Thermal stability of thylakoid membranes isolated from acclimated and non-acclimated wheat (Triticum aestivum L. cv. HD 2329) leaves under irradiation was studied. Damage to the photosynthetic electron transport activity was more pronounced in thylakoid membranes isolated from non-acclimated leaves as compared to thylakoid membrane isolated from acclimated wheat leaves at 35 °C. The loss of D1 protein was faster in non-acclimated thylakoid membrane as compared to acclimated thylakoid membranes at 35 °C. However, the effect of elevated temperature on the 33 kDa protein associated with oxygen evolving complex in these two types of thylakoid membranes was minimal. Trypsin digestion of the 33 kDa protein in the thylakoid membranes isolated from control and acclimated seedlings suggested that re-organisation of 33 kDa protein occurs before its release during high temperature treatment. and A. K. Singh, G. S. Singhal.
The dental pulp represents an easily acces-sible source of adult dental pulp stem cells (DPSCs). The preferred approach to DPSC isolation is enzymatic digestion. However, the duration of the enzymatic activity is crucial. The purpose of this study was to isolate the DPSC populations using this method, characterize their biological properties and proliferation capacity, and to determine their ability to differentiate into mature cells. Before enzymatic
digestion using 0.05% trypsin, we used the homogenization method in order to obtain a fine homogenate from the solid pulp tissue. The stem cells were cultivated in modified cultivation medium for mesenchymal adult progenitor cells containing 2% foetal bovine serum, growth factors and insulin-transferrin-
selenium supplement. We were successfully able to isolate 10 populations of DPSCs. The vitality of DPSCs did not drop below
90 %. However, the DPSCs showed a significant decrease in the relative telomere length number with increasing passaging (P < 0.05). Isolated DPSCs highly expressed the CD markers: CD29, CD44, CD90, CD13, CD73 and CD166. In contrast, CD markers CD31, CD106, CD34 and CD45 were negative or low positive. We confirmed the high osteogenic and chondrogenic potential of the isolated stem cells. Isolated DPSCs did not show signs of cell degeneration or spontaneous differentiation during the entire cultivation. In addition, we were able to shorten the enzyme activity duration, and we were the first to demonstrate trypsin as the enzyme used for the enzymatic digestion method with the viability over 90 % of isolated DPSCs using this method. and Corresponding author: Nela Pilbauerová
Parasitic organisms inhabiting the alimentary canal should permanently resist the destructive action of host digestive enzymes. The intestinal parasites were shown to produce specific protease inhibitors protecting them from proteolysis. However, little is known about this adaptive mechanism in cestodes so far, especially for the tapeworms dwelling inside the fish intestines. Here, we explored the ability to inactivate proteolytic enzymes in the fish tapeworm Eubothrium rugosum (Batsch, 1786) (Bothriocephalidea) parasitising the intestine of wild burbot, Lota lota (Linnaeus). The assays were conducted with different concentrations of commercial trypsin and homogenate of intestinal mucosa both being the sources of proteinases. The incubation of live E. rugosum in trypsin solutions of two different concentrations caused a significant decrease in the enzyme activity. The extent of activity reduction was dependent on trypsin concentration. At the same time, the inhibitory effect of the worm incubation medium turned out to be statistically insignificant. These findings suggest partial adsorption of the enzyme to the tegument surface, with its further inactivation. In contrast to the incubation medium, the worm extract suppressed over 80% of trypsin activity and nearly half of the proteolytic activity in the mucosa homogenate. Notably, the inhibitory activity of the tapeworms hardly depended on their size characteristics. Finally, the research has demonstrated secretion of proteinase inhibitor in E. rugosum, which appears to be essential for its survival in enzymatically hostile environment., Galina I. Izvekova, Tatyana V. Frolova, Evgeny I. Izvekov., and Obsahuje bibliografii
a1_Proteinase-activated receptor-2 (PAR-2) is a ubiquitous surface molecule participating in many biological processes. It belongs to the family of G protein-coupled receptors activated by the site-specific proteolysis of trypsin and similar proteases. Altered function of PAR-2 has been described in different malignant tumors. In the present study, we investigated the expression of PAR-2 in breast cancer surgical specimens and the role of trypsin in breast cancer cell line MDA MB-231 proliferation and metabolism. A total of 40 surgical samples of infiltrative ductal breast cancer and breast cancer cell line were included in this study. We analyzed PAR-2 expression by immunohistochemistry, RT-PCR and western blot. Activation of PAR-2 on cell line MDA MB-231 was measured using calcium mobilization assay determined by flow cytometry. MTT cell metabolism assay and cell count analysis were used to assess the trypsin influence on breast cancer cell line MDA MB-231 proliferation. Immunohistochemical examination showed the expression of PAR-2 in all samples of breast cancer surgical specimens and high levels of cell lines which was confirmed by RT-PCR and western blot., a2_Calcium mobilization assay corroborated the activation of PAR-2 on cell line MDA MB-231 either by trypsin or by an agonistic peptide. Cell metabolism assay and cell count analysis showed significant differences of proliferative activity of breast cancer cells dependent on the presence or absence of trypsin and serum in the culture medium. PAR-2 is expressed by high levels in infiltrative ductal breast cancer tissue specimens. PAR-2 is also strongly expressed in studied breast cancer cell lines. PAR-2 is activated by trypsin and also by agonistic peptide in the model of breast cancer cell line MDA MB-231. Activation of PAR-2 in vitro influences proliferative and metabolic activity of breast cancer cell line MDA MB-231. The action of trypsin is modified by the presence of serum which is a potential source of protease inhibitors., R. Matěj, P. Manďáková, I. Netíková, P. Poučková, T. Olejár., and Obsahuje biblografii a bibliografické odkazy