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The high burden of disease and the long-lasting sequelae following Streptococcus pyogenes (Strep A) infections make the development of an effective vaccine a global health priority. Streptolysin O (SLO), is a key toxin in the complex pathogenesis of Strep A infection. Antibodies are elicited against SLO after natural exposure and represent a key target for vaccine-induced immunity. Here we present the setup and characterization of a hemolysis assay to measure functionality of anti-SLO antibodies in human sera. Assay specificity, precision, linearity, reproducibility, and repeatability were determined. The assay was demonstrated to be highly sensitive, specific, reproducible, linear and performed well in assessing functionality of anti-SLO antibodies induced by exposed individuals. Moreover, different sources of critical reagents, in particular red- blood cells, have been compared and had minimal impact on assay performance. The assay presented here has throughput suitable for evaluating sera in vaccine clinical trials and sero-epidemiological studies to gain further insights into the functionality of infection- and vaccine-induced antibodies., Competing Interests: Declaration of competing interest This work was performed with funds from CARB-X, and with internal funding form GlaxoSmithKline Biologicals SA, who is the sponsor of the study.The work was also supported by funding from the New Zealand Ministry of Health (Te Whatu Ora) as part of an initiative to accelerate Strep A vaccine development for Aotearoa New Zealand. The external funders had no role in the design of the study, in the collection, analyses or interpretation of data, in the writing of the manuscript, or in the decision to publish the results. GSK Vaccines Institute for Global Health Srl is an affiliate of GlaxoSmithKline Biologicals SA. M.C., L.R., L.M, M.I, F.B.S., D.G.M. and O.R. are employees of the GSK group of companies. FBS, MI, DGM, MC, and OR report ownership of GSK shares/share options., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

The pneumolysin (Ply) protein of Streptococcus pneumoniae is composed of four domains and possesses several different but related activities. In this study, recombinant Ply and two truncated forms, Ply domain 1-3 and Ply domain 4 (rPly4), were expressed and characterized regarding their participation in apoptosis, the stimulation of cytokine production, hemolytic activity and virulence. rPly4 activated murine bone marrow-derived dendritic cells in a Toll-like receptor (TLR) 4-dependent manner. The rPly4 alone was able to produce hemolytic activity at high concertation and penetrate the lipid bilayer. We further demonstrated that domain 4 of Ply involved in the virulence of the bacteria in mouse model. In the absence of apoptotic activity, the virulence level caused by rPly4 was similar to that of full length Ply. Our data suggested that domain 4 of Ply alone with TLR4 agonist and hemolytic activity may play roles in virulence of Streptococcus pneumoniae., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

We studied the protein amount and activity of the major virulence factors hemolysin, cysteine protease streptococcal pyrogenic exotoxin B (SpeB), and NAD glycohydrolase (NADase), which are produced by Streptococcus pyogenes type T-25, with a food poisoning outbreak. The three virulence factors were analyzed by activity and amount of protein using supernatants at 2-30 h of culture. All these virulence factors were confirmed by their activity. Streptolysin O (SLO), SpeB, and NADase were immunochemically confirmed at protein level by Western blot analysis. Two hemolytic forms (70 and 60 kDa) of SLO were identified. SpeB was detected as a 44-kDa precursor form and a 30-kDa mature form. NADase was 50 kDa. SLO protein peaked at 8 h of culture, which corresponded with the hemolytic activity peak. Conversion from precursor to SpeB protein peaked at 14 h of culture. The conversion peak corresponded to the activity expression time. Also, mature SpeB protein peaked at 24 h of culture and corresponded to SpeB activity peak. Electrophoretic analysis clarified the relationship between SLO protein and SpeB protein, although amounts of SLO and SpeB have been reported to be inversely proportional to activity. NADase protein peaked at 12 h of culture, but protein level did not correspond to the peak. Because the NADase protein peak was closer to SpeB activity than SLO protein, our results suggested NADase protein was degraded at 12 h of culture. The time course production of these virulence factors is discussed.

A new assay has been developed to measure mitochondrial ATP synthesis of cultured mammalian cells. Cells in a microplate are exposed to streptolysin O to make plasma membranes permeable without damaging mitochondrial function and ATP synthesis is monitored by luciferase. Addition of inhibitors of F₀F₁-ATP synthase (F₀F₁), respiratory chain, TCA cycle and ATP/ADP translocator, as well as knockdown of β-subunit of F₀F₁, resulted in loss of ATP synthesis. Compared with the conventional procedures that need mitochondria fractionation and detergent, this assay is simple, sensitive and suitable for high-throughput analysis of genes and drugs that could affect mitochondrial functional integrity as represented by ATP synthesis activity., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Investigation of MHC class I endogenous antigen processing and presentation is important for understanding non-self immune recognition, vaccine design, immune evasion by pathogens and tumours, and elucidation of the mechanisms of certain auto-immune diseases. To study the MHC class I antigen-processing pathway, the protein antigen of interest has to be expressed inside the cells. To express a desired protein antigen, delivery of protein coding genes into the cell using transfection or infection by a virus carrying the gene of the antigen is a popular method. But there is no satisfactory method using the intact protein as antigen directly for investigation of MHC antigen processing and presentation that can give a quantitative result for the efficiency of antigen processing. Here we describe a novel method to deliver protein antigen directly into the cytosol of cells for quantitative analysis of the efficiency of antigen processing. Adherent mouse fibroblasts, mouse lymphoma cell line RMA, spleen cells and dendritic cells were permeabilized with streptolysin O, and chicken ovalbumin (OVA), a model protein antigen, was successfully introduced into the cells. Mouse fibroblast and RMA cells were able to properly process OVA protein, and OVA-derived peptide OVA(258-265) (or SIINFEKL) was successfully presented via the MHC class I molecule (K(b)) to SIINFEKL-K(b)-specific T cell hybridoma B3Z. As expected, delivery of OVA protein into RMA-S, a cell line with a deficiency in MHC class I antigen processing due to a TAP defect, failed to present the SIINFEKL epitope to B3Z.

RNA interference (RNAi) has been shown to be a valuable tool to specifically target gene expression in a number of organisms becoming an indispensable weapon in the arsenal in functional genomics. In this study, we demonstrate that streptolysin-O (SLO) reversible permeabilisation is an efficient method to deliver small interfering RNAs (siRNAs) to hard-to-transfect human myeloma cell lines. We used published, pre-validated siRNAs for ERK2 and non-silencing siRNA control. We transfected siRNAs into human myeloma cell lines using SLO reversible permeabilisation method. Flow cytometry and western blot analysis were performed to assess the effect of SLO on transfection efficiency and ERK2 knockdown. These experiments demonstrate that SLO reversible permeabilisation method is an efficient and easy-to-use method to deliver siRNAs into human myeloma cell lines. Optimised SLO permeabilisation method showed to transfect >80% of JIM-3, H929, RPMI8226 and U266 cells, with minimal effect on cell viability (<10%) and cell cycle. Equally important, SLO permeabilisation induced a substantial knockdown of ERK2 at the protein level. These studies demonstrate that reversible SLO permeabilisation can successfully be applied to hard-to-transfect human myeloma cell lines to effectively silence genes.

Patch clamping was used to investigate, for the first time, the electrophysiological properties of pores formed by a cholesterol-dependent cytolysin in the membrane of nucleated cells rather than in artificial membranes as previously. Pneumolysin pores in inside-out patches of CHO cell membranes had a broad range of conductance classified into small (<200 pS), medium (>200 pS and <1 nS) and large (>1 nS). A lytic-deficient mutant of pneumolysin (W433F) induced a similar spectrum of channels but there were more small channels than with wild-type (WT) toxin and the number of events was decreased. The WT toxin also induced channels when given to the inside surface of the plasma membrane.

Objectives: The main objective of this study was to investigate the effects of pneumococcal hyaluronidase (0.1-10microg/ml), alone and in combination with pneumolysin (50 and 100ng/ml), on human ciliated epithelium., Methods: Ciliary beat frequency (CBF) and structural integrity of human ciliated respiratory epithelium in vitro were studied using a phototransistor technique and a visual scoring index, respectively., Results: Hyaluronidase per se did not affect either CBF or the structural integrity of the epithelium. However, preincubation of the epithelial strips with hyaluronidase (10microg/ml) for 30min at 37 degrees C significantly potentiated pneumolysin-mediated ciliary slowing and epithelial damage. Hyaluronan, a substrate of hyaluronidase, had no effects on the ciliated respiratory epithelium in concentrations up to 100microg/ml and did not antagonize the injurious effects of pneumolysin on the epithelium. However, preincubation of the epithelial strips with hyaluronan (100microg/ml) was associated with attenuation of the ciliary slowing and epithelial damage induced by incubation of the strips with hyaluronidase (10microg/ml) for 30min at 37 degrees C followed by addition of pneumolysin (50ng/ml)., Conclusions: Although having no direct effects alone, hyaluronidase may contribute to pneumolysin-mediated damage and dysfunction to respiratory epithelium, thereby favoring colonization and subsequently extra-pulmonary dissemination of the pneumococcus.

Previously it was shown that the antimicrobial protein granulysin possesses potent membranolytic activity against Mycobacterium tuberculosis. Here we demonstrate that granF2 and G13, which are two short synthetic peptides derived from granulysin, inhibited the in vitro growth of clinical isolates of both multidrug resistant and drug susceptible strains of M. tuberculosis. Importantly, a particularly high activity against multidrug resistant M. tuberculosis correlated with a reduced growth rate compared to drug susceptible strains. A synergistic antibacterial effect of granF2 was further observed in combination with ethambutol, a compound with a documented effect on cell wall permeability. This finding suggests that granF2 and ethambutol exert their functions at different levels of the mycobacterial surface. Upon infection of macrophages in vitro, granF2 but not G13 efficiently reduced the intracellular growth of multidrug resistant M. tuberculosis in the presence of the pore-forming protein streptolysin O. The apoptotic function of granF2 apparently promoted destruction of host cells whereby the peptide gained access to and killed intracellular bacteria. Thus, a cost of resistance and a subsequent reduced fitness, measured as decreased growth among multidrug resistant strains of M. tuberculosis, could be associated with increased susceptibility to natural immune defense mechanisms, such as antimicrobial peptides of granulysin. However, a robust cell wall and the membrane of cells still provide physical shelter for the bacteria that may spare sensitive M. tuberculosis stains from being killed.

Following the observation that cells are able to recover from membrane lesions incurred by Staphylococcus aureus alpha-toxin and streptolysin O (SLO), we investigated the role of p38 in this process. p38 phosphorylation occurred in response to attack by both toxins, commencing within minutes after toxin treatment and waning after several hours. While SLO reportedly activates p38 via ASK1 and ROS, we show that this pathway does not play a major role for p38 induction in alpha-toxin-treated cells. Strikingly divergent effects of p38 blockade were noted depending on the toxin employed. In the case of alpha-toxin, inhibition of p38 within the time frame of its activation led to disruption of the recovery process and to cell death. In contrast, blockade of p38 in SLO permeabilized cells did not affect the capacity of the cells to replenish their ATP stores.

This study was designed to investigate the effects of the Streptococcus pneumoniae-derived, pro-inflammatory toxin, pneumolysin (8.37 and 41.75 ng/ml), on the oxidative inactivation of alpha-1-protease inhibitor (API) by chemoattractant-activated human neutrophils in vitro. The elastase inhibitory capacity (EIC) of API in supernatants from unstimulated neutrophils, neutrophils treated with pneumolysin only, or with the chemoattractant FMLP (1 microM) only, or the combination of the toxin with FMLP was measured by a colorimetric procedure based on the activity of added porcine elastase. The EIC of API was unaffected by exposure to pneumolysin only, unstimulated neutrophils, or neutrophils treated with pneumolysin only. However, exposure to FMLP-activated neutrophils resulted in a reduction of the EIC of API, which was significantly (P<0.05) augmented by pneumolysin (mean reductions of 16%, 43% and 83% for FMLP only and in combination with 8.37 and 41.75 ng/ml pneumolysin, respectively), and was attenuated by wortmannin (1 microM), an inhibitor of NADPH oxidase, the oxidant-scavenger methionine (100 microM), and depletion of Ca2+ from the cell-suspending medium. These pro-proteolytic interactions of pneumolysin with chemoattractant-activated neutrophils may contribute to the invasiveness of the pneumococcus.

We report a transient modulation of cell fate in fibroblasts briefly exposed to an extract derived from the rat insulin-producing beta cell line, INS-1E. Primary fetal rat fibroblasts were reversibly permeabilized with Streptolysin O, incubated for 1h in a 15,000g INS-1E nuclear and cytoplasmic extract, resealed, and cultured. A first marker of change in cell fate was a reduction of cell and nuclear size within days of exposure to extract such that in some instances the fibroblasts resembled INS-1E cells. Second, two beta cell transcripts, Pdx-1 and insulin, were detected in the fibroblasts for up to 4 weeks. Third, (pro)insulin labeling was detected in 5-30% of the cells for a period of 8-14 days after incubation in extract. These phenotypes were absent from fibroblasts exposed to heat-treated INS-1E extracts, a human fibroblast cell line-derived extract or buffer. The results indicate that the extract of an insulinoma-derived cell line can promote at least a transient modification of cell fate towards a beta cell phenotype in non-beta cells. Because they are easily accessible, cell extracts may represent a practical source of material for investigating the mechanisms of alteration of a nuclear and cellular program.

Phospholipase D (PLD) regulates the polymorphonuclear leukocyte (PMN) functions of phagocytosis, degranulation, and oxidant production. Ceramide inhibition of PLD suppresses PMN function. In streptolysin O-permeabilized PMNs, PLD was directly activated by guanosine 5'-[gamma-thio]triphosphate (GTP gamma S) stimulation of adenosine diphosphate (ADP)-ribosylation factor (ARF) and Rho, stimulating release of lactoferrin from specific granules of permeabilized PMNs; PLD activation and degranulation were inhibited by C2-ceramide but not dihydro-C2-ceramide. To investigate the mechanism of ceramide's inhibitory effect on PLD, we used a cell-free system to examine PLD activity and translocation from cytosol to plasma membrane of ARF, protein kinase C (PKC)alpha and beta, and RhoA, all of which can activate PLD. GTP gamma S-activated cytosol stimulated PLD activity and translocation of ARF, PKC alpha and beta, and RhoA when recombined with cell membranes. Prior incubation of PMNs with 10 microM C2-ceramide inhibited PLD activity and RhoA translocation, but not ARF1, ARF6, PKC alpha, or PKC beta translocation. However, in intact PMNs stimulated with N-formyl-1-methionyl-1-leucyl-1-phenylalamine (FMLP) or permeabilized PMNs stimulated with GTP gamma S, C2-ceramide did not inhibit RhoA translocation. Exogenous RhoA did not restore ceramide-inhibited PLD activity but bound to membranes despite ceramide treatment. These observations suggest that, although ceramide may affect RhoA in some systems, ceramide inhibits PLD through another mechanism, perhaps related to the ability of ceramide to inhibit phosphatidylinositol-bisphosphate (PIP2) interaction with PLD.

Several "soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor" (SNARE) proteins have been identified in rat parotid acinar cells, including VAMP-2, syntaxin 4, and SNAP-23. Furthermore, an association between Munc18c (Munc18-3) and syntaxin 4 has been reported. However, the role of Munc18-3 in secretory granule exocytosis on parotid acinar cells remains unclear. In the present study, we investigated the role of Munc18-3 in rat parotid acinar cells. Munc18-3 was localized on the apical plasma membrane where exocytosis occurs and interacted with syntaxin 4. Anti-Munc18-3 antibody dose-dependently decreased isoproterenol (IPR)-induced amylase release from SLO-permeabilized parotid acinar cells. Furthermore, stimulation of the acinar cells with IPR induced translocation of Munc18-3 from the plasma membrane to the cytosol. Munc-18-3 was not phosphorylated by a catalytic subunit of protein kinase (PK) A but phosphorylated by PKC. Treatment of the plasma membrane with PKC but not PKA induced displacement of Munc18-3 from the membrane. The results indicate that Munc18-3 regulates exocytosis in the acinar cells for IPR-induced amylase release and that phosphorylation of Munc18-3 by PKA is not involved in the mechanism.

We have investigated the effects of pneumolysin and H2O2, putative virulence factors of Streptococcus pneumoniae, on the ciliary beat frequency and structural integrity of human ciliated epithelium in vitro. Human ciliated epithelium was obtained by brushing the inferior nasal turbinate of healthy human volunteers. Ciliary slowing (CS) was measured using a photo-transistor technique and epithelial damage (ED) was documented using a visual scoring index. Effects of recombinant pneumolysin (100 ng/ml), a mutant pneumolysin preparation with markedly reduced haemolytic activity (100 ng/ml) and reagent H2O2 (100 microM) were measured alone and in combination, in the absence and presence of catalase (1000 units/ml). When used individually, both recombinant pneumolysin and H2O2 caused significant (P < 0.05) CS and ED. The effects of H2O2 but not those of pneumolysin were almost completely attenuated by catalase, while the mutant pneumolysin preparation did not cause significant CS or ED. When used in combination, the effects of pneumolysin and H2O2 on CS and ED were additive as opposed to synergistic. These actions of pneumolysin and H2O2 may contribute to the pathogenesis of respiratory tract infections caused by the pneumococcus.

Exocytosis of perforin, subsequent binding of perforin to the target cell membrane, and formation of lytic pores form an important pathway involved in the induction of tumor cell death by cytotoxic effector cells. Here we describe a novel escape mechanism employed by tumor cells to protect themselves from granule-mediated cell death: We were able to demonstrate that the resistance of the human leukemia cell line ML-2 to natural killer (NK)-cell-mediated killing is not caused by impaired NK-cell activation but by resistance against effector molecules contained in the granules of cytotoxic cells. No resistance was observed against other pore-forming agents like complement and streptolysin O. By using the NK-susceptible leukemia cell line K562, we could show that the induction of cell death by cytotoxic granules can be blocked completely by anti-perforin antibodies, indicating that perforin is essentially involved in this process. Flow cytometric data revealed that an impaired binding of perforin on the tumor cell membrane is mainly responsible for target cell resistance, because perforin turned out to bind well on K562 cells but is not able to attach to the surface of ML-2 cells. After impaired binding of perforin was identified as a potential mechanism of tumor cell resistance, leukemia cells from 6 patients with acute myeloid leukemia (AML) were examined. As predicted, AML cells that failed to bind perforin on their surface demonstrated complete resistance toward NK-cell-mediated cytotoxicity. Thus, perforin resistance could represent an important tumor escape mechanism that should be considered when cytotoxic effector cells are used for cellular immunotherapy. (Blood. 2000;96:594-600)

Plasma membrane receptors can undergo translocation in the plane of plasma membrane after binding of polyvalent ligands. Ligand/receptor clusters, named patches, can collect into a polar cap, presumably due to their association with the submembrane actin-based cytoskeleton. We found that the assembly of Fcgamma receptor II caps in human monocytic U937 cells was accompanied by the accumulation of spectrin and actin in the cap region. Permeabilization of cells with streptolysin O rendered capping sensitive to inhibition by phalloidin, an actin filament stabilizing agent. A rabbit antibody directed against the chicken erythrocyte alpha-subunit of spectrin, an actin- and membrane-binding protein, also blocked the capping in a dose dependent manner. The inhibition reached approximately 50% after 20 minutes of cell treatment with the antibody. Anti-alpha-spectrin targeted specifically its submembrane antigen, in contrast to unspecific antibodies which remained dispersed in the cell interior and had no influence on the cap assembly. Our results indicate an active engagement of spectrin and actin filaments in the capping of Fcgamma receptor II., (Copyright 1999 Academic Press.)

Isolated single smooth muscle cells from the fundus of a guinea-pig stomach were permeabilized by use of streptolysin-O (0.5 U/ml). Most of the permeabilized cells responded to 0.6 microM Ca2+, but not to 0.2 microM Ca2+, with a resulting maximal cell shortening to approximately 71% of the resting cell length. These cells were relaxed again by washing with the Ca2+-free solution (2.5 nM free Ca2+) for 3-5 min. Addition of 10 microM acetylcholine (ACh) resulted in both a marked decrease in the concentration of Ca2+ required to trigger a threshold response and an increase in the maximal cell shortening, indicating that the cells retained the muscarinic receptor function. When the cell treated with a protein kinase C (PKC) inhibitor, K-252b (1 microM), for 3 min was exposed to 10 microM ACh in the presence of K-252b, the cell shortened within 2 min with a maximal cell shortening. When the cell shortening was induced by 10 microM ACh plus 1 microM Ca2+ in the presence of K-252b (1 microM) or more selective PKC inhibitors, such as calphostin C (1 microM) or PKC pseudosubstrate peptide (100 microM), the extension of the shortened cells, by washing with the Ca2+-free solution, was significantly inhibited. In contrast, K-252b (1 microM) did not inhibit the relaxation of Ca2+-induced shortened cells. These results suggest that the receptor-mediated activation of PKC in the process of ACh-induced cell shortening plays a role in the subsequent relaxation of the shortened cells.

We tested whether the exposure of human monocytic cells to streptococcal erythrogenic toxins A, B, C and a streptococcal-derived Mitogert BX is associated with synthesis of neopterin in vitro. Neopterin production was not induced when the human myelomono-cytoma cell line THP-1 was stimulated with these toxins, and there was only a slight co-stimulatory effect of streptococcal erythrogenic toxin A together with interferon-gamma stimulation. However, these toxins induced interferon-gamma and further neopterin production in peripheral blood mononuclear cells of three healthy individuals. This neopterin formation could be blocked by anti-human interferon-gamma. From our investigations we conclude that there is no direct effect of streptococcal erythrogenic toxins on neopterin production by monocytic cells. However, the data obtained in peripheral blood mononuclear cell culture imply that these toxins are able to stimulate neopterin production in humans via the induction of huge amounts of interferon-gamma.

Exponentially growing CHO cells exposed to millimolar concentrations of sodium butyrate (SB) for 24 h were treated with AluI using two methods of cell poration, i.e., electroporation and streptolysin O (SLO). Under both conditions, SB was found to induce a 2-4-fold increase in AluI-induced chromosomal aberrations. When cells in monolayer were treated with AluI/SLO, lower concentrations of SB (2.5 mM) and AluI (1-4 U/ml) were required to produce a similar effect as that observed for electroporated cells, demonstrating the differential sensitivity of the two methods. Furthermore, in AluI/SLO-treated cells, a higher percentage of cells was found to show increased frequencies of aberrations per cell, compared to AluI/electroporated cells. The mechanism by which SB modulates the cell response to AluI treatment might involve changes in chromatin configuration thereby increasing the accessibility of AluI to different parts of chromatin.

ACTH-(1-24), 1 microM, enhanced the Ca(2+)-dependent release of [3H]dopamine ([3H]DA) from intact septal synaptosomes by approximately 30%, but had no effect on the release of [3H]noradrenaline ([3H]NA) from intact cortical synaptosomes. Since a strong correlation has been reported between B-50 (phosphorylation) and [3H]NA release from intact or streptolysin-O- (SL-O-) permeated cortical synaptosomes, we investigated whether the effects of ACTH-(1-24) on the release of radiolabelled transmitters are mediated by B-50. We observed that the increment in the release of [3H]DA from SL-O-permeated septal synaptosomes as a result of exposure to a high Ca2+ concentration was much less pronounced than that of the release of [3H]NA from SL-O permeated septal and cortical synaptosomes. ACTH-(1-24) concentration-dependently inhibited [3H]NA release from SL-O-permeated cortical synaptosomes (IC50 value of approximately 10 microM) when ACTH-(1-24) was added 150 s prior to the Ca2+ trigger. Simultaneous addition of ACTH-(1-24), SL-O and Ca(2+)-buffers to cortical synaptosomes did not lead to a change in [3H]NA release at any of the ACTH-(1-24) concentrations tested. ACTH-(1-24) had no effect on B-50 phosphorylation in intact synaptosomes, whereas it concentration-dependently inhibited B-50 phosphorylation in permeated cortical synaptosomes (IC50 value of 100 microM). ACTH-(1-24) inhibited (IC50 value of 10 microM) B-50/calmodulin binding in vitro. We conclude that the effects of high concentrations of ACTH-(1-24) on various biochemical B-50 related parameters are not likely to represent the mechanisms underlying the action of ACTH-(1-24) on neurotransmitter release.

The effects of the phosphatase inhibitors calyculin A and okadaic acid on amylase release from streptolysin-O permeabilized rat pancreatic acini were investigated. Both agents induced similar biphasic effects with moderate potentiation of calcium-stimulated amylase release at medium and strong inhibition at higher concentrations. Calyculin A was thirty times more potent than okadaic acid and at 100 nM totally inhibited calcium-induced amylase release while 3 microM okadaic acid reduced amylase release by 78%. 100nM calyculin A also completely inhibited GTP gamma S-potentiated amylase release and partially inhibited phorbol ester potentiated secretion. The data indicate that inhibition of a serine/threonine phosphatase, probably a type 1 phosphatase, leads to inhibition of calcium-induced amylase release in permeabilized pancreatic acini.

A procedure is described for the poration of living CHO cells with the bacterial cytotoxin streptolysin O (SLO) which allows the introduction into cells of the restriction endonuclease Pvu II to mimic and model the effects of ionising radiation in causing chromosomal damage. The dependence of this clastogenic effect of Pvu II on SLO concentration was measured by assaying the formation of micronuclei in cytokinesis-blocked binucleate cells. The optimum concentration was found to be 0.045 U/ml. Using the micronucleus assay, the time-course of expression of chromosome damage was investigated and found to show a biphasic kinetic with time. Using a sampling time of 30 h, a dose-effect curve for micronucleus induction by Pvu II was generated. Using this optimized SLO treatment protocol, the frequency of metaphase chromosome damage was subsequently investigated and found to be also linearly related to Pvu II concentration and total aberrations were approximately double the frequency of micronuclei. The induction and repair kinetics of DNA double-strand breaks were investigated in CHO cells treated with SLO and Pvu II using the neutral filter elution technique at pH 9.6. The data presented show that SLO can be used as an alternative method for porating cells to allow the introduction of restriction endonucleases into cells.

Disruption of microfilaments in rat basophilic leukemia (RBL) cells by exposure to cytochalasin B is observed to potentiate the rate of antigen-stimulated secretion from these cells. Under these conditions, cytochalasin B is without effect on the antigen-stimulated production of inositol phosphates or 45Ca2(+)-influx. In streptolysin-O-permeabilized RBL cells, cytochalasin B is observed to potentiate the rate of secretion in response both to guanosine 5'-(2-thio)-O-triphosphate (GTP gamma S) and to Ca2+ (buffered between 0.1 and 10 microM). However, under these conditions, cytochalasin B does not affect to antigen-stimulated production of inositol phosphates. Consistent with these data, microfilaments are proposed to regulate a terminal step in exocytosis, in a physiologically relevant manner.

The lytic effect of Clostridium perfringens delta toxin was investigated on goat, human, rabbit, and guinea pig platelets. In contrast to erythrocytes from the latter three species, which are insensitive to the toxin, the platelets were equally lysed by the same amount of toxin. These results suggest the presence of GM2 or GM2-like ganglioside(s) as a specific recognition site of the toxin on platelet plasmic membrane as previously established for sensitive erythrocytes. Plasmic membrane damage of human platelets was evidenced by the release of entrapped alpha-[14C]aminoisobutyric acid used as a cytoplasmic marker. The specific binding of hemolytically active 125I-delta toxin by human and rabbit platelets was practically identical, dose dependent, and inhibitable by GM2. Labeled toxin was also bound by various subcellular organelles separated from rabbit platelets except the 5-hydroxytryptamine (5-HT)-containing dense bodies, suggesting the absence or inaccessibility of GM2 on the surface of the latter organelles. This result correlates with the low amounts of 5-[3H]HT liberated after platelet challenge with delta toxin whereas this mediator was massively liberated upon lysis by the sulfhydryl-activated toxin alveolysin. The levels of M and P forms of phenol sulfotransferase (PST), involved in 5-HT catabolism, were determined in human platelet lysates after challenge with delta toxin, alveolysin, and other disruptive treatments. The low PST-M activities detected after lysis by delta toxin suggest that this isoenzyme is very likely associated to dense bodies in contrast to PST-P which is cytoplasmic. Platelet lysis by the toxin allows easy separation of these organelles.

Adrenal medullary chromaffin cells were permeabilized by treatment with a streptococcal cytotoxin streptolysin O (SLO) which generates pores of macromolecular dimensions in the plasma membrane. SLO did not provoke spontaneous release of catecholamines or chromogranin A, a protein marker of the secretory granule, showing the integrity of the secretory vesicle membrane. However, the addition of micromolar free calcium concentration induced the corelease of noradrenaline and chromogranin A, indicating that secretory products are liberated by exocytosis. Calcium-dependent exocytosis from SLO-permeabilized cells required Mg-ATP and could not occur in the presence of other nucleotides. The pores generated by the toxin were large enough to introduce proteins, e.g., immunoglobulins, but also caused efflux of the cytosolic marker lactate dehydrogenase. Despite this, the cells remained responsive to calcium for up to 30 min after permeabilization, indicating that they retained their secretory machinery. In the search for a functional role of cytoskeletal proteins in the secretory process, we used SLO-permeabilized cells to examine the localization of filamentous actin, using rhodamine-phalloidin, and that of the actin-severing protein, gelsolin, using specific antibodies. It was found that both F-actin and gelsolin were exclusively localized in the subplasmalemmal region of the cell. We examined the relationship between actin disassembly, the elevation of intracellular calcium and secretion in SLO-treated cells. F-Actin destabilizing agents such as cytochalasin D or DNase I were found to potentiate calcium-stimulated release. The maximal effect was observed at low calcium concentrations (1-4 microM) and at the later stages of the secretory response (after 10 min stimulation). In addition, using rhodamine-phalloidin, we observed that calcium provoked simultaneously both cortical actin disassembly and catecholamine release in SLO-permeabilized cells. These results demonstrate that a close relationship exists between the secretory response and actin disassembly and provide further evidence that intracellular calcium controls the subplasmalemmal cytoskeletal actin organization and thereby the access of secretory granules to exocytotic sites.