A Novel Method for Amyloid Detection in Human Tissue Load Using a Fluorescent Dye - Congo Red Analogue.

The aim of the study was to develop a new technology for the detection of amyloid in human tissues based on the fluorescent dye, disodium salt of 2,7-(1-amino-4-sulfo-2-naphthylazo)fluorene (DSNAF). MATERIALS AND METHODS: Synthesis of DSNAF was performed by diazotization of 2,7-diaminofluorene in a stream of argon followed by azo coupling with naphthionic acid. Identification of DSNAF was performed using MALDI mass spectrometry. Human myocardial samples from males and females aged from 85 to 98 years (n=11) were the material for the histochemical study. Myocardial paraffin sections were stained with a 0.1% aqueous solution of Congo red or with an aqueous solution (0.1 or 0.034%) of DSNAF under the same conditions. RESULTS: It has been demonstrated for the first time that a new fluorene-based analogue of Congo red, DSNAF, can be successfully used to identify amyloid deposits in histological sections of human myocardium. In terms of the specificity and intensity of amyloid staining, DSNAF is comparable to Congo red, which is the gold standard for detecting amyloid deposits. The fluorescence intensity of DSNAF when binding to amyloid fibrils is significantly higher than the intensity of Congo red fluorescence (with a lower intensity of background fluorescence of heart muscle tissue). This is especially useful for identifying small deposits of amyloid in the human tissues which is important when using small biopsies. CONCLUSION: The advantages of using DSNAF allow us to consider the developed technology for the detection of amyloid as a new promising method of identifying amyloid deposits in human tissues.

[INTERACTION OF THE DYE CONGO RED WITH FIBRILS OF LYSOZYME, BETA2-MICROGLOBULIN AND TRANSTHYRETIN].

By means of spectrophotometric assay we investigated interaction of the dye Congo red (CR) with fibrils of model proteins--hen egg white lysozyme, recombinant human beta2-microglobulin (b2M) and recombinant human transthyretin (TTR). The commercial dye sample was found to contain a significant amount of impurities. Methods for the dye purification are disclosed and CR molar extinction coefficient at 490 nm (ε490) was determined to be 3.3 x 10(4) M(-1) x cm(-1) at pH above 6.0. Formation of the CR-fibril complex results in changes in the dye visible absorption spectrum. According to the data on titration of fibril solutions with excess of the dye, CR binds to lysozyme fibrils at a ratio of about 5 molecules per protein monomer within fibril structure, to b2M fibrils--about 4 molecules per monomer, to TTR fibrils--about 4 molecules per subunit of the protein.

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.

[Novel compounds increasing chaperone Hsp70 expression and their biological activity].

Hsp70 possesses chaperonic activity, the property associated with the protective function that was demonstrated in experiments on a great number of cell and animal models. Therefore, it seems important to search for the substances able to innocuously elevate the chaperone concentration in an organism cells and tissues. In our work, we screened of more that 60 compounds and found two chemicals, derivatives of shikonin and echinochrome that able to increase the chaperone level in a variety of human cells. It was shown that in human erythroleukemia K562 cells treated with the both substances concomitantly with elevation of Hsp70 level the absolute chaperonic activity was also increased; this can indicate mobilization of the whole cellular chaperonic machinery by above mentioned compounds. Estimating biological activity of the two substances, we demonstrated that treatments of cells by them prior to hard heat stress, hydrogen peroxide or staurosporine reduced cell mortality by 20-50 % depending on a cytotoxic factor. The results show that after simple chemical modifications these compounds might be taken as a basis of pharmaceuticals for therapy of wide range of disorders.