Binding of annexin V to plasma membranes of human spermatozoa: a rapid assay for detection of membrane changes after cryostorage. (1/576)

When the cell membrane is disturbed, phospholipid phosphatidylserine (PS) is translocated from the inner to the outer leaflet of the plasma membrane. This is one of the earliest signs of apoptosis and can be monitored by the calcium-dependent binding of annexin V. Therefore, annexin V-binding, in conjunction with flow cytometry, was used to evaluate the integrity of the sperm plasma membrane after different cryostorage protocols: i.e. 10% (v/v) glycerol; sperm maintenance medium (MM); freezing medium TEST yolk buffer (TYB); or cryostorage without protection (cryoshock). Using a combination of two fluorescent dyes, annexin V and propidium iodide (PI), led to three groups of spermatozoa being identified: (i) viable spermatozoa (annexin V-negative and PI-negative); (ii) dead spermatozoa (annexin V-positive and PI-positive); and (iii) cells with impaired but integer plasma membrane (annexin V-positive and PI-negative). The percentage of vital annexin V-negative spermatozoa increased significantly (P < 0.05) from spermatozoa treated by cryoshock (15.0+/-1.2%) to spermatozoa cryopreserved by TYB (26.6+/-2.2%) via cryopreservation by 10% (v/v) glycerol (19.9+/-1.6%) and by MM (22.2 1.8%) and was associated with the percentage of motile spermatozoa (17.6+/-3.4% by glycerol; 19.6+/-3.7% by MM and 22.6+/-3.9% by TYB; P = 0.0001). Of the spermatozoa, 12-22% were annexin V-positive even though they did not bind to PI, indicating viability before as well as after cryostorage. The percentage of vital annexin V-positive spermatozoa was significantly correlated with different sperm motility parameters (velocity straight linear, r = 0.601, P = 0.018; percentage of linearly motile spermatozoa: r = 0.549, P = 0.034). We, therefore, concluded that annexin V-binding is more sensitive in detecting a deterioration of membrane functions than PI staining, and that a considerable percentage of spermatozoa might have dysfunctional plasma membranes besides dead or moribund cells. Of the cryopreservation protocols tested, TYB yielded the most viable spermatozoa. Therefore, we advocate the use of the annexin V-binding assay for the evaluation of the quality and integrity of spermatozoa.  (+info)

Organophosphorylation of acetylcholinesterase in the presence of peripheral site ligands. Distinct effects of propidium and fasciculin. (2/576)

Structural analysis of acetylcholinesterase (AChE) has revealed two sites of ligand interaction in the active site gorge: an acylation site at the base of the gorge and a peripheral site at its mouth. A goal of our studies is to understand how ligand binding to the peripheral site alters the reactivity of substrates and organophosphates at the acylation site. Kinetic rate constants were determined for the phosphorylation of AChE by two fluorogenic organophosphates, 7-[(diethoxyphosphoryl)oxy]-1-methylquinolinium iodide (DEPQ) and 7-[(methylethoxyphosphonyl)oxy]-4-methylcoumarin (EMPC), by monitoring release of the fluorescent leaving group. Rate constants obtained with human erythrocyte AChE were in good agreement with those obtained for recombinant human AChE produced from a high level Drosophila S2 cell expression system. First-order rate constants kOP were 1,600 +/- 300 min-1 for DEPQ and 150 +/- 11 min-1 for EMPC, and second-order rate constants kOP/KOP were 193 +/- 13 microM-1 min-1 for DEPQ and 0.7-1.0 +/- 0.1 microM-1 min-1 for EMPC. Binding of the small ligand propidium to the AChE peripheral site decreased kOP/KOP by factors of 2-20 for these organophosphates. Such modest inhibitory effects are consistent with our recently proposed steric blockade model (Szegletes, T., Mallender, W. D., and Rosenberry, T. L. (1998) Biochemistry 37, 4206-4216). Moreover, the binding of propidium resulted in a clear increase in kOP for EMPC, suggesting that molecular or electronic strain caused by the proximity of propidium to EMPC in the ternary complex may promote phosphorylation. In contrast, the binding of the polypeptide neurotoxin fasciculin to the peripheral site of AChE dramatically decreased phosphorylation rate constants. Values of kOP/KOP were decreased by factors of 10(3) to 10(5), and kOP was decreased by factors of 300-4,000. Such pronounced inhibition suggested a conformational change in the acylation site induced by fasciculin binding. As a note of caution to other investigators, measurements of phosphorylation of the fasciculin-AChE complex by AChE inactivation gave misleading rate constants because a small fraction of the AChE was resistant to inhibition by fasciculin.  (+info)

In vitro cell cycle arrest, in vivo action on solid metastasizing tumors, and host toxicity of the antimetastatic drug NAMI-A and cisplatin. (3/576)

The effects of NAMI-A (imidazolium trans-imidazoledimethyl sulfoxide-tetrachlororuthenate) are compared with cisplatin on tumor cells cultured in vitro at doses of 1 to 100 microM and on tumor metastases in vivo at maximum tolerated doses. Using mouse tumors that metastasize to the lungs, NAMI-A given i.p. for 6 consecutive days at 35 mg/kg/day, was effective independently of the tumor line being treated and of the stage of metastasis growth. Conversely, cisplatin (2 mg/kg/day for 6 days) was as effective as NAMI-A on MCa mammary carcinoma and TS/A adenocarcinoma and less effective than NAMI-A on Lewis lung carcinoma. Cisplatin reduced body weight gain and spleen weight during treatment and was much more toxic than NAMI-A on liver sinusoids, kidney tubules, and lung epithelium. In vitro NAMI-A caused a transient cell cycle arrest of tumor cells in the premitotic G2/M phase, whereas cisplatin caused a progressive dose-dependent disruption of cell cycle phases. Correspondingly, NAMI-A did not modify cell growth, whereas cisplatin caused a dose-dependent reduction of cell proliferation, as determined by sulforhodamine B test. Thus, NAMI-A, unlike cisplatin, is a potent agent for the treatment of solid tumor metastases as well as when these tumor lesions are in an advanced stage of growth. NAMI-A is endowed with a mechanism of action unrelated to direct tumor cell cytotoxicity, and such mechanism of action is responsible for a reduced host toxicity.  (+info)

Time courses of mammalian cell electropermeabilization observed by millisecond imaging of membrane property changes during the pulse. (4/576)

Time courses of electropermeabilization were analyzed during the electric field application using a rapid fluorescent imaging system. Exchanges of calcium ions through electropermeabilized membrane of Chinese hamster ovary cells were found to be asymmetrical. Entry of calcium ions during a millisecond pulse occurred on the anode-facing cell hemisphere. Entry through the region facing the cathode was observed only after the pulse. Leakage of intracellular calcium ions from electropermeabilized cell in low-calcium content medium was observed only from the anode-facing side. The exchanges during the pulse were mostly due to diffusion-driven processes, i.e., governed by the concentration gradient. Interaction of propidium iodide, a dye sensitive to the structural alteration of membrane, with cell membrane was asymmetrical during electropermeabilization. Localized enhancement of the dye fluorescence was observed during and after the pulsation on the cell surface. Specific staining of a limited anode-facing part of the membrane was observed as soon as the pulse was applied. The membrane fluorescence level increased during and immediately after the pulse whereas the geometry of the staining was unchanged. The membrane regions stained by propidium iodide were the same as those where calcium exchanges occurred. The fraction of the membrane on which structural alterations occurred was defined by the field strength. The density of defects was governed by the pulse duration. Electropermeabilization is a localized but asymmetrical process. The membrane defects are created unequally on the two cell sides during the pulse, implying a vectorial effect of the electric field on the membrane.  (+info)

LY303366 exhibits rapid and potent fungicidal activity in flow cytometric assays of yeast viability. (5/576)

LY303366 is a semisynthetic analog of the antifungal lipopeptide echinocandin B that inhibits (1,3)-beta-D-glucan synthase and exhibits efficacy in animal models of human fungal infections. In this study, we utilized flow cytometric analysis of propidium iodide uptake, single-cell sorting, and standard microbiological plating methods to study the antifungal effect of LY303366 on Saccharomyces cerevisiae and Candida albicans. Our data indicate that an initial 5-min pulse treatment with LY303366 caused yeasts to take up propidium iodide and lose their ability to grow. Amphotericin B and cilofungin required longer exposure periods (30 and 180 min, respectively) and higher concentrations to elicit these fungicidal effects. These two measurements of fungicidal activity by LY303366 were highly correlated (r > 0.99) in concentration response and time course experiments. As further validation, LY303366-treated yeasts that stained with propidium iodide were unable to grow in single-cell-sorted cultures. Our data indicate that LY303366 is potent and rapidly fungicidal for actively growing yeasts. The potency and rapid action of this new fungicidal compound suggest that LY303366 may be useful for antifungal therapy.  (+info)

Excimer laser-induced hydroxyl radical formation and keratocyte death in vitro. (6/576)

PURPOSE: To characterize the type of reactive oxygen species (ROS) produced by excimer photoablation of aqueous solutions and to show the effects of ROS and antioxidants on corneal stromal cells in vitro. METHODS: Electron spin-resonance spectroscopy was performed using the spin-trapping agent 5,5-dimethyl-1-pyrroline N-oxide (DMPO) for the detection of the superoxide anion and the hydroxyl radical in an acellular DMPO solution irradiated with the excimer laser. Hydroxyl radicals were produced by the Fenton reaction in vitro by the mixture of hydrogen peroxide and ferrous iron (Fe2+), and the effects on cultured corneal fibroblasts were observed by fluorescent microscopy using the cell death marker, propidium iodide (PI) and TdT-mediated dUTP nick-end labeling (TUNEL). RESULTS: Excimer photoablation of a 1% DMPO solution produced a species-specific spin-trapping adduct for the hydroxyl radical ('OH), but not for the superoxide anion or other unidentified free radical. The signals were inhibited dose dependently by the hydroxyl radical scavenger dimethylsulfoxide (DMSO) and an L-ascorbic acid analogue, EPCK-1. The production of *OH in the supernatant of cultured rabbit corneal fibroblasts by the Fenton reaction caused an increase in PI (+) and TUNEL (+) cells by 90 minutes, which was significantly inhibited by the addition of DMSO. CONCLUSIONS: Hydroxyl radicals may be partly responsible for stromal fibroblast cell apoptosis after excimer photoablation.  (+info)

Effects of commercial glucose-based peritoneal dialysates on peripheral blood phagocytes apoptosis. (7/576)

BACKGROUND: Variable glucose-lactate-based peritoneal dialysates have negative effects on peritoneal macrophages and peripheral blood leukocytes, reducing the capacity of leukocytes for chemotaxis, bacterial killing. But few reports exist on cell apoptosis. To investigate the effects of glucose-lactate-based peritoneal dialysates on cultured phagocytes (monocytes and neutrophils), we focused on studying phagocyte apoptosis after brief exposure to commercial peritoneal dialysates. METHODS: Cell apoptosis is measured by flow cytometry (FCM) to detect phosphatidylserine (PS) exposure on early apoptotic cells using fluorescein-labeled annexin V. To mimic the composition of dialysate in vivo, where the freshly instilled solution mixes with the residual dialysate from the previous cycle, we performed the experiments using a mixture of fresh and spent dialysate (9:1). In our transient exposure experiments, monocytes and neutrophils were separately incubated in each of the test solutions (1.5% glucose and 4.25% glucose dialysates) for 10 minutes or 30 minutes and afterward separated and resuspended in RPMI 1640 medium and cultured over the indicated time. RESULTS: After exposure to 1.5% glucose dialysates for 10 minutes, monocytes and neutrophils exhibited normally spontaneous apoptosis. After exposure to 4.25% glucose dialysate, monocytes underwent apoptosis increasingly, 21%+/-5.0% versus 9.8%+/-3.6% (p < 0.05) at 24 hours and 47%+/-6.2% versus 16%+/-4.0% (p < 0.01) at 72 hours compared with controls. For neutrophils, the results were discouraging: hypertonic dialysate not only increased apoptosis [65.36%+/-2.6% versus 34.17% +/-8.52% (p < 0.01) at 72 hours], but also induced cell necrosis. When Incubation time was prolonged for 30 minutes, 1.5% dialysate acted like 4.25% dialysate, with the rate of apoptosis increasing rapidly [40%+/-4.0% versus 16%+/-4.0% (p < 0.01) at 72 hours for monocytes, and 66.90%+/-5.6% versus 34.17%+/-8.52% (p < 0.01) at 72 hours for neutrophils]. CONCLUSION: Glucose-lactate-based peritoneal dialysates can induce peripheral blood phagocyte apoptosis in vitro, which indicates that glucose plays an important role in triggering cell apoptosis. Therefore, looking for new, physiologic peritoneal dialysis fluids to replace conventional fluids is reasonable.  (+info)

CD28, IL-2-independent costimulatory pathways for CD8 T lymphocyte activation. (8/576)

We investigate, here, the mechanism of the costimulatory signals for CD8 T cell activation and confirm that costimulation signals via CD28 do not appear to be required to initiate proliferation, but provide survival signals for CD8 T cells activated by TCR ligation. We show also that IL-6 and TNF-alpha can provide alternative costimulatory survival signals. IL-6 and TNF-alpha costimulate naive CD8 T cells cultured on plate-bound anti-CD3 in the absence of CD28 ligation. They act directly on sorted CD8-positive T cells. They also costimulate naive CD8 T cells from Rag-2-deficient mice, bearing transgenic TCRs for HY, which lack memory cells, a potential source of IL-2 secretion upon activation. IL-6 and TNF-alpha provide costimulation to naive CD8 T cells from CD28, IL-2, or IL-2Ralpha-deficient mice, and thus function in the absence of the B7-CD28 and IL-2 costimulatory pathways. The CD8 T cell generated via the anti-CD3 plus IL-6 and TNF-alpha pathway have effector function in that they express strong cytolytic activity on Ag-specific targets. They secrete only very small amounts of any of the cytokines tested upon restimulation with peptide-loaded APC. The ability of the naive CD8 T cells to respond to TCR ligation and costimulatory signals from IL-6 and TNF-alpha provides a novel pathway that can substitute for signals from CD4 helper cells or professional APC. This may be significant in the response to viral Ags, which can be potentially expressed on the surface of any class I MHC-expressing cell.  (+info)