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Purification of enough numbers of circulating eosinophils is difficult because eosinophils account for less than 5% peripheral blood leukocytes. Human eosinophilic leukemia EoL-1 cells have been considered an in vitro source of eosinophils as they can differentiate into mature eosinophil-like cells when incubated with dibutyryl cAMP (dbcAMP) or butyric acid. In this study, the viability and phenotypic maturation of EoL-1 cells stimulated by either dbcAMP or butyric acid were comparatively analyzed. After treatment with 100 microM dbcAMP or 0.5 microM butyric acid, EoL-1 cells showed morphological signs of differentiation, although the number of nonviable EoL-1 cells was significantly increased following butyric acid treatment. Stimulation of EoL-1 cells with 0.5 microM butyric acid more effectively induced the expression of mature eosinophil markers than stimulation with dbcAMP. These results suggest that treatment of EoL-1 cells with 0.5 microM butyric acid for limited duration could be an effective strategy for inducing their differentiation. Considering that expression of CCR3 was not sufficient in EoL-1 cells stimulated with 0.5 microM butyric acid, treatment of the chemically stimulated EoL-1 cells with cytokines, which primarily support eosinophil maturation, would help to obtain differentiated EoL-1 cells with greater functional maturity.
Subject(s)
Humans , Bucladesine , Butyric Acid , Cytokines , Eosinophils , Hypereosinophilic Syndrome , LeukocytesABSTRACT
Objective To study the effect of dualdi butyryl cyclic AMP (dbcAMP)on the proliferation of FTC-133 cell line. Methods FTC-133 cells were normally cultured and divided into control group,dbcAMP treatment group (0.5,1.0,2.0 mmol/L). After FTC-133 cells were treated with dbcAMP (0.5,1.0,2.0 mmol/L)for 24 h or 48 h,the growth activity and growth curve was detected by MTT.Changes of the cell cycle were detected by flow cytometry.The mRNA and protein expression of Raf1 were measured by RT-qPCR and Western blotting.Results Compared with control group,the growth activity of FTC-133 cells was re-duced by different levels of dbcAMP in a dose-time dependence manner.The number of FTC-133 cells was decreased in the S phase and increased in the G2/M phase.The mRNA and protein expression of Raf1 of treatment group were both reduced compared with control group.Conclusion dbcAMP significantly reduced FTC-133 cells proliferation and promoted apoptosis,and which might be involoved by ERK MAPK signalling.
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Objective To study the signaling transduction of dBcAMP induced morphological changes of astrocytes.Methods Morphological changes of primarily cultured mouse astrocytes induced by dBcAMP were studied by immunocytochemical technique.The signaling transduction pathways underlying those changes were further explored using specific inhibitors AG1478 and GM6001.Results Zn2＋-dependent metalloproteinase and epidermal growth factor receptor（EGFR）in astrocytes were involved in the morphological changes of astrocytes induced by dBcAMP.Conclusion dBcAMP-induced transactivation pathways were delineated in the morphological changes of astrocytes.
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The objective of this study was to improve sexed bovine embryo production with sorted sperm in chemically defined conditions by supplementing the IVF medium with db-cAMP. Cumulus-oocyte complexes (COCs) were matured for 18 h in supplemented TCM-199 and fertilized with X- or Y-bearing sperm in the presence of heparin (10 µg/ml), db-cAMP (1 µM) or no treatment (control). Presumptive zygotes were cultured 54 h in g-SOF. From 72 to 144 h post- insemination (hpi) embryos were cultured in c-SOF+NEA and from 144 to 192 hpi embryos were placed in maturation medium without hormones. No significant differences were found among treatments for Y-sperm when compared to controls. A significant (P<0.01) improvement in the proportion of cleaved oocytes was found for X-sperm treated with db-cAMP (70.83%) compared to the Y-sperm inseminated oocytes treated wit db-cAMP (46.37%). Treatment with db-cAMP enabled a better (P<0.05) blastocyst formation rate (19.29%) compared to control (8.47%) and heparin (10.44%). Treatment of db-cAMP significantly increased the rate of blastocysts in X-sperm inseminated oocytes (30.77%) compared to Y-sperm inseminated oocytes treated the same (9.68%) and compared to X- and Y-sperm treated with heparin (5.88% and 15.15%, respectively) and not treated (9.68% and 7.14%, respectively, P<0.05). These results suggest that db-cAMP may prove to be an effective treatment of sorted sperm for in vitro production of female bovine embryos under chemically defined conditions.
El objetivo de este estudio fue mejorar la producción de embriones bovinos con semen sexado bajo condiciones químicamente definidas mediante la suplementación del medio de fecundación con db-cAMP. Los complejos ovocitos cumulus (COCs) fueron madurados por 18 horas en TCM-199 suplementado y fueron fecundados con espermatozoides X o Y en presencia de heparina (10 g/ml), db-cAMP (1 µM) o sin tratamiento alguno (control). Los presuntivos cigotos fueron cultivados por 54 horas en g-SOF. Desde las 72 a las 144 horas post-inseminación (hpi) los embriones se cultivaron en c-SOF+NEA y desde 144 a 192 hpi fueron colocados en medio de maduración pero sin hormonas. No se observaron diferencias entre tratamientos para los oocitos fecundados con espermatozoides Y cuando se compararon a los controles. Se observó una mejora significativa (P<0,01) en la proporción de ovocitos que se dividieron cuando fueron fecundados con espermatozoides X tratados con db-cAMP (70,83%) en comparación con los fecundados con espermatozoides Y tratados con db-cAMP (46,37%). El tratamiento con db-cAMP fue capaz de inducir una mayor (P<0,05) tasa de formación de blastocistos (19.29%) en comparación a los tratamientos control (8,47%) y heparina (10,44%). El tratamiento con db-cAMP incrementó (P<0,05) la tasa de embriones de cuatro células alcanzando el estadio de blastocisto cuando los oocitos fueron fecundados con espermatozoides X (30,77%) en comparación a cuando la fecundación se realizó con espermatozoides Y (9,68%) y en comparación a cuando se llevó a cabo con espermatozoides X o Y en presencia de heparina (5,88% y 15,15%, respectivamente) o en el tratamiento control (9,68% y 7,14%, respectivamente). Estos resultados sugieren, que el db-cAMP puede ser un tratamiento efectivo para el semen sexado, a fin de incrementar la producción in vitro de embriones bovinos hembra bajo condiciones químicamente definidas.
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OBJECTIVE: Melatonin, which is secreted by pineal gland play an important role in the regulation of ovarian function via seasonal rhythm and sleep in most mammals. It also has a role in the protection of cells by removing toxic oxygen free radicals brought about by metabolism. In the present study, effects of melatonin on the mouse oocyte maturation were examined using two different culture conditions provided with 5% or 21% oxygen concentration. MATERIAL AND METHOD: Immature mouse oocytes were obtained from the ovarian follicles of 3~4 weeks old ICR strain mice intraperitoneally injected with 5 I.U. PMSG 44 hour before. Under stereomicroscope, morphologically healthy oocytes with distinct germinal vesicle (GV) were liberated from the graafian follicles and collected using mouth-controlled micropipette. They were then cultured for 17 hour at 37degrees C, 5% CO2 and 21% O2 (95% air) or 5% CO2, 5% O2 and 90% N2. New modified Hank's balanced salt solution (New MHBS) was used as a culture medium throughout the experiments. Effects of melatonin were examined at a concentration of 0.0001 micrometer, 0.01 micrometer or 1.0 micrometer. For the prevention of spontaneous maturation of immature oocytes during culture, dibutyryl cyclic AMP (dbcAMP) and/or hypoxanthine were included in the medium. RESULTS: Under 21% oxygen condition, oocytes cultured in the presence of 0.01 micrometer melatonin showed a significantly higher maturation rates, in terms of germinal vesicle breakdown (95.0% vs 89.0%) and polar body formation (88.1% vs 75.4%), compared to those cultured with 0.0001 micrometer or 1.0 micrometer melatonin. However, no difference was observed in oocytes cultured under 5% oxygen whether they were treated with melatonin or not. In the presence of 0.01 micrometer melatonin, oocytes either cultured under 21% or 5% oxygen exhibited no difference in the polar body formation (85.6% vs 86.7%). However, in the absence of melatonin, oocytes cultured under 21% oxygen exhibited lower polar body formation (74.7%). When oocytes were cultured in the presence of dbcAMP alone or with varying concentrations of melatonin, those treated with both compounds always showed better maturation, i.e., germinal vesicle breakdown and polar body formation, compared to those cultured with dbcAMP alone. At the same concentration of melatonin, however, oocytes exposed to 21% oxygen showed poor maturation than those to 5% oxygen. Similar results were obtained from the experiments using hypoxanthine instead of dbcAMP. CONCLUSION: Based upon these results, it is suggested that melatonin could enhance the meiotic maturation of mouse oocytes under 21% oxygen concentration, and release oocytes from the meiotic arrest by dbcAMP or hypoxanthine regardless of the concentration of oxygen, probably via the removal of oxygen free radicals.
Subject(s)
Animals , Female , Mice , Bucladesine , Free Radicals , Hypoxanthine , Mammals , Melatonin , Metabolism , Oocytes , Ovarian Follicle , Oxygen , Pineal Gland , Polar Bodies , SeasonsABSTRACT
Effects of amrinone and dibutyryl cyclic AMP (DBcAMP) on hemodynamics and myocardial metabolism were studied in 16 patients in the early postoperative period following open-heart surgery. Amrinone was administered continously at 10μg/kg/min for 5 hours and DBcAMP at 12μg/kg/min was infused concomitantly for the last 1 hour. Amrinone increased SVI, and decreased HR, PCWP, arterial systolic blood pressure, double product and central temperature difference significantly. Following concomitant administration of DBcAMP for 1 hour, further increase of CI, O<sub>2</sub> delivery, blood sugar and insulin were observed. Significant decrease of excess lactate from 9.31±3.27 to -1.31±1.83mg/dl (<i>p</i><0.05) showed that anaerobic metabolism improved in the myocardium after amrinone administration, and the effect improved further to -3.64±1.8mg/dl following concomitant administration of DBcAMP. These data demonstrate that amrinone has beneficial effects on hemodynamics and myocardial metabolism, and following supplementary administration of DBcAMP, further improvement in hemodynamics was observed.
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Objective To optimize systems of pig embryo in vitro production. Methods Pig oocytes were matured and fertilized in vitro in different culture mediums. Maturation rate and cleavage rate were analyzed. Results Gonadotropin induced maturation of pig oocytes in vitro , and improved the frequence of oocytes developed into M Ⅱ. The highest maturation and cleavage rates, present in medium with 15 IU/ml FSH+30 IU/ml LH, were 62% and 15% respectively. There was no difference in maturation rates in M199 and NCSU 23(48.2% vs 56.9%). However, difference in cleavage rate was observed in these two mediums(15.7% vs 36%, P