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At least 19 recordsLinked to original sources
PubMed1983

Hirudin from leech heads and whole leeches and "pseudo-hirudin" from leech bodies.

Hirudin from whole leeches behaves like hirudin from leech heads and "pseudohirudin" from leech bodies during isolation and purification by means of acetone and ethanol fractionation, affinity chromatography on trypsin-Sepharose, and isoelectric focusing. However, the antithrombin activity of the hirudin fractions obtained after isoelectric focusing was approximately three times lower than that of the corresponding hirudin fractions from the heads. The "pseudohirudin" fractions had practically no antithrombin activity. In hirudin from whole leeches isoleucine and valine were identified as the N-terminal amino acid. Isoleucine was identified as the dominant amino acid in hirudin from leech heads. The dominant N-terminal amino acid in "pseudohirudin" from leech bodies was valine. The data on antithrombin activity and N-terminal amino acids indicate that hirudin from whole leeches contains admixtures of inactive "pseudohirudin". In contrast to hirudin from leech heads, "pseudohirudin" represents associates of di- and three-isomers. The molecular weight of the "pseudohirudin" molecule is 2000 lower than the molecular weight of hirudin, which corresponds to a difference of 20 amino acid residues, calculated from the total number of amino acid residues in the preparations.

Amino Acid Sequence↗
PubMed1996

Platelet aggregation and coagulation inhibitors in leech saliva and their roles in leech therapy.

Prolonged bleeding by the host after the leech ceases to feed and several reports that the use of leeches restores blood flow in the microcirculation after plastic surgery led us to search for inhibitors of platelet aggregation in Hirudo medicinalis saliva. Dilute leech saliva was collected by phagostimulating starved leeches with a solution of arginine in saline. The saliva is shown to inhibit human platelet aggregation induced by thrombin, collagen, adenosine diphosphate (ADP), epinephrine, platelet activating factor (1-O-alkyl-2-acetyl-sn-3-glycerophophoryl choline [PAF]), and arachidonic acid. We have isolated the PAF inhibitor and found it to be an amphipathic phosphoglyceride. We have also purified apyrase adenosine triphosphate ([ATP] diphosphohydrolase), which inhibits ADP-induced platelet aggregation, and have described collagenase. Besides well-known hirudin, Hirudo saliva contains a potent inhibitor of coagulation factor Xa. We also report antiaggregant and anticoagulant activities in the crop content of the closely related Nile leech, Limnatis nilotica. Anticoagulants of hematophagous species are surveyed. We have used medicinal leeches in plastic surgery for decompression of skin flaps and in patients with postphlebitic syndrome and peripheral arterial occlusions. Preliminary results indicate certain beneficial effects of leech therapy.

Animals↗
PubMed1999

Localization of leech excitatory peptide, a member of the GGNG peptides, in the central nervous system of a leech (Whitmania pigra) by immunohistochemistry and in situ hybridization.

We have recently isolated a myoactive peptide, called leech excitatory peptide, belonging to the GGNG peptide family from two species of leeches, Hirudo nipponia and Whitmania pigra. Immunohistochemistry and in situ hybridization were employed to localize leech excitatory peptide-like peptide(s) and its gene expression in the central nervous system of W. pigra. A pair of neuronal somata were stained by both immunohistochemistry and in situ hybridization in the supraesophageal, subesophageal, and segmental ganglia. In addition, several other neurons showed positive signals by either immunohistochemistry or in situ hybridization in these ganglia. An immunoreactive fiber was observed to run in the anterior root of segmental ganglion 6, which is known to send axons to the sexual organs, though we failed to detect immunoreactivity in possible target tissues. Antiserum specificity was established by enzyme-linked immunosorbent assay using different leech excitatory peptide-related peptides. Leech excitatory peptide elicited muscular contraction of isolated preparations of penis and intestine at concentrations of 10(-8 )M. These results suggest that leech excitatory peptide is a neuropeptide modulating neuromuscular transmission in multiple systems, including regulation of reproductive behavior.

Amino Acid Sequence↗
PubMed1980

[Comparative properties of hirudin from whole leeches and from leech heads and bodies].

During isolation and purification involving fractionation by acetone, ethanol and affinity chromatography on trypsin-Sepharose and isoelectrofocusing hirudin from whole leeches behaves as the inhibitor isolated from leech heads and "pseudohirudin" from leech bodies. The antithrombin activity of hirudin fractions obtained by isoelectrofocusing is about three times less than that of the corresponding hirudin fractions from the heads. The "pseudohirudin" fractions are practically devoid of the antithrombin activity. In hirudin prepared from whole leeches isoleucine and valine were identified as N-terminal amino acids. The antithrombin activity and N-terminal amino acids suggest that hirudin from whole leeches contains admixtures of inactive "pseudohirudin". Hirudin from leech heads purified by complexing with thrombin possesses the activity of 15600 ATNIH units per pg of protein and has isoleucine as an N-terminal amino acid.

Amino Acids↗
PubMed1996

Structural characterization of a novel neuropeptide from the central nervous system of the leech Erpobdella octoculata. The leech osmoregulator factor.

Purification of a material immunoreactive to an antiserum against the C-terminal part of the oxytocin (Pro-Leu-Gly-amide) and present in the central nervous system of the Pharyngobdellid leech Erpobdella octoculata was performed by reversed-phase high performance liquid chromatography combined with both enzyme-linked immunosorbent and dot immunobinding assays for oxytocin. The amino acid sequence of the purified peptide (Ile-Pro-Glu-Pro-Tyr-Val-Trp-Asp) was established by Edman degradation and confirmed by electrospray mass spectrometry measurement. When injected in leeches, purified or synthetic peptides exert an anti-diuretic effect, the most effective ranged between 10 pmol and 1 nmol. They provoked an uptake of water 1-2 h post-injection. Furthermore, electrophysiological experiments conducted in the leech Hirudo medicinalis revealed an inhibition of the potency of Na+ conductances of leech skin by this peptide. Immunocytochemical studies with an antiserum against synthetic oxytocin-like molecule provided the cytological basis for existence of a neuropeptide, since large amounts of immunoreactive neurons were detected in the central nervous systems of E. octoculata. The purified molecule is both different to peptides of the oxytocin/vasopressin family and is a novel neuropeptide in the animal kingdom. It was named the leech osmoregulator factor (LORF). An identification of the proteins immunoreactive to an antiserum against oxytocin performed at the level of both central nervous systems extracts and in vitro central nervous system-translated RNA products indicated that in the two cases, a single protein was detected. These proteins with a molecular masses of, respectively, approximately 34 kDa (homodimer of 17 kDa) for the central nervous systems extracts and approximately 19 kDa for in vitro central nervous system-translated RNA products were not recognized by the antiserum against MSEL- and VLDV-neurophysin (proteins associated to oxytocin and vasopressin), confirming that LORF did not belong to the oxytocin/vasopressin family.

Amino Acid Sequence↗
PubMed1998

Isolation and characterization of a leech neuropeptide in rat brains: coupling to nitric oxide release in leech, rat and human tissues.

The osmoregulator peptide (leech osmoregulatory factor, LORF; IPEPYVWD) was first found in the leech central nervous system (CNS). Given the fact that certain peptides can be found in mammals and invertebrates, e.g., opioid, we examined rat brains to determine if LORF was present. This peptide was found and isolated by successive reversed-phase HPLC purification steps and characterized by electrospray mass spectrometry measurement. It was sequenced by Edman degradation and quantified in different tissues by ELISA. Our results demonstrate the presence of LORF in the hypothalamus, thalamus, and striatum (6 pmol/mg of protein extract) and in other brain areas at lower levels. This octapeptide is also present in the rat duodenum and liver (10 to 14 pmol/mg) and at lower levels in heart, lung, pancreas and caudal spinal cord (< 5 pmol/mg). The testes, adrenals and kidneys have the lowest levels of all the tissues examined (ca. 0.5 pmol/mg of protein). Furthermore, we also demonstrate that LORF is coupled to nitric oxide (NO) release in leech CNS, rat hypothalamus and human saphenous vein in a manner which is inhibited by a nitric oxide synthase inhibitor as well as an antibody directed toward LORF. The study demonstrates that LORF, and its function in relation to NO release, has been conserved over more than 400 million years of evolution.

Animals↗
PubMed1998

Putative leech dopamine1-like receptor molecular characterization: sequence homologies between dopamine and serotonin leech CNS receptors explain pharmacological cross-reactivities.

The biochemical characterization of a serotonin (5HT) receptor and the cloning of a dopamine (DA) receptor in the central nervous system (CNS) of the leech, Theromyzon tessulatum, is presented. Additionally, DA and 5HT binding sites were examined in the CNS by Scatchard analysis which showed a single, relatively high-affinity binding site with a Kd 1.1 nM and a Bmax 126+18 fmol/mg protein for [3H]DA and a Kd 2.1 nM and a Bmax 225 fmol/mg protein for [3H]5HT. The first 88 amino acids of the 5HT receptor, isolated by a 5HT-affinity column followed by anion exchange chromatography and C3 reverse-phase HPLC exhibited a 43% sequence homology with Lymnaea stagnalis 5HT-receptor. The isolated DA receptor revealed a single protein of 45 kDa with an anti-D1-R in Western blot. The first 80 N-terminal amino acid residues and a trypsin digested fragment of 31 residues were obtained, and based on these sequencing data, a molecular biology strategy using reverse transcriptase-polymerase chain reaction, was developed. An amplified 1-kb segment was obtained. The complete deduced sequence of 416 amino acid residues exhibited about 30.6% sequence homology with the vertebrate D1 receptor family. Moreover, we further demonstrate that the leech 5HT and DA receptors also exhibit 30% sequence identity with each other, explaining their pharmacological cross-reactivity. Finally, anti-D1-R immunocytochemistry revealed positive structures in the peripheral and central nervous system, e.g., neurons, sensory fibers and immune cells. This is the first biochemical and molecular characterization of a DA receptor in leeches.

Amino Acid Sequence↗
PubMed1976

The connective tissue coverings of leech peripheral nerves: anatomical evidence for the absence of cerebrospinal fluid in the leech.

The central nervous system of Hirudo medicinalis is contained within a blood vessel, the ventral longitudinal sinus, but the nervous system is separated from the blood by the visceral endothelium. The visceral endothelium possesses many pinocytotic vesicles and basal infoldings and thus appears active whereas the parietal endothelium appears inactive. The junction between the visceral and parietal endothelia is abrupt. Peripheral nerves in this animal, as in vertebrates, are covered by endoneurium, perineurium, and epineurium. The endoneurium is continous with the fibrous tissue of the segmental ganglia. The perineurium, consisting of a single layer of flattened cells that surrounds the peripheral nerve like a sleeve, is not continuous with the endothelium of the ventral sinus, but is separated from it by 5-10 microns. Therefore, at the point where the peripheral nerve joins the segmental ganglion, the extracellular spaces of the central nervous system, the peripheral nervous system, and the body wall are all confluent. Thus, there are only two compartments of the extracellular space in the leech: the blood, which is enclosed by the endothelia of the coelomic sinuses, and the extracellular fluid of the body, which includes the extracellular fluid of the nervous system. There seems to be no equivalent of cerebrospinal fluid in the gnathobdellid leech.

Animals↗
PubMed1986

The primary structure of bdellin B-3 from the leech Hirudo medicinalis. Bdellin B-3 is a compact proteinase inhibitor of a "non-classical" Kazal type. It is present in the leech in a high molecular mass form.

A proteinase inhibitor was isolated from extracts of the leech Hirudo medicinalis by gel filtration and anion exchange chromatography. This inhibitor is similar to the bdellins in that it blocks the activity of trypsin, plasmin and sperm acrosin but has a molecular mass, as estimated by SDS polyacrylamide electrophoresis, of about 20 kDa, whereas the bdellins have molecular masses in the range 5-6 kDa. It is therefore designated as high-molecular mass bdellin B-3 (HMB). The amino-acid sequence of the inhibitor was elucidated as far as position 56. This revealed that the molecule consists of a bdellin B-3 moiety, corresponding to the N-terminal 46 residues, which is then extended at the C-terminus by a polypeptide chain of the composition Asx15, Glx25, Gly6, Val, His26-27 and Lys4. It has been formerly concluded from a partial amino-acid sequence that bdellin B-3 is a Kazal-type inhibitor. However, the complete sequence of bdellin B-3, represented by the N-terminal 46 residues of HMB, discloses that bdellin B-3 is a non-classical Kazal-type inhibitor when the number of amino-acid residues between half-cystines are considered. Presuming that formation of disulfide bridges principally follows the same pattern as in classical Kazal-type inhibitors the bdellin B-3 molecule was modeled based on the known three-dimensional structure of the third ovomucoid domains. This showed that a compact arrangement of the peptide chain of bdellin B-3 is conceivable.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1957

The leech as a potential virus reservoir.

Leeches, fed on swine infected with hog cholera, contained virus for as long as 87 days after their infective blood meals. In three instances, infected leeches apparently transmitted hog cholera virus to susceptible swine in the process of normal feeding. Myxoma virus persisted in leeches for as long as 154 days after the ingestion of a blood meal from rabbits with myxomatosis. Leeches fed consecutively, first on swine with hog cholera, and later on rabbits with myxomatosis, acquired both viruses. In such dually infected leeches, the hog cholera virus persisted for as long as 122 days and the myxoma virus for as long as 110 days, the longest periods tested. Leeches fed consecutively, first on rabbits with myxomatosis, and later on swine with hog cholera, acquired only the myxoma virus. Hog cholera virus could not be demonstrated in such dually fed leeches. Myxoma and hog cholera viruses appeared to be present in about equivalent amounts in the anterior and posterior thirds of the bodies of infected leeches. Myxoma and hog cholera viruses were present in the bloody gut contents of infected leeches but were not demonstrable in the body tissues of these leeches. It seems from the findings presented that leeches are not biological carriers of either myxoma or hog cholera virus but instead carry these two agents mechanically in their gastrointestinal tracts. In doing this, they appear to protect the viruses from various deleterious chemical and physical influences to which they would have been exposed in the open. It is speculated that leeches could play a role in nature in perpetuating the blood-borne viruses of certain diseases in which close association with bodies of fresh water is of epidemiological importance.

Animals↗
PubMed1994

Emergent reuse leech therapy: a better method.

Tissues threatened by venous congestion often can be saved by timely leech therapy. Methods to restimulate sated leeches, particularly emergently, are only poorly described in the nineteenth-century literature. Sated leeches were purged of their blood meals by (1) posterior crop incision, (2) hypertonic saline (3 percent) immersion, (3) gentle finger pressure emesis, or (4) wood ash exposure. Their ability to reattach and refeed with or without serotonin stimulation was evaluated. All 20 leeches (100 percent) purged by posterior crop incision reattached, with 75 percent refeeding. After purging again, 87 percent of these refed leeches reattached, with 46 percent refeeding for a third time. Those leeches which did not initially refeed were exposed to serotonin 10 microM with 100 percent reattaching and 40 percent refeeding. None of the leeches purged by hypertonic saline immersion regurgitation reattached or refed. A single leech purged by finger pressure emesis reattached (20 percent) but did not refeed. After exposure to serotonin, two (40 percent) of each saline and finger pressure group reattached, with neither of the hypertonic saline group refeeding, while both finger pressure-purged leeches refed, consuming a meal 38 percent (+/- 29 percent) of original meal. None of those leeches which refed would reattach or refeed a third time. None of the wood ash-purged leeches reattached or refed even with serotonin exposure. The best method of purging leeches of their blood meals for emergent reuse is by posterior crop incision. Additional refeeding behavior can be achieved by immersion in serotonin 10 microM for 20 minutes.

Analysis of Variance↗