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.

CHARACTERISTICS OF AMYLOID DEPOSITS DETECTED IN THE INTERNAL ORGANS OF mdx MICE.

The dystrophin-deficient mdx mouse is the most commonly used experimental model of Duchenne muscular dystrophy (DMD). Although the amyloid has been shown in the muscle biopsies of patients with different types of muscular dystrophies, there are no data on the amyloid accumulations in the biopsy of DMD patients or mdx mouse. Therefore, the aim of the present study was to testify the hypothesis of probable accumulation of amyloid in the visceral organs of mdx mouse. Specimens of myocardium, kidneys, and liver of male and female mdx mice aged from 2 months to 1.5 years (n = 9) were used in the study. The histochemical staining with Congo red demonstrated amyloid accumulations in the studied organs of the mdx mice. Morphology and localization of the found accumulations were described in details and analyzed. The mass-spectrometric study determined the vitronectin and apolipoprotein A-II as the most probable components of the amyloid accumulations in the mdx mouse.

[Interaction of Arenicin-1 with C1q Protein].

The interaction between arenicin-1, that is an antimicrobial peptide from polychaeta Arenicola marina, and human complement system protein C1q was studied using enzyme-linked receptor sorbent assay and ELISA. We revealed that arenicin-1 and C1q form complex that is stable in high ionic strength condition 0.5 M NaCl. The ability of C1q to interact with arenicin-1 is comparable with the binding activity of C1q towards another antimicrobial peptide, porcine cathelicidin protegrin-1, which has a similar spatial arrangement with arenicin-1. Namely, both arenicin-1 and protegrin-1 form cystine-stabilized antiparallel ß-hairpin structure.

Electrospray ionization mass spectrometry of ionic liquids and determination of their solubility in water.

Electrospray ionization mass spectrometry is used to detect both the cations (C+) and the anions (A-) of ionic liquids (CA). In this study, the ionic liquids are diluted with aqueous methanol before injection. In addition to the main peaks of the parent ions, fragmentation products are observed upon increasing the cone voltage, whereas aggregates of the parent ion with one or more ionic liquid molecules (e.g., C(CA)(n)(+), A(CA)(n)(-)) are observed upon decreasing the cone voltage. The ions of several ionic liquids in a mixture are also detected and the ratios of their concentrations estimated. A method is developed to determine quantitatively the concentration of an ionic liquid in solution by using the cation and anion of another ionic liquid as internal standards. By using this method, the solubilities in water at room temperature (22+/-1 degrees C) of three typical hydrophobic ionic liquids have been determined: 0.70+/-0.08 g L(-1) for methyltributylammonium bis(trifluoromethylsulfonyl)imide (MeBu(3)NNTf(2)), 6.0+/-0.5 g L(-1) for butylmethylpyrrolidinium bis(trifluoromethylsulfonyl)imide (BuMePyrNTf(2)), and 18.6+/-0.7 g L(-1) for 1-butyl-3-methylimidazolium hexafluorophosphate (BMIPF(6)).

Identification of chemical substances by testing and screening of hypotheses. I. General.

The characteristic features and the constituents of an identification procedure for chemical substances are discussed. This procedure is a screening of identification hypotheses followed by experimental testing of each one. The testing operation consists of comparison of the values of the quantities measured with other measurement results or reference data, resulting in the Student's ratio, the significance level, the matching of spectra, etc. The performance and the correctness of identification are expressed as "identification uncertainty", i.e. the probability of incorrect identification. The statistical significance level and other similarity values in spectra, chromatography retention parameters, etc. are the particular measures of uncertainty. Searching of prior data and estimation of the prior probability of the presence of particular compounds in the sample (matrix) to be analysed simplifies the setting up and cancelling of hypotheses during screening. Usually, identification is made by the analyst taking into account measurement results, prior information and personal considerations. The estimation of uncertainty and rules for the incorporation of prior data, make the result of identification less subjective.

Identification of chemical substances by testing and screening of hypotheses. II. Determination of impurities in n-hexane and naphthalene.

The impurities in n-hexane and naphthalene are identified by gas chromatography and mass spectrometry when applying the multiple criteria of identification. The identification uncertainty is expressed by the significance level, the difference in retention indexes and the mass spectra fit value. Identified impurities are substances which are rather abundant in nature/practice/research and rather frequently co-cited with matrix substance in chemical literature. This regularity is discussed with reference to statistical treatment of chemical database as a means to screen identification hypotheses.