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Stressful experiences during the early stages of life can distort normal brain development. In humans, mother-infant interactions may represent a key factor for disease susceptibility which can manifest itself as cognitive and behavioral disorders later in life. The maternal separation (MS) procedure of rat pups represents a useful experimental paradigm to study disturbances in brain function that might occur in response to adverse events during development. MS-subjected animals, as adults, express behavioral and neuroendocrine signs of elevated stress reactivity and cognitive deficits. However, mechanisms by which early life stress exerts its impact on the development and maturation of the brain are poorly understood. This study was aimed at finding the efects of repeated MS on long-term potentiation (LTP) in the lateral amygdala (LA) of adolescent rats. Wistar dams with their offspring were housed under 12:12 h light/dark cycle with food and water available ad libitum. On each of postnatal days (PNDs) 1 - 21 the dams were removed from the maternity cages for 3 h and placed individually in holding cages, while the litter stayed in homecages. Then, the dams were returned to the maternity cages. MS animals, as well as control, animal facility reared rats, were weaned at PND 28 and then housed in groups (4 - 6 animals). For electrophysiological experiments rats between PND35 and PND 50 were used. They were anesthetized and coronal brain slices (450 µm thick) containing the lateral amygdala were cut. Field potentials (FPs) were evoked in LA by the stimulation of the external capsule. LTP was induced using repetitive theta burst stimulation (TBS) protocol. While in slices prepared from control rats FP amplitude, 90 min after TBS, amounted 185.9 ± 17.04 % of baseline, in slices obtained from MS animals LTP was significantly weaker (117.7 ± 7.7%; p=0.0002). These preliminary results indicate that MS stress impairs LTP in the lateral amygdala of adolescent rats.
Thymidylate synthase purified from 5-fluoro-dUrd-resistant mouse leukemia L1210 cells (TSr) was less sensitive to slow-binding inhibition by 5-fluoro-dUMP than the enzyme from the parental cells (TSp), both enzyme forms differing also in sensitivity to several other dump analogues, apparent molecular weights of monomer and dimer, and temperature dependence of the catalyzed reaction. Direct sequencing of products obtained from RT-PCR, performed on total RNA isolated from the parental and 5-fluoro-dUrd-resistant cells, proved both nucleotide sequences to be identical to the mouse thymidylate synthase coding sequence published earlier (NCBI protein database access no. NP_067263). This suggests that the altered properties of TSr are caused by a factor different than protein mutation, presumably posttranslational modification. As a possibility of rat thymidylate synthase phosphorylation has been recently demonstrated (Samsonoff et al. (1997) J Biol Chem 272: 13281), the mouse enzyme amino-acid sequence was analysed, revealing several potential phosphorylation sites. In order to test possible influence of the protein phosphorylation state on enzymatic properties, endogenous TSp and TSr were purified in the presence of inhibitors of phosphatases. Although both enzyme forms were phosphorylated, as shown by electrophoretical separation followed by phosphoprotein detection, the extent of phosphorylation was apparently similar. However, the same two purified enzyme preparations, compared to the corresponding preparations purified in the absence of phosphatase inhibitors, showed certain properties, including sensitivity to the slow-binding inhibition by FdUMP, altered. Thus properties dependence on phosphorylation was indicated.
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