Methodological Approaches to Assessing the Size and Morphology of Microvesicles of Cell Lines.

Morphological properties and the size of microvesicles were assessed using atomic force microscopy, electron microscopy, and granulometric analysis. As these methods require significant numbers of microvesicles, we chose microvesicles derived from cell lines for our research.

[The cytoprotective role of endogenous heat shock proteins during the treatment of patients with acute coronary syndrome].

The study involving 48 patients with the preliminary diagnosis of acute coronary syndrome aged 54-78 years included analysis of clinical, laboratory and instrumental data for subgroups with different definitive diagnosis (myocardial infarction and unstable angina in 28 and 22 patients respectively). On the whole, the mean content of HSP-70 at admission averaged 2.1 ± 0.3 ng/ml and decreased to 1.6 ± 0.4 ng/ml (p < 0.05) after therapy. In the subgroup with myocardial infarction, the HSP-70 level (1.6 ± 0.4 ng/ml) was significantly lower than in patients with unstable angina (2.1 ± 0.3 ng/ml); p < 0.05. The reduced HSP-70 level associated with a severe myocardial lesion gives evidence against the hypothesis of these proteins as markers of myocardial dysfunction whereas a rise in their content at the onset of ischemic process suggests their protective role.

[Biodistribution of the recombinant heat shock protein rhHsp70 in intracranial C6 glioma models in Wistar rats and subcutaneos B16/F10 melanoma in C57BL/6 mice].

For the first time, the biodistribution of recombinant heat shock protein in rhHsp70 rats with grafted intracranial C6 glioma was evaluated. It was assessed using the fluorescent antibody accumulation chaperone rhHsp70 conjugated with fluorochrome Alexa Fluor 555 in tumor cells by intratumoral or intravenous administration. Assessment of the distribution and accumulation of labeled protein was carried out on the model of subcutaneous B16/F10 melanoma in C57BL/6 mice with the use of single-photon emission computer tomography. After 60 minutes after intravenous administration rhHsp70-I123 (20 MBq, 5 mg chaperone) accumulation of the drug mainly in the liver and tumor tissue was showed. The coefficient of the differential accumulation of the labeled protein KDN(tumor/background) was 3.14. It was turned out that comparing the level of fixation of rhHsp70-I123 in the liver and the tumor KDN(tumor/ liver) = 0.76. After 24 hours from the time of injection of rhHsp70-I123 it was observed increase the level of fixation of the labeled protein in the liver and melanoma: KDN(tumor/background) = 3.43; KDN(tumor/liver = 0.78.

Kinetics of chaperone activity of proteins Hsp70 and Hdj1 in human leukemia u-937 cells after preconditioning with thermal shock or compound u-133.

Kinetics of the chaperone activity of proteins Hsp70 and Hdj1 were analyzed in human U-937 promonocytes during their response to heat shock or to treatment with the echinochrome triacetyl glucoside derivative U-133. To measure the chaperone activity of both proteins, a special test was developed for their recognition and binding of a denatured protein. Using this test, the chaperone activity could be concurrently estimated in large numbers of cellular or tissue extracts. We also estimated the contents of both chaperones in cells by immunoblotting. The values for contents of Hsp70 and Hdj1 obtained by two independent test systems coincided, and this suggested that the substrate-binding activity could change proportionally to the chaperone content in the protein mixture. Therefore, the test developed by us can be employed for high throughput screening of drugs activating cellular chaperones. The analysis of quantity and activity of two cellular chaperones during the cell response to heat stress or to the drug-like substance U-133 showed that both factors caused the accumulation of chaperones with similar kinetics. We conclude that the efficiency of drug preconditioning could be close to the efficiency of hyperthermia and that the high activity of chaperones could be retained in human cells for no less than 1.5 days.