Neurite-stimulating effect of Hirudo medicinalis salivary gland secreting factors in organotypic culture of the dorsal root ganglia

Effects of destabilise, bdellin, bdellin A, eglin were investigated in organotypic tissue culture of dorsal root ganglia (DRG) of 10-11-day old chick embryos. Native destabilase and bdellin A, bdellin B and eglin are more active inducing a more intensive neurite growth in DRG as compared with the control. A neurite-stimulating effect of the drug "pyjavit" seems to be associated with destabilase, bdellins and eglin neurite-stimulating activity.     

Recombinant destabilase from the medicinal leech: Preparation and properties

The procedure for obtaining an active recombinant destabilase from the medicinal leech in Escherichia coli cells was developed. The plasmids encoding an analogue of native destabilase, as well as the protein forms carrying polyhistidine sequence at the Cand/or N-terminus of the polypeptide were obtained during the work. The producing strains of different forms of the protein were constructed, the cultivation process was optimized. The conditions of renaturation of destabilase recombinant forms by dialysis and using chromatographic absorbent were selected. The muramidase activity towards cell walls of Micrococcus lysodeikticus bacferia and lytic activity towards E. coli were investigated. The dependence of pH and ionic strength of the solution on the activities was determined. The total antibacterial activity of destabilase towards E. coli was shown.     

Polyfunctionality of destabilase, a lysozyme from a medicinal leech

Experimental data indicating the polyfunctionality of destabilase, a lysozyme from the salivary gland secretion of the medicinal leech, a unique representative of invertebrate lysozymes, were analyzed. The destabilase combines the properties of endo-s-lysyl-y-glutamyl isopeptidase (D-dimer monomerase), lysozyme, and chitinase and simultaneously is a nonenzymatic antimicrobial agent. The polypeptide sequence of lysozyme destabilase is encoded by a family of three genes (Ds1, Ds2, and Ds3). The ability of the enzyme to hydrolyze endoisopeptide bonds formed by transglutaminases, which are detected in many pathological conditions, including thrombosis, is considered from the viewpoint of its further application in practice.     

Destabilase: an enzyme of medicinal leech salivary gland secretion hydrolyzes the isopeptide bonds in stabilized fibrin

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.     

Recombinant Destabilase from Hirudo medicinalis Is Able to Dissolve Human Blood Clots In Vitro

Leeches are amazing animals that can be classified as conditionally poisonous animals since the salivary cocktail they produce is injected directly into the victim, and its components have strictly defined biological purposes, such as preventing blood clot formation. Thrombolytic drugs are mainly aimed at treating newly formed blood clots. Aged clots are stabilized by a large number of isopeptide bonds that prevent the action of thrombolytics. These bonds are destroyed by destabilase, an enzyme of the leech’s salivary glands. Here, we conducted a pilot study to evaluate the feasibility and effectiveness of the use of destabilase in relation to blood clots formed during real pathological processes. We evaluated the isopeptidase activity of destabilase during the formation of a stabilized fibrin clot. We showed that destabilase does not affect the internal and external coagulation cascades. We calculated the dose–response curve and tested the ability of destabilase to destroy isopeptide bonds in natural blood clots. The effect of aged and fresh clots dissolving ability after treatment with destabilase coincided with the morphological characteristics of clots during surgery. Thus, recombinant destabilase can be considered as a potential drug for the treatment of aged clots, which are difficult to treat with known thrombolytics.     

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.     

Polyfunctionality of lysozyme destabilase from the medicinal leech

Experimental data indicating the polyfunctionality of lysozyme destabilase from the salivary gland secretion of the medicinal leech, a unique representative of invertebrate lysozymes, were analyzed. The destabilase combines the properties of endo-?-lysyl-γ-glutamyl isopeptidase (D-dimer monomerase), lysozyme, and chitinase and simultaneously is a nonenzymatic antimicrobial agent. The polypeptide sequence of lysozyme destabilase is encoded by a family of three genes (Ds1, Ds2, and Ds3). The ability of the enzyme to hydrolyze endoisopeptide bonds formed by transglutaminases, which are detected under many pathological conditions, including thrombosis, is considered from the viewpoint of its further application in practice.     

Genes from the medicinal leech (Hirudo medicinalis) coding for unusual enzymes that specifically cleave endo-ɛ(γ-Glu)-Lys isopeptide bonds and help to dissolve blood clots

We previously detected in salivary gland secretions of the medicinal leech (Hirudo medicinalis) a novel enzymatic activity, endo-?(γ-Glu)-Lys isopeptidase, which cleaves isopeptide bonds formed by transglutaminase (Factor XIIIa) between glutamine γ-carboxamide and the ?-amino group of lysine. Such isopeptide bonds, either within or between protein polypeptide chains are formed in many biological processes. However, before we started our work no enzymes were known to be capable of specifically splitting isopeptide bonds in proteins. The isopeptidase activity we detected was specific for isopeptide bonds. The enzyme was termed destabilase. Here we report the first purification of destabilase, part of its amino acid sequence, isolation and sequencing of two related cDNAs derived from the gene family that encodes destabilase proteins, and the detection of isopeptidase activity encoded by one of these cDNAs cloned in a baculovirus expression vector. The deduced mature protein products of these cDNAs contain 115 and 116 amino acid residues, including 14 highly conserved Cys residues, and are formed from precursors containing specific leader peptides. No homologous sequences were found in public databases.     

Differential Age-Dependent Trophic Responses of Nodose,Sensory, and Sympathetic Neurons to Neurotrophins and GDNF: Potencies for Neurite Extension in Explant Culture

The age-dependent trophic responses of sympathetic, sensory, and nodose neurons to the neuro-trophins NGF, BDNF, and NT-3 and to glial cell line-derived neurotrophic factor (GDNF) were examined by an explant culture system. Superior cervical ganglia (SCG), dorsal root ganglia (DRG), and nodose ganglia (NG) were removed from rat embryos (E18), neonatals ( 1 day old), young adults (3–6 months old), and aged adults (>24 months old). The ganglia were cultured with and without each neurotrophic factor; the neurite extension and neurite density were then assessed. The SCG from rats of all ages were significantly influenced by NGF, NT-3, and GDNF; the effects of NT-3 and GDNF were reduced after maturation. The DRG from embryos and neonates were influenced by all neurotrophic factors; however, the effects of BDNF and NT-3 disappeared after maturation. The GDNF showed little effect on adult DRG and no effect on aged DRG. The effect of NGF was preserved over all ages of DRG. The NG from embryonic rats were significantly responsive to BDNF and GDNF; their effects decreased in the neonatal NG, but a minimum effect remained in the aged NG. These results indicate that age-dependent profiles of trophic effects differ extensively among the lineages of the peripheral nervous system and also among the individual neurotrophic factors.     

海参i型溶菌酶基因及其编码产物的结构特点

通过RT-PCR 和 RACE PCR技术,从海参(Stichopus japonicus)体壁中克隆得到一种溶菌酶基因(GenBank：EF036468).生物信息软件分析表明,其中全长cDNA为 713 bp,5′非编码区（UTR）246 bp,3′UTR 29 bp,开放阅读框438 bp,编码145个氨基酸,包括溶菌酶成熟肽124个氨基酸和信号肽21个氨基酸.对海参溶菌酶与多种无脊椎动物的c、g和i型溶菌酶进行分析比较,发现它与i型溶菌酶有较高的同源性,并具有i型溶菌酶高度保守的2个活性位点,即Glu34和Ser50.活性位点附近具有i型溶菌酶的一段特有的氨基酸保守序列MDVGSLSCG(P/Y)(Y/F)QIK,所以推断克隆的海参溶菌酶为i型.另外,通过搜索蛋白保守结构域数据库,发现海参溶菌酶与医用水蛭失稳酶相似性最高,并且这2个酶的三级结构模型也极其相似.因此推测,海参i型溶菌酶具有双功能特性,既能作用于细菌细胞壁的糖苷键使细胞裂解,又具有失稳酶的一些生化功能,能够水解纤维蛋白,这些特点在海参自溶过程中发挥重要的作用.     

An Efficient Method for Dorsal Root Ganglia Neurons Purification with a One-Time Anti-Mitotic Reagent Treatment

Background

The dorsal root ganglia (DRG) neuron is an invaluable tool in axon growth, growth factor regulation, myelin formation and myelin-relevant researches. The purification of DRG neurons is a key step in these studies. Traditionally, purified DRG neurons were obtained in two weeks after exposure to several rounds of anti-mitotic reagent.

Methods and Results

In this report, a novel, simple and efficient method for DRG purification is presented. DRG cultures were treated once with a high-dose anti-mitotic reagent cocktail for 72 hours. Using this new method, DRG neurons were obtained with 99% purification within 1 week. We confirmed that the neurite growth and the viability of the purified DRG neurons have no difference from the DRG neurons purified by traditional method. Furthermore, P0 and MBP expression was observed in myelin by immunocytochemistry in the DRG/SC co-culture system. The formation of mature node of Ranvier in DRG-Schwann cell co-culture system was observed using anti-Nav 1.6 and anti-caspr antibody.

Conclusion and Significance

The results indicate that this high dose single treatment did not compromise the capacity of DRG neurons for myelin formation in the DRG/SC co-culture system. In conclusion, a convenient approach for purifying DRG neurons was developed which is time-saving and high-efficiency.     

The cytochrome P450 inhibitor, ketoconazole, inhibits oxidized linoleic acid metabolite-mediated peripheral inflammatory pain

Background

Painful neuropathy is a common complication of diabetes. Previous studies have identified significant increases in the amount of voltage gated sodium channel isoforms Na_V1.7 and Na_V1.3 protein in the dorsal root ganglia (DRG) of rats with streptozotocin (STZ)-induced diabetes. We found that gene transfer-mediated release of the inhibitory neurotransmitters enkephalin or gamma amino butyric acid (GABA) from DRG neurons in diabetic animals reduced pain-related behaviors coincident with a reduction in Na_V1.7 protein levels in DRG in vivo. To further evaluate the role of Na_V?? subunit levels in DRG in the pathogenesis of pain in diabetic neuropathy, we constructed a non-replicating herpes simplex virus (HSV)-based vector expressing a microRNA (miRNA) against Na_V?? subunits.

Results

Subcutaneous inoculation of the miRNA-expressing HSV vector into the feet of diabetic rats to transduce DRG resulted in a reduction in Na_V?? subunit levels in DRG neurons, coincident with a reduction in cold allodynia, thermal hyperalgesia and mechanical hyperalgesia.

Conclusions

These data support the role of increased Na_V?? protein in DRG in the pathogenesis of pain in diabetic neuropathy, and provide a proof-of-principle demonstration for the development of a novel therapy that could be used to treat intractable pain in patients with diabetic neuropathy.     

P2X receptors in the rat uterine cervix,lumbosacral dorsal root ganglia,and spinal cord during pregnancy

ATP, an intracellular energy source, is released from cells during tissue stress, damage, or inflammation. The P2X subtype of the ATP receptor is expressed in rat dorsal root ganglion (DRG) cells, spinal cord dorsal horn, and axons in peripheral tissues. ATP binding to P2X receptors on nociceptors generates signals that can be interpreted as pain from damaged tissue. We have hypothesized that tissue stress or damage in the uterine cervix during late pregnancy and parturition can lead to ATP release and sensory signaling via P2X receptors. Consequently, we have examined sensory pathways from the cervix in nonpregnant and pregnant rats for the presence of purinoceptors. Antiserum against the P2X₃-receptor subtype showed P2X₃- receptor immunoreactivity in axon-like structures of the cervix, in small and medium-sized neurons in the L6/S1 DRG, and in lamina II of the L6/S1 spinal cord segments. Retrograde tracing confirmed the projections of axons of P2X₃-receptor-immunoreactive DRG neurons to the cervix. Some P2X₃-receptor-positive DRG neurons also expressed estrogen receptor- immunoreactivity and expressed the phosphorylated form of cyclic AMP response-element-binding protein at parturition. Western blots showed a trend toward increases of P2X₃-receptor protein between pregnancy (day 10) and parturition (day 22–23) in the cervix, but no significant changes in the DRG or spinal cord. Since serum estrogen rises over pregnancy, estrogen may influence purinoceptors in these DRG neurons. We suggest that receptors responsive to ATP are expressed in uterine cervical afferent nerves that transmit sensory information to the spinal cord at parturition.     

The hydrolysis of Glu(gamma-epsilon)Lys-amide bonds in D-dimer fragment of fibrin by destabilase from medicinal leech]

A study was made of the dependence of the hydrolysis rate of isopeptide bonds in the D-dimer, the product of limited proteolysis of the stabilized fibrin by plasmin, on the high-molecular substrate concentration catalyzed by destabilase from the medicinal leech. The formation of the reaction product was electrophoretically measured by the increase in the quantity of D-monomer. This dependence was shown to be nonlinear.     

Separation of Monomerizing and Lysozyme Activities of Destabilase from Medicinal Leech Salivary Gland Secretion

Destabilase, endo--(-Glu)-Lys-isopeptidase, was prepared from the salivary gland secretion of the medicinal leech (Hirudo medicinalis). The secretion prepared by the known method of Rigbi et al. (1987) (secretion-K) lacks the destabilase-characteristic highly specific isopeptidase activity (the D-dimer-monomerizing activity) because of its degradation by proteolytic activity (the substrate of Glp-Ala-Ala-Leu-pNA) due to contamination with leech intestinal channel contents. Therefore, we have elaborated a new technique for preparation of a true leech secretion (secretion-I). This secretion is characterized by the complete absence of the leech intestinal channel contents and has no proteolytic activity. For the first time the destabilase-specific D-dimer-monomerizing and lysozyme activities were separated by fractionation of secretion-I by HPLC gel filtration through Superose S-12. For the purified destabilase preparation, these activities were separated by reversed-phase chromatography in an acetonitrile gradient (0-60%) in the presence of 0.1% trifluoroacetic acid. The monomerizing activity of destabilase is responsible for the ability of secretion-I to dissolve stabilized fibrin via isopeptidolysis of - and - fibrin chains bound by -(-Glu)-Lys-isopeptide bonds.