Apoptotic death in erythrocytes of lamprey Lampetra fluviatilis induced by ionomycin and tert-butyl hydroperoxide.

The work examined the effects of Ca2+ overload and oxidative damage on erythrocytes of river lamprey Lampetra fluvialtilis. The cells were incubated for 3h with 0.1-5µM Ca2+ ionophore ionomycin in combination with 2.5mM Ca2+ and 10-100µM pro-oxidant agent tert-butyl hydroperoxide (tBHP). The sensitivity of lamprey RBCs to studied compounds was evaluated by the kinetics of their death. Both toxicants induced dose- and time dependent phosphatidylserine (PS) externalization (annexin V-FITC labeling) and loss of membrane integrity (propidium iodide uptake). Highest doses of ionomycin (1-2µM) increased the number of PS-exposed erythrocytes to 7-9% within 3h, while 100µM tBHP produced up to 50% of annexin V-FITC-positive cells. Caspase inhibitor Boc-D-FMK (50µM), calpain inhibitor PD150606 (10µM) and broad protease inhibitor leupeptin (200µM) did not prevent ionomycin-induced PS externalization, whereas tBHP-triggered apoptosis was blunted by Boc-D-FMK. tBHP-dependent death of lamprey erythrocytes was accompanied by the decrease in relative cell size, loss of cell viability, activation of caspases 9 and 3/7, and loss of mitochondrial membrane potential, but all these processes were partially attenuated by Boc-D-FMK. None of examined death-associated events were observed in ionomycin-treated erythrocytes except activation of caspase-9. Incubation with ionomycin did not alter intracellular K+ and Na+ content, while exposure to tBHP resulted in 80% loss of K+ and 2.8-fold accumulation of Na+. Thus, lamprey erythrocytes appear to be more susceptible to oxidative damage. Ca2+ overload does not activate the cytosolic death pathways in these cells.

Experimental Procedures for Demonstration of MicroRNA Mediated Enhancement of Functional Neuroprotective Effects of Estrogen Receptor Agonists.

Protection of motoneurons is an important therapeutic goal in the treatment of neurological disorders. Recent reports have suggested that specific microRNAs (miRs) could modulate the expression of particular proteins for significant alterations in the pathogenesis of different neurological disorders. Thus, combination of overexpression of a specific neuroprotective miR and treatment with a neuroprotective agent could be a novel strategy for functional protection of motoneurons. The protocols described herein demonstrate that miR-7-1, a neuroprotective miR, can enhance the functional neuroprotective effects of estrogen receptor agonists such as 1,3,5-tris(4-hydroxyphenyl)-4-propyl-1H-pyrazole (PPT), Way 200070 (WAY), and estrogen (E2) in preventing apoptosis in A23187 calcium ionophore (CI) exposed VSC4.1 motoneurons. This article describes the protocols for the cell viability assay, transfection of VSC4.1 motoneurons with miRs, Annexin V/propidium iodide staining for apoptosis, Western blotting, patch-clamp recording of whole-cell membrane potential, and JC-1 staining for detection of mitochondrial membrane potential. Taken together, these protocols are used to demonstrate that miR-7-1 caused significant enhancement of the efficacy of estrogen receptor agonists for functional neuroprotection in VSC4.1 motoneurons.

Artificial oocyte activation with calcium ionophore does not cause a widespread increase in chromosome segregation errors in the second meiotic division of the oocyte.

OBJECTIVE: To study the effect of artificial oocyte activation (AOA) on chromosome segregation errors in the meiotic divisions. DESIGN: Prospective cohort study with historical control. SETTING: Private/academic IVF centers. PATIENT(S): Fifty-six metaphase II oocytes were donated from 12 patients who had undergone IVF between June 2008 and May 2009. INTERVENTION(S): Oocytes were activated by 40 minutes' exposure to 100 µM calcium-ionophore. The activated oocyte was tubed and analyzed by array comparative genomic hybridization and/or single-nucleotide polymorphism genotyping and maternal haplotyping (meiomapping). A control sample of embryos derived from normally fertilized oocytes was included for comparison. MAIN OUTCOME MEASURE(S): Incidence of chromosome segregation errors in artificially activated and normally fertilized oocytes in relation to pronuclear evaluation. RESULT(S): Of 49 oocytes that survived the warming procedure, thirty-nine (79.6%) activated. Most activated normally, resulting in extrusion of the second polar body and formation of a single or no pronucleus (2PB1PN: 30 of 39, 76.9%; or 2PB0PN: 5 of 39, 12.8%). Twenty-seven of these were analyzed, and 16 (59.3%) were euploid, showing no effect of AOA on meiotic segregation. Single-nucleotide polymorphism analysis of normally activated oocytes confirmed normal segregation of maternal chromosomes. No difference in the proportion of meiosis II type errors was observed between artificially activated oocytes (28.6%; 95% confidence interval 3.7%-71.0%) compared with embryos obtained from normally fertilized oocytes (44.4%; 95% confidence interval 13.7%-78.8%). The abnormally activated oocytes, with ≥2PN (4 of 39, 10.3%) were diploid, indicating a failure to coordinate telophase of meiosis II with polar body extrusion. CONCLUSION(S): From this preliminary dataset, there is no evidence that AOA causes a widespread increase in chromosome segregation errors in meiosis II. However, we recommend that it be applied selectively to patients with specific indications.

Melatonin protects mast cells against cytotoxicity mediated by chemical stimuli PMACI: possible clinical use.

Melatonin has documented cytoprotective effects on a wide variety of immune cells. The mechanism of action on mast cells (RBL-2H3) still remains in the dark. We found that melatonin significantly attenuated phorbol 12-myristate 13-acetate plus calcium ionophore A23187 (PMACI)-induced cytotoxicity in a concentration and time-dependent manner. It appears that the effect of melatonin on mast cells is two-fold: dependent (MT1 and MT2) and independent membrane receptors. In conclusion, melatonin treatment reduced the cytotoxicity, mediated by PMACI, and could provide a useful therapeutic option in processes where an excessive activation of mast cells occurs.

New methymycin derivatives of Streptomyces venezuelae ATCC 15439 and their inhibitory effects on human T cell proliferation mediated by PMA/ionomycin.

Two new methymycin derivatives, 3'-demethylmethymycin (1) and 3'-demethyldeoxymethymycin (2), together with seven known ones (3-9), were obtained from the strain Streptomyces venezuelae ATCC 15439. Their structures were determined on the basis of IR, MS, 1D and 2D NMR data. In addition, the inhibitory effects of all the compounds on human T cell proliferation mediated by PMA/ionomycin were evaluated. The data suggested for the first time that methymycin derivatives have potential anti-inflammatory activity.

Involvement of calpain 2 in ionomycin-induced cell death in cultured mouse lens epithelial cells.

PURPOSE: Calpains are calcium-activated, intracellular, non-lysosomal, cysteine proteases that hydrolyze lens crystallins and cytoskeletal proteins. Elevated calcium is a frequent finding in both rodent and human cataracts, and calpain 2 is present in lenses of both species. Lens epithelium forms a critical barrier to influx of calcium, but the role of calpain 2 in lens epithelium is poorly characterized. Thus, the purpose of the present experiment was to determine the role of calpain 2 in lens epithelial cell death. METHODS: Mouse lens epithelial cells (α-TN4) were cultured with the calcium ionophore ionomycin to promote calcium influx. Release of LDH into the culture medium was measured as a general marker of cell death, while necrosis and apoptosis were detected by staining with ethidium homodimer III (EtD-III) or FITC-annexin V. Calpain activity was determined by zymography and immunoblotting for activation-associated, fragments of calpain. Breakdown products of calpain substrate α-spectrin were also detected by immunoblotting as additional markers of calpain activation. RESULTS: Calpain 2 was found to be the major calpain isozyme in α-TN4 cells. Ionomycin caused leakage of LDH into the medium, activation of calpain 2, proteolysis of α-spectrin, and changes in α-TN4 cell morphology and staining characteristic of necrotic cell death. Calpain inhibitor SNJ-1945 significantly inhibited these changes. CONCLUSIONS: The ability of mouse lens epithelium to maintain lens transparency would be compromised by activation of calpain 2 and associated necrotic cell death. Since calpain 2 is ubiquitously present in all animal lenses so far observed, the current results may predict the pathological consequences of calpain 2 activation in animal lenses including those of man.