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PubMed1997

A renin-like enzyme in the leech Theromyzon tessulatum.

We report on the biochemical isolation and characterization of a 32 kDa aspartyl protease from the leech Theromyzon tessulatum. Following a three step purification (gel permeation chromatography, pepstatin A-sepharose affinity column separation followed by reversed-phase HPLC) a renin-like enzyme was purified to homogeneity. The first 124 amino acid residues of the N-terminal part of the purified S-pyridylethylated leech renin exhibits a 26.5-35.5% sequence identity with that of mammals. The 20-81 region of leech renin exhibits a 80% sequence homology with the 175-232 region in mammals. This highly conserved region, which is also found in all aspartic proteases, possesses the aspartyl catalytic residue (D11TGSS). Leech renin hydrolyses at neutral pH and at 37 degrees C the Leu10-Leu11 bond of synthetic porcine angiotensinogen tetradecapeptide yielding the angiotensin I and the Leu11-Val12-Tyr13-Ser14 peptides, with a specific activity of 115 microg AI/min/mg (K[M] 22 microM; K[cat], 2.7). This hydrolysis is inhibited by pepstatin A (IC50: 4.6 microM). Moreover, this enzyme is found on a multiple hormone precursor of 19 kDa which exhibits a specific activity of 850 pmol AI/min/mg of renin. This is the first biochemical characterization of a renin-like enzyme in invertebrates and non-mammalian vertebrates.

Amino Acid Sequence↗
PubMed1999

Hyaluronidase activity in leeches (Hirudinea).

The leech hyaluronoglucuronidase (hyaluronidase I) was identified in Erpobdellidae (Nephelopsis obscura and Erpobdella punctata) and Glossiphoniidae (Desserobdella picta) and historically described from Hirudinidae (Hirudo medicinalis). A second leech hyaluronidase (hyaluronidase II) which hydrolyzed only a few bonds to for hyaluronan oligosaccharides larger than 6500 Da, was found in Glossiphoniidae (Helobdella stagnalis, Glossiphonia complanata, Placobdella ornata, and Theromyzon sp.) and in Haemopidae (Haemopis marmorata). The distribution of the two hyaluronidases in leech occurred in both orders (Arhynchobdellida and Rhynchobdellida) and in macrophagous and haematophagous feeding types whereas the liquidosomatophagous leeches only had hyaluronidase II.

Acetylglucosamine↗
PubMed2004

Leech mycetome endosymbionts are a new lineage of alphaproteobacteria related to the Rhizobiaceae.

Mycetomal organs attached to the esophagus of hematophagous leeches which are known to harbor endosymbiotic bacteria were removed from three species in the leech family Glossiphoniidae. Anatomical observations indicated that placobdellid mycetomes are paired and caecate, inserting into the esophagus posterior to the proboscis. Light and electron microscopy demonstrated that there is a single layer of mycetome epithelial cells harboring gram-negative rods and that these epithelial cells are ultrastructurally distinct from neighboring esophageal epithelial cells. Fluorescent in situ hybridization with eubacterial and alphaproteobacterial probes localized the bacteria solely to the mycetomes both in adult and in unfed juvenile leeches whereas a gammaproteobacterial probe did not yield a bound fluorescencent signal. DNA was isolated from these tissues and subjected to PCR amplification using bacteria-specific primers for 16S and 23S rDNA. Results from sequencing the amplification products and phylogenetic analysis with other Alphaproteobacteria revealed that the bacteria resident in these organs comprise a new genus of Alphaproteobacteria, Reichenowia n. gen., closely related to the nitrogen-fixing, nodule-forming Rhizobiaceae. The three bacterial strains, though different from each other were each other's closest relatives, suggesting a history of close coevolution with their leech hosts.

Alphaproteobacteria↗
PubMed2001

Separation of monomerizing and lysozyme activities of destabilase from medicinal leech salivary gland secretion.

Destabilase, endo-epsilon-(gamma-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 alpha-alpha and gamma-gamma fibrin chains bound by epsilon-(gamma-Glu)-Lys-isopeptide bonds.

Animals↗
PubMed1994

Carbohydrate-binding proteins in the leech: II. Lactose-binding protein LL35 is located to neuronal and muscle subsets and all epithelial cells.

Leech lectin 35 (LL35) is a calcium-independent galactoside-binding protein with a molecular mass of 35 kDa and binding properties similar to those of calcium-independent, galactose-specific lectins found in vertebrates, sponges, and nematodes. LL35 was initially isolated from membranes of the leech CNS; however, large amounts of this lectin were also extracted from the rest of the leech. Using affinity-purified antibodies to LL35, we report the immunocytochemical localization of LL35 in adult and embryonic leech. LL35 is developmentally regulated in epithelial, neuronal, and muscle tissue but is absent from glia. During embryogenesis, LL35 is highly expressed by a subset of sensory neurons, weakly expressed in epithelial cells, and absent from muscle. In the adult, LL35 is still present on the same sensory neurons but has become more abundant in epithelial cells lining the CNS and peripheral organs. LL35 also appeared on a muscle cell specifically located in the CNS but remained absent from peripheral muscle. The developmentally regulated distribution of LL35 in epithelial cells, neurons, and CNS muscles suggests a multifunctional role for this lectin with respect to these different cell types.

Animals↗
PubMed1998

Leech-borne Serratia marcescens infection following complex hand injury.

Leeches are commonly used in the postoperative course of plastic surgical operations where there is venous congestion in a pedicled or free flap. They provide a temporary relief to venous engorgement whilst venous drainage is re-established. It is known that leeches can carry Aeromonas hydrophila infection, and a second or third generation cephalosporin antibiotic has traditionally been given as prophylaxis against infection. We report a new observation that leeches can carry Serratia marcescens and give rise to clinically significant infection. The implication for prophylaxis and treatment of leech-associated cellulitis is discussed.

Adult↗
PubMed1990

Sensitivities of Aeromonas hydrophila cultured from medicinal leeches to oral antibiotics.

Infections associated with medicinal leech application are caused by Aeromonas hydrophila, an organism that resides in the leech gut. We cultured the intestinal tracts of 25 leeches and evaluated the efficacy of oral antibiotics against Aeromonas hydrophila. Cultures of this organism showed no sensitivity to ampicillin and inconsistent sensitivity to cephalothin (equivalent to cephalexin). The Aeromonas hydrophila cultures did show consistent sensitivity to ciprofloxacin, tetracycline, and trimethoprim-sulfamethoxasole. These three antibiotics should be considered if oral antibiotic coverage is used in association with leech application.

Administration, Oral↗
PubMed1972

Regeneration and changes in synaptic connections between individual nerve cells in the central nervous system of the leech.

The central nervous system of the leech has been used for the study of the formation of new synaptic connections by regenerating neurons. In control leeches, individual nerve cells in adjacent ganglia are connected in an orderly and stereotyped manner, with only little variation. In the present experiments, a bundle of axons running between two of the segmental ganglia has been severed and allowed to regenerate. Subsequently, the axons reestablish synaptic connections between certain identified nerve cells in the adjacent ganglia, selectively and accurately. Thus, individual sensory cells in one ganglion show a high degree of neural specificity in reestablishing cell to cell connections with a motor cell in the next ganglion. The performance of the regenerated synapses, however, is significantly altered in a consistent manner. The normal balance between the effects of inhibitory and excitatory innervation in leeches with regenerated synapses is different from that seen in normal leeches, with marked overemphasis on inhibition. Similar alterations have also been seen in a series of ganglia at a distance from the site of the lesion. After the operation, therefore, a widespread modification of synapses occurs along the length of the nerve cord.

Animals↗
PubMed1986

Cloning and expression of a cDNA coding for the anticoagulant hirudin from the bloodsucking leech, Hirudo medicinalis.

Cloned cDNAs have been isolated that encode a variant of hirudin, a potent thrombin inhibitor that is secreted by the salivary glands of the medicinal leech, Hirudo medicinalis. This variant probably corresponds to a form that has been purified from leech heads but differs in amino acid sequence from the hirudin purified from whole leeches. There are at least three hirudin transcripts detectable in leech RNAs that are different in size, site of synthesis, inducibility by starvation, and relationship to hirudin activity. The new hirudin variant predicted by the cDNA and the heterodisperse transcription products suggest a hirudin protein family. The hirudin cDNA was expressed in Escherichia coli under the control of the bacteriophage lambda PL promoter. The recombinant product is biologically active, inhibiting the cleavage by thrombin of fibrinogen and a synthetic tripeptide substrate.

Amino Acid Sequence↗
PubMed1990

Haematemesis due to leech infestation.

We present a case of haematemesis and malaena, severe enough to require blood transfusion, caused by leech infestation from the southern region of Saudi Arabia. The site was the posterior pharyngeal wall. The common mode of presentation in leech endoparasitism is by nasal infestation and recurrent unexplained epistaxis. Leech endoparasitism should be considered as a cause in unexplained haematemesis in areas where aquatic leeches are commonly found.

Animals↗
PubMed2005

Protein mapping of the salivary complex from a hematophagous leech.

The salivary complex of leeches contains many components able to modulate physiological mechanisms, such as coagulation and fibrinolysis, and it is composed by the salivary glands and proboscis, encompassing two different proteomes. The bidimensional electrophoretic pattern of the salivary complex from the Haementeria depressa leech revealed a total of 352 spots, 103 in common with the muscular tissue and 249 exclusive from the salivary complex as detected by silver staining; these spots showed isoelectric points from 3.5 to 9.5 and covered an apparent molecular weight range from 10 to 105 kDa. The following isoforms of proteins were identified by mass spectrometry analysis: antiplatelet protein, myohemerythrin and carbonic anhydrase. Since the leeches were not fed for about 2-3 months to stimulate the secretion of proteins that facilitates the blood metabolism, these most abundant proteins in the salivary complex excised from leeches, are expected to play a role during feeding and might have some anti-hemostatic properties. Furthermore, by zymography, a gelatinolytic and a fibrinolytic protein were identified.

Amino Acid Sequence↗
PubMed1991

Isolation of thrombin inhibitor from the leech Hirudinaria manillensis.

The leech Hirudinaria manillensis belongs to the same family as the medicinal leech Hirudo medicinalis, which has been widely used for the study of hirudin, a specific thrombin inhibitor. A similar inhibitor has now been isolated from the heads of the Hirudinaria leech by acetone/acid extraction and further purified to near homogeneity by ion exchange chromatography followed by affinity chromatography on thrombin-agarose and reverse phase HPLC. The purified material was recovered at about 10-15% yield and had a specific activity of about 12,000-14,000 ATU/mg, similar to other hirudin variants. The inhibitor was shown to be homogenous by sodium dodecyl sulphate/polyacrylamide gel electrophoresis in the presence of 8 M urea with an apparent molecular mass of about 7000 daltons under reducing conditions. Comparison of the anticoagulant effect on human plasma by partial thromboplastin time assay have shown that the inhibitor from Hirudinaria has similar potency as hirudin variant 1 at equivalent dosage. However, it does not cross-react with monoclonal antibodies towards recombinant hirudin variant 1. Comparison of the N-terminal amino acid sequence up to residue 25 also indicates differences at positions 2, 13, 17 and 24 between the two thrombin inhibitors. These findings indicate that the primary anticoagulant present in the leech Hirudinaria is a potent thrombin inhibitor (Bufrudin) with biological activity similar to hirudin, but differs in its structural and immunological properties.

Amino Acid Sequence↗
PubMed1991

Calin--a platelet adhesion inhibitor from the saliva of the medicinal leech.

The saliva of the medicinal leech, Hirudo medicinalis, contains a potent, hitherto unsuspected, inhibitor of collagen-mediated platelet adhesion/aggregation. Calin, of molecular size approximately 65,000 (reduced), has a rapid (1-10 min) effect on collagen which is reflected in its ability to suppress collagen-induced platelet aggregation, as well as adhesion of platelets to collagen-coated microcarrier beads. It also causes flocculation of Type I collagen fibril suspensions. Calin is differentiated from leech collagenase in two ways: (1) by demonstrating, by SDS-PAGE analysis of the products of incubations of Calin with Type I collagen at 37 degrees C, that Calin binds to but does not cleave collagen; and (2) by showing that Calin cannot be purified using the methods used to isolate leech collagenase. Calin's rapid and unusual interaction with collagen makes it a prime candidate for one of the agents that are the causative factors of the prolonged bleeding phenomenon seen after leech bites.

Animals↗
PubMedDate not supplied

Medicinal leech therapy: a case study.

Essential to the outcome of the replantation of digits is adequate arterial inflow and venous outflow. A nonoperative solution to the problem of venous insufficiency is leeching. The leech relieves venous congestion while attached, as the hirudin released by the leech continues to decongest the digit for 1-2 more hours. The emphasis of this article is a case study using leeches for venous congestion as an alternative to surgery.

Aged↗
PubMed2006

Meningococcal purpura fulminans treated with medicinal leeches.

INTRODUCTION: Meningococcal septicemia remains one of the most common infectious causes of admission to a pediatric intensive care unit. Numerous treatment strategies aimed at the thromboembolic complications inducing purpura fulminans and limb/digital ischemia have been attempted, with variable results. The successful use of medicinal leeches for pneumococcal purpura fulminans has been described, and we present a similar case of meningococcal purpura fulminans. PATIENT AND INTERVENTION: A 5-wk-old female infant with meningococcal meningitis and septicemia and progressive purpura fulminans of the left hand was treated with medicinal leeches. Medicinal leeches were applied to the left dorsal hand on a daily basis for 4 consecutive days. RESULT: The swelling and limited functionality visibly improved after 48 hrs, and by 120 hrs, perfusion in the distal phalanges of the thumb and middle finger was evident. Reperfusion of the distal phalanges was not fully sustained, and at 6 wks the plastic surgery department debrided the distal phalanges of her left hand, excluding the thumb. She fully recovered from the meningococcal septicemic shock; the functionality of her left thumb was preserved, and she has limited functionality of her left hand. CONCLUSION: The unique combination of salivary products in leech therapy has theoretical benefits and requires future study.

Bacteremia↗
PubMed1998

Injury-induced expression of endothelial nitric oxide synthase by glial and microglial cells in the leech central nervous system within minutes after injury.

It is known that nitric oxide (NO) is produced by injured tissues of the mammalian central nervous system (CNS) within days of injury. The aim of the present experiments was to determine the cellular synthesis of NO in the CNS immediately after injury, using the CNS of the leech which is capable of synapse regeneration, as a step towards understanding the role of NO in nerve repair. We report that within minutes after crushing the nerve cord of the leech, the region of damage stained histochemically for NADPH diaphorase, which is indicative of nitric oxide synthase (NOS) activity, and was immunoreactive for endothelial NOS (eNOS). On immunoblots of leech CNS extract, the same antibody detected a band with a relative molecular mass of 140,000, which is approximately the size of vertebrate eNOS. Cells expressing eNOS immunoreactivity as a result of injury were identified after freezing nerve cords, a procedure that produced less tissue distortion than mechanical crushing. Immunoreactive cells included connective glia and some microglia. Calmodulin was necessary for the eNOS immunoreactivity: it was blocked by calmodulin antagonist W7 (25 microM), but not by similar concentrations of the less potent calmodulin antagonist W12. Thus in the leech CNS, in which axon and synapse regeneration is successful, an increase in NOS activity at lesions appears to be among the earliest responses to injury and may be important for repair of axons.

Animals↗
PubMed1996

Indigenous bacterial flora of medicinal leeches and their susceptibilities to 15 antimicrobial agents.

Surface bacterial flora, as well as homogenates, of medical leeches, Hirudo medicinalis and Hirudinaria manillensis, were surveyed and the susceptibility of these isolates to 15 antimicrobial agents was examined. Aeromonas spp. were isolated from all leeches, and Pseudomonas fluorescens and other glucose-non-fermenting gram-negative rods (NF-GNR) were frequent isolates. Isolates were highly resistant to cephalosporins but susceptible to carbapenems, aminoglycosides and ofloxacin. The results indicate that prophylaxis with antimicrobial agents active against Aeromonas spp. and NF-GNR is necessary to avoid opportunist infections caused by indigenous leech flora during medical leech therapy on immunocompromised patients.

Aeromonas↗
PubMed1994

FMRFamide-related peptides in the sex segmental ganglia of the Pharyngobdellid leech Erpobdella octoculata. Identification and involvement in the control of hydric balance.

Using enzyme-linked immunosorbent assays, a dot-immunobinding assay and a three-step reverse-phase HPLC separation, four Arg-Phe-amide (RFamide) peptides were purified from sex segmental ganglia extracts of the leech Erpobdella octoculata; FMRFamide, FM(O)RFamide, FLRFamide and GDPFLRFamide. Their amino acid sequences were elucidated by means of a combined approach using antiserum specificity, synthetic-peptide coelution, automated Edman degradation and electrospray mass spectrometry. One of these peptides, GDPFLRFamide, is a novel leech RFamide neuropeptide. Two of the above RFamide peptides are involved in the control of leech hydric balance; one (GDPFLRFamide) is diuretic, the other (FMRFamide) is anti-diuretic. Titration of each purified RFamide peptide indicated a similar amount of each tetrapeptide and of tetrapeptides and heptapeptides. A comparison between RFamide peptides of E. octoculata and molluscs reveals structural similarities supporting the hypothesis for the existence of an ancestral RFamide peptide gene common to leeches and molluscs.

Amino Acid Sequence↗