Effects of calcium ionophore A23187 on proliferation ,cycle and expression of caspase-3 in hepatic stellate cells stimulated by transforming growth factor-β1 / 西安交通大学学报(医学版)

Objective To study the effect of calcium ionophore A23187 on the proliferation , cycle and expression of caspase-3 in rat hepatic stellate cells by stimulated transforming growth factor Bata 1 (TGF-β1 ) . Methods Hepatic stellate cells were cultured in 37 ℃ and 50 mL/L CO2 incubator .The cells were divided into 5 groups:blank group ,TGF-β1 (5 ng/mL) group ,TGF-β1 +low-,medium-and high-dose calcium ionophore A23187 groups:5 ng/mL TGF-β1 stimulation for 24 h ,and then 1μmol/L ,2μmol/L and 4 μmol/L of calcium ion carrier A23187 was added and treated for 24 h .The cells proliferation was detected by MTT .The cell cycle was detected by flow cytometry and the expression of caspase-3 was detected by immunoblotting .Results Different concentrations of calcium ionophore A23187 could significantly inhibit the proliferation of cells ( P< 0 .05 ) . And the dose of calcium ionophore A23187 increased ;RGR in the low-,medium-and high-dose groups was 85 .93% ,61 .71% ,and 48 .43% (P<0 .05) .There was significant difference between the two groups (P<0 .05) .The higher the dose of calcium ionophore A23187 ,the higher the proportion of G1 phase cells ,the lower the ratio of S+ G2 cells ( P<0 .05) ,with significant difference (P<0 .05) .With the increase of calcium ionophore A23187 concentration ,the expression of caspase-3 protein increased significantly ( P< 0 .05 ) . Conclusion Calcium ionophore A23187 prevents hepatic stellate cells from G1 phase to S phase and G2 phase ,inhibits its proliferation and up-regulates the expression of caspase-3 .

Artificial oocyte activation in intracytoplasmic sperm injection cycles using testicular sperm in human in vitro fertilization / 대한생식의학회지

OBJECTIVE: Artificial oocyte activation (AOA) is an effective method to avoid total fertilization failure in human in vitro fertilization-embryo transfer (IVF-ET) cycles. AOA performed using a calcium ionophore can induce calcium oscillation in oocytes and initiate the fertilization process. We evaluated the usefulness of AOA with a calcium ionophore in cases of total fertilization failure in previous cycles and in cases of severe male factor infertility patients with non-motile spermatozoa after pentoxifylline (PF) treatment. METHODS: The present study describes 29 intracytoplasmic sperm injection (ICSI)-AOA cycles involving male factor infertility at Cheil General Hospital from January 2006 to June 2013. Patients were divided into two groups (control, n=480; AOA, n=29) depending on whether or not AOA using a calcium ionophore (A23187) was performed after testicular sperm extraction-ICSI (TESE-ICSI). The AOA group was further split into subgroups according to sperm motility after PF treatment: i.e., motile sperm-injected (n=12) and non-motile sperm-injected (n=17) groups (total n=29 cycles). RESULTS: The good embryo rate (52.3% vs. 66.9%), pregnancy rate (20.7% vs. 52.1%), and delivery rate (10.3% vs. 40.8%) were lower in the PF/AOA group than in the control group. When evaluating the effects of restoration of sperm motility after PF treatment on clinical outcomes there was no difference in fertilization rate (66.6% vs. 64.7% in non-motile and motile sperm, respectively), pregnancy rate (17.6% vs. 33.3%), or delivery rate (5.9% vs. 16.7%) between the two groups. CONCLUSION: We suggest that oocyte activation is a useful method to ensure fertilization in TESE-ICSI cycles regardless of restoration of sperm motility after PF treatment. AOA may be useful in selected patients who have a low fertilization rate or total fertilization failure.

Specific anti-leukemic cell effect mediated by dendritic cells pulsed with acute myelogenous leukemia antigen in vitro / 白血病·淋巴瘤

Objective To explore the effective method for in vitro culture of the dendritic cells(DCs) and the specific anti-leukemic cell effect mediated by dendritic cells pulsed with acute myelogenous leukemia antigen. Methods Bone marrow mononucleas cells (BMMNCs) isolated from AML patients were induced to undergo differentiation with 500 ng/ml A23187 and pulsed with AML antigen. After 96 h, DCs and T cells were co-cultured for 5 to 7 days. The cytotoxic activity of cytotoxic T lymphocyte(CTL) to AML were detected with MTT colorimetry. Results BMMNCs isolated from AML patients treated with 100 ng/ml rhGM-CSF in combination with 500 ng/ml A23187 for 96 h exhibited typical morphology of DCs with rapidly increased expression of CD1a and CD83 (P<0.01). Dendritic cells pulsed with acid eluted tumor antigen peptides group displayed the strongest cytotoxic activity of CTL to AML. There was a significant difference between two groups(P<0.01). Conclusion BMMNCs isolated from AML patients can be successfully induced to DCs with rhGM-CSF and A23187 and dendritic cells pulsed with acid eluted tumor antigen peptides group had the strongest cytotoxic activity of CTL to AML.

Optimization and Limitation of Calcium Ionophore to Generate DCs from Acute Myeloid Leukemic Cells / Journal of the Korean Cancer Association, 대한암학회지

PURPOSE: Calcium ionophore (CI) is used to generate dendritic cells (DCs) from progenitor cells, monocytes, or leukemic cells. The aim of this study was to determine the optimal dose of CI and the appropriate length of cell culture required for acute myeloid leukemia (AML) cells and to evaluate the limitations associated with CI. MATERIALS AND METHODS: To generate leukemic DCs, leukemic cells (4 x 10(6) cells) from six AML patients were cultured with various concentrations of CI and/or IL-4 for 1, 2 or 3 days. RESULTS: Potent leukemic DCs were successfully generated from all AML patients, with an average number of 1.2 x 10(6) cells produced in the presence of CI (270 ng/ml) for 2 days. Several surface molecules were clearly upregulated in AML cells supplemented with CI and IL-4, but not CD11c. Leukemic DCs cultured with CI had a higher allogeneic T cell stimulatory capacity than untreated AML cells, but the addition of IL-4 did not augment the MLR activity of these cells. AML cells cultured with CI in the presence or absence of IL-4 showed increased levels of apoptosis in comparison to primary cultures of AML cells. CONCLUSION: Although CI appears to be advantageous in terms of time and cost effectiveness, the results of the present study suggest that the marked induction of apoptosis by CI limits its application to the generation of DCs from AML cells.

Involvement of throm box aneA2 and tyrosine kinase in the synergistic interaction of platelet activating factor and calcium ionophore A23187 in human platelet aggregation

The present study was carried out to examine the mechanisms of the synergistic interaction of PAF and A23187 mediated platelet aggregation. We found that platelet aggregation mediated by subthreshold concentrations of PAF (5 nM) and A23187 (1 micrometer) was inhibited by PAF receptor blocker (WEB 2086, IC50=0.65 micrometer) and calcium channel blockers, diltiazem (IC50=13 micrometer) and verapamil (IC50=18 micrometer). Pretreatment of platelets with PAF and A23187 induced rise in intracellular calcium and this effect was also blocked by verapamil. While examining the role of the down stream signaling pathways, we found that platelet aggregation induced by the co-addition of PAF and A23187 was also inhibited by low concentrations of phospholipase C (PLC) inhibitor (U73122; IC50 = 10 micrometer), a cyclooxygenase inhibitor (indomethacin; IC50=0.2 micrometer) and inhibitor of TLCK, herbimycin A with IC50 value of 5 micrometer. The effect was also inhibited by a specific TXA2 receptor antagonist, SQ 29548 with very low IC50 value of 0.05 micrometer. However, the inhibitors of MAP kinase, PD98059 and protein kinase C, chelerythrine had no effect on PAF and A23187-induced platelet aggregation. These data suggest that the synergism between PAF and A23187 in platelet aggregation involves activation of thromboxane and tyrosine kinase pathways.

Induction of acrosome reaction by calcium ionophore A23187 in sperm separated by two-layer percoll gradient method.

The aim of this study was to compare thepercentages of sperm with an acrosome reaction between those with and without calcium ionophore A23187 induction after two-layer Percoll gradient separation. Thirty normal semen samples were obtained from the male partners of infertile couples attending the Infertility Clinic at Siriraj Hospital. After the process of sperm separation by two-layer Percoll gradient technique, the final samples samples were divided into 2 portions. An aliquot of 10 ตM of calcium ionophore A23187 was added to one portion to induce an acrosome reaction, while the other portion was used as a control. Fluorescein isothiocyanate-conjugated Pisum sativam agglutinin (FITC-PSA) staining was performed on both specimens and the acrosome reated-sperm were evaluated. The percentage of acrosome-reated sperm in the calcium ionophore A23187 induced group was significantly higher than those of the control group (24.8+6.6vs 15.4+6.0;p < 0.001). It is concluded that calcium ionophore can significantly induce an acrosome reaction on sperm separated by two-layer Percoll gradient technique, and it may be beneficial to add calcium ionophore A23187 to sperm preparation for use in IUI or IVF.

Twin Pregnancy and Delivery After Intracytoplasmic Sperm Injection Followed by Calcium Ionophore with Spermatozoa from a Globozoospermic Man: A Case Report / 대한산부인과학회잡지

Our purpose is to describe a successful twin pregnancy and delivery after intracytoplasmic sperm injection (ICSI) followed by calcium ionophore with spermatozoa from a globozoospermic man. On the second attempt of ICSI, all of eight metaphase II oocytes were fertilized with treatment with calcium ionophore. Day 3 transfer of six normally developing embryos resulted in an ongoing twin pregnancy, and two preterm healthy babies were born in the 33th week of gestation. To the best of our knowledge, this is the first report of pregnancy and delivery after ICSI followed by calcium ionophore with spermatozoa from a globozoospermic man in Korea.

Mapping of Phorbol Ester and Calcium Ionophore - Responsive Sequence Element in the c - jun Promoter in Activated T Cells / 대한면역학회지

No abstract available.

A23187—Channel behaviour: Fluorescence study.

Pyranine entrapped soylipid liposomes have been used as a model system to study the proton transport across membrane in the presence of A23187, a carboxylic ionophore specific for electroneutral exchange of divalent cations. An apparent rate constant (kapp) for transport of protons has been determined from the rate of change of fluorescence intensity of pyranine by stopped flow rapid kinetics in the presence of proton gradient The variation of kapp has been studied as a function of ionophore concentration and the results have been compared with gramicidin—a well known channel former under the similar experimental conditions. The rates thus obtained showed that A23187 is not only a simple carrier but also shows channel behaviour at high concentration of ionophore.

Rescue activation of calcium ionophore A23187 on unfertilized human oocytes after conventional in-vitro fertilization and intracytoplasmic sperm injection / 中国病理生理杂志

AIM: To explore the activation effect of calcium ionophore A23187 on unfertilized human mature oocytes after conventional in-vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI). METHODS: Thirty-seven unfertilized mature oocytes from IVF and 41 after ICSI were included in our experiment. They were incubated in 5 ?mol/L calcium ionophore A23187 for 5 minutes. Second polar body extrusion and pronuclear formation were recorded 12-16 hours later. The activated oocytes were cultured for another 2 days in vitro. RESULTS: Activation rate of unfertilized oocytes from conventional IVF and ICSI were 64.9%(24/37)and 73.2%(30/41), respectively. Among 41 unfertilized oocytes after ICSI treated with A23187, 30 were activated and 24 had 2 polar body (PB) and 2 pronuclear (PN). But for the unfertilized oocytes from conventional IVF only 20% activated oocytes had 2 PN and 2 PB. The percentage difference of oocytes containing 2 PB and 2 PN between the two groups was significant ( P

CALCIUM IONOPHORE STRONGLY INHIBITS THE GENERATION OF EFFECTOR KILLERS FROM PROLIFERATIVE CYTOTOXIC T CELLS / 中国免疫学杂志

The Ly2~+ Cortisone-Resistant Thymocytes(CRT)were cultured in mediumcontaining IL2,CAS and various mitogens for 7 days.The cytolytic activityof effector CTL was assayed by ~(111)In-release cytotoxicity.At the K/T ratio32/1,the cytotoxicity of effector CTL generated from the stimulus of ConA,PMA.Calcium ionophore(CaI)and PMA+CaI was 46%,24%,13% and 5%respectively.This was the first time that the Cal showed a stronger effecton the inhibition of the generation of effector CTL than that of PMA.The inhibition of PMA and CaI on the differentiation of CTL could bepartially reversed by the removal of PMA and CaI-containing medium andreplaced with cytokine-containing fresh medium.The cytolytic activity wasincreased by three fold,but still 2.4 fold lower than that stimulated byConA at the same K/T ratio. The critical cytokine in the replaced medium was IL2 for the differentia-tion of CTL.Supplementation of IFNr,IL4 or CAS could not further increasethe cytotoxicity.However,the cytolytic activity was slightly increased whenIFNr was added at the initiation of culture. Early T cells could be induced to generate antigen-specific cytotoxic Tcells when cultured in the system with our designed protocol,