Zinc is an intracellular signal during sperm activation in Caenorhabditis elegans.

Sperm activation is a rapid and dramatic cell differentiation event that does not involve changes in transcription, and the signaling cascades that mediate this process have not been fully defined. zipt-7.1 encodes a zinc transporter, and zipt-7.1(lf) mutants display sperm-activation defects, leading to the hypothesis that zinc signaling mediates sperm activation in Caenorhabditis elegans. Here, we describe the development of a method for dynamic imaging of labile zinc during sperm activation using the zinc-specific fluorescence probe FluoZin-3 AM and time-lapse confocal imaging. Two phases of dynamic changes in labile zinc levels were observed during sperm activation. Forced zinc entry using the zinc ionophore pyrithione activated sperm in vitro, and it suppressed the defects of zipt-7.1(lf) mutants, indicating that high levels of cytosolic zinc are sufficient for sperm activation. We compared activation by zinc pyrithione to activation by extracellular zinc, the Na+/H+ antiporter monensin and the protease cocktail pronase in multiple mutant backgrounds. These results indicate that the protease pathway does not require zinc signaling, suggesting that zinc signaling is sufficient to activate sperm but is not always necessary.

Efficacy and safety of using premedication with simethicone/Pronase during upper gastrointestinal endoscopy examination with sedation: A single center, prospective, single blinded, randomized controlled trial.

BACKGROUND AND AIM: To investigate the efficacy and safety of premedication with simethicone/Pronase during esophagogastroduodenoscopy (EGD) with sedation. METHODS: Six hundred and ten patients were randomly allocated to two groups based on type of premedication given. Premedication used in the control group was 10 mL lidocaine hydrochloride mucilage (LHM, N = 314) and premedication used in the intervention group was 80 mL simethicone/Pronase solution plus 10 mL lidocaine hydrochloride mucilage (SP/LHM, N = 296). EGD was done under sedation. Visibility scores, number of mucosal areas that needed cleansing, water consumption for cleansing, time taken for examination, diminutive lesions, pathological diagnosis, patients' gag reflex and oxygenation (pulse oximetry) were recorded. RESULTS: SP/LHM has significantly lower total visibility score than LHM (7.978 ± 1.526 vs 6.348 ± 1.097, P < 0.01). During the procedure, number of intragastric areas that needed cleansing and amount of water consumed were significantly less in the SP/LHM than in the LHM group (P < 0.01). In SP/LHM (P = 0.01), endoscopy procedure duration was significantly longer. Although there was no significant difference in rate of detection of diminutive lesions between LHM and SP/LHM, the endoscopist carried out more biopsies in SP/LHM. This led to a higher rate of diagnosis of atrophic gastritis (P = 0.014) and intestinal metaplasia (P = 0.024). There was no significant difference in gag reflex (P = 0.604) and oxygenation during the endoscopy procedure for either group of patients. CONCLUSION: Routine use of premedication with simethicone/Pronase should be recommended during EGD with sedation.

Removal of Listeria monocytogenes dual-species biofilms using combined enzyme-benzalkonium chloride treatments.

The effects of pronase (PRN), cellulase (CEL) or DNaseI alone or combined with benzalkonium chloride (BAC) against Listeria monocytogenes-carrying biofilms were assayed. The best removal activity against L. monocytogenes-Escherichia coli biofilms was obtained using DNaseI followed by PRN and CEL. Subsequently, a modified logistic model was used to quantify the combined effects of PRN or DNaseI with BAC. A better BAC performance after PRN compared to DNaseI eradicating L. monocytogenes was observed. In E. coli the effects were the opposite. Finally, effects of DNaseI and DNaseI-BAC treatments were compared against two different L. monocytogenes-carrying biofilms. DNaseI-BAC was more effective against L. monocytogenes when co-cultured with E. coli. Nonetheless, comparing the removal effects after BAC addition, these were higher in mixed-biofilms with Pseudomonas fluorescens. However, a high number of released viable cells was observed after combined treatments. These results open new perspectives of enzymes as an anti-biofilm strategy for environmental pathogen control.

Quantifying the combined effects of pronase and benzalkonium chloride in removing late-stage Listeria monocytogenes-Escherichia coli dual-species biofilms.

This work presents the assessment of the effectivity of a pronase (PRN)-benzalkonium chloride (BAC) sequential treatment in removing Listeria monocytogenes-Escherichia coli dual-species biofilms grown on stainless steel (SS) using fluorescence microscopy and plate count assays. The effects of PRN-BAC on the occupied area (OA) by undamaged cells in 168 h dual-species samples were determined using a first-order factorial design. Empirical equations significantly (r2 = 0.927) described a negative individual effect of BAC and a negative interactive effect of PRN-BAC achieving OA reductions up to 46%. After treatment, high numbers of remaining attached and released viable and cultivable E. coli cells were detected in PRN-BAC combinations when low BAC concentrations were used. Therefore, at appropriate BAC doses, in addition to biofilm removal, sequential application of PRN and BAC represents an appealing strategy for pathogen control on SS surfaces while hindering the dispersion of live cells into the environment.

Tolerance development in Listeria monocytogenes-Escherichia coli dual-species biofilms after sublethal exposures to pronase-benzalkonium chloride combined treatments.

This study was designed to assess the effects that sublethal exposures to pronase (PRN) and benzalkonium chloride (BAC) combined treatments have on Listeria monocytogenes-Escherichia coli dual-species biofilms grown on stainless steel in terms of tolerance development (TD) to these compounds. Additionally, fluorescence microscopy was used to observe the changes of the biofilm structure. PRN-BAC exposure was carried out using three different approaches and TD was evaluated treating biofilms with a final 100 µg/ml PRN followed by 50 µg/ml BAC combined treatment. Results showed that exposure to PRN-BAC significantly decreased the number of adhered L. monocytogenes (P < 0.05), while E. coli counts remained generally unaltered. It was also demonstrated that the incorporation of recovery periods during sublethal exposures increased the tolerance of both species of the mixed biofilm to the final PRN-BAC treatment. Moreover, control biofilms became more resistant to PRN-BAC if longer incubation periods were used. Regardless of the treatment used, log reduction values were generally lower in L. monocytogenes compared to E. coli. Additionally, microscopy images showed an altered morphology produced by sublethal PRN-BAC in exposed L. monocytogenes-E. coli dual-species biofilms compared to control samples. Results also demonstrated that L. monocytogenes-E. coli dual-species biofilms are able to develop tolerance to PRN-BAC combined treatments depending on way they have been previously exposed. Moreover, they suggest that the generation of bacterial tolerance should be included as a parameter for sanitation procedures design.

Isolation and Culture of Single Cell Types from Rat Liver.

There have been few reports on the simultaneous isolation of multiple liver cell populations thus far. As such, this study was aimed at establishing a protocol for the simultaneous separation of hepatocytes (HCs), hepatic stellate cells (HSCs), liver sinusoidal endothelial cells (LSECs) and Kupffer cells (KCs) from the rat liver and assessing the in vitro culture of these cells. Single-cell suspensions from the liver were obtained by ethylene glycol tetraacetic acid/collagenase perfusion. After low-speed centrifugal separation of HCs, pronase was added to the nonparenchymal cell fraction to eliminate the remaining HCs. Subsequently, HSCs, LSECs and KCs were purified by two steps of density gradient centrifugation using Nycodenz and Percoll in addition to selective attachment. Pronase treatment increased the HSC yield (1.5 ± 0.2 vs. 0.7 ± 0.3 cells/g liver, p < 0.05) and improved LSEC purity (93.6 ± 3.6 vs. 82.5 ± 5.6%, p < 0.01). The isolated cells could also be cultured in vitro. LSEC apoptosis began on day 3 and reached a maximum on day 7. A few surviving LSECs began proliferating and split to form a cobblestone, sheet-like appearance on day 14. The LSECs on day 14 lost fenestrations but retained scavenger function. Thus, viable and purified liver cells were obtained with a high yield from the rat liver using the developed method, which may be useful for studying the physiology and pathology of the liver in the future.

Calcium signaling and the MAPK cascade are required for sperm activation in Caenorhabditis elegans.

In nematode, sperm activation (or spermiogenesis), a process in which the symmetric and non-motile spermatids transform into polarized and crawling spermatozoa, is critical for sperm cells to acquire fertilizing competence. SPE-8 dependent and SPE-8 independent pathways function redundantly during sperm activation in both males and hermaphrodites of Caenorhabditis elegans. However, the downstream signaling for both pathways remains unclear. Here we show that calcium signaling and the MAPK cascade are required for both SPE-8 dependent and SPE-8 independent sperm activation, implying that both pathways share common downstream signaling components during sperm activation. We demonstrate that activation of the MAPK cascade is sufficient to activate spermatids derived from either wild-type or spe-8 group mutant males and that activation of the MAPK cascade bypasses the requirement of calcium signal to induce sperm activation, indicating that the MAPK cascade functions downstream of or parallel with the calcium signaling during sperm activation. Interestingly, the persistent activation of MAPK in activated spermatozoa inhibits Major Sperm Protein (MSP)-based cytoskeleton dynamics. We demonstrate that MAPK plays dual roles in promoting pseudopod extension during sperm activation but also blocking the MSP-based, amoeboid motility of the spermatozoa. Thus, though nematode sperm are crawling cells, morphologically distinct from flagellated sperm, and the molecular machinery for motility of amoeboid and flagellated sperm is different, both types of sperm might utilize conserved signaling pathways to modulate sperm maturation.

Zebrafish (Danio rerio) as a possible bioindicator of epigenetic factors present in drinking water that may affect reproductive function: is chorion an issue?

Emerging organic contaminants have been monitored in stream waters, raw and finished waters and wastewater effluents. Most of these contaminants, such as epigenetic substances, have been detected at very low levels. Unfortunately, their complete monitoring and/or removal are very difficult, given the increasing presence of new contaminants and due to analytical and economic considerations. For this reason, bioindicators are used as an alternative to monitor their presence. To this end, zebrafish is being used to assess certain contaminants in water quality studies. As our long-term aim is to determine if zebrafish (Danio rerio) can be used to detect environmental epigenetic factors in drinking waters with effects on human reproduction, an initial question is whether the chorion could interfere with the possible action of epigenetic factors in two reproductive events: genital ridge formation and migration of the primordial germ cells (PGCs) to these genital ridges. In the first experiment, we attempted to partially degrade the chorion of mid blastula transition (MBT) embryos with pronase, with acceptable survival rates at 5 days post fertilisation (dpf), with the group exposed for 15 min giving the best survival results. As denuded early embryos require a specific culture medium, in the next experiment embryo survival was evaluated when they were cultured up to 5 dpf in drinking waters from six different sources. Results showed a negative effect on embryo survival at 5 dpf from several waters but not in others, thus distorting the survival outcomes. These results suggest using embryos with the chorion intact from the outset when drinking waters from different sources are to be tested.

Efficacy and pharmacological mechanism of pronase-enhanced low-dose antibiotics for Helicobacter pylori eradication.

This study examined the efficacy and pharmacological mechanism of pronase-assisted low-dose antibiotics for eradication of Helicobacter pylori. Mongolian gerbils infected with H. pylori received 7-day treatment (omeprazole, different concentrations of pronase, amoxicillin, and clarithromycin), and the efficacy was assessed using the eradication rate and the colonization of H. pylori. In Mongolian gerbils orally administered pronase, the thickness of the gastric mucous layer (GML) was examined using immunohistochemical and alcian blue staining, and the concentrations of amoxicillin in gastric tissue and serum were detected using high-performance liquid chromatography (HPLC). The eradication rates were 80.0% (12/15) in the high-pronase quadruple group (HPQG) and 86.7% (13/15) in the high-antibiotic group (HAG) (P = 1.000). The antibiotic dose in the HPQG was only 1/20 that in the HAG. Thirty minutes after oral treatment with pronase, the sticky protein of the GML was hydrolyzed, and the GML became thinner. Higher amoxicillin concentrations in both the gastric tissue and serum were observed in the pronase group than in the Am10 group. The concentration of amoxicillin in the Am10-plus-Pr108 group in gastric tissue was 3.8 times higher than in the Am10 group in 5 min. Together, these data suggest that pronase significantly reduced the dose of antibiotics used in H. pylori eradication. The pharmacological mechanism is likely pronase removal of the mucus layer, promoting chemical factor (i.e., gastric acid and pepsinogen) distribution and increasing the antibiotic concentrations in the deep GML, which acted on H. pylori collectively. Thus, pronase may enhance the level of antibiotics for eradication of H. pylori in the clinic.

Spontaneous and pronase-induced HER2 truncation increases the trastuzumab binding capacity of breast cancer tissues and cell lines.

A subgroup of HER2-overexpressing breast tumours co-expresses p95(HER2), a truncated HER2 receptor that retains a functional HER2 kinase domain but lacks the extracellular domain, thus impairing trastuzumab binding. We evaluated p95(HER2) expression in 99 frozen breast carcinoma samples by western blot analysis. The HER2-positive cell line BT474 treated with pervanadate or pronase was used as a positive control for p95(HER2) expression. Immunohistochemistry was performed on parallel formalin-fixed, paraffin-embedded sections of the same case series using antibodies directed against either the intra- or extra-cellular binding domain of HER2. In particular, biotinylated trastuzumab (BiotHER) was used to evaluate the binding capacity of the humanized antibody. To avoid a subjective evaluation of the score values and the percentage of immunostained cells, the slides were scanned and automatically analysed. The number of cases with HER2 overexpression (score 3+) and HER2 gene amplification was higher in the p185(HER2)-positive/p95(HER2)-positive samples than in the p185(HER2)-positive/p95(HER2)-negative group. Automated analysis confirmed a significantly higher percentage of 3+ scored cells in p95(HER2)-positive cases. Conversely, the percentage of 2+ scored cells was higher inp95(HER2)-negative cases. The status of the HER2 extracellular domain was then studied using flow cytometry on BT474 cells after pronase enzymatic digestion using trastuzumab and pertuzumab, while the presence of HER2-HER3 dimers was studied using a proximity-ligation assay. In vitro experiments showed that short-term pronase digestion of BT474 cells produced two HER2 fragments (of 95 and 150 kDa, detectable in tissue specimens as well), increased the binding affinity of trastuzumab, reduced the rate of HER2-HER3 dimers, and did not interfere with pertuzumab-binding capacity. In conclusion, the presence of p95(HER2 as detected by western blot analysis does not compromise the immunohistochemical detection of HER2. Our data suggest that a reduction of the receptor steric hindrance as induced by enzymatic shedding may facilitate the binding capacity of trastuzumab.

Capture and transfer of HIV-1 particles by mature dendritic cells converges with the exosome-dissemination pathway.

Exosomes are secreted cellular vesicles that can be internalized by dendritic cells (DCs), contributing to antigen-specific naive CD4(+) T-cell activation. Here, we demonstrate that human immunodeficiency virus type 1 (HIV-1) can exploit this exosome antigen-dissemination pathway intrinsic to mature DCs (mDCs) for mediating trans-infection of T lymphocytes. Capture of HIV-1, HIV-1 Gag-enhanced green fluorescent protein (eGFP) viral-like particles (VLPs), and exosomes by DCs was up-regulated upon maturation, resulting in localization within a CD81(+) compartment. Uptake of VLPs or exosomes could be inhibited by a challenge with either particle, suggesting that the expression of common determinant(s) on VLP or exosome surface is necessary for internalization by mDCs. Capture by mDCs was insensitive to proteolysis but blocked when virus, VLPs, or exosomes were produced from cells treated with sphingolipid biosynthesis inhibitors that modulate the lipid composition of the budding particles. Finally, VLPs and exosomes captured by mDCs were transmitted to T lymphocytes in an envelope glycoprotein-independent manner, underscoring a new potential viral dissemination pathway.

[A new method for isolation and culture of tracheal epithelial cell of mice].

Since most of the respiratory epithelial cell lines are descended from neoplastic tissues or have been fused with neoplastic cells when being selected to a cell line, their biological behaviors are far different from normal respiratory epithelial cells. Aiming at better reflecting the biological properties of epithelial cells under respiratory pathological conditions, our study probed into a new isolation technique and culture method of mice respiratory epithelial cell. Pronase was applied for cell isolation from mouse trachea, and a modified medium with collagen-coated plate for primary culture. The ciliary movement can be observed under microscope, which demonstrates that the original biological functions of the tracheal epithelial cell have been reserved with our method. The research presents the efficacy, convenience and reliability of the separation technique and culture method established by this study, and laying preferable condition for cell models of respiratory diseases. Meanwhile, this method may be used for other animal models.

In vitro adherence of soluble immune complexes to macrophages.

The adherence of soluble immune complexes to stimulated alveolar macrophages was studied in vitro using HSA-anti-HSA complexes prepared in antigen excess. Those complexes containing more than two molecules of antibody preferentially adhered to macrophages in the absence of complement. Free gammaG in less than physiological concentrations inhibited the adherence of complexes, and the presence of complement did not significantly alter this inhibition. Complexes prepared with reduced and alkylated antibodies showed a decreased adherence. The strength of binding of soluble complexes to macrophages and their efficiency in fixing complement were greater than seen with small aggregates of homologous gammaG. These differences in biological properties were observed even though the immune complexes and aggregates contained comparable numbers of gammaG molecules. The gammaG receptor on rabbit macrophages exhibited species specificity. Pretreatment of macrophages with proteolytic enzymes led to adherence of larger amounts of soluble complexes. These observations suggest that the strength of binding of soluble immune complexes to macrophages and their efficiency in fixing complement are not determined solely by a random summation of individual binding sites. It is proposed that conformational changes in the gammaG antibodies or a specific molecular arrangement in the lattice work of complexes containing large protein antigens may influence the biological properties of the soluble complexes.

Control of B-lymphocyte function. I. Inactivation of mitogenesis by interactions with surface immunoglobulin and Fc-receptor molecules.

Mouse spleen cells were incubated with anti-Ig antibodies for 1 h, washed, exposed to LPS for 1 h, washed, and their DNA synthetic responses assayed 2 days later. It was shown that the 1-h incubation with anti-Ig antibodies produced a profound, internal, and long lasting state of inactivation in the B cells. Experiments with anti-Ig fragments and other ligands showed that the inactivation occurred optimally when both surface Ig molecules and Fc receptors were bound simultaneously. The role of the classical capping and clearing cycle was also investigated. It was shown that capping and clearing were neither necessary nor sufficient for the inactivation to occur, and that the signals, but only secondarily the ligands themselves, were transmitted across the membrane. Capping and clearing were viewed as a natural regulatory mechanism by which the B cell attempts to clear its membrane of perturbations and signals from the exterior.

Antibodies in malarial sera to parasite antigens in the membrane of erythrocytes infected with early asexual stages of Plasmodium falciparum.

Monolayers of human erythrocytes (E) infected with Plasmodium falciparum were briefly fixed with 1% glutaraldehyde and air dried. They were then exposed to sera from patients with P. falciparum malaria or from donors immune to this parasite and tested in an indirect immunofluorescence assay (IFA). Parasites in infected E were made visible by counterstaining with ethidium bromide. Immunofluorescence (IF) was restricted to the surface of infected E. No antibody binding was detected unless the E were dried, suggesting that the relevant antigens were not available on the outer layers of the E surface. Staining over large parts of the E surface was seen already when the merozoite penetrated noninfected cells and was strong in E containing early stages of the parasite (rings, trophozoites). It was weak or absent from E containing schizonts. Antibodies in sera from different parts of Africa, Colombia, or Sweden reacted similarly with E infected with a Tanzanian P. falciparum strain kept in culture for many years and with parasitized E freshly drawn from African, Swedish, or Colombian patients. All sera from residents of a holoendemic area (Liberia) were IFA positive. In contrast, some sera from Colombian or Swedish patients with primary infection gave negative results. The results of the IFA and of an enzyme-linked immunosorbent assay in which fixed and dried E were the targets were well-correlated, suggesting that the same antibodies were detected by these assays. The antigens involved in the IFA were susceptible to pronase but not to trypsin or neuraminidase. E surface IF was inhibited by lysates of infected E, merozoite extracts, or soluble antigens present in P. falciparum culture supernatants but not by lysates of normal E or ghost extracts. The inhibitory antigens were heat stable (100 degrees C, 5 min). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by immunoblotting of either antigen-enriched preparations from culture supernatants or merozoite extracts showed that antibodies eluted from monolayers of infected E reacted consistently with a predominant polypeptide of Mr 155,000 and two to four minor polypeptides of lower molecular weights. Metabolic labeling of the parasites with 75Se-methionine indicated that these antigens were parasite derived. We conclude that the antigens involved in these reactions are released from bursting schizonts or merozoites and are deposited in the E membrane in the course of invasion.(ABSTRACT TRUNCATED AT 400 WORDS)

Role of human decay-accelerating factor in the evasion of Schistosoma mansoni from the complement-mediated killing in vitro.

Decay-accelerating factor (DAF) is a 70-kD membrane glycoprotein that prevents complement (C)-mediated hemolysis by blocking the assembly or accelerating the decay of C3 convertase. Purified DAF is known to incorporate into the membrane of DAF-deficient cells, inhibiting lysis. Since Schistosoma mansoni is a blood-dwelling parasite, we investigated whether DAF can be transferred from human erythrocytes to the worm and protect it against C-mediated killing in vitro. We have found that schistosomula (schla) incubated with normal human erythrocytes (N-HuE), but not with DAF-deficient erythrocytes, become resistant to C damage in vitro. Protected parasites acquire a 70-kD surface protein which can be immunoprecipitated by anti-DAF antibodies. The acquired resistance is abrogated by treatment of N-HuE-incubated parasites with anti-DAF antibody. These results indicate that, in vitro, N-HuE DAF can be transferred to schla, and suggest its participation in preventing their C-mediated killing. This could represent an important strategy of parasites to evade the host's immune response in vivo.

Restriction of gene expression in B lymphocytes and their progeny. III. Endogenous IgA and IgM on the membranes of different plasma cell precursors.

Fluorescent antibody staining with antibodies to the f and g locus allotype markers present on rabbit alpha-chains revealed that the alpha-chain is the heavy chain on the Peyer's patch lymphocytes which previously had been shown to be the precursors of IgA-producing plasma cells. In addition, lymphocytes which had been stripped of membrane Ig with pronase and then cultured overnight to allow the sole expression of endogenous membrane Ig were found to have either the micro-chain or the alpha-chain on their membranes, but not both. These results suggest that most lymphocytes are restricted to the synthesis of one class of heavy chains at a time and that the commitment to synthesizing that particular heavy chain is maintained during the differentiation of lymphocytes into plasma cells. The proportion of lymphocytes with membrane alpha-chains is higher in the Peyer's patch and appendix, two gut-associated lymphoid tissues (GALT), than in other lymphoid tissues. Since the GALT are enriched sources of precursors for IgA-producing plasma cells compared to nongut-associated tissues, the presence of cells bearing membrane alpha-chains correlates well with the relative abilities of these tissues to generate IgA plasma cells.

Influence of erythrocyte membrane components on malaria merozoite invasion.

Chymotrypsin- and Pronase-treated human erythrocytes were refractory to invasion by P. knowlesi merozoites; invasion was not inhibited by trypsin or neurammidase treatment. These data implicate a surface protein other than sialoglycoprotein as the receptor site for merozoites. Invasion of rhesus erythrocytes was unaffected by pretreatment with these enzymes. Differences in membrane structure of erythrocytes from various species may explain the absence of an enzyme effect on rhesus erythrocytes.

Amastigotes of Trypanosoma cruzi escape destruction by the terminal complement components.

We studied the effect of complement on two life cycle stages of the protozoan parasite Trypanosoma cruzi: epimastigotes, found in the insect vector, and amastigotes, found in the mammalian host. We found that while both stages activate vigorously the alternative pathway, only epimastigotes are destroyed. The amounts of C3 and C5b-7 deposited on the amastigotes were similar to those bound to the much larger epimastigotes. Binding of C9 to amastigotes was four to six times less than binding to epimastigotes, resulting in a lower C9/C5b-7 ratio. Although a fairly large amount of C9 bound stably to amastigotes, no functional channels were formed as measured by release of incorporated 86Rb. The bound C9 had the characteristic properties of poly-C9, that is, it expressed a neo-antigen unique to poly-C9, and migrated in SDS-PAGE with an apparent Mr greater than 10(5). The poly-C9 was removed from the surface of amastigotes by treatment with trypsin, indicating that it was not inserted in the lipid bilayer. Modification of amastigote surface by pronase treatment rendered the parasites susceptible to complement attack. These results suggest that amastigotes have a surface protein that binds to the C5b-9 complex and inhibits membrane insertion, thus protecting the parasites from complement-mediated lysis.

Characterization of a membrane pore-forming protein from Entamoeba histolytica.

We describe the partial purification and characterization of a pore-forming material (PEM) from Entamoeba histolytica. The formation of ion channels by PFM was examined in three systems. (a) PFM depolarizes J774 macrophages and mouse spleen lymphocytes as measured by [3H]TPP+ uptake. (b) PFM induces rapid monovalent cation flux across the membrane of phosphatidylcholine-cholesterol vesicles. (c) PFM confers a voltage-dependent conductance to artificial planar bilayers, which is resolved as a summation of opening of individually conducting steps of 67 pS in 0.1 M KCl. Monomers of PFM are functional; however, a preferential aggregation occurs in the planar bilayer. Activity is pronase, trypsin, and heat sensitive and is stable between pH 5-8. PFM is not secreted by unstimulated amoebae but after exposure to the calcium ionophore A23187, concanavalin A, and E. coli lipopolysaccharide, 5-10% of the total cell content of PFM is released into the medium within 5-10 min. High-performance gel filtration results in an approximately 1,000-fold purification of PFM and gives an Mr of 30,000. This protein may play a role in the cytotoxicity mediated by E. histolytica.