Vol 44, No 1 (2018)

Products made of biomaterials, such as heart valve prostheses, vascular grafts, and patches for vascular and intracardiac plastics, are currently used in cardiovascular surgery. The biological tissue used for prosthetics is the alternation of transverse and longitudinal layers of collagen fibers consisting of type I collagen (75%), elastin (<5%), cell elements, as well as glycoproteins, glycosaminoglycans, and other components of the cell matrix. Chemical modifications of components of a biological tissue allow for retention of its natural architectonics and stability of collagen structure over time, while simultaneously increasing the collagen resistance to enzymatic and mechanical destruction and preventing cellular and immune effects on the part of the recipient organism. Proteins in biological tissues are chemically modified (preserved) by the formation of intramolecular and intermolecular cross-links between the amino groups of amino acid residues in collagen molecules. However, cross-linking increases the calcification of biomaterial, making the tissue more rigid and leading to the rupture of the valve flaps, stenosis (reduced clearance), or insufficiency (a decrease in the closure function) of the heart valves. Calcification can also result from specific physiological features of recipient (the patient who received the artificial organ), the nature of the preserving agent, components of the dead cells, defects of collagen structure, cavities in tissues, and the presence of lipids, elastin fibers, glycosaminoglycans, and so on. The factors that induce calcification of the materials used for prosthetic repair and the corresponding methods for its prevention are reviewed. All methods are conventionally divided into three groups: chemical pretreatment of tissues, modification of the preservation method, and posttreatment of preserved tissues with chemical agents. The mechanisms of the processes underlying the effect of chemical agents on the structures of biological tissues are described. The results of their use in clinical practice and prospects for methods still under development and in preclinical trials are discussed, as well as the reasons why some methods have failed. The advantages and disadvantages of various types of treatments are considered. Variants of new methods for chemical modification of biological materials potentially effective in reducing the risk of calcification are proposed.

Severe chronic and sometimes incurable diseases are frequently accompanied by a pain syndrome. Purotoxin-1, isolated from the poison of the Central Asian Geolycosa sp. spider and selectively inhibiting the purinergic P2X3 receptor (which is considered as a target for the control of the pain states), is one potentially highly effective drug with an analgesic effect. To produce the recombinant purotoxin-1, we created four genetically engineered constructions with different carrier proteins for the expression in E. coli: pTRX-PT1, pCBD-PT1, pGyrA-PT1, pDnaB-PT1. The construction with mini-intein DnaB from the Synechocystis sp. was the most efficient. Using the E. coli C3030/pDnaB-PT1 producer strain, the laboratory method, based on which a pilot technology of recombinant purotoxin-1 production was developed as a result of optimization and scaling, was created. Six grams of recombinant PT1 preparation with the confirmed pharmacological purity was developed for preclinical trials.

The angiotensin-converting enzyme (ACE) is a zinc-dependent metalloproteinase widely occurring in the organism; it metabolizes many peptides and plays a key role in blood pressure regulation and vascular remodeling. This enzyme is expressed as a type-1 membrane glycoprotein on the surface of endothelial and epithelial cells, but is also found in a soluble form in biological fluids. In this study, we used purified ACE from lungs, which is mainly produced by endothelial cells of lung capillaries; ACE from heart, produced by endothelial heart cells and, probably, by myofibroblasts; and ACE from seminal fluid, produced by the epithelial cells of the prostate and epididymis. The pattern of binding of a set of 17 mAbs to different conformational epitopes on the surface of two domains of the human ACE significantly differed for ACEs from different organs. This pattern (the conformational “fingerprint” of ACE) reflects the local conformation of the surface of a particular ACE. The differences in the conformational fingerprints of ACEs expressed by different cell types, or even by similar cells but in different organs, can be explained by the posttranslational modification of ACE protein in these organs and, primarily, different glycosylation of N-glycosylation sites Asn25, Asn117, Asn289, Asn666, Asn685, and Asn731. The mass spectrometry of tryptic hydrolyzates of ACEs isolated from different human organs made it possible to reveal, in the composition of different ACEs, N-glycosylation sites that are really occupied by glycans, namely, Asn in positions 82, 117, 416, 648, 666, 685, and 731 in ACE from seminal fluid; Asn in positions 117, 648, 666, and 685 in ACE from lungs; and Asn in positions 117, 480, 666, and 685 in ACE from heart. Differences in the plausible structures of glycans in ACE, in particular, at the Asn666 N-glycosylation site were demonstrated, which can explain the differences in the efficiency of binding of mAbs to ACE from different organs.

The effects of simulation of a geomagnetic storm (GMS) and its components on the calpain family of calcium-dependent proteases of some invertebrate and fish species has been studied. The animals of the experimental groups were exposed to the GMS for 2 h; the control group was kept in conditions of an undisturbed geomagnetic field. It was shown that the calpain family of calcium-dependent proteases of the studied invertebrate and fish representatives reacted to the GMS impact. It was found that the in vivo impact of the GMS and some of its components on the organisms led to a significant calpain activity decrease; the calciumdependent protease specimens isolated from intact invertebrate animals and fish were also significantly inactivated by the impact of these factors. The impact of the GMS on calpain activity was due to the geomagnetic fluctuations within the whole range of a recorded broadband signal. The results complement fundamental ideas about the principles and laws governing the impact of a GMS on living organisms and can be a basis for finding ways to cope with the negative effects of the GMS.

MLO (mildew resistance locus O), which encodes a transmembrane protein 7TM, is considered to be a model plant gene suitable for studying broad-spectrum resistance. It is a negative regulator of powdery mildew resistance and thus has potential applications in plant breeding. In the present paper, a full cDNA sequence encoding MLO was cloned from the leaves of mulberry (Morus multicaulis) based on mulberry expressed sequence tags (EST), homologous cloning technology, and 5′-RLM-RACE using RT-PCR;the sequence was designated MMLO (GenBank accession no. KX683296). The full cDNA was 1943 bp in length with 5′-untranslated region (UTR) of 106 bp, 3′-UTR of 160 bp, and an open reading frame (ORF) of 1677 bp encoding a protein of 558 amino acids. The estimated molecular weight and isoelectric point (pI) of the putative protein were 62.48 kDa and 9.03, respectively. The MMLO protein had Mlo domain and belonged to the Mlo superfamily. Phylogenetic analysis based on the amino acid sequences encoded by the MLO gene from various species showed that mulberry was closely related to Eucalyptus grandis, Ziziphus jujube, and Juglansregia. Quantitative real-time PCR (qRT-PCR) analysis revealed that MMLO was expressed in all the tissues tested, including leaf, bud, fruit, stem, phloem, and xylem in mulberry with the highest expression in the phloem. The expression level of the mRNA increased and significantly changed under drought, cold, and salt stress treatments compared to the normal growth environment. The ORF segment of the MMLO was inserted into the expression plasmid pET-28a(+) to construct a recombinant expression plasmid. SDS-PAGE result revealed that fusion protein was successfully expressed. Overall, these results provide a better understanding of the molecular basis for the signal transduction mechanism during the stress responses in mulberry trees.

New propargylamines were synthesized in 72–75% yields by the interaction of 19-alkynylbetulin and 28-O-propargyl glycinamide of oleanolic acid with N-methylpiperazine under the Mannich reaction conditions. 19-[1-Methyl-4-prop-2-yn-1-yl-piperazine]-20,29,30-trinorbetulin was shown to manifest anticancer activity against one line of leukemia cells and two lines of colon cancer cells, whereas the growth of leukemia cells SR in the presence of 4-(4-methylpiperazin-1-yl)but-2-yn-1-yl-N-(3-hydroxy-28-oxoolean-12-en-28-yl)glycinate was 8%.