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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.

By phagocytosis, macrophages engulf large particles, microorganisms and senescent cells in vesicles called phagosomes. Many internalized proteins rapidly shuttle back to the plasma membrane following phagosome biogenesis. Here, we report a new approach to the study of recycling from the phagosomal compartment: streptolysin O- (SLO) permeabilized macrophages. In this semi-intact cell system, energy and cytosol are required to efficiently reconstitute recycling transport. Addition of GDPbetaS strongly inhibits this transport step, suggesting that a GTP-binding protein modulates the dynamics of cargo exit from the phagosomal compartment. GTPases of the Rab family control vesicular trafficking, and Rab11 is involved in transferrin receptor recycling. To unravel the role of Rab11 in the phagocytic pathway, we added recombinant proteins to SLO-permeabilized macrophages. Rab11:S25N, a negative mutant, strongly diminishes the release of recycled proteins from phagosomes. In contrast, wild type Rab11 and its positive mutant (Rab11:Q70L) favor this vesicular transport event. Using biochemical and morphological assays, we confirm that overexpression of Rab11:S25N substantially decreases recycling from phagosomes in intact cells. These findings show the requirement of a functional Rab11 for the retrieval to the plasma membrane of phagosomal content. SLO-permeabilized macrophages likely constitute a useful tool to identify new molecules involved in regulating transport along the phagocytic pathway.

Objective: We previously demonstrated that, when expressed in COS-7 cells, L-histidine decarboxylase (HDC), which has neither an amino terminal signal sequence nor a hydrophobic membrane anchor, was localized in the endoplasmic reticulum (ER), although its orientation in the membrane remains to be clarified., Methods & Results: Protease digestion and immunofluorescence analyses of the cells, of which plasma membrane was selectively permeabilized, revealed that the amino terminal 50-kDa portion of HDC is hardly accessible to proteases and antibodies added exogenously from the cytosolic side. Green fluorescent protein fused with the carboxyl terminal 20-kDa region of HDC at its carboxyl terminus exhibited the same characteristics as native HDC., Conclusion: These results indicate that HDC is tightly associated with the ER membrane with its carboxyl terminal region exposed on the cytosolic side.

The concept of reprogramming a cell is very intriguing and has immense therapeutic potential. Examples from physiology and developmental biology suggest that it may well be possible. Experimental approaches are beginning to suggest this also, in particular the initially astonishing accomplishment of somatic cell nuclear transfer and cloning. This chapter reviews current strategies and describes emerging methods for the proposition of reprogramming cells with cell extracts.

Objective: To discuss the important role of actin polymerization in calcium ionophore A23187-induced human acrosome reaction and its mechanism., Methods: Each spermatozoon specimen was divided into five groups, treated with A23187 3 micromol/L in Group A, Phalloidin 40 micromol/L and A23187 3 micromol/L in Group B, SLO 0.5 U/ml and A23187 3 micromol/L in Group C, SLO 0.5 U/ ml, Phalloidin 40 micromol/L and A23187 3 micromol/L in Group D, and nothing added in Grpup E. Then the percentage of the human acrosome reaction was assessed with Rhodamine-PSA (10 microg/ml)., Results: The difference of the human spermatozoon acrosome reaction was significant (P < 0.01) among the 5 groups with or without SLO, Phalloidin and calcium ionophore A23187 but not between Groups A and B (P > 0.01)., Conclusion: Phalloidin does not work on the acrosome reaction of intact human spermatozoa, but in an SLO-permeabilized human spermatozoal model, it can obviously decrease the percentage of human spermatozoon acrosome reaction, which indicates that the polymerization of actin plays an important role in the course of human spermatozoon acrosome reaction, and mostly acts on the acrosome inside.

Pneumolysin (PLY) is a major virulence factor of Streptococcus pneumoniae that elicits a variety of proinflammatory responses from cells of the host immune system. Intercellular adhesion molecule 1 (ICAM-1) is a cell adhesion molecule involved in leukocyte trafficking toward inflammatory stimuli in extravascular sites. In this study, we evaluated the effect of PLY on expression of ICAM-1 in THP-1 monocytic cells exposed to S. pneumoniae. Exposure of cells to PLY-expressing S. pneumoniae strain WU2 for 6 h led to significantly higher levels of ICAM-1 message than those in cells exposed to either medium alone or DeltaPLY1, a PLY-negative isogenic mutant of WU2. Cells exposed to purified recombinant PLY also showed a dose-dependent increase in ICAM-1 mRNA compared to cells exposed to medium alone. Exposure to recombinant PLY containing a single amino acid substitution (Trp433-->Phe) that decreases cytolytic activity did not increase ICAM-1 mRNA to levels seen with wild-type PLY. In addition, THP-1 cells exposed to wild-type strain WU2 or D39 had increased ICAM-1 on their surface compared to cells exposed to medium alone or their PLY-negative isogenic mutants DeltaPLY1 and DeltaPLY2, respectively. These data indicate that PLY induces transcription and production of a cell adhesion molecule involved in the inflammatory response that may play a role in pneumococcal infection.

Pneumolysin, the cholesterol-dependent cytolysin of Streptococcus pneumoniae, induces inflammatory and apoptotic events in mammalian cells. Toll-like receptor 4 (TLR4) confers resistance to pneumococcal infection via its interaction with pneumolysin, but the underlying mechanisms remain to be identified. In the present study, we found that pneumolysin-induced apoptosis is also mediated by TLR4 and confers protection against invasive disease. The interaction between TLR4 and pneumolysin is direct and specific; ligand-binding studies demonstrated that pneumolysin binds to TLR4 but not to TLR2. Involvement of TLR4 in pneumolysin-induced apoptosis was demonstrated in several complementary experiments. First, macrophages from wild-type mice were significantly more prone to pneumolysin-induced apoptosis than cells from TLR4-defective mice. In gain-of-function experiments, we found that epithelial cells expressing TLR4 and stimulated with pneumolysin were more likely to undergo apoptosis than cells expressing TLR2. A specific TLR4 antagonist, B1287, reduced pneumolysin-mediated apoptosis in wild-type cells. This apoptotic response was also partially caspase dependent as preincubation of cells with the pan-caspase inhibitor zVAD-fmk reduced pneumolysin-induced apoptosis. Finally, in a mouse model of pneumococcal infection, pneumolysin-producing pneumococci elicited significantly more upper respiratory tract cell apoptosis in wild-type mice than in TLR4-defective mice, and blocking apoptosis by administration of zVAD-fmk to wild-type mice resulted in a significant increase in mortality following nasopharyngeal pneumococcal exposure. Overall, our results strongly suggest that protection against pneumococcal disease is dependent on the TLR4-mediated enhancement of pneumolysin-induced apoptosis.

Antisense and antigene oligonucleotides (ONs) are attractive drugs for gene therapy, but major limiting factors for their routine use are inefficient cellular uptake and low accessibility to the target sites. Adding various lipophilic conjugates to the ON improves intracellular delivery as has been previously reported. We studied the cellular delivery of various ON modifications, as well as their cytosolic and nuclear distribution in mammalian Hep2-EGFP-NLS cell line. We compared uptake efficacy of ON and LNA, both conjugated with cholesterol at the 5' end. All ONs were 3' labeled with fluorescent Cy 5 dye. We made a comparison of the ONs uptake efficacy and the kinetics, both adding ONs to the culture medium, and using streptolysin-O (SL-O) permeabilization. The cellular uptake of each ON used in this study was visualized by fluorescent microscopy. We confirmed the results by FACS analysis. We determined the ratio between initial ON-chol concentration (0.4 microM) and the final amount in nucleus.SL-O can highly improve kinetics of ON delivery; not only into the cytoplasm but also to the nucleus, the presumed site of antigene ON action. The most effective nuclear uptake was observed when ON conjugated with cholesterol (ON-chol) and SL-O was used. Nuclear distribution of ON was reached within few minutes. In contrast, ON simply added to the medium reached cytoplasm only and the process of delivery took several hours., ((Mol Cell Biochem 276: 61-69, 2005).)

Streptococcus pneumoniae is a common cause of septicemia in the immunocompetent host. To establish infection, S. pneumoniae has to overcome host innate immune responses, one component of which is the complement system. Using isogenic bacterial mutant strains and complement-deficient immune naive mice, we show that the S. pneumoniae virulence factor pneumolysin prevents complement deposition on S. pneumoniae, mainly through effects on the classical pathway. In addition, using a double pspA-/ply- mutant strain we demonstrate that pneumolysin and the S. pneumoniae surface protein PspA act in concert to affect both classical and alternative complement pathway activity. As a result, the virulence of the pspA-/ply- strain in models of both systemic and pulmonary infection is greatly attenuated in wild-type mice but not complement deficient mice. The sensitivity of the pspA-/ply- strain to complement was exploited to demonstrate that although early innate immunity to S. pneumoniae during pulmonary infection is partially complement-dependent, the main effect of complement is to prevent spread of S. pneumoniae from the lungs to the blood. These data suggest that inhibition of complement deposition on S. pneumoniae by pneumolysin and PspA is essential for S. pneumoniae to successfully cause septicemia. Targeting mechanisms of complement inhibition could be an effective therapeutic strategy for patients with septicemia due to S. pneumoniae or other bacterial pathogens.

Major barriers separating the blood from tissue compartments in the body are composed of endothelial cells. Interaction of bacteria with such barriers defines the course of invasive infections, and meningitis has served as a model system to study endothelial cell injury. Here we report the impressive ability of Streptococcus pneumoniae, clinically one of the most important pathogens, to induce 2 morphologically distinct forms of programmed cell death (PCD) in brain-derived endothelial cells. Pneumococci and the major cytotoxins H2O2 and pneumolysin induce apoptosis-like PCD independent of TLR2 and TLR4. On the other hand, pneumococcal cell wall, a major proinflammatory component, causes caspase-driven classical apoptosis that is mediated through TLR2. These findings broaden the scope of bacterial-induced PCD, link these effects to innate immune TLRs, and provide insight into the acute and persistent phases of damage during meningitis.

Streptococcus pneumoniae (the pneumococcus) remains one of the major human pathogens and one of the most common causes of community-acquired pneumonia, otitis media, sinusitis, and meningitis. Aside from the threats posed by emerging antibiotic resistance and infection with the human immunodeficiency virus, the mortality rate among those patients with severe pneumococcal disease who receive seemingly appropriate antimicrobial chemotherapy remains unacceptably high. Because of its involvement in the pathogenesis of invasive disease, pneumolysin, one of the best-characterized virulence factors of the pneumococcus, represents not only a potential vaccine target, but also a target for adjunctive therapy to antibiotics in patients with acute pneumococcal disease. In this paper we review the cytolytic and pro-inflammatory properties of pneumolysin and their involvement in subversion of host defenses and extra-pulmonary dissemination of the pneumococcus, as well as strategies, both immunological and pharmacological, which may counter these harmful activities of the toxin.

The endoplasmic reticulum (ER) has a characteristic complex polygonal structure with hallmark three-way junctions in many types of cells. To investigate the mechanisms responsible for maintaining the ER network, we established ER disassembly and reassembly assays in semi-intact Chinese hamster ovary (CHO) cells that constitutively expressed heat shock protein-47 fused to the green fluorescent protein (GFP-HSP47) as an ER marker (the cells are referred to as CHO-HSP cells). Using these assays, we found that maintenance of the ER network required cytosol and adenosine triphosphate/guanosine 5'-triphosphate (ATP/GTP) hydrolysis, but not actin filaments or microtubules. We also showed that the ER network was disrupted upon addition of either N-ethylmaleimide-treated cytosol after washing semi-intact cells with high salt solution or mitotic cytosol in nocodazole-treated semi-intact CHO-HSP cells. The disrupted ER network induced by mitotic cytosol was reformed by the addition of interphase cytosol. In addition, we found that p47, a cofactor of p97, was essential for the maintenance of the ER network, and that phosphorylation of p47 by cdc2 kinase resulted in ER network disruption by mitotic cytosol. Taken together, these results imply that the maintenance of the ER network requires a membrane fusion process mediated by p97/p47, and that cell cycle-dependent morphological changes of the ER network are regulated through phosphorylation/dephosphorylation of p47.

Unlabelled: The aminosteroid U73122 has been established as potent, selective, and cell-permeable inhibitor C-type phosphatidylinositol-specific phospholipases (PI-PLCs), and has been used to define a contribution of PI-PLCs as part of exocytotic signalling pathways in rat peritoneal mast cells (RPMCs). However, doubts have been raised regarding its PI-PLC selectivity of action. Therefore, in the present study, U73122 was tested in RPMCs under experimental conditions allowing to elicit exocytosis PI-PLC independently (streptolysin O [SLO]-permeabilised cells; stimulated by GTPgammaS; in the presence of low concentrations of free Ca2+). The release of [3H]5-hydroxytryptamine ([3H]5-HT) from [3H]5-HT-loaded RPMCs served as measure of secretion. U73122 potently inhibited the exocytotic response induced by 10 microM GTPgammaS (Ca2+: 10(-6) M) in permeabilised cells (IC50: 0.6 microM, n=5) in an insurmountable manner. In intact RPMCs, with a nearly equal potency (IC50: 4 microM, n=4), U73122 also inhibited the PI-PLC-dependent exocytotic response induced by concomitant application of nerve growth factor and lyso-phosphatidylserine (NGF/lyso-PS)., Conclusion: U73122 exerts potent PI-PLC-independent secretostatic effects, limiting its use to define PI-PLC function within exocytotic processes.

Microglial cells express Toll-like receptors (TLRs) recognising exogenous and endogenous ligands. Upon stimulation with agonists of TLR2, TLR4, and TLR9, nitric oxide (NO) and tumor necrosis factor-alpha (TNF-alpha) were released by primary mouse microglial cell cultures. Endotoxin was most potent in stimulating microglia followed by pneumolysin, cytosine-guanosine (CpG) oligodesoxynucleotide (ODN), and Tripalmitoyl-S-glyceryl-cysteine. Maximum stimulation of TLR2, TLR4, and TLR9 resulted in approximately equal amounts of nitric oxide release. Pneumolysin was a potent activator of microglial cells; at high concentrations, it reduced cell viability. No cytotoxicity was noted with the other TLR agonists. Costimulation with maximum concentrations of two TLR agonists did not further increase nitric oxide release. Costimulation with submaximum concentrations was additive or supraadditive, suggesting that even low concentrations of products of infectious agents can lead to microglial activation via TLRs.

Opsonization of apoptotic cells with complement proteins contributes to their clearance by phagocytes. Little is known about the lytic effects of complement on apoptotic cells. Sensitivity of cells treated with anti-Fas antibody (Jurkat cells), staurosporine or etoposide (Raji cells) to lysis by complement was examined. As shown here, early apoptotic cells are more sensitive to lysis by antibody and complement than control cells. More complement C3 and C9 bound to apoptotic than to control cells, even though antibody binding was similar. Enhanced killing and C3/C9 deposition were blocked by benzyloxy-Val-Ala-Asp-fluoromethylketone, a pan-caspase inhibitor. Complement-mediated lysis of early apoptotic cells was also prevented by inhibitors of caspases 6, 8, 9 or 10. In contrast, caspase inhibitors had no effect on the lysis of non-apoptotic Jurkat and Raji cells. Early apoptotic Jurkat cells were also more sensitive to lysis by the pore formers streptolysin O and melittin. Sensitivity of Jurkat Bcl-2 transfectants to lysis by complement was analyzed. Enhanced Bcl-2 expression was associated with reduced C3 deposition and lower sensitivity to complement-mediated lysis. These results demonstrate that at an early stage in apoptosis, following caspase activation, cells become sensitive to necrotic-type death by complement and other pore formers. Furthermore, they suggest that Bcl-2 is actively protecting Jurkat cells from complement-mediated lysis.

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.

Macrophages are resistant to constitutive apoptosis, but infectious stimuli can induce either microbial or host-mediated macrophage apoptosis. Phagocytosis and killing of opsonized pneumococci by macrophages are potent stimuli for host-mediated apoptosis, but the link between pneumococcal killing and apoptosis induction remains undefined. We now show phagocytosis of pneumococci by differentiated human monocyte-derived macrophages (MDM) results in up-regulation of inducible nitric oxide synthase (iNOS) and increased production of NO and reactive nitrogen species. NO accumulation in macrophages initiates an apoptotic program that involves NO-dependent mitochondrial membrane permeabilization, Mcl-1 down-regulation, and caspase activation and results in nuclear condensation and fragmentation. An inhibitor of mitochondrial permeability transition, bongkrekic acid, decreases pneumococcal-associated macrophage apoptosis. Conversely, inhibition of NO production using iNOS inhibitors decreases bacterial killing and shifts the cell death program from apoptosis to necrosis. Pneumolysin contributes to both NO production and apoptosis induction. After initial microbial killing, NO accumulation switches the macrophage phenotype from an activated cell to a cell susceptible to apoptosis. These results illustrate important roles for NO in the integration of host defense and regulation of inflammation in human macrophages.

Ca2+-regulated exocytosis of lysosomes has been recognized recently as a ubiquitous process, important for the repair of plasma membrane wounds. Lysosomal exocytosis is regulated by synaptotagmin VII, a member of the synaptotagmin family of Ca2+-binding proteins localized on lysosomes. Here we show that Ca2+-dependent interaction of the synaptotagmin VII C(2)A domain with SNAP-23 is facilitated by syntaxin 4. Specific interactions also occurred in cell lysates between the plasma membrane t-SNAREs SNAP-23 and syntaxin 4 and the lysosomal v-SNARE TI-VAMP/VAMP7. Following cytosolic Ca2+ elevation, SDS-resistant complexes containing SNAP-23, syntaxin 4, and TI-VAMP/VAMP7 were detected on membrane fractions. Lysosomal exocytosis was inhibited by the SNARE domains of syntaxin 4 and TI-VAMP/VAMP7 and by cleavage of SNAP-23 with botulinum neurotoxin E, thereby functionally implicating these SNAREs in Ca2+-regulated exocytosis of conventional lysosomes.

Small GTPase Rab is a large family of putative membrane trafficking proteins, and each member is thought to regulate a specific type(s) of membrane trafficking. However, little is known about the involvement of Rab protein(s) in secretory granule exocytosis in exocrine cells or the molecular mechanism underlying this process. We show that Rab27B, a closely related isoform of Rab27A that regulates lysosome-related granule exocytosis in cytotoxic T lymphocytes, is abundantly expressed on amylase-containing secretory granules in rat parotid gland acinar cells. We also identify the putative Rab27B effector protein, Slac2-c (Slp homologue lacking C2 domains-c)/MyRIP, which was originally described as a myosin Va/VIIa and actin binding protein, in rat parotid glands. The results of subcellular fractionation, immunoprecipitation and immunohistochemical studies indicate that the Rab27B-Slac2-c complex is formed on secretory granules in vivo. The introduction of either a specific Rab27 binding domain (i.e. a recombinant Slp homology domain of Slac2-b that specifically binds Rab27A/B but not other Rabs) or functionally blocking antibodies that specifically disrupt Rab27B-Slac2-c complex in vitro strongly inhibited isoproterenol-stimulated amylase release from streptolysin O-permeabilized parotid acinar cells. Our results indicate that the Rab27B-Slac2-c complex is an important constituent of secretory granule exocytosis in parotid acinar cells.

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.

Analysis of the function of regulator of G-protein signaling (RGS) protein function and their selectivity of action in vivo is complicated by the expression of multiple RGS proteins in a single cell and requires precise control of cytosolic RGS protein concentration. This article describes two experimental systems using pancreatic acinar cells suitable for such analyses. The first is pancreatic acini permeabilized with streptolysin O, which retains agonist responsiveness while allowing RGS proteins and molecules with molecular masses of up to 25-30 kDa access to the cytosol. The second is a whole cell recording of the Ca(2+)-activated Cl- current of single pancreatic acinar cells as a reporter of [Ca2+]i. This system can be used to introduce to the cytosol any protein of interest, including recombinant RGS proteins and RGS protein-scavenging antibodies. The use of these systems to study the specificity of RGS proteins action, the function of their domains, and the role of RGS proteins in controlling Ca2+ oscillations is discussed.

A functional cysteine-less form of the hamster reduced folate carrier protein was generated by alanine replacement of the 14 cysteine residues. The predicted 12-transmembrane topology was examined by replacing selected amino acids, predicted to be exposed to the extracellular or cytosolic environments, with cysteines. The location of these cysteines was defined by their accessibility to biotin maleimide in the presence or absence of specific blocking agents. Amino acids predicted to be exposed to the extracellular environment (S46C, S179C, L300C, Y355C, and K430C) could be labeled with biotin maleimide; this modification could be blocked by prior treatment with nonpermeable reagents. Amino acids predicted to be within the cytosol (S152C, Cys224, and L475C) could be labeled only after streptolysin O permeabilization. In addition, the cysteine-less reduced folate carrier was exploited to evaluate a potential substrate-binding domain as suggested by previous studies. Nineteen cysteine replacements were generated between residues 39 and 75, a region located between the first and second transmembrane segments. From the biotinylation of these sites and the ability of various reagents to block this labeling, it appears that L41C, E45C, S46C, T49C, I66C, and L70C are exposed to the extracellular environment, whereas Q54C, Q61C, and T63C are slightly less accessible. Cysteines 39, 42, 44, 47, 51, and 73 were inefficiently biotinylated, suggesting that these sites are located in the membrane or within a tightly folded domain of the protein. Furthermore, biotinylation of cysteines 41, 46, 49, 70, and 71 could be prevented by prior treatment with either methotrexate or folinic acid, indicating that these sites form part of a substrate-binding pocket.

Investigations of lipid-mediated signalling pathways are often limited by a lack of methods for the intracellular delivery of lipid messengers. We established a procedure for the transient permeabilization of astrocytes by an oxygen-insensitive mutant of streptolysin-O (SLO) to investigate the participation of the phospholipase D (PLD) signalling pathway in astroglial cell proliferation. Exogenous PLD, when incubated in the presence of SLO, caused an increase in DNA synthesis (measured by thymidine incorporation) which was completely suppressed by ethanol (0.3%, v/v). In parallel experiments, phosphatidic acid also induced a dose-dependent mitogenic response which, however, was not affected by the presence of ethanol. Phosphatidic acid was more effective in this assay than diacylglycerol but its effect was sensitive to the protein kinase inhibitor Ro 31-8220. Our findings provide direct evidence that disruption of the PLD signalling pathway by ethanol is sufficient to suppress astroglial proliferation, an effect that might contribute to the inhibition of brain growth in alcoholic embryopathy.

Background: The round window membrane (RWM) acts like a barrier for topically applied substances into the middle ear preventing diffusion into the perilymphatic fluid compartment., Material and Method: In an animal model, modulation of the permeability of the RWM was attempted using Streptolysin-O (SLO) in various concentrations and sodium hyaluronate. Thereafter, the effect of intratympanically applied Lidocain 2 % on hearing threshold measured by auditory brainstem response was tested for Lidocain 2 % and Lidocain 2 % in combination with SLO or sodium hyaluronate., Results: The results show that both, SLO and sodium hyaluronate, influence the effect of Lidocain 2 % on hearing thresholds as an indirect sign for changes of the permeability of the RWM. However SLO by itself in low concentrations decreased auditory thresholds in some animals while this was never observed with sodium hyaluronate., Conclusions: Our findings preclude SLO and favor sodium hyaluronate as a modulator of RWM permeability in therapeutic trials.

In mammalian cells, inhibition of translation interferes with synthesis of the lipid-linked oligosaccharide (LLO) Glc3Man9GlcNAc2-P-P-dolichol as measured with radioactive sugar precursors. Conflicting hypotheses have been proposed, and the fundamental basis for this regulation has remained elusive. Here, fluorophore-assisted carbohydrate electrophoresis (FACE) was used to measure LLO concentrations directly in cells treated with translation blockers. Further, LLO biosynthetic enzymes were assayed in vitro with endogenous acceptor substrates using either cells gently permeabilized with streptolysin-O (SLO) or microsomes from homogenized cells. In Chinese hamster ovary (CHO)-K1 cells treated with translation blockers, FACE did not detect changes in concentrations of Glc3Man9GlcNAc2-P-P-dolichol or early LLO intermediates. These results do not support earlier proposals for feedback repression of LLO initiation by accumulated Glc3Man9GlcNAc2-P-P-dolichol, or inhibition of a GDP-mannose dependent transferase. With microsomes from cells treated with translation blockers, there was no interference with LLO initiation by GlcNAc-1-P transferase (GPT), mannose-P-dolichol synthase, glucose-P-dolichol synthase, or LLO synthesis in vitro, as reported previously. Surprisingly, inhibition of all of these was detected with the SLO in vitro system. Additional experiments with the SLO system showed that the three transferases shared a limited pool of dolichol-P that was trapped as Glc3Man9GlcNAc2-P-P-dolichol by translation arrest. Overexpression of GPT was unable to reverse the effects of translation arrest on LLO initiation, and experiments with FACE and the SLO system showed that overexpressed GPT was not functional in vivo, although it was highly active in microsomal assays. Thus, the combined use of the SLO in vitro system and FACE showed that LLO biosynthesis depends upon a limited primary pool of dolichol-P. Physical perturbation associated with microsome preparation appears to make available a secondary pool of dolichol-P, masking inhibition by translation arrest, as well as activating a nonfunctional fraction of GPT. The implications of these results for the organization of the LLO pathway are discussed.

The effects that the Streptococcus pneumoniae-derived, proinflammatory toxin, pneumolysin (8.37 and 41.75 ng/mL), has on the production of interleukin (IL)-8 and tumor-necrosis factor (TNF)-alpha by human neutrophils have been investigated in vitro. Total and extracellular IL-8 and TNF-alpha were assayed by enzyme-linked immunosorbent assay, and flow cytometry and colorimetric procedures were used to detect intracellular cytokine and cytokine messenger RNA, respectively. Treatment of neutrophils with pneumolysin either alone or in combination with the chemotactic tripeptide, N-formyl-l-methionyl-l-leucyl-l-phenylalanine (1 microM), resulted in a time-dependent (maximal at 6 h) increase in synthesis and release of IL-8 but not of TNF-alpha, which was associated with increased expression of IL-8 messenger RNA transcripts and was abrogated by either cycloheximide (10 microg/mL) or depletion of Ca(2+) from the cell-suspending medium. These interactions between the toxin and neutrophils may contribute to the exaggerated pulmonary inflammatory responses caused by pneumolysin-producing strains of the pneumococcus.

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.

Ryanodine receptor (RyR) phosphorylation by protein kinase A (PKA) may be important in modulating resting sarcoplasmic reticulum (SR) Ca2+ release, especially in heart failure. However, clear cellular data on PKA-dependent modulation of cardiac RyRs is limited because of difficulty in distinguishing between PKA effects on RyR, phospholamban (PLB), and Ca2+ current. To clarify this, we measured resting Ca2+ sparks in streptolysin-O permeabilized ventricular myocytes from wild-type (WT) and PLB knockout (PLB-KO) mice and transgenic mice expressing only double-mutant PLB (PLB-DM) that lacks the regulatory phosphorylation sites (S16A/T17A). In WT myocytes, cAMP dramatically increased Ca2+ spark frequency (CaSpF) by 2- and 3-fold when [Ca2+] was clamped at 50 and 10 nmol/L (and the SR Ca2+ content also rose by 40% and 50%). However, in PLB-KO and PLB-DM, neither CaSpF nor SR Ca2+ load was changed by the addition of 10 micromol/L cAMP (even with phosphatase inhibition). PKA activation also increased Ca2+ spark amplitude, duration, and width in WT, but not in PLB-KO or PLB-DM. RyR phosphorylation was confirmed by measurements of 32P incorporation on immunoprecipitated RyR. In intact resting myocytes, PKA activation increased CaSpF 2.8-fold in WT, but not in PLB-KO, confirming results in permeabilized myocytes. We conclude that the PKA-dependent increase in myocyte CaSpF and size is entirely attributable to PLB phosphorylation and consequent enhanced SR Ca2+ load. PKA does not seem to have any appreciable effect on resting RyR function in these ventricular myocytes. Moreover, the data provide compelling evidence that elevated intra-SR [Ca2+] increases RyR gating independent of cytosolic [Ca2+] (which was clamped).

Streptolysin O (SLO), archetype of a cholesterol-binding bacterial cytolysin, forms large pores in the plasma membrane of mammalian cells. We have recently reported that when a limited number of pores are generated in a cell, they can be sealed in a Ca++-dependent process. Here, we show that resealing is followed by the release of IL-6 and IL-8 from keratinocytes and from endothelial cells, both relevant targets for SLO attack. Production of cytokines by these cells was preceded by activation of transcription factor nuclear factor kappaB, which thus emerges as a common denominator of stress responses to various pore-forming agents, including alpha-toxin of Staphylococcus aureus and complement. Furthermore, we show that activation and cytokine release in response to an agent that forms a pore in the plasma membrane do not depend on paracrine effects, because supernatants of cells perforated by SLO did not activate bystander cells. The study provides definitive evidence that a transient transmembrane pore suffices to trigger productive transcriptional activation in a target cell.

The Streptococcus pneumoniae pore-forming toxin, pneumolysin, is an important virulence factor in pneumococcal pneumonia. The effect of pneumolysin on human lung epithelial and monocyte cell viability was compared. Pneumolysin caused a dose-dependent loss of viability of human lung epithelial (A549 and L132) and monocyte (U937 and THP-1) cell lines. Analysis of the dose-response curves revealed similar log 50% inhibitory concentration (pIC(50)) values for A549, L132, and THP-1 of 0.12+/- 0.1, 0.02+/- 0.04, and 0.12+/- 0.13 hemolytic units (HU), respectively, but U937 cells showed a significantly greater pIC(50) of 0.42+/- 0.12 HU. Differentiation of A549 and L132 with phorbol ester or THP-1 with gamma interferon had no effect on their sensitivity to pneumolysin. However, a significant decrease in the potency of pneumolysin against U937 cells followed gamma interferon treatment. The Hill slopes of the inhibition curves were greater than unity, indicating that pneumolysin may act with positive cooperativity. Analysis of pneumolysin-treated THP-1 cells by electron microscopy revealed membrane lesions of between 100 and 200 nm in diameter.

Retrovirus Gag proteins are synthesized on free ribosomes, and are sufficient to govern the assembly and release of virus particles. Like type C retroviruses, human T-cell leukemia virus type 1 (HTLV-1) assembles and buds at the plasma membrane. After immunofluorescence staining, HTLV-1 Gag proteins appear as punctuated intracellular clusters, which suggests that they are associated either with intracellular membranes or with the plasma membrane. However, colocalization experiments using a panel of markers demonstrated that Gag proteins were not associated with the membranes involved in the secretory or endocytosis pathway. Small amounts of Gag proteins were detected at the plasma membrane and colocalized with the envelope glycoproteins. Moreover, Gag proteins were excluded from streptolysin-O permeabilized cells and in this respect behaved like cytoplasmic proteins. This suggests that the trafficking of HTLV-1 Gag proteins through the cytoplasm of the host cell is independent of any cell membrane system.

Pneumolysin (PLY), an important virulence factor of Streptococcus pneumoniae, is known to exert various effects on the host immune cells, including cytokine induction, in addition to its known cytolytic activity as a member of the thiol-activated cytolysins. It is of interest to determine whether cytolytic activity is involved in triggering the cytokine production. In this study, we constructed full-length recombinant PLY and noncytolytic truncated PLYs with C-terminal deletions to examine the response of spleen cells to these PLY preparations. When cytolytic activity was blocked by treatment with cholesterol, full-length PLY was capable of inducing gamma interferon (IFN-gamma) production. Truncated PLYs that originally exhibited no cytolytic activity were also active in IFN-gamma induction. Therefore, the IFN-gamma-inducing ability of PLY appeared to be independent of the cytolytic activity. Furthermore, IFN-gamma-inducing preparations were also capable of inducing nitric oxide synthase expression and nitric oxide (NO) production, and the addition of neutralizing antibody to IFN-gamma abolished the NO production. These results clearly demonstrated that PLY is capable of inducing IFN-gamma production in spleen cells by a mechanism different from pore formation and that the induced IFN-gamma stimulates NO production. These findings were discussed with reference to the contribution of PLY to the virulence of S. pneumoniae in vivo.

The oxygen-stable hemolysin streptolysin S (SLS) of Streptococcus pyogenes is encoded in part by the pel/sagA gene product. Antibodies to a synthetic peptide from the C terminus of the Pel/SagA open reading frame inhibited hemolysis mediated by both culture supernatants from multiple M serotypes of S. pyogenes isolates or a commercially available SLS preparation. Analysis of the SLS-mediated hemolytic reaction demonstrated that it was temperature- and concentration-dependent. Like complement-mediated hemolysis it conforms to the prediction of a one-hit mechanism of hemolysis. A number of intermediates in the SLS-mediated hemolysis of sheep erythrocytes could be distinguished. SLS could bind to erythrocytes below 17 degrees C; however, lysis could only occur at temperatures >23 degrees C. Following binding of SLS and washing, a papain-sensitive intermediate could be distinguished prior to insertion of the SLS complex into the erythrocyte membrane, which resulted in formation of a transmembrane pore and led to irreversible osmotic lysis of the cell. These intermediates were similar to those described previously during complement-mediated hemolysis.

In the preceding study (see accompanying paper), we showed by a variety of different techniques that intracellular mature vaccinia virus (vaccinia IMV) is unexpectedly complex in its structural organization and that this complexity also extends to the underlying viral core, which is highly folded. With that analysis as a foundation, we now present different thin-section electron microscopy approaches for analyzing the IMV and the processes by which it is assembled in infected HeLa cells. We focus on conventional epoxy resin thin sections as well as cryosections to describe key intermediates in the assembly process. We took advantage of streptolysin O's ability to selectively permeabilize the plasma membrane of infected cells to improve membrane contrast, and we used antibodies against bone fide integral membrane proteins of the virus to unequivocally identify membrane profiles in thin sections. All of the images presented here can be rationalized with respect to the model put forward for the assembly of the IMV in the accompanying paper.

We used flow cytometry and confocal immunofluorescence microscopy to study the localization of Salmonella typhimurium in spleens of infected mice. Animals were inoculated intragastrically or intraperitoneally with S. typhimurium strains, constitutively expressing green fluorescent protein. Independently of the route of inoculation, most bacteria were found in intracellular locations 3 days after inoculation. Using a panel of antibodies that bound to cells of different lineages, including mononuclear phagocyte subsets, we have shown that the vast majority of S. typhimurium bacteria reside within macrophages. Bacteria were located in red pulp and marginal zone macrophages, but very few were found in the marginal metallophilic macrophage population. We have demonstrated that the Salmonella SPI-2 type III secretion system is required for replication within splenic macrophages, and that sifA(-) mutant bacteria are found within the cytosol of these cells. These results confirm that SifA and SPI-2 are involved in maintenance of the vacuolar membrane and intracellular replication in vivo.

Antisense oligonucleotides provide a powerful tool in order to determine the consequences of the reduced expression of a selected target gene and may include target validation and therapeutic applications. Methods of predicting optimum antisense sites are not always effective. We have compared the efficacy of antisense oligonucleotides, which were selected in vitro using random combinatorial oligonucleotide libraries of differing length and complexity, upon putative target sites within TNFalpha mRNA. The relationship of specific target site accessibility and oligonucleotide efficacy with respect to these parameters proved to be complex. Modification of the length of the recognition sequence of the oligonucleotide library illustrated that independent target sites demonstrated a preference for antisense oligonucleotides of a defined and independent optimal length. The efficacy of antisense oligonucleotide sequences selected in vitro paralleled that observed in phorbol 12-myristate 13-acetate (PMA)-activated U937 cells. The application of methylphosphonate:phosphodiester chimaeric oligonucleotides to U937 cells reduced mRNA levels to up to 19.8% that of the untreated cell population. This approach provides a predictive means to profile any mRNA of known sequence with respect to the identification and optimisation of sites accessible to antisense oligonucleotide activity.

Tumor necrosis factor (TNF)-alpha and interleukin (IL)-1beta are potent activators of the transcription factor NF-kappaB, induced during inflammatory conditions. We have previously shown that both secretory and cytosolic phospholipase A(2) (PLA(2)) are involved in TNF-alpha- and IL-1beta-induced NF-kappaB activation. In this study, we have addressed the mechanism of PLA(2) involvement with respect to downstream arachidonic acid (AA) metabolites and the functional coupling between PLA(2)s mediating NF-kappaB activation. We show that in addition to inhibitors of secretory and cytosolic PLA(2)s, 5-lipoxygenase inhibitors attenuate TNF-alpha- and IL-1beta-stimulated NF-kappaB activation. Exogenous addition of leukotriene B(4) (LTB(4)) restored NF-kappaB activation reduced by 5-lipoxygenase inhibitors or an LTB(4) receptor antagonist, thus identifying LTB(4) as a mediator in signaling to NF-kappaB. TNF-alpha- and IL-1beta-induced AA release from cellular membranes was accompanied by phosphorylation of cytosolic PLA(2). Inhibitors of secretory PLA(2) and of 5-lipoxygenase/LTB(4) functionality markedly reduced AA release and nearly completely abolished cytosolic PLA(2) phosphorylation. This demonstrates that secretory PLA(2), through 5-lipoxygenase metabolites, is an essential upstream regulator of cytosolic PLA(2) and AA release. Our results therefore suggest the existence of a functional link between secretory and cytosolic PLA(2) in cytokine-activated keratinocytes, providing a molecular explanation for the participation of both secretory and cytosolic PLA(2) in arachidonic acid signaling and NF-kappaB activation in response to proinflammatory cytokines.

We have developed a novel method to examine [3H]-noradrenaline and [14C]-glutamate release from the same sample of streptolysin-O (SLO) perforated rat cortical synaptosomes. Ca2+ -dependent [3H]-noradrenaline and [14C]-glutamate release was examined at different temperatures and was found to be greater at 30 degrees C than at 25 degrees C. Ca2+ -dependent release of [3H]-noradrenaline is more ATP dependent than Ca2+ -dependent release of [14C]-glutamate. No significant reuptake of either neurotransmitter by the perforated synaptosomes was detected, indicating all the synaptosomes were indeed perforated. Incubations with 1 mM ouabain, a specific Na+,K+ -ATPase inhibitor, slightly increased Ca2+ -dependent release of both neurotransmitters. [3H]-noradrenaline is released from large dense-core vesicles and [14C]-glutamate is released from small clear synaptic vesicles, so one can directly compare and contrast neurotransmitter release mechanisms between large dense-core vesicles and small clear synaptic vesicles using this preparation.

Vibrio cholerae cytolysin (VCC) forms oligomeric pores in lipid bilayers containing cholesterol. Membrane permeabilization is inefficient if the sterol is embedded within bilayers prepared from phosphatidylcholine only but is greatly enhanced if the target membrane also contains ceramide. Although the enhancement of VCC action is stereospecific with respect to cholesterol, we show here that no such specificity applies to the two stereocenters in ceramide; all four stereoisomers of ceramide enhanced VCC activity in cholesterol-containing bilayers. A wide variety of ceramide analogs were as effective as D-erythro-ceramide, as was diacylglycerol, suggesting that the effect of ceramide exemplifies a general trend of lipids with a small headgroup to augment the activity of VCC. Incorporation of these cone-shaped lipids into cholesterol-containing bilayers also gave similar effects with streptolysin O, another cholesterol-specific but structurally unrelated cytolysin. In contrast, the activity of staphylococcal alpha-hemolysin, which does not share with the other toxins the requirement for cholesterol, was far less affected by the presence of lipids with a conical shape. The collective data indicate that sphingolipids and glycerolipids do not interact with the cytolysins specifically. Instead, lipids that have a conical molecular shape appear to effect a change in the energetic state of membrane cholesterol that in turn augments the interaction of the sterol with the cholesterol-specific cytolysins.

Exposure to pneumolysin (8.37 and 41.75 ng/ml) caused a calcium-dependent increase in the generation of prostaglandin E(2) and leukotriene B(4) by both resting and chemoattractant-activated human neutrophils in vitro. These interactions of pneumolysin with neutrophils may result in dysregulation of inflammatory responses during pneumococcal infection.

The pore-forming toxin streptolysin O (SLO) can be used to reversibly permeabilize adherent and nonadherent cells, allowing delivery of molecules with up to 100 kDa mass to the cytosol. Using FITC-labeled albumin, 10(5)-10(6) molecules were estimated to be entrapped per cell. Repair of toxin lesions depended on Ca(2+)-calmodulin and on intact microtubules, but was not sensitive to actin disruption or to inhibition of protein synthesis. Resealed cells were viable for days and retained the capacity to endocytose and to proliferate. The active domains of large clostridial toxins were introduced into three different cell lines. The domains were derived from Clostridium difficile B-toxin and Clostridium sordelli lethal toxin, which glycosylate small G-proteins, and from Clostridium botulinum C2 toxin, which ADP-ribosylates actin. After delivery with SLO, all three toxins disrupted the actin cytoskeleton to cause rounding up of the cells. Glucosylation assays demonstrated that G-proteins Rho and Ras were retained in the permeabilized cells and were modified by the respective toxins. Inactivation of these G-proteins resulted in reduced stimulus-dependent granule secretion, whereas ADP-ribosylation of actin by the C. botulinum C2-toxin resulted in enhanced secretion in cells. The presented method for introducing proteins into living cells should find multifaceted application in cell biology.

Pneumolysin, a major virulence factor of the human pathogen Streptococcus pneumoniae, is a soluble protein that disrupts cholesterol-containing membranes of cells by forming ring-shaped oligomers. Magic angle spinning and wideline static (31)P NMR have been used in combination with freeze-fracture electron microscopy to investigate the effect of pneumolysin on fully hydrated model membranes containing cholesterol and phosphatidylcholine and dicetyl phosphate (10:10:1 molar ratio). NMR spectra show that the interaction of pneumolysin with cholesterol-containing liposomes results in the formation of a nonbilayer phospholipid phase and vesicle aggregation. The amount of the nonbilayer phase increases with increasing protein concentration. Freeze-fracture electron microscopy indicates the coexistence of aggregated vesicles and free ring-shaped structures in the presence of pneumolysin. On the basis of their size and analysis of the NMR spectra it is concluded that the rings are pneumolysin oligomers (containing 30-50 monomers) complexed with lipid (each with 840-1400 lipids). The lifetime of the phospholipid in either bilayer-associated complexes or free pneumolysin-lipid complexes is > 15 ms. It is further concluded that the effect of pneumolysin on lipid membranes is a complex combination of pore formation within the bilayer, extraction of lipid into free oligomeric complexes, aggregation and fusion of liposomes, and the destabilization of membranes leading to formation of small vesicles.

The effects of pneumolysin on the proinflammatory activity of human neutrophils, as well as on cation fluxes in these cells, have been investigated. Superoxide production, release of elastase, CR3 expression, phospholipase A2 activity, and alterations in membrane potential were measured by use of lucigenin-enhanced chemiluminescence and colorimetric, flow cytometric, radiometric, and spectrofluorimetric procedures, respectively; and cation fluxes were measured by use of 45Ca2+ and 86Rb+ and by fura-2 spectrofluorometry. Pneumolysin at concentrations >1.67 ng/mL caused influx of Ca2+ and increased phospholipase A2 activity and CR3 expression, which were associated with enhanced superoxide production and release of elastase after activation of the cells with the chemotactic tripeptide FMLP. At the same concentrations, pneumolysin caused efflux of K+ and membrane depolarization. The effects of pneumolysin on cation fluxes were not attributable to inhibition of Ca2+-adenosine triphosphatase (ATPase) or Na+, K+-ATPase. Pneumolysin potentiates the proinflammatory activities of neutrophils by a pore-forming mechanism resulting in Ca2+ influx.

The Lec35 gene product (Lec35p) is required for utilization of the mannose donor mannose-P-dolichol (MPD) in synthesis of both lipid-linked oligosaccharides (LLOs) and glycosylphosphatidylinositols, which are important for functions such as protein folding and membrane anchoring, respectively. The hamster Lec35 gene is shown to encode the previously identified cDNA SL15, which corrects the Lec35 mutant phenotype and predicts a novel endoplasmic reticulum membrane protein. The mutant hamster alleles Lec35.1 and Lec35.2 are characterized, and the human Lec35 gene (mannose-P-dolichol utilization defect 1) was mapped to 17p12-13. To determine whether Lec35p was required only for MPD-dependent mannosylation of LLO and glycosylphosphatidylinositol intermediates, two additional lipid-mediated reactions were investigated: MPD-dependent C-mannosylation of tryptophanyl residues, and glucose-P-dolichol (GPD)-dependent glucosylation of LLO. Both were found to require Lec35p. In addition, the SL15-encoded protein was selective for MPD compared with GPD, suggesting that an additional GPD-selective Lec35 gene product remains to be identified. The predicted amino acid sequence of Lec35p does not suggest an obvious function or mechanism. By testing the water-soluble MPD analog mannose-beta-1-P-citronellol in an in vitro system in which the MPD utilization defect was preserved by permeabilization with streptolysin-O, it was determined that Lec35p is not directly required for the enzymatic transfer of mannose from the donor to the acceptor substrate. These results show that Lec35p has an essential role for all known classes of monosaccharide-P-dolichol-dependent reactions in mammals. The in vitro data suggest that Lec35p controls an aspect of MPD orientation in the endoplasmic reticulum membrane that is crucial for its activity as a donor substrate.

This paper studies the endocytosis of ricin at the apical pole of polarized MDCK II cells after permeabilization of the cells basolaterally with streptolysin O. Ricin endocytosis after the addition of cytosol with an ATP-regenerating system was 2-3-fold higher than after the addition of a transport medium. A similar increase in ricin endocytosis was obtained by reconstitution of dialyzed cytosol with the nonhydrolyzable GTP analog, GTP gamma S, in the presence of an ATP-regenerating system. The nonhydrolyzable GDP analog, GDP beta S, did not increase ricin uptake. In contrast to the data obtained with ricin, GTP gamma S was found to inhibit apical transferrin uptake in MDCK II cells transfected with the human transferrin receptor, and the data thus imply that GTP gamma S supports clathrin-independent endocytosis. Electron microscopy (EM) demonstrated that free endocytic vesicles were formed from the apical pole of permeabilized MDCK II cells in the presence of GTP gamma S and that both a ricin-HRP conjugate, HRP, and cationized gold were endocytosed. Ricin endocytosis in the presence of intact cytosol, as well as GTP gamma S-stimulated ricin uptake, was inhibited by Clostridium botulinum C3 transferase, an enzyme found to inactivate Rho proteins. The data demonstrate that apical clathrin-independent endocytosis functions in the presence of GTP gamma S, and suggest that one or more of the small GTP binding proteins of the Rho family is involved in regulation of the apical clathrin-independent endocytosis in MDCK II cells.

Pneumococci cause damage to the ear in otitis media and in association with bacterial meningitis. The pathogenesis of injury involves host response to cell wall and pneumolysin. Release of cell wall, particularly during antibiotic-induced bacterial lysis, leads to an influx of leukocytes and subsequent tissue injury. The signal transduction cascade for this response is becoming defined and includes CD14, Toll-like receptor 2, NFkB, and cytokine production. The second source of injury is the cytotoxicity of the pore forming toxin, pneumolysin. Decreasing the sequelae of otitis can be achieved by an increased understanding of the site-specific mechanisms of pneumococcal-induced inflammation.