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Öğe Analysis of the Embryonic Growth Supporting Fractions of Extra Embryonic Coelomic Fluid (EECF)(1997) Karabulut, A. K.; Layfield, R.; Ülger, H.; Pratten, M. K.[Abstract not Available]Öğe Growth-Promoting Effects of Different Fractions of Extra-Embryonic Coelomic Fluid on Embryonic Development(Wıley, 2000) Karabulut, A. K.; Layfield, R.; Pratten, M. K.In the early stages of embryonic development, many growth-promoting molecules must be provided by the maternal system. These factors may be supplied locally to the embryo, by the decidua, the placenta, or the yolk sac. In this study the growth-promoting potential of extra-embryonic coelomic fluid (EECF) and its fractions was investigated. The embryonic requirement of growth-promoting molecules may be studied by reducing the growth-supporting capacity of serum. Thus, ultrafiltration of rat serum was carried out for 8h using Millipore filters with a molecular weight exclusion of 30 kDa. Rat embryos at 9.5 days of age were cultured for 8 days for anembryonic yolk sacs, and then EECF was collected and divided into three different molecular weight fractions by ultrafiltration. Rat embryos were cultured for 48 h in whole rat serum and the serum retenate (which has low growth-supporting capacity) in the presence and absence of EECF, its fractions, or in EECF only. Embryos grown in retenate showed severe growth retardation, and the addition of EECF significantly improved embryonic growth. The fraction which contained the molecules with molecular weight between 10 and 30 kDa had significantly more effect on embryonic development than the other fractions. This fraction of EECF was analysed by gel electrophoresis. Three of the four protein bands observed in this fraction were identified by amino-terminal sequencing as alpha-fetoprotein precursor (22 kDa), apolipoprotein Al precursor (24kDa) and fetal haemoglobin Y2 chain /14kDa), none of which are likely to be responsible for the growth-promoting activity. To further investigate growth-promoting proteins, EECF was Western-blotted to nitrocellulose membranes and probed with antisera against rat prolactin, epidermal growth factor, insulin-like growth factors I and II and human placental lactogen. No immunoreactive bands were detected in the EECF, suggesting that either these proteins are not present or are present at levels too low to be detected. Although the growth-promoting effect of the EECF was demonstrated in this study, the molecules responsible remain uncharacterized.Öğe Protection by Free Oxygen Radical Scavenging Enzymes Against Salicylate-İnduced Embryonic Malformations in Vitro(Pergamon-Elsevier Science Ltd, 2000) Karabulut, A. K.; Ülger, H.; Pratten, M. K.Salicylates are among the oldest and most widely used drugs and are known to lead to foetal death, growth retardation and congenital abnormalities in experimental animals. In this study, the effects of acetyl salicylic acid (ASA), salicylic acid (SAL) and sodium salicylate (NaSAL) on early organogenesis and the interaction of these molecules with free radicals has been investigated. Postimplantation rat embryos were cultured in vitro from day 9.5 of gestation for 48 hr. ASA, SAL and NaSAL were added to whole rat serum at concentrations between 0.1 and 0.6 mg/ml. Also, the lowest effective concentration of ASA for all parameters (0.3 mg/ml) and the same concentration of NaSAL and SAL was added to the culture media in the presence of superoxide dismutase (SOD) (30 U/ml) or glutathione (0.5 mu mol/ml). The growth and development of embryos was compared and each embryo was evaluated for the presence of any malformations. When compared to growth of control embryos, the salicylates decreased all growth and developmental parameters in a concentration-responsive manner. There was also a concentration-related increase in overall dysmorphology, including the incidence of haematoma in the yolk sac and neural system, open neural tube, abnormal tail torsion and the absence of fore limb bud. When SOD was added in the presence of ASA, growth and developmental parameters were improved and there was a significant decrease in the incidence of malformations. Addition of SOD also decreased the incidence of malformations in the presence of SAL, but did not effect the growth and developmental parameters of SAL and NaSAL. There was no significant difference between the embryos grown in the presence of these three molecules on the addition of glutathione. The effects of salicylates might involve free oxygen radicals by the non-enzymatic production of the highly teratogenic metabolites 2,3- and 2,5-dihydroxybenzoic acid. An enhanced production of these metabolites in embryonic tissues may be directly related to the increased risk of congenital malformations.Öğe Species-Specificity of Growth-Promoting Effects of Prolactin During Rat Embryogenesis(WILEY, 1998) Karabulut, A. K.; Pratten, M. K.In the early stages of embryonic development, many growth-promoting molecules must be provided by the maternal system. The molecules involved in growth processes may be either hormones or growth factors, or molecules that interact with such factors. The pregnancy related hormone, prolactin (PRL, MW 23 kDa) has been implicated in the control of embryonic growth. The growth-promoting potential of PRL and its species-specificity was investigated by culturing 9.5 d rat embryos in vitro for 48 h in depleted serum in the presence and absence of PRL from 3 different species. The growth-supporting capacity of the serum was reduced by removal of low molecular weight molecules by prolonged filtration of the serum using filters with a molecular weight exclusion of 30 kDa. This method provided a 'semidefined' medium (retenate) in which embryonic growth and development was significantly reduced, demonstrating that the low molecular weight fraction of serum may contain some growth-promoting factors. Addition of PRL (0.4-25.6 ng/ml) from different species (human, sheep and rat) to retenate significantly improved embryonic growth and development, suggesting that the developing embryo may utilise PRL. Amongst PRLs, rat PRL was found to be active at much lower concentrations than either of the other molecules, and human PRL had more effect in low concentrations than sheep PRL suggesting a species-specificity for this hormone. It may be that the PRL receptors of the rat embryos have greater affinity for the rat hormone as different responses for hormones from different species have been shown. These findings suggest that embryos may be able to utilise maternally derived PRL during organogenesis.Öğe Visualisation of the Uptake of Prolactin (PRL) in Rat Embryonic Tissues(1999) Karabulut, A. K.; Ulger, H.; Pratten, M. K.Objective: Evidences implicate roles for prolactin (PRL) in the regulation of embryonic growth. To clarify the roles of PRL in rat embryogenesis we examined the uptake and expression of the hormone in embryonic tissues. Methods: Nine and a half day postimplantation rat embryos were cultured in vitro for 44 h in rat serum and serum depleted of low molecular weight molecules (retenate). The embryos were transferred to M199 for the last 4 h, and 12.8 ng/ml rat PRL was added to culture medium for different times (4 h - 15 min) and/or different temperatures (37°C and 4°C). As a control tissue, pituitary glands from 11.5 and 18.5d pregnant rats were used. Embryos and tissues were then examined by an indirect immunofluorescence protocol. Results: The pituitary glands showed positive immunoreactivity for anti-PRL antibody whilst there was no stain in the control brain tissue. Immunoreactivity was observed in embryos grown in rat serum, and intensity was much greater in the presence of additional rat PRL, whilst there was no immunoreactivity detected in those grown in retenate only. Shorter incubations and incubations at 37°C caused a greater immunoreactivity for PRL, suggesting that this is an active and temperature dependent metabolic process. Conclusion: These results show the uptake and distribution of PRL by the yolk sac and embryonic tissues which might be interpreted for the presence of PRL receptors.
