Browsing by Author "Sabana Khatun"

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Calcium ionophore-induced egg activation and apoptosis are associated with the generation of intracellular hydrogen peroxide
(2008) S.K. Chaube; Sabana Khatun; S.K. Misra; T.G. Shrivastav
The present study was designed to investigate whether calcium ionophore-induced activation and apoptosis are associated with the generation of hydrogen peroxide (H2O2) in rat eggs cultured in vitro. Culture of metaphase-II (M-II) arrested eggs in Ca2+/Mg2+-deficient medium did not induce egg activation, while a second polar body was observed in 20% of eggs when cultured in Ca2+/Mg2+-supplemented medium. In Ca2+/Mg2+-deficient medium, lower concentrations of calcium ionophore (0.2,0.4 and 0.8 μm) not only induced egg activation in a dose-dependent manner but also generation of intracellular H2O2 (84.40 ± 0.50 ng/egg) when compared to control eggs (80.46±1.34 ng/egg). The higher concentration of calcium ionophore (1.6 μm) induced apoptosis and pronounced generation of intracellular H2O2 (92.43±0.93 ng/egg) in treated eggs. Conversely, cell-permeant antioxidant such as 2(3)-tert-butyl-4-hydroxyanisole (BHA) reduced intracellular H2O2 level (81.20±1.42 ng/ egg) and protected against calcium ionophore-induced morphological changes characteristics of egg activation and apoptosis. These results clearly suggest that calcium ionophore-induced activation and apoptosis are associated with the generation of intracellular H2 O2 in rat eggs.
Development of ELISA for measurement of progesterone employing 17 - OH-P-HRP as enzyme label
(2009) Sabana Khatun; Seema Nara; Vinay Tripathi; Kiran Rangari; Shail K. Chaube; Kiran P. Kariya; Suman Kumar; Tulsidas G. Shrivastav
The present study was aimed to develop a highly specific and sensitive Enzyme Linked Immunosorbent Assay (ELISA) to measure progesterone in human serum using a heterologous combination of immunogen and enzyme conjugate. The antiserum was raised against Progesterone-3-O-carboxymethyloxime bovine serum albumin (P-3-O-CMO-BSA) in New Zealand white rabbits. The enzyme conjugate was prepared by labeling 17 - hydroxy-progesterone-3-O-carboxymethyloxime (17 - OH-P-3-O-CMO) with Horseradish Peroxidase (HRP) to form 17 - OH-P-3-CMO-HRP. A Checkerboard assay was performed to determine the working dilutions of antiserum and enzyme conjugate. Dose-response studies were carried out by incubating 100L enzyme conjugate along with 50L of standards in the primary antibody coated wells for 1 hour.
Extracellular calcium protects against verapamil-induced metaphase-II arrest and initiation of apoptosis in aged rat eggs
(2009) S.K. Chaube; Anima Tripathi; Sabana Khatun; S.K. Mishra; P.V. Prasad; T.G. Shrivastav
Non-specific L-type calcium channel blockers, such as verapamil (≥50 μM), induce metaphase-II (M-II) arrest and apoptosis in aged rat eggs cultured in Ca2+-deficient medium. However, the effects of extracellular Ca2+ on verapamil-induced M-II arrest and apoptosis have not yet been reported. We have demonstrated that postovulatory aging induced exit from M-II arrest by extruding a second polar body, a morphological sign of spontaneous egg activation (SEA). Verapamil inhibited SEA and induced egg apoptosis in a dose-dependent manner in Ca2+-deficient medium. The initiation of apoptotic features was observed at 50 μM of verapamil. Extracellular Ca2+ (1.80 mM) reduced intracellular H2O2 level, bax protein expression, caspase-3 activity, DNA fragmentation and protected against 50 μM, but not higher concentrations of ≥100 μM in verapamil-induced egg apoptosis. These results suggest that extracellular Ca2+ ions have a role during SEA and protect against verapamil-induced apoptosis in aged rat eggs. © 2009 International Federation for Cell Biology.
Generation of hydrogen peroxide mediates hanging death-induced neuronal cell apoptosis in the dentate gyrus of the rat brain
(2013) Sabana Khatun; Shail K. Chaube; Chandra N. Bhattacharyya
Present study was aimed to find out whether hanging death (HD) induces generation of reactive oxygen species (ROS) and neuronal cell apoptosis in the dentate gyrus (DG) region of rat brain. Permanent global brain ischemia was generated by HD in experimental rats and the brain was isolated after 0, 1, 2, 3, 4, 5, 6, 9, 12 and 24h post- HD and cervical dislocation (CD). The histology, hydrogen peroxide (H2O2) concentration, catalase, caspase-9 and caspase-3 activities and DNA fragmentation were analyzed in neuronal cells of DG region of the brain. Permanent global brain ischemia generated due to HD induced generation of H2O2 as well as catalase activity. The increased level of H2O2 was associated with the increased caspase-9 and caspase-3 activities. The increased caspase-3 activity induced neuronal cell apoptosis during early period (0-9h) of HD as compare to CD group. The neuronal cells necrosis was observed only 12h post-HD, while CD induced necrosis as early as 3h post-CD and the histoarchitecture of DG region was gradually disrupted after 6h of CD. In conclusion, data of the present study suggest that the permanent global brain ischemia induces neuronal cell apoptosis during early period of HD through ROS-mediated pathway, while CD induces neuronal cell necrosis and disruption of the histoarchitecture of the DG region. Thus, neuronal cell apoptosis may be used to develop a cellular marker to find out the exact timing of HD. © 2013 Elsevier Inc.
Intracellular levels of hydrogen peroxide and nitric oxide in oocytes at various stages of meiotic cell cycle and apoptosis
(2009) Anima Tripathi; Sabana Khatun; A.N. Pandey; S.K. Mishra; Radha Chaube; T.G. Shrivastav; S.K. Chaube
The objective was to find out the functional roles of hydrogen peroxide (H2O2) and nitric oxide (NO) during various stages of meiotic cell cycle and apoptosis in rat oocytes. For this purpose, 30 oocytes from each stage such as diplotene, metaphase-I (M-I), metaphase-II (M-II) and apoptosis were collected and intracellular H2O2, total nitrite level and inducible nitric oxide synthase (iNOS) expression were analysed. This study demonstrated that generation of a tonic level of H2O2 induces meiotic resumption in diplotene-arrested oocytes and further increase may lead to apoptosis. Conversely, reduction in iNOS expression and total nitrite level are associated with meiotic resumption in diplotene-arrested oocytes, but induce apoptosis in aged oocytes. These results suggest that generation of a tonic level of H2 O2, reduced iNOS expression and total nitrite level are associated with meiotic resumption, while more generation of H2O2 and sustained reduced total nitrite level are linked with oocyte apoptosis in rat.
Melatonin protects against clomiphene citrate-induced generation of hydrogen peroxide and morphological apoptotic changes in rat eggs
(2011) Anima Tripathi; Karuppanan V. Premkumar; Ashutosh N. Pandey; Sabana Khatun; Surabhi Kirti Mishra; Tulsidas G. Shrivastav; Shail K. Chaube
The present study was aimed to determine whether clomiphene citrate-induces generation of hydrogen peroxide in ovary, if so, whether melatonin could scavenge hydrogen peroxide and protect against clomiphene citrate-induced morphological apoptotic changes in rat eggs. For this purpose, forty five sexually immature female rats were given single intramuscular injection of 10 IU pregnant mare's serum gonadotropin for 48 h followed by single injections of 10 IU human chorionic gonadotropin and clomiphene citrate (10 mg/kg bw) with or without melatonin (20 mg/kg bw) for 16 h. The histology of ovary, ovulation rate, hydrogen peroxide concentration and catalase activity in ovary and morphological changes in ovulated eggs were analyzed. Co-administration of clomiphene citrate along with human chorionic gonadotropin significantly increased hydrogen peroxide concentration and inhibited catalase activity in ovary, inhibited ovulation rate and induced egg apoptosis. Supplementation of melatonin reduced hydrogen peroxide concentration and increased catalase activity in the ovary, delayed meiotic cell cycle progression in follicular oocytes as well as in ovulated eggs since extrusion of first polar body was still in progress even after ovulation and protected against clomiphene citrate-induced egg apoptosis. These results clearly suggest that the melatonin reduces oxidative stress by scavenging hydrogen peroxide produced in the ovary after clomiphene citrate treatment, slows down meiotic cell cycle progression in eggs and protects against clomiphene citrate-induced apoptosis in rat eggs. © 2011 Elsevier B.V. All rights reserved.
p53 activation and mitochondria-mediated pathway are involved during hanging deathinduced neuronal cell apoptosis in dentate gyrus region of the rat brain
(SpringerOpen, 2013) Sabana Khatun; Shail K. Chaube; Chandra N. Bhattacharyya
The goal of this study was to understand the molecular event in the brain caused by hanging death (HD). Animals were subjected to either cervical dislocation (CD) or HD. Brain was collected at various times (0, 1, 3, 6 and 12 h) after death. Brain expression of p53 and Bax, cytochrome c concentration, caspases activity and DNA fragmentation were analyzed. Compared to that of CD, HD increased p53 and Bax proteins expressions, cytochrome c concentration, caspases activity and DNA fragmentation during the early period (0-6 h) of HD, whereas CD induced necrosis 3 h post- CD and thereafter. These data support that HD induces neuronal cell apoptosis, in part, through mitochondria-mediated pathways. These data also suggest that neuronal apoptosis could be a potential marker and an aid to forensic science of HD. © 2013 Khatun et al.