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PubMed1996

Purification, characterization and biological evaluation of recombinant leech-derived tryptase inhibitor (rLDTI) expressed at high level in the yeast Saccharomyces cerevisiae.

An efficient expression/purification procedure has been developed which allows the production of pure, biologically active recombinant leech-derived tryptase inhibitor (rLDTI), originally found in the leech Hirudo medicinalis. The gene for LDTI was generated synthetically from three overlapping oligonucleotides by PCR synthesis. LDTI was expressed in the yeast Saccharomyces cerevisiae under the control of the copper-inducible CUP1 promoter and fused to the invertase signal sequence (SUC2). The entire expression cassette was inserted into the yeast high-copy vector pDP34. Appropriate host strains transformed with the expression plasmid secreted rLDTI into the medium upon copper addition. Proteinchemical analysis of the secreted rLDTI revealed exclusively inhibitor with the correct N-terminal sequence. Up to 60% of the rLDTI, however, appeared to be modified by glycosylation and the unglycosylated material showed heterogeneity at the C-terminus. Besides full-length rLDTI, truncated rLDTI species lacking either the terminal Asn46 or in addition the penultimate Leu45 were isolated. The C-terminally truncated variants were eliminated using a S. cerevisiae host strain disrupted in the structural genes of carboxypeptidases yscY and ysca, thus identifying these proteases as being responsible for the degradation of rLDTI. Mature rLDTI was purified in high yields from the culture supernatant of the carboxypeptidase-deficient yeast strain by cation-exchange chromatography and reverse-phase HPLC. The recombinant protein is at least 98% pure, based on HPLC and capillary electrophoresis, and is fully biologically active. Structural identity with the authentic leech protein was confirmed by sequence analysis and molecular-mass determination. The purified protein was tested for its ability to inhibit tryptase and trypsin in vitro and to interfere with the tryptase-induced proliferation of human fibroblasts and keratinocytes. Recombinant LDTI appears to be as potent as the authentic leech protein, exhibiting Ki-values of approximately 1.5 nM and approximately 1.6 nM against human tryptase and bovine trypsin, respectively. The tryptase-induced proliferation of human fibroblasts and keratinocytes was inhibited with half-maximum values of approximately 0.1 nM and approximately 1 nM, respectively. The availability of the recombinant material will allow evaluation of the concept of tryptase inhibition in various disease models and to test the therapeutic potential of LDTI in mast-cell-related disorders.

Amino Acid Sequence↗
PubMed1987

The regulation of intracellular pH by identified glial cells and neurones in the central nervous system of the leech.

1. Double-barrelled, neutral-carrier pH-sensitive micro-electrodes were used to measure the intracellular pH (pHi) and the pHi regulation of neuropile glial (n.g.) cells and of identified neurones of the leech Hirudo medicinalis. 2. The distribution of H+ in the n.g. cells and in the neurones was found not to be in electrical equilibrium. The mean pHi of the n.g. cells was 6.87 +/- 0.13 (mean +/- S.D. of mean here and for all following data n = 27) in HEPES-buffered leech saline (pHo = 7.4) and 7.18 +/- 0.19 (n = 13) in 2% CO2-11 mM-HCO3(-)-saline. The mean pHi was 7.28 +/- 0.1 (n = 20) in Retzius neurones and 7.32 +/- 0.15 (n = 12) in noxious neurones in HEPES-buffered leech saline, and 7.20 +/- 0.15 (n = 10) and 7.27 +/- 0.16 (n = 6) in 2% CO2-11 mM-HCO3(-)-buffered saline in these two types of neurones, respectively. 3. The cytoplasmic buffering power, as calculated by the change in pHi following the change from 2% CO2-11 mM-HCO3- to 5% CO2-22 mM-HCO3- in the leech saline, was 20-30 mM/pH unit in the n.g. cells and between 12 and 33 mM/pH unit in the neurones. 4. The recovery of pHi in n.g. cells from an experimentally induced acidification (addition and removal of 20 mM-NH4Cl) was dependent on the presence of external Na+. Independent of the buffer system used, pHi recovery was inhibited when external Na+ was exchanged by N-methyl-D-glucamine. Amiloride (2-3 mM) reduced the rate of pHi recovery by about 50% in these n.g. cells. 5. In CO2-HCO3(-)-free saline, or in the presence of the anion exchange blocker 4-acetamido-4'-isothiocyanostilbene-2, 2'-disulphonic acid (SITS, 0.5 mM), pHi recovery from an acid load was often slowed by up to 50% in n.g. cells. This suggests that there is a significant contribution of a HCO3(-)-dependent membrane transport to pHi regulation in n.g. cells. 6. When a HEPES-buffered saline was exchanged by a 2% CO2-11 mM-HCO3(-)-buffered saline, the pHi of n.g. cells increased by 0.31 pH units. This alkaline shift was reversible upon removal of the CO2-HCO3- and was absent in the Na+-free saline. It was not inhibited by 1 mM-furosemide or by 0.5 mM-SITS.(ABSTRACT TRUNCATED AT 400 WORDS)

4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfo↗
PubMed2006

Culture-independent characterization of the digestive-tract microbiota of the medicinal leech reveals a tripartite symbiosis.

Culture-based studies of the microbial community within the gut of the medicinal leech have typically been focused on various Aeromonas species, which were believed to be the sole symbiont of the leech digestive tract. In this study, analysis of 16S rRNA gene clone libraries confirmed the presence of Aeromonas veronii and revealed a second symbiont, clone PW3, a novel member of the Rikenellaceae, within the crop, a large compartment where ingested blood is stored prior to digestion. The diversity of the bacterial community in the leech intestinum was determined, and additional symbionts were detected, including members of the alpha-, gamma-, and delta-Proteobacteria, Fusobacteria, Firmicutes, and Bacteroidetes. The relative abundances of the clones suggested that A. veronii and the novel clone, PW3, also dominate the intestinum community, while other clones, representing transient organisms, were typically present in low numbers. The identities of these transients varied greatly between individual leeches. Neither time after feeding nor feeding on defibrinated blood caused a change in identity of the dominant members of the microbial communities. Terminal restriction fragment length polymorphism analysis was used to verify that the results from the clone libraries were representative of a larger data set. The presence of a two-member bacterial community in the crop provides a unique opportunity to investigate both symbiont-symbiont and symbiont-host interactions in a natural model of digestive-tract associations.

Animals↗
PubMed2002

Endosymbiotic bacteria in the esophageal organ of glossiphoniid leeches.

We characterized the intracellular symbiotic bacteria of the hematophagous glossiphoniid leeches Placobdelloides siamensis and a Parabdella sp. These leeches have a specialized structure called an "esophageal organ," the cells of which harbor bacterial symbionts. From the esophageal organ of each species, a 1.5-kb eubacterial 16S rRNA gene segment was amplified by PCR, cloned, and sequenced. Diagnostic PCR detected the symbiont in the esophageal organ and intestine. Phylogenetic analysis of the 16S rRNA gene(s) demonstrated that the symbionts from the leeches formed a monophyletic group in a well-defined clade containing endosymbiotic bacteria of plant sap-feeding insects in the gamma-subdivision of the Proteobacteria. The nucleotide compositions of the 16S rRNA gene from the leech symbionts were highly AT biased (53.7%).

AT Rich Sequence↗
PubMed2004

Dynamics and reproducibility of a moderately complex sensory-motor response in the medicinal leech.

Local bending, a motor response caused by mechanical stimulation of the leech skin, has been shown to be remarkably reproducible, in its initial phase, despite the highly variable firing of motoneurons sustaining it. In this work, the reproducibility of local bending was further analyzed by monitoring it over a longer period of time and by using more intact preparations, in which muscle activation in an entire body segment was studied. Our experiments showed that local bending is a moderately complex motor response, composed of a sequence of four different phases, which were consistently identified in all leeches. During each phase, longitudinal and circular muscles in specific areas of the body segment acted synergistically, being co-activated or co-inhibited depending on their position relative to the stimulation site. Onset and duration of the first phase were reproducible across different trials and different animals as a result of the massive co-activation of excitatory motoneurons sustaining it. The other phases were produced by the inhibition of excitatory and activation of inhibitory motoneurons, and also by the intrinsic relaxation dynamics of leech muscles. As a consequence, their duration and relative timing was variable across different preparations, whereas their order of appearance was conserved. These results suggest that, during local bending, the leech neuromuscular system 1) operates a reduction of its available degrees of freedom, by simultaneously recruiting groups of otherwise antagonistic muscles and large populations of motoneurons; and 2) ensures reliability and effectiveness of this escape reflex, by guaranteeing the reproducibility of its crucial initial phase.

Animals↗
PubMed2001

Functional analyses of the leech swim oscillator.

The oscillations that underlie swimming movements in the leech arise from a series of identified concatenated circuits within the ventral nerve cord. In the intact nerve cord, ascending and descending intersegmental interactions via axons within the lateral connectives function both to generate robust oscillations throughout the cord and to establish an anterior-to-posterior phase delay among segmental oscillators. We addressed two questions about this system. First, do the intrasegmental swim circuits in each ganglion function as a single oscillator or do they comprise a pair of coupled oscillators? Second, what are the relative strengths of the ascending and descending intersegmental interactions between the segmental oscillators? Experiments were carried out on semi-intact leeches (Hirudo medicinalis) and on isolated leech nerve cords in which "Z-cut" ganglia were generated by cutting one lateral connective nerve anterior and the contralateral connective nerve posterior to the target ganglion. In these Z-cut ganglia, all rhythmic ascending intersegmental input is conveyed via one lateral connective while rhythmic descending input is conveyed via the contralateral connective. We found that rhythmic bursting recorded from the left and right sides of Z-cut ganglia had identical cycle periods with no phase difference, despite strong intersegmental inputs with differing periods from the two swimming ends of the preparations. We conclude that the swim circuits within individual leech ganglia act as single units. Moreover, we determined through correlation and Fourier spectral analyses, that the functional strengths of ascending and descending intersegmental inputs to Z-cut ganglia located in the middle of the nerve cord are approximately equal.

Animals↗
PubMed2000

Segmentation of the central nervous system in leech.

Central nervous system (CNS) in leech comprises segmentally iterated progeny derived from five embryonic lineages (M, N, O, P and Q). Segmentation of the leech CNS is characterized by the formation of a series of transverse fissures that subdivide initially continuous columns of segmental founder cells in the N lineage into distinct ganglionic primordia. We have examined the relationship between the N lineage cells that separate to form the fissures and lateral ectodermal and mesodermal derivatives by differentially labeling cells with intracellular lineage tracers and antibodies. Although subsets of both lateral ectoderm and muscle fibers contact N lineage cells at or near the time of fissure formation, ablation experiments suggest that these contacts are not required for initiating fissure formation. It appears, therefore, that this aspect of segmentation occurs autonomously within the N lineage. To support this idea, we present evidence that fundamental differences exist between alternating ganglionic precursor cells (nf and ns primary blast cells) within the N lineage. Specifically, ablation of an nf primary blast cell sometimes resulted in the fusion of ipsilateral hemi-ganglia, while ablation of an ns primary blast cell often caused a 'slippage' of blast cells posterior to the lesion. Also, differences in cell behavior were observed in biochemically arrested nf and ns primary blast cells. Collectively, these results lead to a model of segmentation in the leech CNS that is based upon differences in cell adhesion and/or cell motility between the alternating nf and ns primary blast cells. We note that the segmentation processes described here occur well prior to the expression of the leech engrailed-class gene in the N lineage.

Animals↗
PubMed1994

Molecules that become redistributed during regeneration of the leech central nervous system.

A search has been made for molecules other than laminin that change their distribution during axonal regrowth after injury. Two monoclonal antibodies, generated against a protein extract of leech central nervous system (CNS), stain distinct regions of leech CNS and, following lesions, show changes in distribution with time. 1. Monoclonal antibody NP17 stained two bands of M(r) 80 x 10(3) and 60 x 10(3) on Western blots of protein extracted from whole CNS. On cryosections of leech CNS, staining was predominantly within the neuropile. 2. A second antibody, mAb CT16, stained a collagen-associated molecule of the extracellular matrix. It labelled the outer capsule, which surrounds the neuronal cell packets, and the inner capsule, which envelopes the neuropile. In the connectives, CT16 immunoreactivity was restricted to the connective capsule and was not associated with nerve fibres or glia. 3. When the connectives were crushed, immunocytochemical studies revealed changes in distribution of both mAb NP17 and mAb CT16. After 3 days, a time when fibres begin to sprout and form connections, an increase in NP17 immunoreactivity appeared at the site of the lesion. Staining levels remained elevated for several weeks. In contrast, CT16 immunoreactivity did not change for several days after damage. After 10 days, fibre-like staining appeared at the site of the crush; for several weeks it continued to spread throughout the connective. 4. These results show that regeneration of the leech CNS involves the redistribution of at least two molecules. Using monoclonal antibodies, these two molecules, which are situated in distinct regions of the CNS, have been visualized at different stages of the repair process. It has been shown that they alter their distribution at distinct times during regeneration.

Animals↗
PubMed1996

An increase in activity of serotonergic Retzius neurones may not be necessary for the consummatory phase of feeding in the leech Hirudo medicinalis.

During the consummatory phase of feeding, in which blood is ingested, medicinal leeches display a characteristic set of behaviours: they extend their jaws, are less responsive to sensory input, produce mucus, relax the body wall and exhibit waves of peristalsis that can run the length of the body. Earlier reports suggested that this pattern of behaviour is orchestrated by serotonin released from Retzius cells in response to the appropriate sensory stimulation of the lip. We have developed a semi-intact preparation in which only the nervous system in the posterior half of the leech was exposed. The front half of the leech was free to explore, bite through and feed until satiated from a blood-filled sausage casing while continuous intracellular and extracellular recordings were made from identified cells and the nerve roots of the exposed segments. Prior to attachment of the animal to the feeding device, the firing frequency of the Retzius cell increased transiently during spontaneous movements or tactile stimuli to its front or posterior end. In contrast, Retzius cell activity decreased after the anterior sucker attached to the membrane of the feeding device at about the time when ingestion was initiated. Increased activity of Leydig cells, which are known to modulate several circuits in the leech, was also associated with exploration. However, unlike that of Retzius cells, the activity of Leydig cells increased significantly following the onset of consumption. These results suggest that increased activity of Retzius cells in midbody ganglia is not a prerequisite for the consummatory phase of feeding and raises questions regarding the role of serotonin in regulating this behaviour.

Animals↗
PubMed2006

Encoding and decoding touch location in the leech CNS.

Spike times encode stimulus values in many sensory systems, but it is generally unknown whether such temporal variations are decoded (i.e., whether they influence downstream networks that control behavior). In the present study, we directly address this decoding problem by quantifying both sensory encoding and decoding in the leech. By mechanically stimulating the leech body wall while recording from mechanoreceptors, we show that pairs of leech sensory neurons with overlapping receptive fields encode touch location by their relative latencies, number of spikes, and instantaneous firing rates, with relative latency being the most accurate indicator of touch location. We then show that the relative latency and count are decoded by manipulating these variables in sensory neuron pairs while simultaneously monitoring the resulting behavior. Although both variables are important determinants of leech behavior, the decoding mechanisms are more sensitive to changes in relative spike count than changes in relative latency.

Action Potentials↗
PubMed2006

Field evaluation of deet, Repel Care, and three plant based essential oil repellents against mosquitoes, black flies (Diptera: Simuliidae) and land leeches (Arhynchobdellida: Haemadipsidae) in Thailand.

Diethyl methyl benzamide, or deet, a commercial plant-based repellent (Repel Care), and essential ils from 3 species of plants (finger root rhizomes, guava leaves, and turmeric rhizomes), steam distillated and formulated as insect repellents, were evaluated in the field on human volunteers against hematophagous mosquitoes, black flies, and land leeches in Thailand. Field trials were conducted against wild mosquitoes in Bang Bua Thong District, Nonthaburi Province, and in the Thap Lan National Park Headquarters, Nadee District, Pranchinburi Province; anthroophilic black flies (Diptera: Simuliidae) at the Forestry Fire Control Station in Doi Inthanon National Park, Chomthong district, Chiang Mai Province; and land leeches (Arhynchobdellida: Haemadipsidae) in the Khao Yai National Park, Pak Chong District, Nakhon Ratchasima Province. The 3 experimental plant-based essential oil formulations as well as Repel Care and deet provided complete protection from mosquito landing and biting for up to 9 h (duration of the experiment). Similar results were obtained with the 5 products against black flies, providing 100% protection for 9 h but 96-82% protection after 10 and 11 h posttreatment. The 5 repellent products also provided 100% protection against land leeches for at least 8 h. Thi is the 1st report of repellency of plant-based repellents against black flies and land leeches in Thailand. The identification and availability of inexpensive sources of plant-based oils, i.e., finger root rhizomes, guava leaves, and turmeric rhizomes providing long-lasting repellency against blood-sucking organisms are promising leads into commercial production of relatively safe and effective repellents.

Aedes↗
PubMedDate not supplied

[Invasion of fish with leeches (Fam. Piscicolidae) in reservoirs of the Volga river].

It is shown for the first time that during 50 years of the Rybinsk reservoir's existence the abundance of parasite leeches (Piscicolidae) has increased, and the species composition has become richer. In 70 the leech Caspiobdella fadejewi (Epstein) was registered here as a mass species. This species became a real competitor to Piscicola geometra (L.) inhabiting the Volga river in all times. In early '90s the leech Acipenserobdella volgensis (Zykoff) appeared in the Rybinsk reservoir. Till 1987 this species was not found to the north from the Kuibyschev reservoir. An increase in number of fish leeches and an extension of their specific composition occurred not only in the Rybinsk reservoir but in the other reservoirs of the Volga cascade as well.

Animals↗
PubMed2002

Cellular localization of a chromogranin B-like derived peptides in leeches.

Using immunocytochemistry techniques, we demonstrated specific immunostaining with antibodies rose against fragment (547-560) and (614-626) of bovine chromogranin B (CGB) at the level of brain and the tegument of the leech Theromyzon tessulatum. The used of these antibodies on leech sections revealed immunopositive labeling in neurons and glial cells of the central nervous system (CNS), and epidermal glandular cells of the tegument. Co-localization between two antibodies rose against different fragment of bovine CGB have been demonstrated in neurons and glial cells of leech CNS like in vertebrates. Finally, the whole of the data showed for the first time the presence in leeches of CGB like derived peptides.

Animals↗
PubMed2002

Evidence for sequential decision making in the medicinal leech.

Decision making can be a complex task involving a sequence of subdecisions. For example, we decide to pursue a goal (e.g., get something to eat), then decide how to accomplish that goal (e.g., go to a restaurant), and then make a sequence of more specific plans (e.g., which restaurant to go to, how to get there, what to order, etc.). In characterizing the effects of stimulating individual brain neurons in the isolated nervous system of the leech Hirudo medicinalis, we have found evidence that leeches also make decisions sequentially. In this study, we describe a pair of interneurons that elicited locomotory motor programs, either swimming or crawling, in isolated nerve cords. In semi-intact animals, stimulating the same neurons also produced either swimming or crawling, and which behavior was produced could be controlled experimentally by manipulating the depth of saline around the intact part of the leech. These same neurons were excited and fired strongly when swimming or crawling occurred spontaneously or in response to mechanosensory stimulation. We conclude that these brain interneurons help to decide on locomotion (i.e., they are "locomotory command-like neurons") and that the ultimate behavior is determined downstream, in a part of the decision-making hierarchy that monitors stimuli related to the depth of fluid surrounding the leech.

Animals↗
PubMed1992

Leeches to hirulogs and other thrombin-directed antithrombotics.

Leeches have been used for various medicinal purposes since before written history. Bloodletting and leeching declined with the advent of modern medicine. Nevertheless, the European medicinal leech has made a comeback in reattachment and plastic surgery. The antithrombotic substance of this leech is the small protein, hirudin, which has recently been cloned and advanced as an antithrombotic. From speculating how hirudin interacted with thrombin and before knowledge of the crystallographic structures of hirudin-thrombin complexes, the bridge-binding double-ligand concept was born and led to the highly specific thrombin inhibitors of the hirulog class. Like heparin, hirulogs and recombinant hirudins are not orally active but should fill needs where heparin and its derivatives have shortcomings. On the other hand, they most likely will be supplanted by small-molecule thrombin inhibitors when sufficiently specific and nontoxic ones are found. Other approaches to antithrombotic therapy include modulating cellular functions of thrombin. Because thrombin has central bioregulatory functions in thrombosis and hemostasis, as well as wound healing, it is an attractive target for antithrombotic intervention.

Amino Acid Sequence↗
PubMed2005

Leech bite: a rare gynecologic emergency.

Vaginal bleeding as the result of a leech bite is a rare occurrence. We report 2 cases of vaginal bleeding in young girls that resulted from a leech bite and required treatment. Clinical presentation and management for young girls is described. Health professionals working in rural areas where leech infestation is common should be aware that children are at risk for leech bites in the genital region; a high index of suspicion is of great help to make an early diagnosis and ensure prompt treatment.

Animals↗
PubMed1999

[The role of eye-like spots of parasitic leeches (Hirudinea, Piscicolidae) in searching for fish-hosts].

Piscicola geometra (L.), Piscicola respirans Troschel, 1850, and Caspiobdella fadejewi (Epshtein, 1968) find their hosts (fishes) by reacting to their shadow. Eye-like spots whose number ranges from 8 to 16 depending on a species, located on the posterior sucker are the main receptors perceiving the decrease in light intensity and thus enabling an immedaite reaction to the host's shadow. They are distributed on the perimeter of the sucker, are absent only from the section that is constantly shaded by the leech body. Experiments with a simulated shadow reveled considerable differences in the threshold light doses between the studied species: P. geometra - 12 lux, P. resprans - 4 lux and C. fadejewi - 2.5 lux. Below these values the leeches do not perceive the decrease in light intensity and do not make host-searching movements. The smallest decreases in light intensity, at which the leeches still make host-searching movement, are: P. geometra - 5 lux, P. resprans - 2 lux, and C. fadejewi- 1.5 lux. In case of P. respirans and C. fadejewi such small decreases in light intensity (2 - 1.5 lux) may be caused only by small fishes which, according to the results of the experiment, should not be infested. This is confirmed by field observations - small fishes are mostly free from parasites. Large fishes (casting a large shadow) have much more numerous leeches.

Animal Structures↗
PubMed1989

Isolation and structural characterization of a potent inhibitor of coagulation factor Xa from the leech Haementeria ghilianii.

The present work reports the discovery and characterization of an anticoagulant protein in the salivary gland of the giant bloodsucking leech, H. ghilianii, which is a specific and potent inhibitor of coagulation factor Xa. The inhibitor, purified to homogeneity, displayed subnanomolar inhibition of bovine factor Xa and had a molecular weight of approximately 15,000 as deduced by denaturing SDS-PAGE. The amino acid sequence of the first 43 residues of the H. ghilianii derived inhibitor displayed a striking homology to antistasin, the recently described subnanomolar inhibitor of factor Xa isolated from the Mexican leech, H. officinalis. Antisera prepared to antistasin cross-reacted with the H. ghilianii protein in Western Blot analysis. These data indicate that the giant Amazonian leech, H. ghilianii, and the smaller Mexican leech, H. officinalis, have similar proteins which disrupt the normal hemostatic clotting mechanisms in their mammalian host's blood.

Amino Acid Sequence↗