[Extract of Hirudo medicinalis leeches: biogenic substance for the creation of effective drugs].

Investigations of the extract of medicinal leech Girulux established parameters that enable one to verify the authenticity of related drugs using the characteristics of their UV spectra the and anticoagulant and antithrombin activity. The limits of pH must range from 5.5 to 7.5. Pilot-scale series of medical leech extract are in compliance with terms of "microbiological purity" quality characteristics. The natural storage has been used to assess the temporal stability of leech extract. The obtained characteristics showed that the activity of extract was retained after 3-year storage.

Main group catalysed reduction of unsaturated bonds.

This Perspective highlights the recent developments in the reduction of unsaturated substrates catalysed by main group element compounds. Various activation modes are discussed and comparison with relevant examples from transition metal systems is made when possible. Main group element catalysis offers significant advantages through its lower cost and more benign environmental impact and has now reached the point where it can successfully compete with the more common catalysis based on precious transition metals.

A stable prostacyclin-like substance produced by the medicinal leech Hirudo medicinalis.

The medicinal leech Hirudo medicinalis produces a low-molecular mass compound with properties similar to those of prostacyclin. It extracted with organic solvent, had affinity to 6-keto-PGF1alpha antibodies, inhibited human platelet aggregation induced in vitro by thrombin (by 50% at 4 pg/ml), and caused hypotension and secretion of plasminogen (t-PA) into the blood stream of rats. A main distinction from prostacyclin is stability of the substance due to covalent binding with the polypeptide chain of destabilase. Because of the high aggregability of destabilase, the molecules of the protein-lipid complex are organized into micelles that can change their spatial orientation depending on the nature of the solvent. Incorporation of hirudin and blood plasma kallikrein inhibitor into the micelle structure causes the formation of liposomes (with a molecular mass of the structural monomer 25 kDa). This complex with polypeptides provides not only stability but also rapid transmembrane penetration. The pure prostacyclin-like substance has a molecular mass of 391 Da and can be produced on destruction of the destabilase polypeptide chain.

Destabilase complexes--natural liposome produced by medicinal leeches Hirudo medicinalis.

Electrophoretic analysis of destabilase preparation demonstrates the presence of protein combinations with MW 12.3, 25 and 50 kD. Fraction (MW 12.3 D) is a monomer of destabilase aggregation having properties of micellar proteins and represents a stable lipid-protein complex, where the role of lipid component is played by the stable analogue of prostacyclin (MW 391 D). The synthesis of a low molecular fraction of destabilase is fulfilled with bacteria--symbiont of leeches Aeromonas hydrophila. When the destabilase (MW 12.3 kD) contacts with blood a process of complexe formation is triggered with hirudin and blood plasma kallikrein inhibitor, forming a stable 'destabilase complex' (DC; MW 25 kD), possessing also a high aggregation capacity. Polymer forms of the destabilase complex form a liposome changing its spatial orientation depending on the nature of the solvent. Such structural organization provides a high stability of DC components and a rapid penetration through cellular membranes (transmembrane transfer) and it also provides prophylactic antithrombotic action in the case of peroral application to animals, due to the blockade of vascular platelets (inhibition of platelet aggregation by prostacyclin analogue) and plasmic (inhibition of thrombin activity and blood plasma kallikrein) links of the hemostasis process. Destabilase fraction with MW 50 kD is a dimer of the destabilase complex. As a result of DC destruction (liposome), hirudin, prostacycline analogue and blood plasma kallikrein inhibitor are released.

Use of the medicinal leech in the treatment of ear diseases.

Medicinal leeches (Hirudo medicinalis) and the native secretion of their salivary glands diluted with saline (5 times) were successfully used for the treatment of some ear diseases: tinnitus caused by inner-ear affections, acute external otitis and chronic otitis media. The effect of diluted leech saliva injected into the region of the mastoid process by microelectrophoresis was 25-30% lower than that of the medicinal leeches.

Destabilase, the novel epsilon-(gamma-Glu)-Lys isopeptidase with thrombolytic activity.

The salivary gland secretion of the leech Hirudo medicinalis contains the enzyme destabilase which hydrolyses epsilon-(gamma-Glu)-Lys cross-links in stabilized fibrin. Accumulation of Glu residues instead of the original Gln residues leads to spontaneous depolymerization of destabilized fibrin. L-gamma-Glu-p-nitroanilide; L-gamma-Glu-dansylcadaverine and isopeptide epsilon-(gamma-Glu)-Lys are low-molecular-weight substrates of destabilase. Destabilase probably exists in molecular forms of molecular weight 50,000, 25,000 and 12,300. The protein part of destabilase is covalently bound to a lipid component of molecular weight 390, which has little cross-reactivity to 6-keto-prostaglandin F1 alpha antiserum. The lipid component ensures the hydrophobic properties of destabilase, inhibition of platelet aggregation, protection from proteolysis and absorption from the intestine into blood during oral administration to experimental animals. It also ensures the protective antithrombotic effect. Almost total (80-100%) thrombolysis of preformed thrombus in the rat was achieved by destabilase 70-100 h after i.v. injection or oral administration.

[Hydrolysis of the isopeptide epsilon-(gamma-glutamyl)-lysine by destabilase from the medicinal leech Hirudo medicinalis]. / Gidroliz izopeptida epsilon-(gamma-glutamil)-lizina destabilazoi iz meditsinskoi piiavki Hirudo medicinalis.

Using amino acid analysis, the ability of destabilize to hydrolyze the epsilon-(gamma-Glu)-Lys isopeptide bond was demonstrated. Incubation of the epsilon-(gamma-Glu)-Lys isopeptide with the enzyme was accompanied by a decrease of the amount of the isopeptide and an increase of equimolar amounts of lysine and glutamic acid. Complete hydrolysis of the isopeptide was observed after 96 hour incubation with destabilize. It was supposed that the isopeptide is a less specific substrate for destabilize compared to L-gamma-Glu-pNA.

[Kinetics of L-gamma-Glu-pNA hydrolysis by destabilase, the enzyme from the medicinal leech Hirudo medicinalis]. / Kinetika gidroliza L-gamma-Glu-pNA destabilazoi, fermentom iz piiavki Hirudo medicinalis.

The molecular mass of destabilase isolated from the medicinae leech Hirudo medicinalis was found to be equal to 12.3 kDa. A kinetic analysis of the sole presently known synthetic substrate, L-gamma-Glu-pNA, showed that the enzyme is relatively stable to heating (5 min, 70 degrees C); the pH optimum lies at 7.0-8.5. The enzyme has a specific activity of 0.15 x 10(-9) mol.s-1.mg-1; Km = 2.2 x 10(-4) M, kcat is 3.53 x 10(-3) s-1 (pH 8.0, 37 degrees C).

[The effect of preparations of Hirudo medicinalis leeches on DNA methylation in the rat liver]. / Vliianie preparatov iz piiavok Hirudo medicinalis na metilirovanie DNK pecheni krys.

The effect of the salivary gland secretion and dialysable part of the homogenate of the leeches Hirudo medicinalis on the methylation of DNA in the rat liver after the intraperitoneal injection and perfusion of isolated liver has been analysed. The maximum concentration of 5-methylcytosine is observed 1 h later the injection of preparations: for the salivary gland secretion the increase is 39%, for the dialysate of leech homogenate is 28%. The 5-methylcytosine content increases on 28% after the perfusion of isolated liver with the leech saliva and after the dialysate of the leech homogenate--on 20%. No other changes in DNA content is observed. It is suggested that the DNA-methylation of the liver cells is due to the penetration of biologically active substances produced by the medical leech into the cell-targets accompanied by the forming of corresponding ligand-receptor complexes.

[Effect of medicinal leech preparations on indicators of atherogenesis]. / Vliianie preparatov iz meditsinskikh piiavok na pokazateli aterogeneza.

Medicinal leech salivary gland secretion, administered intravenously to rats on an atherogenic diet, reduces fibrinogen level and plasma heparin tolerance and diminishes lipid deposits in the intima of large vessels. Long-term chronic oral administration of dried medicinal leech powder to rats, kept on an atherogenic diet, reduces the area of local edema in the intima of large vessels, although blood lipid composition, fibrinogen level and plasma heparin tolerance remain basically unchanged, as compared to respective values in the control animals. The salivary gland secretion reduces the 3H-thymidine incorporation into the human atherosclerotic intimal cell culture.

[Inhibition of kallikrein from human plasma by salivary gland secretion and extracts of the medicinal leech Hirudo medicinalis]. / Ingibirovanie kallikreina plazmy krovi cheloveka sekretom sliunnykh zhelez i ékstraktom iz meditsinskoi piiavki Hirudo medicinalis.

It has been shown for the first time that the salivary gland secretion of the medicinal leech (Hirudo medicinalis) contains a human blood plasma kallikrein inhibitor which is capable of blocking the amidolytic activity of the enzyme in an irreversible manner (with D-Pro-Phe-Arg-pNA as substrate) and which also suppresses the kininogenase activity of kallikrein. The inhibition of the amidolytic activity of highly purified kallikrein preparations from human blood plasma obeys the pseudo-first order kinetics. The experimental results suggest that in the salivary-gland secretion of H. medicinalis the inhibitor concentration exceeds by one order of magnitude that in whole leech homogenate extracts, which indicates that the inhibitor biosynthesis may be localized in leech salivary glands.

Thrombolytic agents of the preparation from the medicinal leeches.

The thrombolytic effect of an extract from medicinal leeches orally administered to rats was shown. This effect depends on the number of administrations and on the concentration of extract. It is due to the leech prostaglandins and enzyme destabilase. After the extraction of prostaglandins by ethylacetate the thrombolytic effect diminished by 45%. It is supposed that the leech-prostaglandins, like prostacyclin and its stable analogous, induce the release of tissue plasminogen activator from vessel walls. The thrombolytic effect of destabilase was demonstrated in experiments in vitro. We observed the phenomenon of increasing of glutaminase activity of citrate blood and the appearance of amidolytic and destabilase activity.

Antithrombotic effect of preparations from the leeches Hirudo medicinalis.

Antithrombotic effect of leech salivary gland secretion was maximal after intravenous administration into rats and was slightly decreased in cases of peroral administration. Blood from leech intestinal tract and leech homogenate exhibited less distinct antithrombotic action. Effect of these preparations was maintained after peroral administration. The antithrombotic effect of the leech preparations did not depend on their antithrombic activity caused by hirudin. These leech preparations appear to elongate a period of blood plasma recalcification caused by kallikrein inhibitor as well as apparently due to their capacity to inhibit aggregation of the thrombocytes.

[Mechanisms of inhibition of vascular-platelet hemostasis by salivary gland secretion of the medicinal leech Hirudo medicinalis]. / Mekhanizmy ingibirovaniia sosudisto-trombotsitarnogo gemostaza sekretom sliunnykh zhelez meditsinskikh piiavok Hirudo medicinalis.

The mechanism of inhibition of the vascular-platelet stage of hemostasis by medicinal leech salivary gland secretion was studied. It was shown that the secretion blocks platelet adhesion on the surface of collagens belonging to different genetic classes, inhibits the primary attachment of platelets and completely suppresses their spreading on collagen surface. Whatever its antithrombin activity, the leech secretion inhibits platelet aggregation stimulated by various inductors, e. g., ADP, prostaglandin endoperoxide analog U-46619, Ca2+ ionophore A23187, arachidonic acid. The secretion possessing the antithrombin activity causes a greater inhibition of the thrombin-stimulated aggregation than that devoid of this activity. Leech secretion stimulates adenylate cyclase of platelet membranes in a receptor-mediated fashion and increases the level of cAMP. The active substance is a low molecular weight, thermostable trypsin-resistant fraction of the secretion. Stimulation of adenylate cyclase is not mediated by adenosine receptors. It is supposed that the mechanism of this activating effect involves platelet prostaglandin receptors.

[Antithrombotic effect of salivary gland secretion and other preparations from the leech Hirudo medicinalis after intravenous and peroral administration to rats]. / Antitromboticheskoe deistvie sekreta sliunnykh zhelez i drugikh preparatov iz piiavki Hirudo medicinalis pri vnutrivennom i peroral'nom vvedenii krysam.

Antithrombotic effect of leech salivary gland secretion was maximal after intravenous administration into rats and was slightly decreased in cases of peroral administration. Blood from leech intestinal tract and leech homogenate exhibited less distinct antithrombotic action. Effect of these preparations was maintained after peroral administration. The antithrombotic effect of the leech preparations did not depend on their antithrombic activity caused by hirudin. These leech preparations appear to elongate a period of blood plasma recalcification caused by kallikrein inhibitors as well as apparently due to their capacity to inhibit aggregation of the thrombocytes. Relatively low molecular mass substances, penetrating through pores of cellophan membrane during dialysis, were responsible for the antithrombotic effect of the preparations studied.

[Destabilase: an enzyme of medicinal leech salivary gland secretion hydrolyzes the isopeptide bonds in stabilized fibrin]. / Destabilaza-ferment sekreta sliunnykh zhelez meditsinskikh piiavok gidrolizuet izopeptidnye sviazi v stabilizirovannom fibrine.

The salivary gland secretion of the leech Hirudo medicinalis contains an enzyme termed by us as destabilase, which hydrolyzes the epsilon-(gamma-glutamyl)-lysine bonds as a result of fibrin stabilization by factor XIIIa in the presence of Ca2+. This hydrolysis, apart from the original lysine and glutamine, is characterized by an appearance of lysine and glutamic acid residues. The accumulation of glutamic acid residues leads to spontaneous depolymerization of the destabilized fibrin. As a result, fluid "spots" of destabilized fibrin depolymerization (DFD) begin to appear at the sites of leech secretion application on the surface of stabilized fibrin plates. The DFD activity of the leech salivary gland secretion manifests itself only in case of stabilized fibrin and increases with an increase in the stabilization degree. Treatment of leech secretion with diisopropylfluorophosphate does not affect the enzyme activity, which is completely blocked by monoiodoacetate. The mechanism of action of leech salivary gland secretion and the enzyme isolated from it, i. e., destabilase, was studied, using a synthetic chromogenic substrate - p-nitroanilide-gamma-glutamic acid. The amidolytic activity of leech salivary gland secretion is 2.2 +/- 0.18 nkat/ml, Km(app) for destabilase is 0.6 X 10(-5) M, V = 5.4 X 10(-3) mol/min.