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  • 1
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Abstract: Conditions are described for controlled plasma membrane permeabilization of rat pheochromocytoma cells (PC12) and cultured bovine adrenal chromaffin cells by Streptolysin O (SLO). The transmembrane pores created by SLO invoke rapid efflux of intracellular 86Rb+ and ATP, and also permit passive diffusion of proteins, including immunoglobulins, into the cells. SLO-permeabilized PC12 cells release [3H]dopamine in response to micromolar concentrations of free Ca2+. Permeabilized adrenal chromaffin cells present a similar exocytotic response to Ca2+ in the presence of Mg2+/ ATP. Permeabilized PC12 cells accumulate antibodies against synaptophysin and calmodulin, but neither antibody reduces the Ca2+-dependent secretory response. Reduced tetanus toxin, although ineffective when applied to intact chromaffin cells, inhibits Ca2+-induced exocytosis by both types of permeabilized cells studied. Omission of dithiothreitol, toxin inactivation by boiling, or preincubation with neutralizing antibodies abolishes the inhibitory effect. The data indicate that plasma membrane permeabilization by Streptolysin O is a useful tool to probe and define cellular components that are involved in the final steps of exocytosis.
    Type of Medium: Electronic Resource
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  • 2
    Electronic Resource
    Electronic Resource
    s.l. : American Chemical Society
    Biochemistry 33 (1994), S. 7477-7484 
    ISSN: 1520-4995
    Source: ACS Legacy Archives
    Topics: Biology , Chemistry and Pharmacology
    Type of Medium: Electronic Resource
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  • 3
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Vibrio cholerae cytolysin (VCC) is an oligomerizing pore-forming toxin that is related to cytolysins of many other Gram-negative organisms. VCC contains six cysteine residues, of which two were found to be present in free sulphydryl form. The positions of two intramolecular disulphide bonds were mapped, and one was shown to be essential for correct folding of protoxin. Mutations were created in which the two free cysteines were deleted, so that single cysteine substitution mutants could be generated for site-specific labelling. Employment of polarity-sensitive fluorophores identified amino acid side-chains that formed part of the pore-forming domain of VCC. The sequence commenced at residue 311, and was deduced to form a β-barrel in the assembled oligomer with the subsequent odd-numbered residues facing the lipid bilayer and even-numbered residues facing the lumen. Pro328/Lys329 were tentatively identified as the position at which the sequence turns back into the membrane and where the antiparallel β-strand commences. This was deduced from fluorimetric analyses combined with experiments in which the pore was reversibly occluded by derivatization of sulphydryl groups with a bulky moiety. Our data support computer-based predictions that the membrane-permeabilizing amino acid sequence of VCC is homologous to the β-barrel-forming sequence of staphylococcal cytolysins and identify the β-barrel as a membrane-perforating structure that is highly conserved in evolution.
    Type of Medium: Electronic Resource
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  • 4
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    Journal of interventional cardiology 11 (1998), S. 0 
    ISSN: 1540-8183
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: The concept that oxidation is the major single event underlying the transformation of LDL to a proinflammatory molecule dominates the world literature. An alternative hypothesis on the pathogenesis of atherosclerosis will be presented here. We have found that nonoxidative, enzymatic modification of LDL with ubiquitous enzymes also transforms the molecule to an atherogenic moiety. Enzymatically altered LDL (E-LDL) shares major properties in common with lipoproteins that have been isolated from atherosclerotic lesions. It activates complement and is recognized by a scavenger receptor on human macrophages, thus inducing foam cell formation. Uptake of E-LDL is accompanied by induction of MCP−1 synthesis and secretion. In contrast, E-LDL does not stimulate IL-1 or TNF-production and is only a weak inducer of IL-6. Monoclonal antibodies were produced that recognize neoepitopes on E-LDL, but that do not react with native or oxidized LDL. With the use of these antibodies, extensive deposition of E-LDL in very early atherosclerotic lesions was demonstrated. Activated complement components colocalized with E-LDL, corroborating the concept that subendothelially deposited LDL is enzymatically transformed to a complement activator. The relevance of unhalted complement activation was revealed by the finding that C6-deftciency markedly protected against development of diet-induced atherosclerosis in rabbits. Thus, our hypothesis departs from mainstream atherosclerosis research and derives from the recognition that extracellular exposition of free cholesterol by itself confers proinflammatory properties onto LDL. The degrading enzymes probably are present in the extracellular matrix, so the only requirement for atherogenesis to occur is the deposition of large amounts of LDL. Oxidative processes or infections probably play only minor roles, and reduction of LDL plasma levels will predictably represent the single most important prophylactic measure against development and progression of atherosclerosis.
    Type of Medium: Electronic Resource
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  • 5
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Staphylococcal alpha-toxin forms homo-oligomeric channels in lipid bilayers and cell membranes. Here, we report that electrophysiological monitoring of single-channel function using a derivatized cysteine substitution mutant allows accurate determination of the subunit stoichiometry of the oligomer in situ. The electrophysiological phenotype of channels formed in planar lipid bilayers with the cysteine replacement mutant I7C is equal to that of the wild type. When pores were formed with I7C, alterations of several channel properties were observed upon modification with SH reagents. Decreases in conductance then occurred that were seen only as negative voltage was applied. At the level of single channels, these were manifest as stepwise changes in conductance, each step most probably reflecting modification of a single SH group within the oligomer. Because seven steps were observed, the functional channel formed by alpha-toxin in planar lipid membranes is a heptamer.
    Type of Medium: Electronic Resource
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  • 6
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Staphylococcal α-toxin forms heptameric pores that render membranes permeable for monovalent cations. The pore is formed by an amphipathic β-barrel encompassing amino acid residues 118–140 of each subunit of the oligomer. Human fibroblasts are susceptible to α-toxin but are able to repair the membrane lesions. Thereby, toxin oligomers remain embedded in the plasma membrane and exposed to the extracellular medium. In this study, we sought to detect structural changes occurring in the pore-forming sequence during lesion repair. Single cysteine substitution mutants were labelled with the environmentally sensitive fluorochrome acrylodan and, after mixing with wild-type toxin, incorporated into hybrid heptamers on fibroblast membranes. Formation of the lipid-inserted β-barrel was accompanied by characteristic fluorescence emission shifts. After lesion repair, the environment of the residues at the outer surface of the β-barrel remained unchanged, indicating continued contact with lipids. However, the labelled residues oriented towards the channel lumen underwent a green to blue shift in fluorescence, indicating reduced exposure to water. Pore closure proceeded in the presence of calmodulin inhibitors and of microtubule disruptors; however, it was prevented by cytochalasin D and by inhibitors of lipid metabolism. Our findings reveal the existence of a novel mechanism of membrane repair that may consist in constriction of the inserted proteinaceous pore within the lipid bilayer.
    Type of Medium: Electronic Resource
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  • 7
    Electronic Resource
    Electronic Resource
    Springer
    Medical microbiology and immunology 181 (1992), S. 117-126 
    ISSN: 1432-1831
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract We describe a simple and rapid bioassay for estimating fungicidal activity of Amphotericin B in human serum using flow cytometry. The method exploits the fact that Candida albicans damaged by Amphotericin B show a decrease in size and take up propidium iodide to exhibit a red fluorescence after deoxycholate treatment. These phenomena display characteristic dose dependencies, and their assessment permits serum fungicidal activity to be broadly grouped into three categories: (1) subfungicidal; (2) fungicidal; and (3) strongly fungicidal. In normal human serum, these three categories correspond to Amphotericin B concentrations of 0 ≤ 0.5 μg/ml, 0.75–1.5 μg/ml, and 〉2 μg/ml, respectively. Pilot analysis of serum samples obtained from four patients undergoing Amphotericin B therapy confirmed the feasibility of using the flow cytometric assay for estimating drug fungicidal activity ex vivo. The method is very simple, generates results within 5 h, and could prove useful for monitoring therapy with this effective but toxic drug.
    Type of Medium: Electronic Resource
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  • 8
    Electronic Resource
    Electronic Resource
    Springer
    Medical microbiology and immunology 184 (1995), S. 37-44 
    ISSN: 1432-1831
    Keywords: Pore-forming toxin ; Vibrio cholerae ; Membrane damage ; Membrane repair
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract V. cholerae El Tor cytolysin is a secreted, water-soluble protein of M r 60,000 that may be relevant to the pathogenesis of acute diarrhea. In this communication, we demonstrate that the toxin binds to and oligomerizes in target membranes to form SDS-stable aggregates of M r 200000–250000 that generate small transmembrane pores. Pores formed in erythrocytes were approximately 0.7 nm in size, as demonstrated by osmotic protection experiments. Binding was shown to occur in a temperature-independent manner preceding the temperature-dependent oligomerization step. Pores were also shown to be formed in L929 and HEp-2 cells, human fibroblasts and keratinocytes, albeit with highly varying efficacy. At neutral pH and in the presence of serum, human fibroblasts were able to repair a limited number of lesions. The collective data identify V. cholerae El Tor cytolysin as an oligomerizing toxin that damages cells by creating small transmembrane pores.
    Type of Medium: Electronic Resource
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  • 9
    ISSN: 1432-1831
    Keywords: Key wordsHelicobacter pylori ; VacA ; CagA ; Vacuolating cytotoxin ; Bacterial density
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Heterogeneity of the Helicobacter pylori vacA gene may be associated with bacterial virulence and presentation. In this study, the possible correlation between vacA genotypes and gastric histopathology was investigated. Using a modified one-step polymerase chain reaction (PCR)-based method, 122 of 131 H. pylori isolates obtained from 63 of 67 patients from Germany were classified into distinct vacA genotypes according to their signal sequence (s1 or s2) and their midregion alleles (m1 or m2). A possible subtype of m1, now alluded to as m3, was identified in one-third of the isolates. Signal sequence s1 was significantly associated with higher H. pylori density but not with gastric inflammation parameters as compared with s2. Compared with m2, m1 initially appeared to correlate with higher mononuclear cell scores in corpus, although not with H. pylori density. Upon differentiation between m1 and m3, however, only the latter was associated with the high cell scores. Moreover, m3 also correlated with a higher antral H. pylori density. Positive cagA status correlated significantly with vacA signal sequence s1, and higher gastric mononuclear cell scores and corpus neutrophil score. H. pylori density was always associated with enhanced gastric neutrophil and corpus mononuclear cell scores. These data indicate a significant association of specific vacA genotypes with enhanced bacterial density and gastric inflammation. PCR-based identification of the respective alleles can now easily be performed in the diagnostic laboratory using a one-step PCR assay.
    Type of Medium: Electronic Resource
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  • 10
    Electronic Resource
    Electronic Resource
    Springer
    Annals of hematology 60 (1990), S. 309-318 
    ISSN: 1432-0584
    Keywords: Complement cytolysis ; C5b—9 complex ; Immunopathology ; Membrane damage ; Pathophysiology
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary The terminal complement sequence is initiated upon cleavage of C5 with liberation of C5a anaphylatoxin, and involves the assembly of macromolecular C5b—9 complexes either on cell surfaces or in plasma. Cell-bound C5b—9 complexes generate transmembrane pores that can cause cell death, or they can elicit secondary cellular reactions triggered, for example, by passive flux of calcium ions into the cells. In vivo functions of the fluid-phase SC5b—9 complex have not yet been defined, but the identity of S-protein with vitronectin (serum spreading factor) provokes the anticipation that significant biological functions of this complex do exist. The terminal complement sequence may fulfil protective functions when it is triggered on alien cells that are marked for destruction. Dysregulation in the complement sequence may, however, result in detrimental attack by C5b—9 on autologous cells. Examples include not only autoimmune disease states, but also the activation of complement on dead or dying cells, and bystander attack on blood cells during cardiopulmonary bypass. Methods for detecting and quantifying C5b—9 are outlined, and the potential usefulness of such assays in clinical research is discussed.
    Type of Medium: Electronic Resource
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