Experimental rat models for Hashimoto's thyroiditis.

PURPOSE: Hashimoto's thyroiditis (HT) is an autoimmune thyroid disease characterized by T lymphocyte-mediated destruction of thyroid follicles. To study the pathogenesis of HT and the efficacy of new substances for its treatment, an easily obtained and adequate to the human disease experimental model is needed. The aim of our study was to find out whether it is possible to induce experimental autoimmune thyroiditis (EAT) similar to Hashimoto's thyroiditis by injecting with thyroglobulin (Tg) without using agents that enhance its thyroiditogenicity and without taking into account the genetic sensitivity of animals. METHODS: Wistar rats were immunized with freshly isolated rat Tg or porcine Tg. In 8 weeks, histological studies of the thyroid and parathyroid glands were performed. Thyroid function and total serum calcium level were also evaluated. RESULTS: Immunization with both rat and porcine freshly isolated Tg caused T lymphocytic infiltration of the thyroid gland, thyroid follicle atrophy and degradation in Wistar rats. EAT caused by porcine Tg was characterized by greater severity than EAT induced with rat Tg. In 55% of rats with porcine Tg-induced EAT, oxyphilic metaplasia was detected in the parathyroid glands. In addition, low total serum calcium was observed in these rats. CONCLUSION: Two rat models of autoimmune thyroiditis were obtained. EAT caused in Wistar rats by immunization with rat Tg is similar to Hashimoto's thyroiditis. EAT induced with porcine Tg was accompanied by oxyphil cell metaplasia in the parathyroids and hypocalcemia.

Floating marine macro litter in the Black Sea: Toward baselines for large scale assessment.

The Black Sea is a semi-enclosed basin subject to major anthropogenic pressures, including marine litter and plastic pollution. Due to numerous large rivers draining into the basin and a population settled along the coast, the region could accumulate significant amounts of floating litter over time. Until now, only limited field data were available, and litter quantities and distribution remained unknown. In this study, floating marine macro litter (FMML) was assessed at the regional Black Sea scale for the first time, showing relatively high litter densities across the basin that reached a weighted mean of 81.5 items/km2. Monitoring data revealed an accumulation of floating items offshore in the eastern part of the basin, resembling on a small scale a 'garbage patch', where litter items were trapped, showing elevated densities in comparison to their surrounding areas. Most of these items were made of plastic materials (ca. 96%) and included large numbers of plastic and polystyrene fragments of small size ranges (2.5-10 cm). Harmonised field data collection through consistent and regular monitoring programmes across the region is essential to establish baselines and thresholds for large scale assessment at international level.

Nanoparticles of water-soluble dyads based on amino acid fullerene C60 derivatives and pyropheophorbide: Synthesis, photophysical properties, and photodynamic activity.

Synthesis, spectral properties, and photodynamic activity of water-soluble amino acid fullerene C60 derivatives (AFD) and four original AFD-PPa dyads, obtained by covalent addition of dye pyropheophorbide (PPa) to AFD, were studied. In aqueous solution, these AFD-PPa dyads form nanoassociates as a result of self-assembly. In this case, a significant change in the absorption spectra and strong quenching of the dye fluorescence in the structure of the dyads were observed. A comparison of superoxide or singlet oxygen generation efficiency of the studied compounds in an aqueous solution showed the photodynamic mechanism switching from type II (singlet oxygen generation of the native dye) to I type (superoxide generation of dyads). All dyads have pronounced phototoxicity on cells Hela with IC50 9.2 µM, 9.2 µM, 12.2 µM for dyads Val-C60-PPa, Ala-C60-PPa and Pro-C60-PPa, respectively. Such facilitation of type I photodynamic mechanism could be perspective against hypoxic tumors.

Is Kidney Transplantation From a COVID-19-Positive Deceased Donor Safe for the Recipient?

INTRODUCTION: In recent months, the number of kidney transplants from deceased donors has declined significantly. One of the reasons is the possibility of infection of the recipient with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Determining the risk of transmission of coronavirus disease 2019 (COVID-19) with a donor organ is very important for developing a kidney transplantation policy during a pandemic. MATERIALS AND METHOD: We present cases of kidney transplantation from COVID-19-positive deceased donors to 2 dialysis patients 49 and 45 years old. One of them was on hemodialysis for 28 months; the other received continuous ambulatory peritoneal dialysis (CAPD). Both patients received only basic immunosuppression, including tacrolimus, methylprednisolone, and mycophenolic acid. No antilymphocyte agents were used for induction therapy. RESULTS: Cold ischemia time was 22 and 21 hours, respectively. One recipient had delayed graft function with increasing of urine output on day 8; another had immediate function. Both patients had no febrile and no other symptoms of acute respiratory disease during their hospital stay. No abnormalities on the chest x-ray were seen. No serum anti-SARS-CoV-2 IgM and IgG were detected before and during 6 weeks after surgery. Repeated nasopharyngeal swabs real-time reverse transcription polymerase chain reaction (rRT-PCR) were negative during the period. Both recipients were discharged 5 weeks after surgery with serum creatinine levels of 122 and 91 mcmol/L, respectively. CONCLUSION: Today we have no evidence of the possibility of transmission of COVID-19 from a SARS-CoV-2 positive donor to a kidney recipient. We also have no reason to suspect kidney damage by COVID-19 in a deceased donor at normal serum creatinine level.

Dual Character of Reactive Oxygen, Nitrogen, and Halogen Species: Endogenous Sources, Interconversions and Neutralization.

Oxidative stress resulting from accumulation of reactive oxygen, nitrogen, and halogen species (ROS, RNS, and RHS, respectively) causes the damage of cells and biomolecules. However, over the long evolutionary time, living organisms have developed the mechanisms for adaptation to oxidative stress conditions including the activity of the antioxidant system (AOS), which maintains low intracellular levels of RONS (ROS and RNS) and RHS. Moreover, living organisms have adapted to use low concentrations of these electrophiles for the regulation of cell functions through the reversible post-translational chemical modifications of redox-sensitive amino acid residues in intracellular effectors of signal transduction pathways (protein kinases and protein phosphatases), transcription factors, etc. An important fine-tuning mechanism that ensures involvement of RONS and RHS in the regulation of physiological processes is interconversion between different reactive species. This review focuses on the complex networks of interacting RONS and RHS types and their endogenous sources, such as NOX family of NADPH oxidases, complexes I and III of the mitochondrial electron transport chain, NO synthases, cytochrome P450-containing monooxygenase system, xanthine oxidoreductase, and myeloperoxidases. We highlight that kinetic parameters of reactions involving RONS and RHS determine the effects of these reactive species on cell functions. We also describe the functioning of enzymatic and non-enzymatic AOS components and the mechanisms of RONS and RHS scavenging under physiological conditions. We believe that analysis of interactions between RONS and relationships between different endogenous sources of these compounds will contribute to better understanding of their role in the maintenance of cell redox homeostasis as well as initiation and progression of diseases.

[THERMAL STABILITY AS A PROGNOSTIC INDICATOR OF CONSERVATION OF LIVE EMBRYONIC SMALLPOX VACCINE (TEOVAC) DURING STORAGE].

AIM: Determination of values of coefficients of thermal stability of TEOVac for prognosis of conservation of the vaccine (specific biological activity) during the process of warranty period storage. MATERIALS AND METHOD: TEOVac (masticatory tablets) in primary packaging was kept at increased temperature (accelerated and stress-tests) and at the conditions established by PAP for the preparation (long-term tests). Biological activity of the vaccine was determined by titration on 12-day chicken embryos. RESULTS: A correlation between the value of coefficients of thermal stability and conservation of the prepared series of the condition preparation at the final date of storage was experimentally established. CONCLUSION: Coefficients of thermal stability could be used as a prognostic indicator of quality of the produced pelleted formulation of the preparation for evaluation of conservation of the vaccine during warranty period storage.

Alpha-fetoprotein: a renaissance.

Alpha-fetoprotein (AFP) is a major mammalian embryo-specific and tumor-associated protein that is also present in small quantities in adults at normal conditions. Discovery of the phenomenon of AFP biosynthesis in carcinogenesis by G. Abelev and Yu. Tatarinov 50 years ago, in 1963, provoked intensive studies of this protein. AFPs of some mammalian species were isolated, purified and physico-chemically and immunochemically characterized. Despite the significant success in study of AFP, its three-dimensional structure, mechanisms of receptor binding along with a structure of the receptor itself and, what is the most important, its biological role in embryo- and carcinogenesis remain still obscure. Due to difficulties linked with methodological limitations, research of AFP was to some extent extinguished by the 1990 s. However, over the last decade a growing number of investigations of AFP and its usage as a tumor-specific biomarker have been observed. This was caused by the use of new technologies, primarily, computer-based and genetic engineering approaches in studying of this very important oncodevelopmental protein. Our review summarizes efforts of different scientific groups throughout the world in studying AFP for 50 years with emphasis on detailed description of recent achievements in this field.

Correlation between biological activity and conformational dynamics properties of tetra- and pentapeptides derived from fetoplacental proteins.

In this work, using molecular dynamics simulation, we study conformational and dynamic properties of biologically active penta- and tetrapeptides derived from fetoplacental proteins such as alpha-fetoprotein, pregnancy specific ß1-glycoprotein, and carcinoembryonic antigen. Existence of correlation between flexibility of peptide backbone and biological activity of the investigated peptides was shown. It was also demonstrated that flexibility of peptide backbone depends not only on its length, but also on the presence of reactive functional groups in amino acid side chains that participate in intramolecular interactions. Peptides that demonstrate similar biological effects in regulation of proliferation of lymphocytes and expression of differentiation antigens on their surface (LDSYQCT, PYECE, YECE, and YVCE) are characterized by rigidity of their peptide backbone. Increased backbone flexibility in peptides PYQCE, YQCE, SYKCE, YQCT, YQCS, YVCS, YACS, and YACE is correlated with decreased biological activity. Conformational mobility of amino acid residues does not depend on physicochemical properties only, but also on intramolecular interactions. So, evolutionary restrictions should exist to maintain such interactions in the environment of functionally important sites.

Influence of intramolecular interactions on conformational and dynamic properties of analogs of heptapeptide AFP(14-20).

Conformational and dynamic properties of proteins and peptides play an important role in their functioning. However, mechanisms that underlie this influence have not been fully elucidated. In the present work we computationally constructed analogs of heptapeptide AFP(14-20) (LDSYQCT) - one of the biologically active sites of human α-fetoprotein (AFP) - to study their conformational and dynamic properties using molecular dynamics simulation. Analogs were obtained by point substitutions of amino acid residues taking into account differences in their physicochemical properties and also on the basis of analysis of amino acid substitutions in the AFP(14-20)-like motifs revealed in different physiologically active proteins. It is shown that changes in conformational mobility of amino acid residues of analogs are due to disruption or arising of intramolecular interactions that, in turn, determine existence of steric restrictions during rotation around covalent bonds of the peptide backbone. Substitution of an amino acid by another one with significant difference in physicochemical properties may not lead to remarkable changes in conformational and dynamic properties of the peptide if intramolecular interactions remain unchanged.

Dynamic proteomics in modeling of the living cell. Protein-protein interactions.

This review is devoted to describing, summarizing, and analyzing of dynamic proteomics data obtained over the last few years and concerning the role of protein-protein interactions in modeling of the living cell. Principles of modern high-throughput experimental methods for investigation of protein-protein interactions are described. Systems biology approaches based on integrative view on cellular processes are used to analyze organization of protein interaction networks. It is proposed that finding of some proteins in different protein complexes can be explained by their multi-modular and polyfunctional properties; the different protein modules can be located in the nodes of protein interaction networks. Mathematical and computational approaches to modeling of the living cell with emphasis on molecular dynamics simulation are provided. The role of the network analysis in fundamental medicine is also briefly reviewed.

Lamin B content in chromatin fractions after purification of nuclear matrix from cells of different types.

We analyzed the content of lamin B, one of the main proteins of the nuclear membrane, in different chromatin fractions obtained during purification of the nuclear matrix from different cell types. Depending on cell type and nuclear matrix preparation technique, lamin B was found in different not associated with matrix chromatin compartments. This effect was observed after chromatin extraction with ammonium chloride after nucleolysis and after chromatin extraction with sodium chloride before nuclease treatment. These findings suggest that the structure of the nuclear matrix is destabilized in certain extraction procedures and that studies of subcompartmentalization of nuclear macromolecules require additional control of nuclear matrix integrity.

Cell adhesion proteins and alpha-fetoprotein. Similar structural motifs as prerequisites for common functions.

This review summarizes and analyzes data on structural and functional relationships between cell adhesion proteins and alpha-fetoprotein (AFP), which play an important role in embryo- and carcinogenesis and act in synergism with growth factors. These two groups of proteins are mosaic, multimodular, and polyfunctional, and each of their modules can function independently through binding with its specific membrane receptor. Most cell adhesion proteins contain modules similar to epidermal growth factor (EGF) and also their repeats, which determine the involvement of these proteins in regulation of cell proliferation, differentiation, and apoptosis. These EGF-like modules are found to include short motifs similar to the fragment LDSYQCT of human AFP. Both direct and inverted AFP-like motifs are linked through a consensus octapeptide motif CXXGY/FXGX. Such AFP-like motifs of cell adhesion proteins and the tripeptide RGD found in AFP may be structural prerequisites for common functions of these groups of nonhomologous and unrelated proteins.

Conformational dynamics of human alpha-fetoprotein-derived heptapeptide LDSYQCT analogs.

Conformational dynamics of a biologically active fragment of alpha-fetoprotein, the heptapeptide LDSYQCT, and its analogs obtained by site-directed substitutions of amino acid residues were studied. The conformational dynamics of the peptide were conservative under the substitutions Y17F, Y17S, and D15E. Substitutions C19A and S16V resulted only in local changes in the dynamic behavior of the peptide. Chemical modification of cysteine (C19) or dimerization of the peptide by producing a disulfide bond between cysteine residues of two parallel peptide chains, as well as the substitutions C19G, C19S, Q18E, and D15N changed a set of possible conformations and dynamic behavior of all amino acid residues. The most significant changes were caused by substitution of uncharged amino acid residues by charged ones, and vice versa.

Structural and functional mapping of alpha-fetoprotein.

Alpha-fetoprotein (AFP) is a major mammalian oncofetal protein, which is also present in small quantities in adults. It is a member of the albuminoid gene superfamily, which consists of AFP, serum albumin, vitamin D binding protein, and alpha-albumin (afamin). Although physicochemical and immunological properties of AFP have been well-studied, its biological role in embryo- and carcinogenesis and in adult organisms as well as mechanisms underlying its functioning remain unclear. During the recent decades, the biological role of AFP has been evaluated by identification of its functionally important sites. Comparison of primary structure of AFP and some physiologically active proteins revealed similarity of some polypeptide regions. This has been used for prediction of AFP functions (i.e., its multifunctionality). Localization of functionally important sites followed by determination of their amino acid composition and type of biological activity has provided valuable information for structural-functional mapping of AFP. Some peptide fragments of AFP have been synthesized and tested for biological activity. This review summarizes data on structural-functional interrelationships. We also describe functionally important AFP sites found by various groups during the last decade of structural-functional mapping of AFP with experimentally confirmed and putative biologically active sites.

The identification of vicinally substituted cyclohexane isomers in their mixtures by 1H and 13C NMR spectroscopy.

The radical addition reactions of organobromine compounds, XBr (X = CH2COOMe, PhCH2, CHBr2 and CCl3) with cyclohexene afforded mixtures of cis/trans isomer pairs of 1-X-2-Br-cyclohexanes. In addition to benzyl benzoyloxy derivatives are formed also, when benzoyl peroxide is used as an initiator. Owing to the great difficulties in separating these cis/trans isomer pairs, they are identified directly in their mixtures by NMR spectroscopy. In addition to one-dimensional (ID) 1H, proton decoupled 13C and DEPT-135, also two-dimensional (2D) 13C-13C INADEQUATE as well as 1H-13C HMQC experiments have been used in assigning the signals of each compound in their mixtures. The identification of each isomer was based on comparison of experimental 3J(H,H) coupling constants with theoretical ones based on the well-known Karplus type relationship. The more stable conformation for each isomer was estimated using the semiempirical AM1 molecular orbital method. The calculations support the isomer pair elucidations.

Human alpha-fetoprotein and its purification by chromatography on immobilized estrogens.

Human alpha-fetoprotein (AFP) was isolated from human abortive tissue by biospecific chromatography on immobilized estrogens. The most effective sorbents were: estrone-0-3-hemisuccinyl-hexamethylenediamine-Sepharose CL 4B and diethylstilbestrol-diasoanisole-sulfonyl-oxyethyl-Sepharose CL 4B. As elution solution the most optimum was 10% buffered aqueous butanol. Taking into consideration the data obtained, one can conclude that AFP in human biological fluids is bound to immobilized estrogens. Butanol extraction deestrogenizes AFP, and as a result human AFP acquires affinity to immobilized estrogens. During rechromatography on immobilized diethylstilbestrol, it was possible to obtain AFP preparations of about 95% purity. The present results provide the opportunity to work out new methodological approaches to human AFP isolation using biospecific chromatography on immobilized estrogens.