Concentrations Of Ionomycin Research Articles

Особенности постлучевых нарушений аккумуляции ионов кальция во внутриклеточных депо тромбоцитов в различные сроки постлучевого периода

Calcium ions are an important intracellular regulator; an increase in their concentration in the cytoplasm plays a key role in determining the functional activity of platelets associated with platelet aggregation and the release reaction. The main way to reduce the concentration of free calcium ions in the platelet cytoplasm is the activation of Ca2+-ATPases of intracellular depositing structures and the plasma membrane, which reduce the cytoplasmic content of calcium due to its reuptake in the depot (resequestration) and the release of Ca2+ from the cell. Platelets express two Ca2+-ATPase isoforms in different depositor structures (SERCA2b and SERCA3) and two plasma membrane Ca2+-ATPase isoforms (PMCA1b and 4b). The SERCA2b isoform, which is Ca2+-ATPase of the dense tubular system, is inhibited by thapsigargin. The SERCA3 isoform present in acidic organelles has low sensitivity to thapsigargin, but high sensitivity to tBHQ (2,5-di-(tert-butyl)-1,4-hydroquinone). When stimulated with micromolar concentrations of thapsigargin and ionomycin, which cause complete depletion of the calcium depots of the tubular system and acid organelles, differences in the levels of accumulated calcium in the norm and in the post-radiation period are found in a calcium-free medium: an increase in calcium concentration by 1.4 times is noted on the 3rd day. In the structures of the tubular system of platelets, under the action of nmolar concentrations of thapsigargin, which inhibit SER-CA2b Ca2+-ATPase of tubular systems, a change in the content of deposited calcium in irradiated animals was revealed with an increase on the 3rd day. Under conditions of inhibition of Ca2+-ATPase SERCA3 in a calcium-free medium in platelets on the 3rd day after irradiation at a dose of 1 Gy, a more intense release of calcium ions was also noted compared to the control. On the 10th, 30th, 90th day, there were no statistically significant differences in these indicators. In the calcium-containing medium, a more significant intake of calcium ions from the outside into the cytoplasm was observed due to the activation of depot-controlled mechanisms in the post-radiation period, especially on the 3rd and 10th days.

Modified kinetics of generation of reactive species in peripheral blood of patients with type 2 diabetes

Level of reactive species in blood is an important pathogenic factor in diabetes mellitus leading to dysfunctions of vascular endothelial and smooth muscle cells and coagulation system abnormality. A massive release of reactive species (respiratory burst), catalyzed by NADPH oxidase in blood phagocytes, is not well understood in diabetes. The work aimed to study kinetics of response to microbial particles in blood to specify changes in regulatory mechanisms of generation of reactive species in patients with type 2 diabetes. Production of reactive species in blood and isolated granulocytes was measured by luminol-dependent chemiluminescence. Respiratory burst was initiated by serum opsonized zymosan in blood samples and phorbol ester in cell samples. Kinetic parameters were calculated from experimental kinetic curves of chemiluminescence intensity. ROC curve analysis and mathematical modeling were used to reveal the most significant predictors and clarify specific mechanisms of NADPH oxidase activation. It was shown that kinetic parameters of response to opsonized zymosan (lag-time, response rate, amplitude, production of reactive species) were higher in blood of patients than controls. Amplitude and response rate were the most statistically significant predictors for distinguishing patients and controls at high glucose. It indicated NADPH oxidase activation was the target of hyperglycemia. Mathematical modeling showed hyperglycemia increased stability of NADPH oxidase complex, decreased synchronization of its assembling and elevated neutrophil capacity to phagocytosis in patients. Weak or no dependence of response kinetics on ionomycin concentration was shown in patients indicating changed Ca2+-dependent mechanism of NADPH oxidase activation. Hyperglycemia in type 2 diabetes causes disturbances in mechanisms of NADPH oxidase activation associated with both phagocytosis and the state of intracellular signaling systems, including Ca2+-dependent. We suggest that NADPH oxidase in blood granulocytes can be a promising target for clinical intervention improving management of diabetic complications associated with inflammation.

Ionomycin-induced mouse oocyte activation can disrupt preimplantation embryo development through increased reactive oxygen species reaction and DNA damage

Oocyte activation induced by calcium oscillations is an important process in normal fertilization and subsequent embryogenesis. In the clinical-assisted reproduction, artificial oocyte activation (AOA) is an effective method to improve the clinical outcome of patients with null or low fertilization rate after ICSI. However, little is known about the effect of AOA on preimplantation embryo development in cases with normal fertilization by ICSI. Here, we used ionomycin at different concentrations to activate oocytes after ICSI with normal sperm and evaluated energy metabolism and preimplantation embryo development. We found that a high concentration of ionomycin increased the frequency and amplitude of calcium oscillation patterns, affecting the balance of mitochondrial energy metabolism, leading to increased reactive oxygen species (ROS) and decreased ATP. Eventually, it increases DNA damage and decreases blastocyst formation. In addition, the addition of vitamin C to the culture medium ameliorated the increase in ROS and DNA damage and rescued the abnormal embryo development caused by excessive ionomycin activation. This study provides a perspective that the improper application of AOA may have adverse effects on preimplantation embryo development. Thus, clinical AOA treatment should be cautiously administered.

Abstract A74: T cell activation standardization for therapeutic assay development

Abstract Introduction: Several cancer immunotherapy strategies rely on T-cell activation (1). While T-cell activation mechanisms are well established, there is a dearth of quantified standardization of these activation pathways. Standardization with quantified end-points [induced by known activators and pathways] allows the development of useful assays to evaluate emerging immunotherapies. Our initial work has focused on T-cell activation. Materials and Methods: Activation of the Jurkat T-cell line was induced by the combined action of ionomycin and PMA (phorbol 12-myristate 13-acetate) (2). Jurkat cells were cultured in 24 well plates in RPMI 1640 media (with 10% FBS and 1% Penicillin/Streptomycin) at a density of 106 cells/ml. We looked at a wide concentration range for both activators (500 - 1500 ng/ml for ionomycin and 10 - 100 ng/ml for PMA), in order to identify their most synergistic combination for T-cell activation as quantified by biomarker expression (i.e. IL-2 ELISA). The negative controls were 1% DMSO (used for solubilizing the activators), and PBS (phosphate buffered saline). For a specific combination of 1000 ng/ml ionomycin and 100 ng/ml PMA, the designed time points were 1 h, 2 h, 4 h, 6 h, 8 h, and 24 h. The cells and supernatant were preserved at the end of these time-points. The viability of the cells was tested using the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) assay and the IL-2 production was detected using ELISA (Enzyme-Linked ImmunoSorbent Assay). Results: In addition to identifying the optimal activating concentrations of ionomycin and PMA, the obtained results characterized the time-course of maximal Jurkat cell activation over a 24 h period. During this 24 h period, the IL-2 levels and the cell proliferation rates progressively increased before starting to plateau. The production of IL-2 started increasing at 4 h from 157.6 pg/ml and continued to go up until 24 h to 687.6 pg/ml. Cell proliferation decreased a little during the first 2 h and increased from 57% to 114% (of the PBS control) between 8 to 24 h. Conclusions and Discussion: Our work establishes a standardized basis to determine the extent and the time-course of maximal T-cell activation through a given mechanism - in this case through the mechanism of calcium ionophore induced (ionomycin) and phorbol ester induced (PMA) T-cell activation (3). Such quantified standardization and maximization of T-cell activation in turn presents a cell-culture model (or assay) to evaluate candidate therapeutics designed to work through the same activation pathway. In addition to T-cells, we are working on the quantified standardization of the activation of NK-cells (4) and dendritic cells (5), with activation measured by the end-points of biomarker expression profiling (ELISA/Luminex), proliferation (MTT assay), microscopy and flow cytometry (CD receptor expression profiling).

P-71 - Influx of calcium ions accelerates neurite degeneration via induction of microtubule destabilization

Reactive oxygen species (ROS) attack several living organs and induce neuronal cell death. Previously, we found that treatment with a low concentration of hydrogen peroxide induced neurite degeneration prior to the cell death. The neurites showed abnormal morphologies including beads formation, shrinkage and fragmentation. This phenomenon also observed in hippocampus of the normal-aged and vitamin E-deficient mice. In order to clarify the mechanism of neurite degeneration, we measured lipid peroxidative products in hydrogen peroxide-treated cells. The LOOH levels were significantly increased compared to the untreated group. If cell membranes are oxidized by ROS, large parts of receptors and ion channels may be also damaged. Here, we show the elucidation of the relationship between calcium influx and neurite degeneration in cultured neurons. Treatment with an ionomycin (calcium ionophore) induced neuronal cell death in a concentration- and a time-dependent manner. Treatment with a low concentration of ionomycin induced neurite degeneration via influx of calcium ions, mitochondrial-dependent superoxide production and mitochondrial membrane oxidations. These phenomena were recorded by a time-lapse live imaging system. Finally, these neurite degeneration prevented by antioxidant vita.

Culture conditions affect Ca2+ release in artificially activated mouse and human oocytes.

Inconsistent fertilisation and pregnancy rates have been reported by different laboratories after application of ionomycin as a clinical method of assisted oocyte activation (AOA) to overcome fertilisation failure. Using both mouse and human oocytes, in the present study we investigated the effects of ionomycin and Ca2+ concentrations on the pattern of Ca2+ release and embryonic developmental potential. In the mouse, application of 5μM ionomycin in potassium simplex optimisation medium (KSOM) or 10µM ionomycin in Ca2+-free KSOM significantly reduced the Ca2+ flux and resulted in failure of blastocyst formation compared with 10μM ionomycin in KSOM. Increasing the Ca2+ concentration up to three- or sixfold did not benefit mouse embryonic developmental potential. Similarly, 10μM ionomycin-induced rise in Ca2+ in human oocytes increased with increasing total calcium concentrations in the commercial medium. Remarkably, we observed significantly reduced mouse embryo development when performing AOA over a period of 10min in Quinn's AdvantageTM Fertilisation medium (Cooper Surgical) and IVFTM medium (Vitrolife) compared with Sydney IVF COOK cleavage medium (Cook Ireland), using the same sequential culture system from the post-activation stage to blastocyst formation stage in different AOA groups. In conclusion, concentrations of both ionomycin and Ca2+ in culture media used during AOA can have significant effects on Ca2+ release and further embryonic developmental potential.

Nuclear translocation and function of Calcyclin (S100A6)-binding-protein/Siah-1 interacting protein in response to intracellular calcium concentration by ionomycin in SW480 cells

Objective To exploring the nuclear translocation of calcyclin(S100A6)-binding-protein (CacyBP)/SIP in response to elevated intracellular calcium concentration ([Ca2+ ]i) by ionomycin in SW480 cells. And the effect of different [Ca2+ ]i on proliferation and apoptosis in SW480 cells. Methods Immunofluorescence staining was used to detected the localization of CacyBP/SIP treated with inonmycin in SW480 cells. The cell survival rate was detected by methyl thiazol tetrazolium (MTT). Cell cycle and apoptosis was detected by flow cytometry. Results Different concentration of ionomycin were treated with colon cancer SW480 cells and probed by immunocytochemistry staining using anti-CacyBP/SIP antibodies. CacyBP/SIP was distributed throughout the cytoplasm in untreated cells. In ionomycin-treated cells, CacyBP/SIP was also detected in the cytoplasm treated with 1 μmol/L and 2 μmol/L ionomycin. However, 5 μmol/L ionomycin caused a significant translocation of endogenous CacyBP/SIP from the cytoplasm to nucleus. While 10 μmol/L of ionomycin induces CacyBP/SIP retranslocation to cytoplasm and nucleus. The ionomycintreated cells showed no significant effect on cell proliferation by the MTT assay (P=172.918). The results of the cell apoptosis analysis by FACS similarly indicate that ionomycin has no effect on the SW480 cells early apoptosis rate [(0.747±0.049)%] and total apoptosis [(2.383±0.068)%, P=79.618]. Conclusion Although elevating [Ca2+ ]i by ionomycin has affected on the nuclear translocation of CacyBP/SIP, it has no affected on the proliferation and apoptosis in SW480 cells. Key words: Colon cancer; Calcium; Nuclear translocation; Proliferation; Apoptosis

Abstract 4594: Induction of apoptosis by anti-CD20 antibodies requires the induction of EGR-1 and calcium influx

Abstract Background: Anti-CD20 monoclonal antibodies (mAbs) are an essential component of the treatment of patients with CD20-positive non-Hodgkin's lymphoma and chronic lymphocytic leukemia (CLL). Anti-CD20 mAbs mediate their antitumor effects by activating the immune system or by direct apoptotic signaling in target cells. In a previous preclinical study, we have shown that treatment of B-lymphoma cell lines with anti-CD20 mAbs, rituximab and obinutuzumab, resulted in upregulated expression of the transcription factor early growth factor -1 (EGR-1) (Dalle et al. 2011). However, the role of EGR-1 in response to passive immunotherapies has not been explored so far. Furthermore, EGR-1 has been described as a calcium (Ca2+) regulated transcription factor and CD20 is hypothesized to regulate transmembrane Ca2+ flux. In this study we aim to assess the role of EGR-1 and Ca2+ flux in the cytotoxic activity of anti-CD20 mAbs. Methods: EGR-1 modulation and cell death induction by anti-CD20 mAbs rituximab and obinutuzumab were investigated in cells expressing endogenous and exogenous CD20. The cytotoxic effect of anti-CD20 mAbs was evaluated in SCID mice and in B-lymphoma cell lines overexpressing EGR-1 or knocked down for EGR-1. The impact of anti-CD20 mAbs on Ca2+ flux was investigated by flow cytometry using Indo-1 AM stained cells. Ca2+ channel blocker agent nifedipine was used to investigate the role of Ca2+ flux on obinutuzumab efficacy. Results: EGR1 expression is rapidly upregulated in CD20+ cells following rituximab and obinutuzumab exposure. Decreasing EGR-1 expression by shRNA abolished the direct cytotoxic effect of obinutuzumab both in vitro and in vivo, indicating that EGR-1 is required for CD20-mediated apoptosis. Additionally, the overexpression of EGR-1 resulted in enhanced cytotoxic activity of obinutuzumab both in vitro and in vivo. Rescuing EGR-1 expression in EGR-1 knocked-down cells restored sensitivity to obinutuzumab. Moreover, our results indicate that both rituximab and obinutuzumab could induce calcium influx in the presence of suboptimal concentrations of ionomycin. Blocking Ca2+ flux with the calcium channel blocker nifedipine or the Ca2+ chelating agent EGTA abolished EGR-1 induction by anti-CD20 mAbs. In vivo, nifedipine treatment interfered with obinutuzumab antitumor activity against established Granta (a human mantle cell lymphoma line) xenografts in SCID mice. Conclusion: EGR-1 plays a major role in the direct cytotoxic activity of anti-CD20 monoclonal antibodies and should be evaluated as a new biomarker to predict response to anti-CD20 treatment. Our data also show that calcium channel blockers interfere with the antitumor activity of obinutuzumab in preclinical models. Citation Format: Ivana Spasevska, Jade Villé, Kamel Chettab, Eva-Laure Matera, Charles Dumontet. Induction of apoptosis by anti-CD20 antibodies requires the induction of EGR-1 and calcium influx [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4594. doi:10.1158/1538-7445.AM2017-4594

Ionomycin-induced calcium influx induces neurite degeneration in mouse neuroblastoma cells: analysis of a time-lapse live cell imaging system

Reactive oxygen species induce neuronal cell death. However, the detailed mechanisms of cell death have not yet been elucidated. Previously, we reported neurite degeneration before the induction of cell death. Here, we attempted to elucidate the mechanisms of neurite degeneration before the induction of cell death using the neuroblastoma N1E-115 cell line and a time-lapse live cell imaging system. Treatment with the calcium ionophore ionomycin induced cell death and neurite degeneration in a concentration- and time-dependent manner. Treatment with a low concentration of ionomycin immediately produced a significant calcium influx into the intracellular region in N1E-115 cells. After 1-h incubation with ionomycin, the fluorescence emission of MitoSOXTM increased significantly compared to the control. Finally, analysis using a new mitochondrial specific fluorescence dye, MitoPeDPP, indicated that treatment with ionomycin significantly increased the mitochondrial lipid hydroperoxide production in N1E-115 cells. The fluorescence emissions of Fluo-4 AM and MitoPeDPP were detected in the cell soma and neurite regions in ionomycin-treated N1E-115 cells. However, the emissions of neurites were much lower than those of the cell soma. TBARS values of ionomycin-treated cells significantly increased compared to the control. These results indicate that ionomycin induces calcium influx into the intracellular region and reactive oxygen species production in N1E-115 cells. Lipid hydroperoxide production was induced in ionomycin-treated N1E-115 cells. Calcium influx into the intracellular region is a possible activator of neurite degeneration.

Propofol-induced Inhibition of Catecholamine Release Is Reversed by Maintaining Calcium Influx.

Propofol (2,6-diisopropylphenol) is one of the most frequently used anesthetic agents. One of the main side effects of propofol is to reduce blood pressure, which is thought to occur by inhibiting the release of catecholamines from sympathetic neurons. Here, the authors hypothesized that propofol-induced hypotension is not simply the result of suppression of the release mechanisms for catecholamines. The authors simultaneously compared the effects of propofol on the release of norepinephrine triggered by high K-induced depolarization, as well as ionomycin, by using neuroendocrine PC12 cells and synaptosomes. Ionomycin, a Ca ionophore, directly induces Ca influx, thus bypassing the effect of ion channel modulation by propofol. Propofol decreased depolarization (high K)-triggered norepinephrine release, whereas it increased ionomycin-triggered release from both PC12 cells and synaptosomes. The propofol (30 μM)-induced increase in norepinephrine release triggered by ionomycin was dependent on both the presence and the concentration of extracellular Ca (0.3 to 10 mM; n = 6). The enhancement of norepinephrine release by propofol was observed in all tested concentrations of ionomycin (0.1 to 5 μM; n = 6). Propofol at clinically relevant concentrations promotes the catecholamine release as long as Ca influx is supported. This unexpected finding will allow for a better understanding in preventing propofol-induced hypotension.

Characterization of cannabidiol-induced increases in CD25 and FOXP3 expression on CD4+ T cells (IRC11P.421)

Abstract Cannabis sativa-derived cannabinoid compounds are being investigated as potential immunosuppressive agents and important drug candidates for immune related diseases. Our previous data demonstrated that under conditions of suboptimal T cell activation, cannabidiol (CBD) could increase interleukin-2 (IL-2) production. Initially this result was surprising given that cannabinoids are immunosuppressive, but IL-2 can also contribute to regulatory T cell (Treg) induction. We tested the hypothesis that part of the mechanism for immunosuppression by CBD is induction of a Treg population in response to suboptimal activation. Splenocytes were treated with CBD or vehicle (VH; 0.1% ethanol) for 30 minutes, then stimulated with low concentrations of PMA and ionomycin (Io). CBD induced IL-2, TGF-β, CD25 and FOXP3 on CD4+ T cells in response to 4 nM PMA/0.05 µM Io after an overnight stimulation. Further characterization of this effect demonstrated that CBD also induced CD25 and FOXP3 on CD4+ T cells after a 5-day incubation with 1 nM PMA/0.01 µM Io. Interestingly, while CBD robustly induced IL-2 after an overnight incubation with 4 nM PMA/0.05 µM Io, the IL-2 in the culture supernatant dropped by day 5. Overall, these data suggest that the mechanism by which CBD is immunosuppressive might involve induction of Tregs in response to suboptimal activation of T cells.

Identification of cell populations in splenocytes of Nile Tilapia (Oreochromis niloticus) and functional parameters of lymphocytes through flow cytometry.

Identification of cell populations in splenocytes of Nile Tilapia (Oreochromis niloticus) and functional parameters of lymphocytes through flow cytometry.

Specific Activation Requirements of Zona-Free Sheep Oocytes before and after Somatic Cell Nuclear Transfer

In this study, the effect of the steps involved in zona-free somatic cell nuclear transfer (SCNT) on oocyte transcripts was investigated in sheep. To establish the reliable combined electrical-chemical activation for zona-free oocytes, oocytes were first exposed to an electrical pulse and then treated with 18 chemical activation regimens designed through modifying duration and concentration of ionomycin and 6-dimethyl aminopurine (6-DMAP), which is routinely used for SCNT. Electrofusion-mediated nuclear transfer significantly reduced transcript abundances of CCNB1, POU5F1, NPM2, GMMN, and CX43 compared to intact oocytes. Maximum parthenogenetic blastocyst development was obtained when oocytes were submitted to electric pulse and then to (1) 5 μM ionomycin for 5 or 2.5 min, both followed by 2 h of incubation with 6-DMAP (41.7±1.1, and 42.4±1.4%, respectively), (2) 5 μM ionomycin for 1 min+6-DMAP for 4 h (43.1±1.4%), and (3) 2.5 μM ionomycin for 1 min+6-DMAP for 2 h (42.4±1.4%), with significant differences compared to all the other groups. Statistical assessment of interactions between duration and concentration of ionomycin and duration of 6-DMAP exposure revealed that (1) concentration of ionomycin may be a more important factor than its duration, (2) both a long exposure period and a low concentration of ionomycin had marked decreasing effects on parthenogenetic development of zona-free oocytes, and (3) high duration of exposure to 6-DMAP can reduce parthenogenetic development. Despite an activation preference of parthenogenetic oocytes, a significantly higher rate of cloned blastocyst development was observed when reconstructed oocytes were activated with 5 μM ionomycin for 5 min rather than 2.5 μM ionomycin for 1 min (8.8±2.5 vs. 1.25±2.2%). These results suggested that SCNT steps have determining effects on oocyte transcripts and activation preferences of the reconstituted oocytes compared to intact counterparts. In this sense, reconstituted oocytes may need a higher concentration of ionomycin for a longer period than intact oocytes.

Pharmacological Potentiators of TRPM5 Channel Activity

TRPM5 belongs to the ‘melastatin’ transient receptor potential subfamily, fluxes mainly Na+, and is activated by cytosolic Ca2+ increases (< 1 μM). TRPM5 activates and deactivates rapidly even during sustained Ca2+ rises, which leads to transient membrane depolarization kinetics. Only the Ca2+ ionophore ionomycin yielded significant and transient membrane potential (MP) signals in HEK293 cells stably expressing TRPM5. Various agonists for endogenous Gq‐GPCRs induced significant Ca2+ signals, but did not or only weakly activate TRPM5. Five tool potentiators were assessed using two TRPM5‐expressing cell clones, and various pharmacological concentrations of the indirect stimulator ionomycin. For both cell lines, potencies and efficacies of potentiators were best using an EC80 (rather than an EC20) concentration of ionomycin. This indirectly indicates that maximal cytosolic Ca2+ increases lead only to submaximal TRPM5 activation. EC60–80 concentrations of ionomycin yielded acceptable MP assay statistics, and a focused compound library was tested to find further and more potent TRPM5 potentiators. 114 TRPM5 potentiator hits were found (EC50 values: 0.11 –40 μM), and 67 thereof confirmed in a patch clamp assay. 38 of these novel TRPM5 potentiators increased currents over 100%. This work was supported by the Novartis Institutes for BioMedical Research (NIBR).

Complex Actions of Ionomycin in Cultured Cerebellar Astrocytes Affecting Both Calcium-Induced Calcium Release and Store-Operated Calcium Entry

The polyether antibiotic ionomycin is a common research tool employed to raise cytosolic Ca(2+) in almost any cell type. Although initially thought to directly cause physicochemical translocation of extracellular Ca(2+) into the cytosol, a number of studies have demonstrated that the mechanism of action is likely to be more complex, involving modulation of intrinsic Ca(2+) signaling pathways. In the present study we assessed the effect of ionomycin on primary cultures of murine cerebellar astrocytes. Ionomycin concentrations ranging from 0.1 to 10μM triggered a biphasic increase in cytosolic Ca(2+), consisting of an initial peak and a subsequent sustained plateau. The response was dependent on concentration and exposure time. While the plateau phase was abolished in the absence of extracellular Ca(2+), the peak phase persisted. The peak amplitude could be lowered significantly by application of dantrolene, demonstrating involvement of Ca(2+)-induced Ca(2+)-release (CICR). The plateau phase was markedly reduced when store-operated Ca(2+)-entry (SOCE) was blocked with 2-aminoethoxydiphenyl borate. Our results show that ionomycin directly affects internal Ca(2+) stores in astrocytes, causing release of Ca(2+) into the cytosol, which in turn triggers further depletion of the stores through CICR and subsequently activates SOCE. This mechanistic action of ionomycin is important to keep in mind when employing it as a pharmacological tool.

Specific activation requirements of in vitro‐matured sheep oocytes following vitrification‐warming

Oocyte vitrification and assisted oocyte activation have increasingly important roles in assisted reproductive technology. Yet, an important area of concern with matured oocyte cryobiology is that elements of oocytes intimately involved in metaphase-II arrest may be modified by cryopreservation. By comparing different cellular characteristics of unvitrified, vitrified-warmed, and unvitrified-activated oocytes, the present study investigated how vitrification-warming process may affect developmental competence of in vitro-matured sheep oocytes following parthenogenetic activation. Structural, ultrastructural, and molecular analyses indicated that the characteristics of vitrified-warmed oocytes vastly differed from fresh oocytes, instead resembling unvitrified-activated oocytes. For unvitrified oocytes, the highest blastocyst yield (41.8 ± 0.6%) was achieved using the maximum ionomycin concentration (5 µM), and importantly, the duration of ionomycin treatment was not of utmost importance at this concentration. In contrast, the maximum blastocyst yield of vitrified-warmed oocytes (28.4 ± 1.4%) was achieved with a minimal duration of ionomycin treatment (1 min), and further extending the duration dramatically reduced developmental potential of vitrified-warmed oocytes. These results suggested that vitrified-warmed oocytes may need an activation protocol different from unvitrified oocytes. In this respect, unvitrified oocytes were more sensitive to the concentration rather than the duration of ionomycin treatment when compared with vitrified oocytes, which were sensitive to the treatment duration. These results may provide a platform to improve the potential applications of vitrified oocytes in medicine and agriculture.

Minocycline Suppresses Activation of Nuclear Factor of Activated T Cells 1 (NFAT1) in Human CD4+ T Cells

Minocycline is a tetracycline family antibiotic that has anti-inflammatory and immunomodulatory properties. These properties have shown promise in the treatment of conditions such as rheumatoid arthritis, Huntington disease, and multiple sclerosis. As lymphocyte activation is involved in the pathogenesis of many of these diseases, T cells are postulated to be a primary target in minocycline therapy. Previous studies have demonstrated attenuation of CD4(+) T cell activation by minocycline, but a specific mechanism has not been elucidated. In this study, we investigated the effect of minocycline on the activity of three key transcription factors regulating CD4(+) T cell activation: NF-κB, AP-1 (activator protein 1), and NFAT (nuclear factor of activated T) cells. Our data demonstrate that minocycline selectively impairs NFAT-mediated transcriptional activation, a result of increased phosphorylation and reduced nuclear translocation of the isoform NFAT1. Minocycline increased the activity of the NFAT kinase GSK3 and decreased intracellular Ca(2+) flux, both of which facilitate NFAT1 phosphorylation. These findings provide a novel mechanism for minocycline induced suppression of CD4(+) T cell activation and may better inform the application of minocycline as an immunomodulatory agent.

Regulation of T Cell Activation and Anergy by the Intensity of the Ca2+Signal in Cooperation with Other Signals

The results of our previous in vitro study indicated that the intensity of the Ca²+ signal could determine T cell activation and anergy. We show here that the T cell response of mice that had been treated with cyclosporine A during oral tolerance induction was higher than that of control mice, indicating that the Ca²+ signal could also determine T cell activation and tolerization in vivo. However, T cell activation was not apparent at any concentration of ionomycin, although a low dose of anti-CD3 monoclonal antibody (mAb) induced activation, while a high dose induced anergy in vitro. These results indicate that the balance between the Ca²+ signal and other signals which can also be induced by anti-CD3 stimulation, but not the actual intensity of the Ca²+ signal or the presence of co-stimulation, played an important role in regulating T cell activation and anergy.

Matrine Induces Cell Anergy in Human Jurkat T Cells through Modulation of Mitogen-Activated Protein Kinases and Nuclear Factor of Activated T-Cells Signaling with Concomitant Up-Regulation of Anergy-Associated Genes Expression

Induction of immunotolerance has become a new strategy for treating autoimmune conditions in recent decades. However, so far there is no ideal therapeutics available for clinical use. Medicinal herbs are a promising potential source of immunotolerance inducers. In the current study, we sought first to optimize conditions for a validated cellular model of human Jurkat cells; and then used this model to screen bioactive compounds derived from medicinal plants for inducing T cell anergy in comparison with the effect of well-known T cell anergy inducer, ionomycin. The results showed that passage of the cells, and concentration and stimulation time of ionomycin on the cells could influence the ability of T cell anergy induction. Matrine, a small molecule derived from the root of Sophora flavescens AIT., was demonstrated to be effective in inducing T cell anergy in human Jurkat cells. The cells exposed to matrine showed markedly decreased mRNA expression of interleukin-2, an indicator of T cell anergy, when the cells were stimulated by antigens, anti-OKT3 plus anti-CD28. Mechanistic study showed that ionomycin and matrine could up-regulate the anergy-associated gene expressions of CD98 and Jumonji and activate nuclear factor of activated T-cells (NFAT) nuclear translocation in absence of cooperation of AP-1 in Jurkat cells. Pre-incubation with matrine or ionomycin could also shorten extracellular signal-regulated kinase (ERK) and suppress c-Jun NH(2)-terminal kinase (JNK) expression on the anergic Jurkat cells when the cells were stimulated with anti-OKT-3 plus anti-CD28 antibodies. Thus, matrine is a strong candidate for further investigation as a T cell immunotolerance inducer.

NK 1 receptor-mediated endothelium-dependent relaxation and contraction with different sensitivity to post-receptor signaling in pulmonary arteries

In rabbit intrapulmonary arteries, substance P (SP) has been reported to induce endothelium-dependent relaxation (EDR) and endothelium-dependent contraction (EDC) via tachykinin NK 1 receptors, and endothelium-independent contraction (EIC) via tachykinin NK 2 receptors. The present study pharmacologically examined whether these opposite responses (EDR and EDC) are mediated by the same NK 1 receptor. Five tachykinin agonists, including septide, a reportedly atypical NK 1 agonist, caused concentration-dependent EDR in the presence of NK 2 antagonist (SR-48968) + TXA 2 synthetase inhibitor (ozagrel), which blocked EIC and EDC, in pre-contracted arteries, and concentration-dependent EDC in the presence of NK 2 antagonist (SR-48968) + nitric oxide synthase inhibitor ( l-N G-nitro-arginine methyl ester), which blocked EIC and EDR, in non-contracted arteries. The EC 50 values of these agonists for EDR were smaller than those for EDC, indicating that the affinities of NK 1 agonists to NK 1 receptors are different between EDR and EDC. However, the rank order of their potency for EDR and EDC was the same: SP = septide > SP methyl ester (SPME) > neurokinin A > neurokinin B. [Ala 5, β-Ala 8]-α-neurokinin fragment 4–10 (NK 2 agonist) and senktide (NK 3 agonist) caused no responses. Two structurally different NK 1 antagonists, CP-99994 and SR-140333, shifted the concentration–EDC and –EDR curves of SPME, a selective NK 1 agonist, and septide rightward and suppressed their maximal responses in a similar concentration-dependent manner, indicating that the affinities of NK 1 antagonists to NK 1 receptors are similar between EDR and EDC. U-73122, a phospholipase C inhibitor, and thapsigargin, 2,5-di-tert-butylhydroquinone, and ruthenium red, all intracellular Ca 2+ release blockers, inhibited SP-induced EDR and EDC. Effective concentrations of ionomycin (Ca 2+ ionophore) causing EDR were also lower than those causing EDC. Taken together, SP-induced EDR and EDC are mediated by activation of the same NK 1 receptor followed by an increase in intracellular Ca 2+, and sensitivity to Ca 2+ may be higher in the EDR than EDC pathway.