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Human peripheral blood monocytes were reproducibly shown to lyse a variety of tumor cells in a 3- to 4-hr ⁵¹Cr release assay. Ficoll-Hypaque-purified mononuclear cells were suspended in medium supplemented with either 10% autologous serum or fetal calf serum (PCS). With either serum, highly purified (97–99%) and viable (>99%) monocyte suspensions were obtained by EDTA-reversible adherence to plastic surfaces which had been precoated with autologous serum. When used as effectors in cytotoxicity assays, the monocytes recovered from mononuclear cells suspended in FCS-supplemented medium exhibited higher cytolytic activity and were therefore used for further studies. Using FCS for both coating the plates and supplementing the suspension medium resulted in monocytes with low cytolytic activity. Tumor cell lysis measured by ⁵¹Cr release was detected within 2 hr of incubation and increased gradually with time. The level of lysis was dependent on the effector/target ratio and the tumor target cell employed. The involvement of natural killer lymphocytes in the observed tumoricidal activity was excluded. Detection of cytotoxic activity in a short-term assay will be very helpful in further studies of the mechanism of tumor cell killing by human monocytes since potential complicating effects of long-term in vitro cultivation will be minimized.     

Mechanism of lysosomal enzyme release from Mercenaria mercenaria granulocytes: a scanning electron microscope study

A scanning electron microscope study of Mercenaria mercenaria granulocytes at 1 and 2 hr postchallenge with a 0.02-ml Millipore-filtered, sterile sea water suspension of heat-killed Bacillus megaterium at a concentration of 4 × 10⁶ bacteria/ml revealed that (1) granulocytes challenged with bacteria revealed more and larger lysosomes protruding from their surfaces than those of the control groups; (2) release of intact lysosomes from granulocytes into serum occurred concurrently with phagocytosis; (3) enzymes released from discharged lysosomes acted on bacterial cell walls causing their partial degradation, thereby enhancing endocytosis; and (4) degranulation is a normal process but is greatly enhanced when stimulated by phagocytosis of bacteria. This study also demonstrated that lysosomes budded off from the plasma membrane of granulocytes into serum, although the actual biochemical mechanism(s) that causes the detachment of lysosomes from the plasma membrane and the subsequent lysis of the double-membraned lysosomes to release the hydrolases remains unresolved.     

Lymphocyte-mediated cytotoxicity to autologous cells infected with measles virus. II. Specificity of the cytotoxic reaction and characterization of the effector cells involved.

The mechanism of lymphocyte-mediated cytotoxicity to cells infected with measles virus was investigated. Cytotoxicity was measured in a direct assay, immediately after the isolation of lymphocytes from human peripheral blood; mononuclear leukocytes, infected with measles virus in vitro, served as autologous target cells. Virus-specific cytotoxicity required the presence of both IgG antibodies against measles virus and of effector lymphocytes. The effector lymphocytes had Fc receptors and were mainly present in a fraction of non-T lymphocytes. Monocytes were not cytotoxic but rather inhibitory. These results indicate that lysis of virus-infected cells in this direct assay is due to antibody-dependent cellular cytotoxicity (ADCC), caused by K cells. Control experiments showed that the virus-infected target cells were sensitive to incubation with human serum or IgG, resulting in a nonspecific increase of ⁵¹Cr release; however, this did not affect the results of K-cell cytotoxicity. Maximal virus-specific lysis by ADCC did not reach the level obtained by complement-dependent cytotoxicity. Possible explanations for this difference are discussed.     

Human anti-peptidoglycan-IgG-mediated opsonophagocytosis is controlled by calcium mobilization in phorbol myristate acetate-treated U937 cells

Recently, we demonstrated that human serum amyloid P component (SAP) specifically recognizes exposed bacterial peptidoglycan (PGN) of wall teichoic acid (WTA)-deficient Staphylococcus aureus ΔtagO mutant cells and then induces complement-independent phagocytosis. In our preliminary experiments, we found the existence of human serum immunoglobulins that recognize S. aureus PGN (anti-PGNIgGs), which may be involved in complement-dependent opsonophagocytosis against infected S. aureus cells. We assumed that purified serum anti-PGN-IgGs and S. aureus ΔtagO mutant cells are good tools to study the molecular mechanism of anti-PGN-IgG-mediated phagocytosis. Therefore, we tried to identify the intracellular molecule(s) that is involved in the anti-PGN-IgG-mediated phagocytosis using purified human serum anti-PGN-IgGs and different S. aureus mutant cells. Here, we show that anti-PGN-IgG-mediated phagocytosis in phorbol myristate acetate-treated U937 cells is mediated by Ca²⁺ release from intracellular Ca²⁺ stores and anti-PGN-IgGdependent Ca²⁺ mobilization is controlled via a phospholipase Cγ-2-mediated pathway. [BMB Reports 2015; 48(1): 36-41]     

Stabilization and functional studies of high-molecular-weight murine lymphotoxins

High levels of lymphotoxin-like activity (LT) were found in supernatants from secondarily stimulated immune mouse splenocytes activated with concanavalin A (Con A) in vitro. Splenocytes obtained from C57Bl/6 mice immune to the P815 mastocytoma were restimulated in vitro with mitomycin C-treated P815 cells, and then stimulated with Con A. High levels of unstable LT activity are rapidly (2–4 hr) released by these lectin-stimulated splenocytes. The introduction of a crosslinking agent, glutaraldehyde, was found to stabilize this LT activity and allowed us to perform more defined biochemical studies and to examine the functional activities of the LT classes. The lytic activity in these supernatants resided in the high-molecular-weight classes, termed Complex (Cx > 200,000 daltons) and alpha-heavy (α_H 130,000–160,000 daltons). It was found that the Cx and α_H LT classes from the secondarily stimulated immune splenocytes cause lysis of allogeneic target cells, P815 and EL-4, in a 16-hr ⁷⁵Semethionine release assay, and in some cases, this lysis was specific for the sensitizing target cell.     

Genetic targeting of Card19 is linked to disrupted NINJ1 expression,impaired cell lysis,and increased susceptibility to Yersinia infection

Cell death plays a critical role in inflammatory responses. During pyroptosis, inflammatory caspases cleave Gasdermin D (GSDMD) to release an N-terminal fragment that generates plasma membrane pores that mediate cell lysis and IL-1 cytokine release. Terminal cell lysis and IL-1β release following caspase activation can be uncoupled in certain cell types or in response to particular stimuli, a state termed hyperactivation. However, the factors and mechanisms that regulate terminal cell lysis downstream of GSDMD cleavage remain poorly understood. In the course of studies to define regulation of pyroptosis during Yersinia infection, we identified a line of Card19-deficient mice (Card19^lxcn) whose macrophages were protected from cell lysis and showed reduced apoptosis and pyroptosis, yet had wild-type levels of caspase activation, IL-1 secretion, and GSDMD cleavage. Unexpectedly, CARD19, a mitochondrial CARD-containing protein, was not directly responsible for this, as an independently-generated CRISPR/Cas9 Card19 knockout mouse line (Card19^Null) showed no defect in macrophage cell lysis. Notably, Card19 is located on chromosome 13, immediately adjacent to Ninj1, which was recently found to regulate cell lysis downstream of GSDMD activation. RNA-seq and western blotting revealed that Card19^lxcn BMDMs have significantly reduced NINJ1 expression, and reconstitution of Ninj1 in Card19^lxcn immortalized BMDMs restored their ability to undergo cell lysis in response to caspase-dependent cell death stimuli. Card19^lxcn mice exhibited increased susceptibility to Yersinia infection, whereas independently-generated Card19^Null mice did not, demonstrating that cell lysis itself plays a key role in protection against bacterial infection, and that the increased infection susceptibility of Card19^lxcn mice is attributable to loss of NINJ1. Our findings identify genetic targeting of Card19 being responsible for off-target effects on the adjacent gene Ninj1, disrupting the ability of macrophages to undergo plasma membrane rupture downstream of gasdermin cleavage and impacting host survival and bacterial control during Yersinia infection.     

Phage-induced lysis enhances biofilm formation in Shewanella oneidensis MR-1

Shewanella oneidensis MR-1 is capable of forming highly structured surface-attached communities. By DNase I treatment, we demonstrated that extracellular DNA (eDNA) serves as a structural component in all stages of biofilm formation under static and hydrodynamic conditions. We determined whether eDNA is released through cell lysis mediated by the three prophages LambdaSo, MuSo1 and MuSo2 that are harbored in the genome of S. oneidensis MR-1. Mutant analyses and infection studies revealed that all three prophages may individually lead to cell lysis. However, only LambdaSo and MuSo2 form infectious phage particles. Phage release and cell lysis already occur during early stages of static incubation. A mutant devoid of the prophages was significantly less prone to lysis in pure culture. In addition, the phage-less mutant was severely impaired in biofilm formation through all stages of development, and three-dimensional growth occurred independently of eDNA as a structural component. Thus, we suggest that in S. oneidensis MR-1 prophage-mediated lysis results in the release of crucial biofilm-promoting factors, in particular eDNA.     

Inhibition of human NK-induced cell lysis and soluble cell-lytic molecules with anti-human LT antisera and various saccharides

The present study examines and compares the cytolysis of K-562 and MOLT-4 cells mediated by human natural killer (NK) cells from fresh peripheral blood and lymphotoxins (LT) derived from human lymphoid cell populations after lectin stimulation in vitro. Lymphotoxins were obtained from 5-hr concanavalin A (Con A)-restimulated human peripheral blood lymphocytes (PBL) which were precultured for 5 days in medium and fetal calf serum or with allogeneic human B-lymphoid cell lines. Two classes of probes were employed in both direct (cell) and indirect (supernatant) induced target-cell lysis: (a) various saccharides and (b) antibodies reactive with human LT forms. Two sugars, N-acetylglucosamine and α-methylmannoside, were able to inhibit direct cell lysis of both MOLT-4 and K-562 target cells. However, saccharide inhibition was distinct for each type of target even when effector cells were obtained from the same donor. These same saccharides were also able to inhibit 20–30% of the total LT activity in a supernatant for L-929 cells and 50–90% of the lytic activity on MOLT-4 cells. Anti-human F(ab′)₂ (IgG) and rabbit anti-α₂ LT sera blocked direct cell lysis of MOLT-4 and K-562 targets in 50% of the experiments. The anti-α₂ LT serum only recognizes a portion of the LT forms in these supernatants. These results reveal that, while both direct and indirect cell lysis are complex phenomena, they may both occur in some cases by a common mechanism(s).     

The human LT system. V. A comparison of the relative lytic effectiveness of various MW human LT classes on 51Cr-labeled allogeneic target cells in vitro: enhanced lysis by LT complexes associated with Ig-like receptor(s).

The present studies examine the in vitro cell-lytic capacity of various molecular weight (MW) human lymphotoxin (LT) classes obtained from lectin-activated normal or immune lymphocytes on allogeneic target cells. The findings reveal that the high-MW complex class of LT is up to 100 times more effective than the smaller MW LT forms (α, β, and γ) in causing lysis of various allogeneic cell types including lymphoid cells in vitro. Moreover, the data suggest that lectin-stimulated alloimmune cells (MLC sensitized) release complex LT forms in association with a specific antigen-binding receptor(s), and that these complexes are from 3 to 10 times more effective on the sensitizing target cell than complexes obtained from lectin-stimulated nonimmune cells. Positive evidence that complex-induced lysis involved LT was indicated by the finding that lysis was completely neutralized by incubation with heterologous antisera directed against a refined human α₂-LT subclass (anti-α₂) and partially neutralized with anti-human Fab₂′ serum. These findings support the concept that LT molecules may represent a system of related cell-lytic molecules. While the smaller MW forms are only weakly lytic by themselves, they can be assembled into highly lytic complexes which may be focused or directed by an antigen-binding receptor(s).     

Inhibition of the lytic phase of murine T-cell-mediated alloimmune cytotoxicity by a rat antiactivated T-cell antiserum

Antisera produced in rats by immunization with alloimmune murine C57Bl/6 anti-P815 splenic lymphocytes or purified T cells activated in vitro by coculture with phytohemagglutinincoated L-929 cells were found to inhibit the in vitro cytolytic action of in vivo and in vitro alloimmune C57Bl/6 anti-P815 cytotoxic T cells in a 4-hr chromium-51 release assay. The rat anti-murine-activated lymphocyte (anti-MAL) or antiactivated T-cell (anti-ATC) serum inhibited lysis in the absence of exogenously added complement activity and were not directly cytotoxic to CTL. Absorption of anti-MAL with target cells P815, L-929, EL-4, and normal C57Bl/6 lymphocytes removed a limited amount of the CTL-inhibitory activity. In contrast, lectin-activated alloimmune lymphocytes fully absorbed the inhibitory activity indicating these antisera preferentially recognize unique antigenic determinants associated with the activated CTL cell surface. The anti-ATC was found to block alloimmune lysis by CTL from several inbred mouse strains suggesting these antisera recognized antigenic determinants of a common lytic mechanism. A kinetic analysis of the inhibitory activity of the anti-MAL on the CTL reaction scheme revealed this antiserum inhibited lysis at a post-Ca²⁺-dependent step, presumably during the target cell lytic phase. This result suggests the rat antiserum can neutralize the CTL lytic mechanism.     

A sensitive and specific tritium release assay for dopamine-beta-hydroxylase (DbetaH) in serum.

The release of tritium from [7-³H₂]dopamine was investigated as a possible procedure for the assay for dopamine-β-hydroxylase (DβH) in rat and human serum. The release was found to have the same characteristics as those deseribed previously for DβH in serum; for example, an optimum rate of reaction at pH 5.0 or an enhancement of release with agents such as Cu²⁺ ions and N-ethylmaleimide which are known to inactivate endogenous inhibitors of DβH in serum. Tritium release was blocked by the DβH inhibitor fusaric acid but not by inhibitors of other dopamine-metabolizing enzymes in serum. Incubation of ¹⁴C-labeled dopamine along with [7-³H₂]dopamine revealed that, under the standard assay conditions, the formation of [¹⁴C]norepinephrine was accompanied by release of one of the two tritium atoms on the 7-carbon. It was concluded that the procedure provided a simple and sensitive assay of DβH activity in serum.     

Effect of quercetin on human polymorphonuclear leukocyte lysosomal enzyme release and phospholipid metabolism

Quercetin inhibited in a concentration-dependent manner the release of beta-glucuronidase from human polymorphonuclear leukocytes stimulated with zymosan-activated serum. ³H-arachidonic acid-prelabelled polymorphonuclear leukocytes released ³H-arachidonic acid upon stimulation with zymosan-activated serum and this was associated with a decrease of radioactivity in the phospholipid fraction as determined by thin layer chromatography. Quercetin inhibited the release of ³H-arachidonic acid. These observations suggest that the zymosan-activated serum stimulus activates phospholipase A₂ and that phospholipase A2 is inhibited by quercetin. Thus, quercetin alters polymorphonuclear leukocyte phospholipid metabolism and responses to stimulation.     

MECHANISMS OF LYSOSOMAL ENZYME RELEASE FROM HUMAN LEUKOCYTES : I. Effect of Cyclic Nucleotides and Colchicine

In order to study mechanisms underlying selective enzyme release from human leukocytes during phagocytosis, the effects were studied of compounds which affect microtubule integrity or the accumulation of cyclic nucleotides. Human leukocytes selectively extrude lysosomal enzymes (β-glucuronidase) from viable cells during phagocytosis of zymosan or immune complexes, or upon encounter with immune complexes dispersed along a non-phagocytosable surface such as a millipore filter. In each circumstance, lysosomal enzyme release was reduced by previous treatment of cells with pharmacological doses of drugs which disrupt microtubules (e.g. 10^-3–10^-5 M colchicine) or with agents which affect accumulation of adenosine 3'5'-monophosphate (cAMP) (e.g. 10^-3 M cyclic nucleotides and 2.8 x 10^-4–2.8 x 10^-6 M prostaglandin E (PGE) and A (PGA) compounds). Preincubation of cells with 5 µg/ml cytochalasin B resulted in complete inhibition of zymosan ingestion, but not of adherence of zymosan particles to plasma membranes or selective enzyme release. In this system, in which enzyme release was independent of particle uptake, preincubation of cells with colchicine, vinblastine, dibutyryl cAMP, or PGE₁ also reduced extrusion of lysosomal enzymes. When cell suspensions were incubated with membrane-lytic crystals of monosodium urate (MSU), cytoplasmic as well as lysosomal enzymes were released with subsequent death of the cells. However, enzyme release followed phagocytosis of crystals (as measured by enhanced C-1 oxidation of glucose) and was due to "perforation from within" of the lysosomal membrane, rather than lysis by crystals of the plasma membrane. Enzyme release after MSU ingestion was also reduced when cells were treated with pharmacological doses of the test agents. When cells were killed by Triton X-100, acting on the plasma membrane, C-1 oxidation of glucose was abolished and enzyme release could not be inhibited pharmacologically. These observations suggest that lysosomal enzyme release from human phagocytes can be an active process which accompanies plasma membrane stimulation, is independent of cell death, and may be controlled by cyclic nucleotides and agents which affect microtubules.     

Characterization of human cholesterol side chain cleavage enzyme (EC 1.14.15x) of human term placental mitochondria

The cholesterol side chain cleavage enzyme (EC 1.14.15x) in mitochondria of a human term placenta was partially characterized. Enzyme activity was determined by separation of [26-¹⁴C]-cholesterol and [5-¹⁴C]-isocaproic acid formed by side chain cleavage. Since the amounts of unlabeled cholesterol were too large, a K_M of cholesterol could not be determined. The apparent K_M value of NADPH is 6.25 × 10⁻⁴ M. A pH optimum was found at pH 9.5 (Tris-buffer) and a temperature optimum at 40°C. The metal ions Sr²⁺ and Ba²⁺ showed no inhibition at 1 and 10 mM and a moderate inhibition at 100 mM. In low concentrations (1 mM), Mg²⁺ and Ca²⁺ slightly stimulated the enzyme whereas in higher concentrations (100 mM) an inhibitory effect was observed. A strong inhibition was achieved with 1 mM Zn²⁺, Cd²⁺, Cu²⁺ and by 10 and 100 mM Fe²⁺, Mn²⁺, Co²⁺ and Ni²⁺. During preincubation of the enzyme without radioactive substrate, a rapid loss in enzyme activity in relation to enzyme concentration was observed (initial activity = 100%) (preincubation time in hours): 0.5 h (97%), 1 h (55%) and 1.5 h (34%). A dose-dependent inhibition of the enzyme by the following proteins was achieved: bovine serum protein, human serum protein, human immunoglobulin G and ovalbumin. Furthermore, a dose-dependent inhibition was found with the membrane lipids lecithin and sphingosine.     

Trypanosoma brucei: some properties of the cytotoxic reaction induced by normal human serum.

The trypanocidal activity of normal human serum has been studied in vitro using Trypanosoma brucei as the test organism. The variables affecting the rate and extent of lysis, such as time, temperature, serum concentration, and pleomorphism of trypanosomes, are described. Trypanocidal titers of serum and serum fractions were quantitatively determined under standardized incubation conditions. Inactivation and/or removal of components of both the classical and alternate pathways of complement activation had no effect on the trypanocidal properties of human serum. The active factor was nondialyzable, present in plasma at equivalent levels to that in serum, and not removed by absorption with IgG fractions of antisera against human IgM or α₂-macroglobulin. The trypanocidal factor could be inactivated by heat (65 C), dithiothreitol, urea, and trypsin. Gel filtration studies indicated that the trypanocidal activity eluted as a single protein with a molecular weight of about 500,000.     

Characterisation of a secretory antigen from Toxoplasma gondii and its role in circulating antigen production

Hughes H. P. A. and van Knapen F. 1982. Characteristics of a secretory antigen from Toxoplasma gondii and its role in circulating antigen production. International Journal for Parasitology12: 433–437. In vitro culture of RH Toxoplasma gondii in HEp2 cells was found to yield an antigen, of mol. wt. 324,000 dallons, which is one of the components of circulating antigen (CAg). Hydrophobic interaction electrophoresis of ¹²⁵I labelled solubilised parasites has shown that this antigen, in common with the other CAg component, is of intracellular origin. Cyclophosphamide had no effect on either parasite proliferation or on secretion of antigen in vitro. Although immune lysis appears to be the major pathway of CAg release in vivo, secretion by the parasite may be important in the expression of CAg in serum and body fluids immediately following infection.     

Antibody response to technetium-99m-labeled sheep erythrocytes in mice treated with anti-lymphocyte serum and concanavalin A.

In the present series of experiments we have studied the effects of anti-lymphocyte serum (ALS) and concanavlin A (Con A) on the immune response to technetium-99m-labeled sheep erythrocytes (SRBC) and have related this to the localization and persistence of antigen at the site of induction and antibody synthesis. The number of ^99mTc-labeled SRBC in the spleen and liver was quantified by gamma scintillation counting and the cellular kinetics of the splenic antibody response was determined by means of the hemolytic plaque technique. After injection of normal rabbit serum (NRS)-treated control mice with 4 × 10^899mTc-labeled SRBC, the number of cells localizing in the spleen ranged from a high of 4.2 × 10⁶ on Day 1 to a low of 1.7 × 10⁶ on Day 4, while the number in the liver ranged from a high of 68.8 × 10⁸ on Day 1 to 18.6 × 10⁶ on Day 4. The number of splenic plaque-forming cells (PFC) increased from 321–429 on Day 1 to 93,000–101,000 PFC on Day 4 and this was paralled by a rise in serum hemagglutinin and hemolysin titers. In mice treated with ALS on the other hand, splenic localization initially was increased 10-fold, hepatic localization was unchanged, and the antibody response was markedly suppressed. Splenic PFC ranged from approximately 100 between days 1 and 3 and increased to only 500 on Day 4. Mice which received Con A on Day — 1 had a reduction in splenic PFC which ranged from 150 on Day 1 to 1900 on Day 4. Splenic localization of ^99mTc-labeled RBC initially was three- to fourfold greater than that in NRS-treated mice and then decreased to control levels. The increased numbers of SRBC detected in the spleens of immunosuppressed mice at the time of peak response can be attributed to decreased in vivo lysis by reduced numbers of splenic antibody-producing cells.     

Lysis of virus-infected target cells by antibody-dependent cellular cytotoxicity. I. General requirements of the reaction and temporal relationship between lethal hits and cytolysis.

The effect of various physical and chemical parameters on the cytotoxic reaction was studied in a ⁵¹Cr-release assay in order to analyze the mechanism by which human blood mononuclear cells (MC) damage antibody-sensitized target cells infected with herpes simplex virus. Centrifugation of the target cell-MC mixture consistently increased the velocity of the reaction. In addition, uncentrifuged target cell-MC cultures showed a sigmoidal kinetic curve of ⁵¹Cr release with an initial lag phase of at least 10 min, whereas ⁵¹Cr release in centrifuged cultures followed a linear pattern with time without an initial lag. These findings indicate that direct contact between target and effector cells is necessary for cytotoxicity to occur. The reaction as a whole was temperature dependent, proceeding well at 37 °C and not at all at 4 °C. Incubation of the MC at 46 °C for 10 min abolished their cytotoxic potential without affecting their viability; similar heating of the target cells did not affect their background isotope release or sensitivity to the lytic process. Heating target cell-MC mixtures at 46 °C for 10 min thus provided a tool by which the temporal relationship between the mounting of “lethal hits” and specific isotope release, or cell lysis, could be studied. Using this technique, we observed virtually simultaneous occurrence of lethal hits and cell lysis, measured at various intervals between 10 and 360 min postincubation. Likewise, we were unable to demonstrate a transient period of increased osmotic fragility in target cells after contact with MC but before actual cell lysis. Taken together, these findings imply either that cell lysis, as indicated by ⁵¹Cr release, results from a sudden nonosmotic injury to the target cell membrane or, alternatively, osmotic damage leading to ⁵¹Cr release occurs too rapidly to be detected by the methods employed in this study. These findings imply either a qualitative or a quantitative difference between antibody-dependent cellular cytotoxicity (ADCC) mediated by K cells and cytotoxicity mediated by sensitized T cells.The cytotoxic reaction was completely inhibited by 10 mM EDTA and did not occur in a Ca²⁺- and Mg²⁺-free medium. Neither Ca²⁺ nor Mg²⁺ alone produced as much cytotoxicity as the two cations in tandem; in addition, when added to the culture medium in suboptimal amounts, the two cations were either additive or synergistic. These observations suggest that both cations are necessary in ADCC and also that there may be separate Ca²⁺- and Mg²⁺-dependent events in the lytic pathway.     

Hydrogen peroxide release from human blood platelets

The release of hydrogen peroxide from human blood platelets after stimulation with particulate membrane-perturbing agents has been determined by fluorescence using scopoletin as the detecting agent. Platelet suspensions containing less than 1 polymorphonuclear leukocyte/10⁸ platelets showed a significant release of hydrogen peroxide (6.11 nmol/10⁹ platelets per 20 min, S.D., 0.26, n=9) after addition of zymosan or latex particles, compared to unstimulated platelets. The release of hydrogen peroxide was only observed when the scopoletin was added to the platelet suspensions during the stimulation. Any attempt to determine hydrogen peroxide release in the supernatant at the end of the incubation with zymosan or latex failed. A NADH-dependent production of hydrogen peroxide was observed by measuring the difference of oxygen uptake in the presence and absence of catalase (500 units), which was not inhibited by potassium cyanide (1 mM). By this method the NADH-dependent cyanide-insensitive peroxide production and release was 6.0 nmol/10⁹ platelets per 20 min from resting platelets (S.D., 2, n=6) vs. 15 nmol/10⁹ platelets per 20 min from stimulated platelets (S.D., 2, n=6).     

Assay and properties of the hemolysis activity of pure venom from the nematocysts of the acontia of the sea anemone Aiptasia pallida

Pure venom from the acontial nematocysts of the sea anemone Aiptasia pallida was isolated and an assay for the hemolysis activity of the venom devised. The assay is rapid, sensitive, and reproducible. Venom concentrations as low as 0.1 μg protein/ ml were accurately assayed. The properties of the hemolysis activity were analyzed using techniques similar to those used to study enzyme-catalyzed reactions. The biochemical events underlying venom-induced lysis required the direct participation of millimolar levels of Ca²⁺. The slight variability of the apparent K_m for Ca²⁺ at different venom concentrations appeared to be due to the release of some material(s) from lysing cells. Both Sr²⁺ and Mg²⁺ weakly substituted for Ca²⁺. Inhibition of lysis by EDTA was reversed by Ca²⁺. Small monovalent cations, such as Na⁺ or K⁺, appeared to be involved in the venom-induced alteration of the red cell membrane so that lysis could occur. The venom's hemolysis activity was stabilized in solution only if the concentration of the venom proteins was high while also in the presence of at least the equivalent of 0.15 m NaCl.