis by RNase H. Enzyme inhibition by CPHM was found to be highly specific for HIV-1 reverse transcriptase; little or no inhibition of DNA strand transfer or DNA polymerase activity was observed with MLV or AMV reverse transcriptase, T7 DNA polymerase, or DNA polymerase I. Examination of additional 4-chlorophenylhydrazones showed that the dicarboxylic acid moiety of CPHM is essential for activity, suggesting its important role for enzyme binding. Consistent with the role of the dicarboxylic acid in inhibitor function, Mg(2+) was found to chelate directly to CPHM with a K(d) of 2.4 mM. Together, these studies suggest that the inhibitor may function by binding to enzyme-bound divalent metal cofactors.


Subject(s)
Anti-HIV Agents/chemistry , DNA, Viral/chemistry , HIV Reverse Transcriptase/antagonists & inhibitors , Hydrazones/chemistry , Malonates/chemistry , Reverse Transcriptase Inhibitors/chemistry , Animals , Avian Myeloblastosis Virus/enzymology , Binding Sites , DNA Primers/chemistry , HIV Reverse Transcriptase/chemistry , Humans , Hydrazones/chemical synthesis , Hydrolysis , Kinetics , Magnesium/chemistry , Malonates/chemical synthesis , Mice , Moloney murine leukemia virus/enzymology , Ribonuclease H/antagonists & inhibitors , Ribonuclease H/chemistry , Species Specificity , Spectrophotometry, Ultraviolet , Substrate Specificity , Templates, Genetic , Thymine Nucleotides/chemistry
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