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PubMed1998

Amino-acid-sequence determination and biological activity of tessulin, a naturally occurring trypsin-chymotrypsin inhibitor isolated from the leech Theromyzon tessulatum.

We purified a new trypsin-chymotrypsin inhibitor, designated tessulin, from the rhynchobdellid leech Theromyzon tessulatum. This 9-kDa peptide was purified to apparent homogeneity by gel-permeation and anion-exchange chromatographies followed by reverse-phase HPLC. The structure of tessulin was determined by reduction, S-beta-pyridylethylation, trypsin digestion, automated Edman degradation and matrix-assisted laser desorption mass spectrometry (m/z 8985 Da). The 81-amino-acid peptide possesses 16 cysteines and exhibits a 16% sequence similarity with antistasin-type inhibitors. Tessulin inhibits trypsin (Ki 1 pM) and chymotrypsin (Ki 150 pM) and exhibits no activity with thrombin, factor Xa, cathepsin G and elastase. This is the first trypsin-chymotrypsin inhibitor isolated from leeches that does not inhibit elastase or cathepsin G, except for cytin and therin. Furthermore, tessulin, in conjunction with other serine-protease inhibitors isolated from Theromyzon (therin, theromin), significantly diminishes the level of human granulocyte and monocyte activation induced by lipopolysaccharides (10 microg). The combined level of inhibition is higher than that of aprotinin, another serine-protease inhibitor used biomedically. Thus, tessulin may be clinically significant in reducing inflammatory events.

Adjuvants, Immunologic↗
PubMed1999

Stretch-activated cation channels of leech neurons: characterization and role in neurite outgrowth.

The goal of this study was to characterize the stretch-activated ion channels (SACs) of adult identified neurons of the leech Hirudo medicinalis and to test the role of SACs in neurite outgrowth of isolated cells. Using cell-attached patch recording, we established that SACs are densely distributed in the growth cone membrane of cultured neurons. In excised patches, we found that these channels are permeable to Ca2+, as well as to monovalent cations. The channels are blocked by the extracellular application of gadolinium (Gd3+), amiloride and gentamicin. Amiloride and gentamicin, respectively, induce a partial and complete voltage-dependent block. Time-lapse video recordings of neurite outgrowth from single cultured neurons were used to study the effects of blocking SACs with gentamicin. Within 20 h of plating in the presence of the aminoglycoside, the total length of neuronal arborization was significantly greater than that measured in its absence. The amount of assembled axon per unitary surface area remained constant over 40 h and did not differ significantly with or without gentamicin. Our findings show that SACs of leech neurons admit Ca2+, are densely distributed in the growth cone membrane and exhibit typical pharmacological features of mechanotransducer ion channels. In addition, our data suggest that these cation channels participate in the early interaction between growing neurites and culture substrate.

Amiloride↗
PubMed1999

Peptide-mediated glial responses to leydig neuron activity in the leech central nervous system.

Neuronal activity may lead to a variety of responses in neighbouring glial cells; in general, an ensemble of neurons needs to be active to evoke a K+- and/or neurotransmitter-induced glial membrane potential change. We have now detected a signal transfer from a single neuromodulatory Leydig neuron to the giant neuropil glial cells in the central nervous system of the leech Hirudo medicinalis. Activation of a Leydig neuron, two of which are located in each segmental ganglion, elicits a hyperpolarization in the giant neuropil glial cells. This hyperpolarization could be mimicked by bath application of the peptide myomodulin A (1 nM-1.0 microM). Myomodulin-like immunoreactivity has recently been found to be present in a set of leech neurons, including Leydig neurons (Keating & Sahley 1996, J. Neurobiol., 30, 374-384). The glial responses to Leydig neuron stimulation persisted in a high-divalent cation saline, when polysynaptic pathways are suppressed, indicating that the effects on the glial cell were direct. The glial responses to myomodulin A application persisted in high-Mg2+/low-Ca2+ saline, when chemical synaptic transmission is suppressed, indicating a direct effect of myomodulin A on the glial membrane. The glial hyperpolarization evoked by myomodulin A was dose dependent (EC50 = 50 nM) and accompanied by a membrane conductance increase of approximately 25%. Ion substitution experiments indicated that myomodulin A triggered a Ca2+-independent K+ conductance. Thus, our results suggest, for the first time, direct signal transmission from an identified modulatory neuron to an identified glial cell using a myomodulin-like peptide.

Animals↗
PubMed2000

Different types of response to foreign antigens by leech leukocytes.

We used morphological and immunocytochemical approaches to characterize and to show the behavior of cells involved in leech inflammatory responses. Leeches were injected with bacterial lipopolysaccharide, fluoresceinated yeasts, sulfate spheres and ciliates (Protozoa). Shortly after injection, migrating cells appeared in the area of injection. The response of the cells occurred in relation to the injected micro or macro antigens. Each injection first provoked a migration of cells towards the non-self material. Afterwards, different responses (degranulation, phagocytosis, encapsulation, melanization) occurred. The migrating cells involved in these series of processes have a similar behavior and are characterized by CD markers of macrophages, NK cells and granulocytes, which are typical of many invertebrates and vertebrates.

Animals↗
PubMed2000

Lipopolysaccharide-dependent induction of leech leukocytes that cross-react with vertebrate cellular differentiation markers.

We have designed experiments to characterise leech leukocytes that mediate inflammatory responses. Shortly after inflicting injury to the body wall in the presence of lipopolysaccharides, many cells resembling macrophages, NK cells and granulocytes of vertebrates and many invertebrates migrated to the lesioned area. Nuclei of migrating cells incorporated bromodeoxyuridine. Using human monoclonal antibodies, macrophage-like cells were positive for CD25, CD14, CD61, CD68, CD11b and CD11c. NK-like cells were positive for CD25, CD56, CD57 and CD16, and granulocytes were positive for CD11b and CD11c. In blots of leech extracts, the CD25 monoclonal antibody recognised a band of about 55 kD; the CD56 monoclonal antibody, two bands of about 140 and 210 kD; the CD57 monoclonal antibody, two bands of about 106 and 70 kD; the CD14 monoclonal antibody, a band of about 50 kD; the CD16 monoclonal antibody, a band of about 60 kD. CD61 and CD68 both recognised a band of about 110 kD; CD11b recognised a band of 200 kD, and CD11c, a band of 180 kD.

Animals↗
PubMed1989

Individual microglia move rapidly and directly to nerve lesions in the leech central nervous system.

Small cells called microglia, which collect at nerve lesions, were tracked as they moved within the leech nerve cord to crushes made minutes or hours before. The aim of this study was to determine whether microglia respond as a group and move en masse or instead move individually, at different rates, and whether they move along axons directly to the lesion or take another route, such as along the edges of the nerve cord. Cell nuclei in living nerve cords were stained with Hoechst 33258 dye and observed under dim ultraviolet illumination using fluorescence optics, a low-light video camera, and computer-assisted signal enhancement. Muscular movements of the cord were selectively reduced by bathing in 23 mM MgCl2. Regions of nerve cord within 300 microns of the crush were observed for 2-6 hr. Only a fraction of microglia, typically less than 50%, moved at any time, traveling toward the lesion at speeds up to 7 microns/min. Cells were moving as soon as observation began, within 15 min of crushing, and traveled directly toward the lesion along axons or axon tracts. Movements and roles of leech microglia are compared with their vertebrate counterparts, which are also active and respond to nerve injury.

Animals↗
PubMed1994

Cell fates in leech embryos with duplicated lineages.

We have examined the fates of the progeny of supernumerary embryonic stem cells (O/P teloblasts) generated by microinjecting polyadenylic acid into newborn O/P teloblasts in embryos of the leech, Helobdella triserialis. In normal development, each O/P teloblast generates a rostrocaudal column of daughter cells (primary blast cells) that contribute distinct segmentally iterated O or P sets of epidermal and neural progeny to the mature leech. Previous results suggest that primary blast cells derived from ipsilateral pairs of O/P teloblasts are equipotent and equivalent at birth; that they and their progeny assume distinct O or P fates according to hierarchical and position-dependent interactions; and that the P fate is the primary, or default, fate and the O fate is the secondary fate. In the work presented here, one O/P teloblast was experimentally induced to undergo a supernumerary equal division, and the developmental fates of the progeny of the three (two "duplicate" and one "nonduplicate") ipsilateral O/P teloblasts were determined at stages 8 and 10. We find that some supernumerary O/P teloblasts produce supernumerary P progeny, whereas others generate supernumerary O progeny. When three O/P-derived bandlets are present, bandlets derived from the duplicate O/P teloblasts give rise to progeny of the same (O or P) fate. When the nonduplicate bandlet is absent, the duplicate bandlets assume distinct O and P fates. These results suggest that ipsilateral sister O/P teloblasts, while equipotent, might not be equivalent.

Animals↗
PubMed1995

Structural characterization of a diuretic peptide from the central nervous system of the leech Erpobdella octoculata. Angiotensin II Amide.

Purification of a material immunoreactive to an antiserum against angiotensin II and present in the central nervous system of the pharyngobdellid leech Erpobdella octoculata was performed by reversed-phase high pressure liquid chromatography combined with both enzyme-linked immunosorbent assay and dot immunobinding assays for angiotensin II. Establishment of the amino acid sequence by Edman degradation, electrospray, and fast atom bombardement mass spectrometry measurements and enzymatic treatment by carboxypeptidase A indicated that this "central" angiotensin II-like material, the first one fully characterized in the animal kingdom, is an angiotensin II amide. This finding constitutes also the first biochemical characterization of a peptide of the angiotensin family in an invertebrate. Synthetic angiotensin II amide exerts, when injected in leeches, a diuretic effect and is, 1 and 2 h postinjection, 100-fold more potent than vertebrate angiotensin II. An identification of the proteins immunoreactive to an antiserum against angiotensin II performed at the level of both central nervous system extracts and in vitro central nervous system-translated RNA products indicated that in the two cases, two proteins were detected. Their molecular masses, which were, respectively, approximately 14 and approximately 18 kDa for the central nervous system extracts and approximately 15 and approximately 19 kDa for in vitro central nervous system-translated RNA products, differ from that of angiotensinogen (approximately 60 kDa), the precursor of vertebrate angiotensin II.

Amino Acid Sequence↗
PubMed1996

Purification, sequence analysis, and cellular localization of a prodynorphin-derived peptide related to the alpha-neo-endorphin in the rhynchobdellid leech Theromyzon tessulatum.

Cells immunoreactive to an antiserum specifically directed against vertebrate alpha-Neo-endorphin (alpha-NE) were detected in the internal wall of anterior and posterior suckers of the rhynchobdellid leech Theromyzon tessulatum. These cells have morphological and ultrastructural characteristics close to the "releasing gland cells" of adhesive organs. The epitope recognized by anti-alpha-NE was contained in granules having a diameter of 0.2-0.3 microm. Previous works involving the brain of this leech demonstrate the existence of approximately 14 neurons immunoreactive to the anti-alpha-NE. Following an extensive purification including high pressure gel permeation and reversed-phase high performance liquid chromatography, epitopes contained in both suckers and central nervous system were isolated. Purity of the isolated peptides was controlled by capillary electrophoresis. Their sequences were determined by a combination of automated Edman degradation, electrospray mass spectrometry measurement, and coelution experiments in reversed-phase high performance liquid chromatography with synthetic alpha-NE. The results demonstrate that epitopes recognized by the anti-alpha-NE in the suckers and the central nervous system are identical to vertebrate alpha-NE (YGGFLRKYPK). This finding constitutes the first biochemical characterization of a prodynorphin-derived peptide in invertebrates. Moreover the isolation of this peptide in the annelida establishes the very ancient phylogenetic origin of alpha-NE as well as its conservation in evolution.

Amino Acid Sequence↗
PubMed1998

A carboxypeptidase inhibitor from the medical leech Hirudo medicinalis. Isolation, sequence analysis, cDNA cloning, recombinant expression, and characterization.

A novel metallocarboxypeptidase inhibitor was isolated from the medical leech Hirudo medicinalis. Amino acid sequence analysis provided a nearly complete primary structure. which was subsequently verified and completed by cDNA cloning using reverse transcriptase-polymerase chain reaction/rapid amplification of cDNA end techniques. The inhibitor, called LCI (leech carboxypeptidase inhibitor), is a cysteine-rich polypeptide composed of 66 amino acid residues. It does not show sequence similarity to any other protein except at its C-terminal end. In this region, the inhibitor shares the amino acid sequence -Thr-Cys-X-Pro-Tyr-Val-X with Solanacea carboxypeptidase inhibitors, suggesting a similar mechanism of inhibition where the C-terminal tail of the inhibitor interacts with the active center of metallocarboxypeptidases in a substrate-like manner. This hypothesis is supported by the hydrolytic release of the C-terminal glutamic acid residue of LCI after binding to the enzyme. Heterologous overexpression of LCI in Escherichia coli, either into the medium or as an intracellular thioredoxin fusion protein, yields a protein with full inhibitory activity. Both in the natural and recombinant forms, LCI is a tightly binding, competitive inhibitor of different types of pancreatic-like carboxypeptidases, with equilibrium dissociation constants Ki of 0.2-0.4 x 10(-9) M for the complexes with the pancreatic enzymes A1, A2, and B and plasma carboxypeptidase B. Circular dichroism and nuclear magnetic resonance spectroscopy analysis indicate that recombinant LCI is a compactly folded globular protein, stable to a wide range of pH and denaturing conditions.

Amino Acid Sequence↗
PubMed2004

Molecular characterization of two novel antibacterial peptides inducible upon bacterial challenge in an annelid, the leech Theromyzon tessulatum.

Two novel antimicrobial peptides named theromacin and theromyzin were isolated and characterized from the coelomic liquid of the leech Theromyzon tessulatum. Theromacin is a 75-amino acid cationic peptide containing 10 cysteine residues arranged in a disulfide array showing no similarities with other known antimicrobial peptides. Theromyzin is an 86-amino acid linear peptide and constitutes the first anionic antimicrobial peptide observed in invertebrates. Both peptides exhibit activity directed against Gram-positive bacteria. Theromacin and theromyzin cDNAs code precursor molecules containing a putative signal sequence directly followed by the mature peptide. The enhancement of theromacin and theromyzin mRNA levels has been observed after blood meal ingestion and upon bacterial challenge. In situ hybridization revealed that both genes are expressed in large fat cells in contact with coelomic cavities. Gene products were immunodetected in large fat cells, in intestinal epithelia, and at the epidermis level. In addition, a rapid release of the peptides into the coelomic liquid was observed after bacterial challenge. The presence of antimicrobial peptide genes in leeches and their expression in a specific tissue functionally resembling the insect fat body provide evidence for the first time of an antibacterial response in a lophotrochozoan comparable to that of holometabola insects.

Amino Acid Sequence↗
PubMed2000

Therostasin, a novel clotting factor Xa inhibitor from the rhynchobdellid leech, Theromyzon tessulatum.

Therostasin is a potent naturally occurring tight-binding inhibitor of mammalian Factor Xa (K(i), 34 pm), isolated from the rhynchobdellid leech Theromyzon tessulatum. Therostasin is a cysteine-rich protein (8991 Da) consisting of 82 amino acid residues with 16 cysteine residues. Its amino acid sequence has been determined by a combination of techniques, including Edman degradation, enzymatic cleavage, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) on the native and s-beta-pyridylethylated compound. Sequence analysis reveals that it shares no significant homology with other Factor Xa inhibitors except for the putative reactive site. Moreover, it contains a signature pattern for proteins of the endothelin family, potent vasoconstrictors isolated in mammal and snake venom. Therostasin cDNA (825 bp) codes for a polypeptide of 82 amino acid residues preceded by 19 residues, representing a signal peptide sequence. As for the other known inhibitors of Factor Xa, therostasin is expressed and stored in the cells of the leech salivary glands.

Amino Acid Sequence↗
PubMedDate not supplied

Unexpected result in the etiological approaching to an anemic case: a leech infestation.

One of the causes of the childhood anemia is gastrointestinal system bleeding, which rarely results from parasites. The authors report on a 3-year-old boy with severe anemia and a history of hematemesis. While they were investigating the cause of the anemia and the hematemesis, a leech showing itself in the nasal passage led to a quick and exact treatment. Leeches should be considered for differential diagnosis of anemia and gastrointestinal system bleeding in the children.

Anemia↗
PubMed1995

Calsensin: a novel calcium-binding protein expressed in a subset of peripheral leech neurons fasciculating in a single axon tract.

The mAb lan3-6 recognizes a cytosolic antigen which is selectively expressed in the growth cones and axons of a small subset of peripheral sensory neurons fasciculating in a single tract common to all hirudinid leeches. We have used this antibody to clone a novel EF-hand calcium-binding protein, calsensin, by screening an expression vector library. A full-length clone of 1.1 kb identified by the antibody was isolated and sequenced. In situ hybridizations with calsensin probes and antibody staining using new polyclonal antisera generated against calsensin sequence demonstrate that calsensin indeed corresponds to the lan3-6 antigen. Calsensin consists of 83 residues with a calculated molecular mass of 9.1 kD that contains two helix-loop-helix domains. The calcium-binding domains are likely to be functional in vivo since a fusion protein derived from the calsensin clone binds 45Ca2+ in vitro. Immunoaffinity purification experiments with the lan3-6 antibody shows that a large 200,000 M(r) protein selectively copurifies with calsensin in two different leech species. These results suggest that calsensin may be functioning as a trigger protein which interacts with the larger protein. These data are consistent with the hypothesis that calsensin may mediate calcium-dependent signal transduction events in the growth cones and axons of this small group of sensory neurons which fasciculate in a single axon tract.

Amino Acid Sequence↗
PubMed1984

A large fragment approach to DNA synthesis: total synthesis of a gene for the protease inhibitor eglin c from the leech Hirudo medicinalis and its expression in E. coli.

A DNA containing the coding sequence for the proteinase inhibitor protein, eglin c, from the leech Hirudo medicinalis has been obtained by enzymatic assembly of chemically synthesized DNA fragments. The synthetic gene consists of a 232 base-pair fragment containing initiation and termination codon signals with restriction enzyme recognition sites conveniently placed for cloning into a plasmid vector. Only six oligonucleotides from 34 to 61 bases in length, sharing pairwise stretches of complementary regions at their 3'-termini, were prepared by phosphotriester solid-phase synthesis. The oligomers were annealed pairwise and converted into double stranded DNA fragments by DNA polymerase I mediated repair synthesis. The fragments were assembled by ligation, and the synthetic gene was expressed in high yield in E. coli under the transcriptional control of the E. coli tryptophan promoter. The expression product was purified to homogeneity and was shown to have similar physicochemical and identical biological properties as the authentic protein isolated from the leech.

Amino Acid Sequence↗
PubMed1992

The use of medicinal leeches in the salvage of flaps with venous congestion.

The use of medicinal leeches (Hirudo medicinalis) for the salvage of tissues with venous congestion has been intermittent over the last two centuries. During the last decade, interest in leech therapy has undergone a resurgence. Hirudo's tricuspid bite injects a highly potent anticoagulant. The site usually bleeds for 1 to 2 hours and under special circumstances may bleed for up to 24 hours. This collective series presents four patients in whom immediate postoperative venous congestion threatened the survival of three tissue replants and one latissimus dorsi flap. Two of the replants were completely salvaged; the other two failed due to infection. Hirudo medicinalis is an important adjunct in preventing flap or replant failure secondary to venous congestion, but its use is associated with significant risks.

Adolescent↗
PubMed1995

Successful use of leeches in the treatment of purpura fulminans.

A case of purpura fulminans secondary to pneumococcal septicemia is presented in an 8-month-old girl. The purpuric lesions on the fingers of both hands, as well as on the lower extremities, were treated by the local application of medicinal leeches. There was nearly complete salvage of the threatened tissues and the baby made a complete recovery. The possible mechanisms by which the leeches may have contributed to the clinical salvage are discussed.

Animals↗