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The purpose of the study was to estimate the influence of temperature increase on Hsp70 induction in M. haemolytica serovar 1 strains. Three wildtype M. haemolytica strains, obtained from calves respiratory tracts and incubated at a temp of 41.5°C for 2 hours were used as the research material. Analyses of particular fractions were carried out by SDS-PAGE electrophoresis and identification of obtained proteins by immunoblotting (Western blotting) using polyclonal rabbit anti Hsp70 antibodies. The first step was to separate the capillaries in gradient pH 5/7 and 3/10 which was carried out in two-dimensional electrophoresis. The second step was carried out in SDS-PAGE electrophoresis using 4% stocking and 12% resolving gels. An analysis of SDS-PAGE electrophoresis revealed additional protein fractions, displaying positive reactions with anti-Hsp70 antibodies. The presence of these proteins was observed both in membrane and cytoplasmatic bacterial cell fractions. The molecular weight of the obtained proteins ranged between 77.5-79 kDa. The additional protein fractions were present in membrane fractions between molecules, weighed 22-26 kDa, as well as displaying a positive reaction with anti-Hsp70 antibodies. The electrophoregrams obtained in 2D electrophoresis revealed the presence of additional spots in membrane, cyto- and periplasmatic fractions. The obtained results suggest the potential for M. haemolytica strains to produce Hsp70 during stress induced by temperature increase.
au is a microtubule-associated protein important for the assembly and stabilization of microtubules. Six tau isoforms are produced in the central nervous system from one single gene as a result of the alternative splicing of exons 2, 3 (N-terminal part) and exon 10 (C-terminal part). The shortest isoform (2-3-10-, 0N 3R) is characteristic for fetal brains, whereas the remaining (2+3-10-, 1N 3R; 2+3+10-, 2N 3R; 2-3-10+, 0N 4R; 2+3-10+, 1N 4R; 2+3+10+, 2N 4R) for adult brains. The aim of the study was to establish a profile of tau protein variants in the C57BL/6J mouse frontal cortex during the aging process. The total RNA was isolated from tissues, followed by reverse transcription and PCR reaction. It was found that the sequence encoded by exon 10 was absent in the youngest 5-day old newborns (isoform 3R), while it was present in 21, 70 and 140-day old animals (isoform 4R). The most abundant isoform in 5-day old mice was 1N and accounted for 66% of the total tau protein. The percentage of 1N isoforms lowered with age and was 31% in 140-day old animals. The total percentage of 0N isoforms was 11% in 5-day old mice and was approximately threefold lower than in each of the older groups. It may be concluded that alternative splicing of the tau protein undergoes age-dependent regulation in the mouse brain cortex.
The main aim of the study was to investigate the intracellular localization of the following calcium-binding proteins: parvalbumin, calbindin and calretinin. 15 sexually mature chinchilla males (about 1.5 years old) were used in the examination. The hippocampus was collected from each immediately after the slaughter, fixed and properly prepared for immunohistochemical examinations. Peroxidase-anti-peroxidase (PAP) reaction was carried out using specific antibodies against parvalbumin and calbindin D28k, as well as calretinin. Our own examination results have shown cytoplasmic as well as nuclear reactions in the examined regions of the hippocampal areas (CA1-CA4) and dentate gyrus. Only in the CA2 area was no nuclear reaction observed for the examined proteins, as well as in the CA1 area for calretinin. Intracellular localization of calcium-binding proteins proves that regulatory functions of parvalbumin, calbindin and calretinin lead to neuronal plasticity, i.e. to a change of their activity. Therefore, calcium-binding proteins may be indirectly involved in the regulation of metabolic processes affecting basic vital functions of neurons.
Glutathione S-transferase pi (GST pi) inactivates a large variety of toxic, electrophilic compounds. The substrates of GST pi include environmental toxins and intracellular reactive oxygen species, factors significant in the pathogenesis of neurodegenerative diseases. The aim of the present study was to investigate the expression of glutathione S-transferase pi in transgenic mouse models of neurodegeneration on both the mRNA and protein levels. Experiments were conducted on the frontal cortex of transgenic B6-C3H hybrids SOD1, Cra1 and SOD1/Cra1, aged 70 and 140 days. The SOD1 mice express a human SOD1ᴳ⁹³ᴬ mutation, the Cra1 strain carries mutation in the cytoplasmic dynein heavy chain 1 (Dnchc1), and the double heterozygote SOD1/Cra1 mice show a delayed disease progression as well as an increased lifespan compared with the SOD1 strain. A wild strain of mice were used as a control. The expression of GST pi mRNA in younger mice (age 70 days) was found to be similar in all studied groups of animals. In older (aged 140 days) controls and Cra1 mice the GST pi expression was at a similar level and it did not significantly differ from younger animals. In SOD1 and SOD1/Cra1 strains, the mRNA-GST pi expression was lower when compared to 140-day-old controls and the Cra1 strain. Moreover, it was significantly lower than in corresponding 70-day-old animals. A decrease in the GST pi expression on the mRNA level was accompanied by a decrease in the protein level. High and unchanged GST pi expression in the frontal cortex of Cra1 mice indicates that the antioxidant-detoxification system plays an important role in protection against neurodegeneration. A significant decrease of GST pi expression in the frontal cortex of SOD1 and SOD1/Cra1 mice at the symptomatic stage of the disease suggests that the expression of this enzyme is related more to the G93A mutation in the SOD1 gene than to the efficient axonal transport.
Pojęto próbę oceny wpływu warunków hodowli na ekspresję białek powierzchniowych pięciu gatunków gronkowców koagulazoujemnych. Badania przeprowadzone na 31 szczepach gronkowców pochodzących od nowo#rodków hospitalizowanych na Oddziale Intensywnej Terapii pozwoliły na analizę podobieństw tych białek w obrębie danego szczepu hodowanego na różnych podłożach.
Ectromelia virus (ECTV) is a member of the Orthopoxvirus genus of the Poxviridae family. It is a causative agent of mouse pox in genetically sensitive mice strains of H-2ᵈ (e.g. BALB/c) and H-2ᵃ (eg.A, A/J) haplotypes. Mouse pox virus is a well recognized model for studying generalized viral infections in natural hosts. The aim of this study was to determine the role of heat shock proteins (namely hsp90, hsp70 and hsp2) during the replication of the Moscow strain of ECTV (ECTV-MOS) in BALB/c mice. The internal organs of mice are important sites for primary virus replication whereas ECTV penetration into the brain may seriously influence mechanisms supervising immune and nervous systems cooperation. Based on studies carried out in BALB/c mice infected with ECTV-MOS, it was found that the hsp (hsp90, hsp70 and hsp27) expression in brain cells reach peak values during the incubation period and clinical manifestation of mouse pox but the relative numbers of hsp⁺ cells in the brains decreased during the recrudescence of the infection to values observed in the control mice. The high expression of hsp (hsp90, hsp70 and hsp27) on oligodendrocytes in BALB/c mice during the incubation and clinical periods may reflect the protective action of heat shock proteins within the brain.
Apoptosis is the process involved in embryonic development and homeostasis maintenance of developed organisms. Furthermore, apoptosis is the mechanism of NK and T cells (CTL) cytotoxicity. The family of bcl-2 proteins plays a crucial role in regulation of cell death and consists both of proteins with pro-apoptotic (Bax, Bad, Bid, Bik), and anti-apoptotic (Bcl-2, Bcl-xL) activity. The present work was aimed at elucidating the role of Bax and Bcl-2 expression in pathogenesis of mousepox caused by virulent Moscow strain of Ectromelia virus (ECTV-MOS) in sensitive animals - BALB/c mice (H-2ᵈ). BALB/c mice were infected via footpad with ECTV-MOS and at 5, 10 and 15 day of infection, lymph nodes, livers and spleens were isolated and subjected to further research. The tests included measurement of: apoptosis using TUNEK assay, caspase-9 activity and bcl-2 and Bax expression. The results showed: (a) influence of ECTV-MOS upon expression of Bcl-2/Bax and subsequent transduction of mitochondrial apoptotic pathway and (b) the role of Bcl-2 protein in suppression of apoptosis in Mø depending on specific immunological environment of infected organs.
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