Investigation of Telomerase activity and hTERT gene expression in MCF7 cells treated with papaverine

Introduction: Telomere maintenance is essential for the continued proliferation of dividing cells, and is implicated in chromosome stability and cell immortalization. Telomerase activity, that allows cancer cells to maintain their telomeric DNA for an indefinite replicative capacity, is an attractive target against cancer. A well known benzylisoquinoline alkaloid, papaverine, we focused on to evaluate its antiproliferaive effects on breast cancer MCF7 cells

Methods: Cytotoxicity of the commercially available pure compound papaverine HCl [Sigma] was determined by MTT assay. A modified quantitative real-time polymerase chain reaction [PCR]-based telomerase repeat amplification protocol [TRAP] was used to estimate relative telomerase activity in papaverine-treated cells in comparison with the untreated control cells. Relative expression level of the catalytic subunit of telomerase [hTERT] gene was estimated using real time reverse transcription-PCR [RT-PCR]

Results: IC50 concentration of papaverine after 48 hours treatment was measured to 120 micromolar. At this concentration telomerase activity showed a considerable decrease [almost 70% in comparison with untreated control cells], in a concentration dependent manner. Quantitative real-time RT-PCR experiments indicated a similar reduction in transcription level of hTERT gene under treatment with papaverine

Conclusion: Papaverine is a potent natural compound in suppression of cancer cell immortality most probably by anti-telomerase activity. It is a valuable putative compound for further development of promising anti-cancer agents

[Telomerase activity in breast cancer cells under treatment with crocin]

Crocin is a major constituent of saffron, the dried stigmas of Crocus sativus L., which is used mainly as a herbal medicine or a food coloring agent around the world. Novel publications reporting a cancer preventive effect for crocin motivated us to evaluate telomerase activity, the main cause of immortality in cancer cells, under treatment with crocin. IC50 concentration of crocin was estimated in MCF-7 cell line, a breast adenocarcinoma cancer model, by MTT [3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide] test after 48 hours of treatment. A conventional telomere repeat amplification protocol [TRAP] assay and a real-time quantitative telomere repeat amplification protocol [qTRAP] assay were used to estimate relative telomerase activity in crocin-treated cells in comparison with untreated control cells. Telomerase activity in the treated cells with different concentrations of crocin up to IC50 showed an increment after administration of very low doses of crocin, whereas higher concentrations of crocin remarkably inhibited the enzyme in a dose-dependent manner. IC50 values of crocin reduced by 85% in comparison with untreated control cells. qTRAP estimations show a good correlation with the conventional assay results. Antiproliferative effect of crocin in cancer cells is probably due to strong inhibition of telomerase activity

Frequency of syncope in patients with accessory atrioventricular connection

Syncope in patients with Wolff-Parkinson-White [WPW] syndrome is related to rapid reciprocating tachycardia or rapid ventricular response over the accessory pathway during atrial fibrillation [AF]. The aim of this retrospective study is to evaluate the frequency of syncope in patients with WPW syndrome. We reviewed the records of 150 consecutive patients with WPW syndrome. There were 20 patients [13.3%] who reported at least one episode of syncope and 130 patients [86.7%] without such a history. Syncope is relatively frequent in patients with WPW. Patient with WPW syndrome who has experienced this symptom should be thoroughly evaluated

Serological study of bovine viral respiratory diseases in dairy herds in Kerman province, Iran

Respiratory disorders are major concern for dairy cattle industry. Viruses and bacteria in combination with stress play a key role in triggering acute respiratory infections. The most important viral agents are bovine viral diarrhoea virus [BVDV], bovine herpes virus type 1 [BHV-1], bovine respiratory syncytial virus [BRSV], parainfluenza virus type 3 [PIV-3] and bovine adenovirus [BAV]. This cross-sectional study was conducted to evaluate the serological status of BVDV, BHV-1, BRSV, PIV-3 and BAV in dairy herds in Kerman province, Iran. From June to November 2007, 181 serum samples were collected from 1-3-year-old cattle from 15 industrial dairy farms in Kerman province using cluster sampling. The samples were tested by commercial indirect ELISA kits. Antibodies were detected against BVDV, BHV-1, BRSV, PIV-3 and BAV in 77.90, 30.39, 100, 100 and 100% of serum samples, respectively. All farms were positive at least for one of these viruses and antibodies against all the 5 viruses were detected in 4 [26.66%] herds among 15 dairy farms. According to the present study, BVDV, BRSV, PIV-3 and BAV are common viruses in dairy herds in Kerman province

arrangement of microtubules in embryos derived from mice young, old and reconstructed oocytes

To study the structure and distribution of microtubules in embryos derived from young, old and reconstructed oocytes. Embryos obtained from old [50 embryos], young [50 embryos] and reconstructed oocytes [10 embryos] were studied by immunocytochemistry. The microtubule structures of the embryos were studied by using fluroscent microscopy with FITC-PI filter and polyclonal antibody against alfa tubulin. The spindle structure of MII young oocyte and the obtained embryos were normal with the suitable condensation. There was no contact between chromosome and spindle in old Oocytes as well as the obtained embryos, in addition, the spindle was extended in old group. In reconstructed embryos, thin and scattered filaments were observed. This study reveals that the arrangement of microtubules in reconstructed embryos was caused by repeating of injection and oocyte manipulation. Also, interactions between karyoblast, cytoplasm and microtubuls may not be suitable. This may be caused by low fertilization in these oocytes

[Effect of direct current pulsation on maturation of mouse oocyte and development of fetus]

Introduction: In this study the immature mouse oocytes [Germinal Vesicles: GV], which were arrested in metaphase [MI], were activated with DC pulses and the effect of DC pulse frequency on immature oocyte activation and their subsequent in vitro development were studied

Material and Methods: Immature oocytes successfully passed the meiosis processes. That is, germinal vesicle stage of oocytes changed to germinal vesicle breakdown [GVB] and finally the first polar body extruded and reached metaphse II, [MII] and formation of 2-cell, 4-cell, 8-cell embryos. Immature Oocytes were separated from NMARY mice [4-6 week old] ovary in different phases. They were placed in M2 medium droplet and then activated with DC pluse [50V, 30micros]

Results: Immature oocytes [GV] began meiosis resumption and GVs changed to GVB and extruded first polarbody and some of them reached metaphase II. After 24 hours evaluation by inverted microscope was performed. Ovulated oocytes were inseminated with the capacitded epididymal sperm of the same strain of mice. One to four pulses with a duration of 30micros induced more oocyte activation [67% to 89%], maturation [68 to 77%] and embryo formation [44% to 84%]. Embryo formation increased significantly with more than two DC pulses [55-88%] compared with a frequency of less than two [51%] groups

Conclusion: This study revealed that electroactivation is helpful for in-vitro maturation, fertilization, embryo formation and development in female infertility, especially in those with irrgular secretion of follicle stimulating and luteinizing hormone [FSH, LH]

[Developmental potential of human fragmented embryos]

Introduction: The aim of this investigation was to study the developmental potential of human fragmented embryos to blastocyst stage and to evaluate the developed blastocysts in the term of size, cellularity, the type of inner cell mass [ICM], trophoetoderm [TE] and the number of dead cells

Material and Methods: Four to eight cell fragmented human embryos were obtained from the embryology lab of Royan Institute. Embryos were scored according to the degree of fragmentation, using invert microscope, into four groups [group I: embryos with least fragmentation, group II: embryos with small fragment among blastomers, group III: embryos with large fragment approximately in the size of blastomers and group IV: embryos with necrotic fragments]. Each group was cultured in rS2 medium and their development was recorded to the day six [embryos were cultured in rS1 for first 3 days].In this study the size and the quality of developed blastocyst and number of blastomer in the ICM and TE were evaluated. The number of dead cells in each blastocyst [apoptotic or necrotic] were counted using TUNEL staining and the results were analyzed statistically by chi2 and ANOVA

Results: High percentage of embryos from group IV had developmental block at 2- 4 cell stage. In addition, a high percentage of embryos from all groups stopped their development at 8-cell to morolla stage. The rate of blastocyst formation of embryos from group I and II was higher than group III and IV. The results showed that size of embryos from group IV was statistically lower than groups I and II. Blastocyst quality [the number of blastomers in ICM] of embryos from group I and II was better than group III and IV. Differential staining results suggested that the ratio of ICM to TE in embryos from each group is not statistically different compared to other groups. But the mean total number of blastomer and the mean number of ICM and TE of embryos from groups I and II was higher than the other two groups. The results of TUNEL staining showed that embryos with high number and size of fragmentation at 4-cell stage had a high number of apoptotic and necrotic cell

Conclusion: Embryos with high fragmentation have reduced developmental potential, blastocyst size, low quality blastocyst, low quality ICM and TE, and low number of total cell number. In addition the number of dead cell was higher