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Background: Since bactericidal properties of some lysozymes are independent of their glycosidase activity, we have investigated this phenomenon for destabilase-lysozyme (DL) from medicinal leech (Hirudo medicinalis). Methods: Glycosidase activity was determined on Micrococcus luteus, non-enzymatic antibacterial activity of heat-treated DL and of synthetic peptides α1, α2 and α3 (fragments of its primary structure) on M. luteus, Escherichia coli, Bacillus brevis and Streptomyces chrysomallus. Results: Glycosidase activity disappeared after the heating of native DL at 100°C for 40 min. Antibacterial activity of heat-treated DL for M. luteus MDMSU128 and MDMSU140 expressed as minimal inhibitory concentration was 9.8·10–8 and 12·10–8M, respectively, and to E. coli MDMSU52 11·10–8M. Antibacterial activity of synthetic peptide α1 for M. luteus MDMSU128 and for E. coli MDMSU52 was 8.3·10–5 and 4.9·10–5M, respectively. Conclusion: DL is the first invertebrate lysozyme with combined enzymatic and non-enzymatic antibacterial action. Copyright © 2006 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Introduction: Known thrombolytic agents either break peptide bonds in the fibrin molecule or act as plasminogen activators, which also results in peptide bond cleavage. In thrombi, fibrin molecules are known to be cross-linked by isopeptide bonds, the formation of which is mediated by factor XIIIa. In this work, we studied the dissolution of thrombi via isopeptide bond cleavage using a recombinant destabilase. Destabilase is an enzyme secreted from the medicinal leech salivary gland. This enzyme exhibits muramidase (lysozyme) activity, in addition to endo-ε-(γ-Glu)-Lys-isopeptidase activity, which is responsible for isopeptide bond cleavage., Methods: Venous (jugular vein) and arterial (carotid artery) thrombosis was induced in rats. Rats were intravenously injected with both recombinant destabilase produced in Escherichia coli and a commercial streptokinase preparation. After 24 h, the weight and degree of cross-linking in the thrombi were analysed. Amidolytic activity in rat blood serum was measured in order to evaluate destabilase levels in the blood., Results: Destabilase was definitively shown to cause a 47.6% and 74.6% decrease in the weight of venous and arterial thrombi, respectively. The enzyme proved to be more efficient at dissolving thrombi compared to streptokinase. The combined administration of destabilase and streptokinase has a greater effect than the injection of individual enzymes. Destabilase reduces fibrin stabilization in thrombi., Conclusion: Cumulatively, we find that the medicinal leech destabilase is a more efficient thrombolytic agent for dissolving thrombi, which could help increase the overall effectiveness of conventional thrombolytic drugs., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The purpose. Identifying the capacity of the medicinal leech novel original recombinant thrombolytic preparation Destabilase-Lysozyme-2 to inhibit the blood platelet aggregation., Methods: Gene of destabilase-lysozyme. ds2 (mlDL-Ds2 ), was cloned in E.coli cells. Recombinant protein was isolated in denaturing conditions using metal-chelate chromatography followed by denaturation of the polypeptide by rapid dilution in exact accordance with the procedure described by Kurdyumov A.S. et al. ( 2016, Russian Journal of Bioorganic Chemistry, v.42, s. 42-52). Blood was collected from the jugular vein of 18 horses. The functional status of platelets in the presence of different destabilase-lysozyme concentrations were evaluated for their aggregation in Platelet Rich Plasma ( PRP) and in Washed Platelet suspension (WP) using aggregometers Chrono-Log-700 and Сhrono-Log-560, USA560, США. As used aggregation inducers of ADP, collagen type III and human thrombin., Results: First demonstrated the ability of newly synthesized (Kurdyumov A.S. et al. 2016, Russian Journal of Bioorganic Chemistry, v42, s. 42-52) thrombolytic recombinant enzyme destabilase-lyzosyme to inhibit more than 40% of ADP-stimulated PRP aggregation and ADP- stimulated aggregation of horse blood washed platelets., Conclusion: The ability of destabilase-lyzosyme -2 to inhibit platelets aggregation extends biological properties of recombinant thrombolytic enzyme, pre-clinical trials which resulted in the end of 2015.

From the highly purified but lowly active recombinant protein Destabilas-Lysozyme (Dest-Lys) by use cation-exchange column TSK CM 3-SW chromatography, it was separated non-active fraction IV, contained 90% of protein. Fractions I, II and III, represented proteins with lysozyme and isopeptidase activities. Their lysozyme activity correlates with the activity of natural Des-Lys. The ratio of the activities in fractions I - III is such, that maximal lysozyme activity is concentrated in fraction III, isopeptidase - in fraction I. It is discussed the possibility of Dest-Lys different functions regulation is depended on the formation of protein complex forms.

The state of elderly patients arterial wall after the putting of one medicinal leech was estimated by use hardwarily software system "Angioscan-01". There was compared the effect of one medicinal leech on indicators of vasomotor function of endothelium of small resistance arteries and of middle arteries of muscular type. Stickiness index and augmentation index were determined in order to evaluate the medicinal leech effect on the rigidity state of arterial wall. It is shown that the putting of one leech stimulates the improving of endothelium vasomotor function and of normalization arterial wall stickiness. It is supposed the participation in this process the secretion of the medicinal leech salivary cells, which, as has been shown recently, is able to activate e-NOS and n-NOS in human endothelium culture (HUVEC) and increase NO level. Elevation of share stress during occlusion test is also stimulated NO production in vascular endothelium.

The medicinal leech salivary cell secretion (SCS) may stimulate NO-production in cultures of human endothelium cells (HUVEC) and rat cardiomiocytes (RCM). This effect was detected using a NO specific reagent, - the complex Cu2+ with a fluorescein derivative (Cu-Fl). NO had also been detected in the cells by fluorescent electronic microscopy and determined quantitatively in the cells and in culture fluid by the fluorescence method. SCS stimulated NO synthesis in HUVEC cells (but not in RCM) is accompanied by NO release into intercellular space. Localization of NO synthesis centers is presented and it is shown that the increase in NO levels during the SCS action on HUVEC and RCM is associated with the increase in the activity of eNOS/nNOS, but not iNOS. In endothelial cells SCS activates nitrosylation processes, assessed by the increase of nitrite-ions in the culture medium. It is therefore important to use Cu-Fl, other than Griss-reagent, during the first hour of analysis of NO synthesis. The NO-depended mechanism of SCS action on endothelial cells might be a factor in providing of its positive action in hirudotheraphy.

Salivary gland secretions of three species of the medicinal leech differ in the level of lysozyme peptidoglycan-lysing activity. Using the synthetic fluorogenic substrate, 4-methyl-umbelliferyl tetra N-acetyl-beta-chitotetraosid, the glycosidase activity (as one of peptidoglycan-lysing activities) of salivary gland secretion of three species of the medicinal leech was quantitatively evaluated in comparison with egg lysozyme. It is supposed, that lysozyme activity of the leech secretions is determined not only by 5 isoforms of destabilase-lysozyme, but by some other enzymes which can utilize this substrate. These may be lysozymes other than i- (invertebrate) lysozymes (such as destabilase-lysozyme, or related enzymes).