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1.
Antimicrob Agents Chemother ; 56(4): 1979-84, 2012 Apr.
Article in English | MEDLINE | ID: mdl-22252818

ABSTRACT

The aim of this study was to determine the potential application of N-chlorotaurine (NCT), N,N-dichloro-2,2-dimethyltaurine (NVC-422), and N-monochloro-2,2-dimethyltaurine (NVC-612) as catheter lock solutions for the prevention of catheter blockage and catheter-related bloodstream infections by testing their anticoagulant and broad-spectrum antimicrobial activities in human blood. NCT, NVC-422, NVC-612, and control compounds were serially diluted in fresh human blood to evaluate the effects on prothrombin time, activated partial thromboplastin time, thrombin time, fibrinogen, and direct thrombin inhibition. Quantitative killing assays against pathogens, including methicillin-resistant Staphylococcus aureus, Escherichia coli, and Candida albicans, were performed in the presence of heparin and human blood. NCT and NVC-612 (1.38 mM each) and 1.02 mM NVC-422 prolonged prothrombin time (Quick value, 17 to 30%), activated partial thromboplastin time 3- to 4-fold to 76 to 125 s, and thrombin time 2- to 4-fold to 34 to 68 s. Fibrinogen decreased from 258 to 283 mg/dl (range of controls) to <40 mg/dl. No direct thrombin inhibition was observed by NVC-422 or NVC-612. Heparin did not influence the bactericidal activity of NCT. The microbicidal activities of NCT, NVC-422, and NVC-612 were maintained in diluted human blood. NCT, NVC-612, and NVC-422 have broad-spectrum antimicrobial activity in blood and anticoagulant activity targeting both intrinsic and extrinsic pathways of the coagulation system. These properties support their application as catheter lock solutions.


Subject(s)
Anti-Bacterial Agents/blood , Anti-Bacterial Agents/pharmacology , Anticoagulants/blood , Anticoagulants/pharmacology , Antifungal Agents/blood , Antifungal Agents/pharmacology , Taurine/analogs & derivatives , Blood Coagulation/drug effects , Blood Coagulation Tests , Buffers , Candida albicans/drug effects , Escherichia coli/drug effects , Heparin/pharmacology , Humans , In Vitro Techniques , International Normalized Ratio , Methicillin-Resistant Staphylococcus aureus/drug effects , Microbial Sensitivity Tests , Solutions , Taurine/blood , Taurine/pharmacology , Thrombin/antagonists & inhibitors
2.
J Biol Chem ; 276(23): 20589-96, 2001 Jun 08.
Article in English | MEDLINE | ID: mdl-11279185

ABSTRACT

Clustering seems to be employed by many receptors for transmembrane signaling. Here, we show that acid sphingomyelinase (ASM)-released ceramide is essential for clustering of CD95. In vitro and in vivo, extracellularly orientated ceramide, released upon CD95-triggered translocation of ASM to the plasma membrane outer surface, enabled clustering of CD95 in sphingolipid-rich membrane rafts and apoptosis induction. Whereas ASM deficiency, destruction of rafts, or neutralization of surface ceramide prevented CD95 clustering and apoptosis, natural ceramide only rescued ASM-deficient cells. The data suggest CD95-mediated clustering by ceramide is prerequisite for signaling and death.


Subject(s)
Ceramides/metabolism , Signal Transduction , fas Receptor/metabolism , Apoptosis , Cell Membrane/metabolism , Cells, Cultured , Humans , Sphingolipids/metabolism
3.
Am J Physiol Renal Physiol ; 279(4): F605-15, 2000 Oct.
Article in English | MEDLINE | ID: mdl-10997910

ABSTRACT

Ion fluxes and volume changes of the whole cell as well as of organelles belong to the hallmarks of apoptosis; however, the molecular mechanism regulating these changes is only poorly characterized. Several ion channels in the plasma membrane, in particular the N-type K(+) channel, the chloride channel cystic fibrosis conductance regulator, and an outward rectifying chloride channel, as well as the mitochondrial permeability transition pore, have been implicated to be involved in signal transduction cascades regulating apoptosis. Furthermore, Bcl-2-like proteins have been suggested to function, at least in part, as ion channels, because they display some homology to bacterial pore-forming toxins. In contrast to the demonstration of the involvement of these different ion channels in apoptosis, the molecular consequences regulated by these ion channels, and finally triggering apoptosis, are almost completely unknown.


Subject(s)
Apoptosis/physiology , Animals , Ceramides/physiology , Humans , Ion Channels/physiology , Signal Transduction/physiology , Sphingomyelin Phosphodiesterase/physiology , fas Receptor/physiology
4.
Biochem Biophys Res Commun ; 272(2): 395-9, 2000 Jun 07.
Article in English | MEDLINE | ID: mdl-10833425

ABSTRACT

Expression of CD95 ligand on parenchymal, epithelial, or tumor cells has been suggested to downregulate the immune response and to control lymphocyte activation. Suppression might be mediated by induction of apoptosis or by inhibition of Ca(2+) channels upon CD95 triggering. We, therefore, aimed to employ this model to modify the immune response to an antigen presented to cytotoxic T cells by antigen-presenting MC57 cells. This model would be very useful to specifically downregulate the immune response to autoantigens in autoimmune situations. However, cytotoxic T cell lines tested in the present study were resistant to CD95 ligand expression on antigen-presenting MC57 cells. In addition, coincubation of the lymphocytes with antigen presenting cells failed to block cytotoxicity mediated by the T lymphocytes. We, therefore, conclude that single expression of CD95 ligand on antigen-presenting cells is insufficient to specifically downregulate an immune response by CD8(+-)triggered immune response.


Subject(s)
Cytotoxicity, Immunologic/immunology , Membrane Glycoproteins/immunology , T-Lymphocytes, Cytotoxic/immunology , fas Receptor/immunology , Animals , Antigen-Presenting Cells/immunology , Antigen-Presenting Cells/metabolism , Apoptosis , Cell Line , Coculture Techniques , Fas Ligand Protein , Flow Cytometry , Humans , Jurkat Cells , Membrane Glycoproteins/analysis , Membrane Glycoproteins/genetics , Mice , Mice, Inbred C57BL , Models, Immunological , Transfection
5.
Proc Natl Acad Sci U S A ; 96(24): 13795-800, 1999 Nov 23.
Article in English | MEDLINE | ID: mdl-10570152

ABSTRACT

Calcium influx through store-operated calcium release-activated calcium channels (CRAC) is required for T cell activation, cytokine synthesis, and proliferation. The CD95 (Apo-1/Fas) receptor plays a role in self-tolerance and tumor immune escape, and it mediates apoptosis in activated T cells. In this paper we show that CD95-stimulation blocks CRAC and Ca(2+) influx in lymphocytes through the activation of acidic sphingomyelinase (ASM) and ceramide release. The block of Ca(2+) entry is lacking in CD95-defective lpr lymphocytes as well as in ASM-defective cells and can be restored by retransfection of ASM. C2 ceramide, C6 ceramide, and sphingosine block CRAC reversibly, whereas the inactive dihydroceramide has no effect. CD95-stimulation or the addition of ceramide prevents store-operated Ca(2+) influx, activation of the transcriptional regulator NFAT, and IL-2 synthesis. The block of CRAC by sphingomyelinase metabolites adds a function to the repertoire of the CD95 receptor inhibiting T cell activation signals.


Subject(s)
Calcium Channel Blockers , Calcium Channels/metabolism , Calcium Signaling , Sphingolipids/metabolism , Sphingomyelin Phosphodiesterase/metabolism , T-Lymphocytes/metabolism , fas Receptor/metabolism , Animals , Calcium/metabolism , Ceramides/metabolism , Humans , Jurkat Cells , Lymphocyte Activation , Mice , Mice, Inbred C57BL , Tumor Cells, Cultured
6.
Oncogene ; 18(35): 4983-92, 1999 Sep 02.
Article in English | MEDLINE | ID: mdl-10490833

ABSTRACT

Induction of apoptosis is a hallmark of cytostatic drug and radiation-induced cell death in human lymphocytes and lymphoma cells. However, the mechanisms leading to apoptosis are not well understood. We provide evidence that ionizing radiation induces a rapid activation of caspase-8 (FLICE) followed by apoptosis independently of CD95 ligand/receptor interaction. The radiation induced cleavage pattern of procaspase-8 into mature caspase-8 resembled that following CD95 crosslinking and resulted in cleavage of the proapoptotic substrate BID. Overexpression of dominant-negative caspase-8 interfered with radiation-induced apoptosis. Caspase-8 activation by ionizing radiation was not observed in cells genetically defective for the Src-like tyrosine kinase Lck. Cells lacking Lck also displayed a marked resistance towards apoptosis induction upon ionizing radiation. After retransfection of Lck, caspase-8 activation and the capability to undergo apoptosis in response to ionizing radiation was restored. We conclude that radiation activates caspase-8 via an Lck-controlled pathway independently of CD95 ligand expression. This is a novel signaling event required for radiation induced apoptosis in T lymphoma cells.


Subject(s)
Apoptosis/radiation effects , Caspases/metabolism , Lymphocyte Specific Protein Tyrosine Kinase p56(lck)/metabolism , Signal Transduction/radiation effects , fas Receptor/metabolism , Antibodies , BH3 Interacting Domain Death Agonist Protein , Carrier Proteins/metabolism , Caspase 8 , Caspase 9 , Caspases/genetics , Enzyme Activation/radiation effects , Enzyme Precursors/genetics , Enzyme Precursors/metabolism , Fas Ligand Protein , Genes, Dominant/genetics , Humans , Jurkat Cells , Kinetics , Lymphocyte Specific Protein Tyrosine Kinase p56(lck)/genetics , Membrane Glycoproteins/genetics , Membrane Glycoproteins/metabolism , Mutation , Protein Processing, Post-Translational/radiation effects , RNA, Messenger/analysis , RNA, Messenger/genetics , Radiation, Ionizing , Solubility , Transfection
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