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PubMed1994

Structure of leech derived tryptase inhibitor (LDTI-C) in solution.

The three-dimensional solution structure of the leech derived tryptase inhibitor form C (LDTI-C), an inhibitor of 46 amino acids which contains 3 disulfide bridges, has been determined using 2D NMR spectroscopy. The 3D structure was determined on the basis of 262 interresidue interproton distance constraints derived from nuclear Overhauser enhancement measurements and 25 phi angles, supplemented by 3 psi and 15 chi 1 angles. The core of LDTI-C is very well defined and consists of a short 3(10)-helix-loop and a short two-stranded antiparallel beta-sheet between residues 13-14 and 20-21. The N-terminus is fixed to the core by two disulfide bridges, while the C-terminus is connected to the beta-sheet via the third disulfide bridge. The binding loop in LDTI exhibits lowest energy conformations belonging to the canonical conformation of serine proteinase inhibitors.

Amino Acid Sequence↗
PubMed1995

Isolation and structural characterization of enkephalins in the brain of the rhynchobdellid leech Theromyzon tessulatum.

This paper reports the purification of four peptides related to enkephalins from the brain of the leech Theromyzon tessulatum. After reverse-phase HPLC purification, the sequence of the enkephalins (YGGFM, YGGFL, FM, FL) was established by a combination of automated Edman degradation, electrospray mass spectrometry measurement, and co-elution experiments in reverse-phase HPLC with synthetic peptides. ELISA titrations performed on each purified peptide indicated that the major amount was borne by the leucine-enkephalin. The ratio of leucine-enkephalin and methionine-enkephalin of 2:1 is in line with previous immunocytochemical data obtained on T. tessulatum brains. The presence of enkephalins in T. tessulatum, an animal belonging to the oldest group of coelomate metazoans (the Annelida) establishes the very ancient phylogenetic origin of opioids and their conservation in the course of evolution.

Amino Acid Sequence↗
PubMed1996

Enzyme from the medicinal leech (Hirudo medicinalis) that specifically splits endo-epsilon(-gamma-Glu)-Lys isopeptide bonds: cDNA cloning and protein primary structure.

Earlier we detected a novel enzymatic activity in salivary gland secretion of the medicinal leech, splitting isopeptide bonds between the glutamine gamma-carboxamide and lysine epsilon-amino group. This activity is due to destabilase. We described its partial amino acid sequence and sequences to two closely related cDNAs, but none of them perfectly matched the protein isolated. Here we report the isolation and sequence peculiarities of the third cDNA of the family as well as the complete sequence of the destabilase protein. The inferred mature protein product of this cDNA matches the independently determined destabilase protein sequence. It contains 115 amino acid residues including 14 highly conserved Cys residues and is formed from a precursor containing specific leader peptide.

Amino Acid Sequence↗
PubMed1986

[Evidence of apparent vasopressin and oxytocin peptides in the brain of the leech Rhynchobdelle Theromyzon tessulatum (O.F.M.)].

Vasopressin- and oxytocin-immunoreactive cells have been demonstrated in the brain of the leech Theromyzon tessulatum. A mapping of their localization in the different compartments of the brain has been undertaken. The cells immunohistochemically identified have been compared to previously described cell types defined by classical staining methods for neurosecretory material. Preliminary results obtained with high performance liquid chromatography confirm the presence in brain homogenates of substances with chromatographic properties similar to that of vertebrate nonapeptides. The possible role of these vasopressin- and oxytocin-like substances in osmoregulation is discussed.

Animals↗
PubMed1986

A potent factor in extracts of the skin of the Australian frog, Pseudophryne coriacea--II. Stimulation of the leech helical muscle in vitro and in vivo.

Extracts of the skin of Pseudophryne coriacea displayed a powerful stimulant action on the leech helical muscle, both in vitro and in vivo. In the isolated dorsal muscle, the extract caused the appearance of vigorous phasic movements, accompanied by rapid increase in tonus, up to intense spasm. Hyoscine, physostigmine, hexamethonium, tubocurarine and alpha-bungarotoxin did not affect the response to the extract; tetrodotoxin, nifedipine and 5-hydroxytryptamine (5-HT) produced a partial blockade. In the intact animal, the extract at first potently stimulated the musculature, evoking the appearance of a succession of incoordinated, spastic movements, with twisting and rolling of the body of the animal. Stimulation was followed by paralysis and death. It is suggested that the pumiliotoxin-like alkaloid of Pseudophryne coriacea, responsible for these effects, acts directly on the helical muscle.

Alkaloids↗
PubMed1989

Unimpaired RNA synthesis in neurons and epithelial cells in a freshwater leech exposed to the organophosphate insecticide chlorpyrifos.

Chlorpyrifos (Dursban) is an organophosphate insecticide widely used mainly for control of mosquito larvae. This study used the dominant freshwater leech Nephelopsis obscura as a representative aquatic invertebrate to test toxicity of chlorpyrifos on a nontarget species. RNA synthesis in neurons of the cerebral ganglion, epithelial cells of the intestine, and the tegument of small immature (50-70 mg) and large mature (300-400 mg) N. obscura was examined histochemically with acridine orange fluorochrome after exposure to chlorpyrifos in concentrations of 16, 32, 64, 128, and 144 ppb for as long as 6 weeks. The maximum environmental concentration of this insecticide in lake water, when used properly, is 4.25 ppb. There was no mortality, and no behavioral changes were observed in experimental animals, except for transient curling and cutaneous mucus secretion at the highest concentrations of chlorpyrifos. No pathological changes were observed in the intensity of RNA fluorescence or in the distribution of RNA within the cytoplasm of neurons or epithelial cells in any specimens, unlike N. obscura exposed to anoxia, hyperoxia, or salinity. Chlorpyrifos in the concentrations studied does not appear to adversely affect nucleic acid metabolism in N. obscura.

Animals↗
PubMed1994

Mutational analysis of antistasin, an inhibitor of blood coagulation factor Xa derived from the Mexican leech Haementeria officinalis.

Antistasin is a Factor Xa inhibitor that is present in the salivary glands of the Mexican leech Haementeria officinalis. The antistasin protein consists of 119 amino acids, of which residues 1-55 (domain I) are 56% similar to residues 56-110 (domain II). Of the nine C-terminal amino acids (residues 111-119; domain III), four are positively charged. The reactive site for Factor Xa is located in domain I. In this study we assessed the role of separate domains and of individual amino acids in the reactive site for the inhibition of Factor Xa. A series of mutants was constructed and expressed in Chinese hamster ovary (CHO) cells. In vitro chromogenic assays for Factor Xa show that domain I is sufficient for inhibition of Factor Xa. Domains II and III neither contain any intrinsic Factor Xa inhibitory activity, nor contribute to the activity of domain I. Furthermore, domain II does not become a Factor Xa inhibitor by partially adaptating its sequence towards that of the reactive site in domain I. Mutation of the cysteine at position 33 is not crucial for Factor Xa inhibition, suggesting a relatively rigid reactive site loop structure.

Amino Acid Sequence↗
PubMed1992

A potent inhibitor of platelet activating factor from the saliva of the leech Hirudo medicinalis.

Leech saliva is shown to contain protein platelet aggregation inhibitors and a range of selective low molecular weight (LMW) aggregation inhibitors. Gel filtration on Bio-Gel P-2 (cut-off kDa) yields a protein fraction (Fr. I) and three LMW fractions. Fr. I inhibits aggregation induced by collagen, ADP, epinephrine and arachidonic acid. Of all the fractions, only one, Fr. II (LMW) specifically inhibits aggregation induced by platelet activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphorylcholine). Fr. II also inhibits thrombin-induced platelet aggregation. Fr. III inhibits aggregation induced by ADP, epinephrine and arachidonic acid, and Fr. IV only that induced by arachidonic acid. Fr. II also inhibits PAF- and thrombin-induced thromboxane generation in platelets, but does not inhibit arachidonic acid-induced thromboxane generation. Efforts to separate the anti-PAF from the anti-thrombin activity have been unsuccessful. The inhibition may therefore be due to a single inhibitor, though it may also be due to several inhibitors. Fr. II also inhibits superoxide anion production in formyl Met-Leu-Phe (fMLP)- and ionophore 23187- stimulated neutrophils. This may be due to the inhibition of the effects of PAF generated within the cell. Preliminary results suggest that the Fr. II inhibitor(s) is (are) amphipathic. The interaction of platelets with PAF and their interaction with the inhibitor(s) are mutually exclusive, and the inhibition may be competitive.

Animals↗
PubMed1982

Cell clones and segmentation in leech development.

Cell lineage tracer dyes rhodamine-D-peptide and fluorescein-D-peptide were used to study the development of segmentation in embryos of the leech Helobdella triserialis. The earliest overt manifestation of segmentation is in the mesodermal cell layer of the germinal bands, where a repeating pattern of nearly isomorphic mesoblast clusters is seen at an early stage of development. With joint use of both lineage tracers, it is shown that each mesoblast cluster is a clone derived from a single primary mesoblast, as well as being the precursor to an adult mesodermal hemisegment. Not all of the primary mesoblasts give rise to hemisegments, however. Cell clones derived from the first primary mesoblasts produced contribute to the nonmetameric prostomial region, whereas the last primary mesoblasts produced appear to be supernumerary.

Animals↗
PubMed1993

Effect of the tail ganglion on swimming activity in the leech.

In the medicinal leech, Hirudo medicinalis, isolated segmental nerve cords are capable of generating swimming activity. The role played by the head and tail ganglia in regulating the expression of swimming activity by the segmental nerve cord was evaluated by comparing swimming activity in nerve cord preparations with and without the head and tail ganglia attached. Several swim properties were examined, including length of induced swim episodes, ability to initiate swim episodes, swim cycle period, and phase. We found that, in general, the presence of the tail ganglion attached to isolated nerve cords countered the effects produced by the head ganglion on swimming activity. Moreover, we observed that the tail ganglion itself provides excitatory drive to the swim generating system. Thus, the inputs from the head and tail ganglia influence significantly the expression of swimming activity.

Animals↗
PubMed1993

A lower bound on the detectability of nonassociative learning in the local bending reflex of the medicinal leech.

Studies of neural mechanisms of learning and memory have focused on large changes at identified synapses. However, memory in distributed processing reflexes could involve widely distributed engrams characterized by small changes at every synapse in the network. To investigate this possibility, we used a neural network optimization algorithm to construct distributed engrams for nonassociative conditioning in a model of the local bending reflex of the medicinal leech (Hirudo medicinalis). The model comprised 4 sensory neurons, 10 to 40 interneurons, 8 motor neurons, and up to 480 connections. Synaptic connections in the model were first optimized to reproduce the amplitude and time course of motor neuron synaptic potentials recorded during local bending. This network, which represented the naive state before conditioning, was then reoptimized to the habituated or sensitized state. Following reoptimization, the memory for nonassociative learning was encoded by small changes dispersed across the entire network, and each change made only a small contribution to the learning. Moreover, because the changes were small, resolution of a few tenths of a millivolt, or 3-5% of an average synaptic potential, would be needed to account for half of the nonassociative learning. These results show how difficult distributed engrams can be to detect and provide a likely lower bound on the detectability of nonassociative learning in this and related networks.

Animals↗
PubMed1992

Monitoring neuronal activity during discrete behaviors: a crawling, swimming and shortening device for tethered leeches.

An apparatus is described which facilities continuous electrophysiological recordings in segmental ganglia of leeches while the animal performs different behavioral patterns: crawling, swimming or shortening. Both the behaviors and neuronal recordings are monitored by a video camera system and can be evaluated simultaneously. The device is well suited to investigate questions of behavioral choice and neuronal decision-making mechanisms in these animals. It can also be modified to study the neural control of behavior in other aquatic animals, such as snails.

Animals↗
PubMed1995

Maintenance of Fura-2 fluorescence in glial cells and neurons of the leech central nervous system.

Identified glial cells and neurones of the leech central nervous system (CNS) were injected iontophoretically with the calcium indicator dye Fura-2 to measure intracellular Ca2+, while simultaneously recording the membrane potential using a double-barrelled theta-type microelectrode. Both glial cells and neurones responded with Ni(2+)-sensitive Ca2+ transients upon membrane depolarization, indicating Ca2+ influx through voltage-gated Ca2+ channels. In contrast to neurones, the glial cells showed a rapid loss of fluorescence with a half-time of 6.3 +/- 1.8 min (n = 6) after dye injection. Both kinetics and amplitudes of the stimulus-induced Ca2+ transients were affected by this rapid dye loss. The anion exchange inhibitor probenicid (2 mM) significantly reduced, but did not prevent, the loss of Fura-2 fluorescence, suggesting that some dye left the glial cell via an anion exchanger. In order to compensate this fluorescence loss, we injected Fura-2 throughout the experiment. Under this condition, similar Ca2+ transients could be elicited repeatedly for more than 1 h. In Retzius neurones single injections of Fura-2 yielded enough intracellularly trapped dye to allow measurement of intracellular Ca2+ for up to 30 min after the end of injection without large decrease in absolute fluorescence.

Animals↗
PubMed1994

Simultaneous multisite recordings and stimulation of single isolated leech neurons using planar extracellular electrode arrays.

Planar extracellular electrode arrays provide a non-toxic, non-invasive method of making long-term, multisite recordings with moderately high spatial frequency (recording sites per unit area). This paper reports advances in the use of this approach to record from and stimulate single identified leech neurons in vitro. A modified enzyme treatment allowed identified neurons to be extracted with very long processes. Multisite extracellular recordings from the processes of such isolated neurons revealed both the velocity and direction of action potential propagation. Propagation in two cell types examined was from the broken stump towards the cell body (antidromic). This was true for spontaneous action potentials, action potentials produced by injecting current into the cell body and extracellular stimulation of the extracted process via a planar extracellular electrode. These results extend previous findings which have shown that the tip of the broken stump of extracted neurons has a high density of voltage-activated sodium channels. Moreover they demonstrate the applicability of extracellular electrode arrays for recording the electrical excitability of single cells.

Action Potentials↗
PubMed1993

The computational leech.

The local bending reflex of the leech computes a well-defined sensorimotor input-output function in which each of several unique patterns of sensory input elicits a unique pattern of motoneuron activity. Interneurons in the reflex respond to most input patterns and contribute to most motor patterns, suggesting a distributed processing mechanism for the reflex. This suggestion is supported by models in which connection strengths are adjusted by a neural network optimization algorithm to reproduce the local bending input-output function. In addition, computational parallels between the local bending network and the perceptron, a major class of artificial neural networks, brings the functional role of local bending interneurons into question and suggests new physiological experiments.

Animals↗
PubMed1995

Isolation of a renin-like enzyme from the leech Theromyzon tessulatum.

This article reports the purification of a renin-like enzyme (an aspartyl protease) from head parts of the leech Theromyzon tessulatum. After four steps of purification including gel permeation and anion exchange chromatographies followed by reversed-phase HPLC, this enzyme was purified to homogeneity. The renin-like enzyme (of 32 kDa) 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 peptide as products, with a specific activity of 1.35 pmol AI/min/mg (Km 22 microM; Kcat 2.7). The hydrolysis of angiotensinogen is inhibitable at 90% by pepstatin A (IC50 = 4.6 microM), consistent with a renin activity. This is the first biochemical evidence of renin-like enzyme in invertebrates.

Amino Acid Sequence↗
PubMed1989

Ammonia formation in the medicinal leech, Hirudo medicinalis--in vivo and in vitro investigations.

1. The excretion of N compounds was investigated in leeches fed various test solutions. 2. Ingestion was followed by a striking increase of NH3 release exhibiting a characteristic time-course. 3. The NH3 excreted resulted from the degradation of N compounds present in the test solutions. 4. Formation of NH3 from proteins was inhibited by kanamycin, but was unaffected in the case of amino acids. 5. Symbiotic microorganisms do not significantly contribute to NH3 formation. 6. Glutamate dehydrogenase and AMP deaminase are the enzymes most likely to be responsible for NH3 formation in Hirudo.

Amino Acids↗
PubMed1990

Ionic conductances in two types of sensory neurons in the leech, Macrobdella decora.

1. Ion conductances were investigated in two kinds of leech sensory neurons (PM and N1 cells) which differ in their membrane excitability and action potential. 2. In the PM cell body excitable membrane behavior is dominated by only two currents, a sodium current (INa) and a delayed rectifier (IK). 3. In contrast, in the N1 cell INa and IK is supplemented with the presence of a transient potassium current, IA. 4. A comparison between INa and IK in the two cell types did not reveal any significant difference in activation and inactivation kinetics of either current between neurons. 5. Thus, the properties and presence of the A-current in the N1 cell and not in the PM cell may account for the functional difference in excitability of the two kinds of neurons.

Action Potentials↗