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PubMed1989

[Effect of medicinal leech preparations on indicators of atherogenesis].

Medicinal leech salivary gland secretion, administered intravenously to rats on an atherogenic diet, reduces fibrinogen level and plasma heparin tolerance and diminishes lipid deposits in the intima of large vessels. Long-term chronic oral administration of dried medicinal leech powder to rats, kept on an atherogenic diet, reduces the area of local edema in the intima of large vessels, although blood lipid composition, fibrinogen level and plasma heparin tolerance remain basically unchanged, as compared to respective values in the control animals. The salivary gland secretion reduces the 3H-thymidine incorporation into the human atherosclerotic intimal cell culture.

Administration, Oral↗
PubMed1989

Antistasin, a leech-derived inhibitor of factor Xa. Kinetic analysis of enzyme inhibition and identification of the reactive site.

Antistasin is a 119-amino acid protein isolated from the salivary glands of the Mexican leech Haementeria officinalis. The determination of the primary structure of antistasin revealed that the protein is highly disulfide-bonded with a 2-fold internal homology. Antistasin exhibits a potent anticoagulant activity purportedly due to the selective inhibition of Factor Xa (Tuszinsky, G. P., Gasic, T. B., and Gasic, G. J. (1987) J. Biol. chem. 262, 9718-9723). In the present study a detailed kinetic analysis of the inhibitory interaction between antistasin and Factor Xa was performed. In addition, the specificity of antistasin was examined by testing its ability to inhibit a variety of serine proteinases. Utilizing purified antistasin and a tripetidyl p-nitroanilide substrate, antistasin was shown to act as a reversible inhibitor of Factor Xa which exhibits slow-tight binding kinetics. Antistasin reacts stoichiometrically with Factor Xa with inhibition displaying a mixed, primarily competitive type. The inhibition is partial in the presence of Ca2+ and becomes complete in the absence of Ca2+. The estimated dissociation constant for the enzyme-inhibitor complex is between 0.31 and 0.62 nM. After binding to Factor Xa, antistasin is cleaved at a single site to yield a modified inhibitor. Automated gas-phase sequence analysis of the modified inhibitor indicates the arginine residue at position 34 in antistasin occupies the P1 position of the reactive site. These data indicate that the leech has evolved a highly selective and potent inhibitor of coagulation Factor Xa that shares several mechanistic similarities with other serine proteinase inhibitors.

Amino Acid Sequence↗
PubMed1986

Evidence for GABA as a neurotransmitter in the leech.

In the leech, Hirudo medicinalis, the inhibitory motor neurons to the longitudinal muscles in the body wall, cells 1 and 2, are linked via central inhibitory synapses to the excitatory motor neurons innervating the same muscles. Examination of these synapses showed that the inhibitors are GABAergic according to several electrophysiological and pharmacological criteria. Presynaptic release of neurotransmitter during passage of depolarizing current into the inhibitors, as well as direct application of GABA to the excitor cell bodies, hyperpolarizes the postsynaptic excitor. Moreover, both synaptic and extrasynaptic GABA receptors of the excitors are specifically blocked by the GABA antagonist bicuculline methiodide. The inhibitors, dissected from the ganglion and grown in culture, synthesize GABA when exposed to the GABA precursor glutamate, whereas the excitors do not synthesize detectable levels of GABA under these same conditions. The innervation and neurotransmitter sensitivity of the longitudinal muscles in the body wall of the glossiphoniid leeches Haementeria ghilianii and H. officinalis were examined in order to determine if the inhibitory neurotransmitter at the neuromuscular junction is GABA. Individual muscle fibers are innervated by both inhibitory and excitatory motor neurons in a manner such that the inhibitory and excitatory nerve terminals and neurotransmitter receptors are spatially and electrically separate. Intracellular recordings taken from the muscle fibers reveal a resting potential of about -70 mV. The amplitude of the spontaneous inhibitory junctional potentials (IJPs) falls to zero at a polarization of about -65 mV and reverses in sign at the normal resting potential.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

Development of neurotransmitter metabolism in embryos of the leech Haementeria ghilianii.

We have investigated the development of neurotransmitter metabolism in embryos of the glossiphoniid leech Haementeria ghilianii. The neurotransmitter content of dissected embryonic tissues was measured by means of radioenzymatic assays, while the presence of neurotransmitters in individual identified neurons was detected by means of immunocytochemical and monoamine histofluorescence techniques. The capacity for synthesis of neurotransmitters was measured by incubating dissected embryonic tissues in radiolabeled neurotransmitter precursors. A specific neurotransmitter uptake system present in some neurons was detected by means of an autoradiographic technique. At an early stage of development of the nervous system, when most neurons are just beginning process outgrowth, the nerve cord acquires the capacity to synthesize ACh, 5-HT, and GABA from their immediate precursors, and contains ACh. Moreover, 5-HT-immunoreactive neurons and neurons that are capable of GABA uptake can be identified. Dopamine-containing neurons are first detected by their histofluorescence at a slightly later stage, after process outgrowth is under way. As development continues, the content of and capacity for synthesis of these neurotransmitters increase, as does the number of neurons capable of GABA uptake. During the earlier stages of development, ACh content exceeds 5-HT content, which in turn exceeds dopamine content. By the end of embryogenesis, however, 5-HT and dopamine contents have greatly increased relative to ACh content, with 5-HT content exceeding ACh content by a factor of 2. Of the neurotransmitters thus far studied, 5-HT is present in the highest amount in the juvenile and adult nerve cord. Our results indicate that in the development of the leech nervous system neurotransmitter metabolism is one of the first neuronal characters to differentiate and that the subsequent levels of the different neurotransmitters are differentially regulated.

Acetylcholine↗
PubMed1988

Embryonic assembly of a complex muscle is directed by a single identified cell in the medicinal leech.

The present study examines the morphological development of a highly organized muscle layer in the leech Hirudo medicinalis, in an effort to characterize those factors that are important in directing its assembly. The tubular body wall of the leech contains 3 major muscle layers that are anatomically distinct: an inner layer of longitudinal muscle, an outer layer of circular muscle, and a grid of oblique muscle sandwiched between them. The oblique muscle layer appears later in development than the other 2 and is preceded by several days by the development of a single, complex cell (here called the comb, or C-cell) whose shape strongly resembles the organization of the oblique muscle grid. There is a bilateral, mirror-image pair of C-cells in each segment. The C-cell has a central, longitudinally oriented soma and projects about 35 fine, parallel processes both medially and laterally at approximately 45 degrees to the long axis. Using a combination of intracellular and antibody labels, it was found that individual muscle cells align themselves with these processes to form correctly oriented fascicles during development. Photoablation of the C-cell at early stages resulted in the complete absence of all oblique muscle fascicles that would have corresponded to that cell; therefore, this discrete muscle-associated cell is considered to be an identified "muscle organizer." Such cellular organizers may direct muscular and neuromuscular assembly in many species.

Animals↗
PubMed1988

[Retraction of processes of remaining cells of the leech nervous system caused by neuron destruction].

Individual mechanosensory neurons in the leech segmental ganglia were eliminated in vivo by intracellular Pronase injection. 7-20 days later Lucifer Yellow was injected into mechanosensory neurons of the same modality in isolated ganglia. There was a clear evidence of retraction of neuronal processes. It is suggested that the leech nervous system structure is not necessarily fixed and can be changed after the death of individual neurons.

Animals↗
PubMed1987

Cell lineage, cell death, and the developmental origin of identified serotonin- and dopamine-containing neurons in the leech.

The nervous system of the glossiphoniid leech includes segmentally iterated neurons that contain serotonin (5-HT) and dopamine. These have been investigated in Helobdella triserialis, Theromyzon rude, and Haementeria ghilianii. Five types of 5-HT neurons are identified by immunocytochemistry in the abdominal ganglia of the ventral nerve cord: the bilaterally paired Retzius, anteromedial, ventrolateral and dorsolateral neurons, and the unpaired posteromedial (pm) neuron. Three types of bilaterally paired dopamine neurons are identified by glyoxylic acid-induced fluorescence in the segmental body wall: MD, LD1, and LD2. Each left or right half of the segmental complement of the leech nervous system is known to develop from 6 distinct ectodermal primary blast cells (ns, nf, o, p, qs, and qf). To identify the blast cells of origin of the 5-HT and dopamine neurons, fluorescent cell lineage tracers were injected into the various precursors of the blast cells in early (stage 6) embryos. The embryos were then raised until their 5-HT and dopamine neurons could be scored (stage 11) for the presence or absence of lineage tracer. We find that the Retzius, anteromedial, and posteromedial 5-HT neurons are derived from the ns blast cell, while the ventrolateral and dorsolateral 5-HT neurons are derived from the nf blast cell. The unpaired pm 5-HT neuron arises as one of a bilateral pair of neurons, of which one later dies. Whether the left or right pm neuron survives in any given ganglion is the consequence of some form of competitive interaction between cells derived from the left and right n primary blast cells, possibly between the left and right pm neurons themselves. We find that, of the dopamine neurons, the LD1 neuron is derived from the o blast cell, the LD2 neurons from the p blast cell, and the MD neuron from one of the 2 kinds of q blast cells. These results show that the 5-HT and dopamine neurons arise from 5 different primary blast cells in a highly determinate manner, and they support the view that cells of a similar phenotype need not be closely related in the developmental cell lineage tree.

Animals↗
PubMed1987

Nasal leech infestation of man.

We present a case in a male patient from Indonesia of nasal leech infestation by Dinobdella ferox that had gone unnoticed for at least three months. The possibility of leech endoparasitism should not be overlooked in people presenting with epistaxis or hemoptysis and a history of recent contact with fresh water lakes or streams in tropical regions.

Child↗
PubMed1986

Regeneration of a distinctive set of axosomatic contacts in the leech central nervous system.

The nociceptive sensory neurons (N cells) in the leech Hirudo medicinalis contact other neurons through conventional synapses in the neuropile and through baskets of processes that wrap the somata of particular cells. These axosomatic contacts are made with the pressure (P) and N sensory neurons in the adjacent segmental ganglia, but not with cells within the same ganglion as the wrapping cell. Physiological evidence indicates that these contacts may be synaptic, although conventional synapses have not been observed with electron microscopy. After they have been injured, lateral N cell processes can grow into the anterior adjacent ganglion and regenerate somatic contacts. In general, regenerated N cell processes wrap the same somata as do intact N cells, but they often wrap fewer somata. However, six of 14 regenerated N cells also wrapped the soma of a small posterior cell that was contacted in only one of 120 normal ganglia examined. It thus appears that the growing processes of an injured N cell select certain cell somata to wrap, but that the selection is somewhat broader than that in the embryonic leech.

Action Potentials↗
PubMed1985

Developmental arborization of sensory neurons in the leech Haementeria ghilianii. II. Experimentally induced variations in the branching pattern.

The sharp, nonoverlapping boundaries of the major and minor receptive fields of the mechanosensory neuron Pv of the leech, as well as the mutual exclusion during embryonic development of growing axon branches belonging to the same Pv cell, have suggested that peripheral axon arborization of these neurons is constrained by a process of neuronal self-avoidance. To provide a direct experimental test of this proposal, the development of the major and minor receptive fields of the Pv neuron was studied in embryos of the leech, Haementeria ghilianii, after surgically preventing or delaying the outgrowth of the axon branches which establish only a minor or only the major field of that neuron. As predicted by the proposal of self-avoidance, interference with the outgrowth of a minor field axon branch resulted in the spread of the major field axon branch into what is normally minor field territory. Conversely, similar interference with the establishment of the major field resulted in the spread of the minor field axon branches into what is normally major field territory. The findings presented here indicate that neuronal self-avoidance does play a significant role in the development of mechanosensory receptive field structure but suggest also that the detailed pattern of arborization of the sensory axons is guided by prespecified pathways of only ephemeral availability or recognizability.

Animals↗
PubMed1985

Pioneering and pathfinding by an identified neuron in the embryonic leech.

Numerous investigations of pathfinding by embryonic neurons, including many leech neurons, have demonstrated that pathfinding is precise. Neurons project the correct number of growth cones which grow to their target areas by making specific choices along the way. However, one leech mechanosensory neuron, the dorsal P (PD) neuron, is unusual in the sense that it initially projects an excessive number of growth cones. One of the growth cones will form the peripheral axon while the others are eliminated. This suggests that PD is one of the earliest neurons to project a peripheral axon, i.e., it may pioneer a peripheral nerve, and that it searches with its multiple growth cones for an external cue which can guide it to its target area. Examination of the early PD axon with light and electron microscopy reveals that it indeed is the first growth cone in its nerve and that it grows in contact with a large non-neuronal (DV) cell until it reaches its target area. The DV cell has a unique morphology and location: a large cell body with thin flat processes extending from the edge of the ganglion to the target area of the PD. It is also present with its unique morphology prior to axonal outgrowth by the PD neuron. These features suggest that the DV cell may be an attractive substrate and/or axonal guidance cue for the PD peripheral axon and therefore for the entire peripheral nerve.

Animals↗
PubMed1977

[Morphologic changes in the axo-dendritic synapses of the leech following excitatory conduction block in a medium with decreased ionic concentration].

Reactive changes in the synaptic apparatus of the leech neuropile were studied in low ionic strength media using supravital methylene blue staining. Special preparation developed in the laboratory permitted obtaining clearly identified axodendritic synapses in the leech. It was shown that the reactive changes were pronounced during a decrease in the electrical conductance and block in the abdominal chain. Certain correlations between the structural alterations in axodendritic synapses and phasic reactive changes in axosomatic synapses were observed.

Animals↗
PubMed1984

Antithrombin, antitrypsin and antichymotrypsin activities of the salivary gland secretion and intestinal chyme of medicinal leeches. Antichymotrypsin activity of partially purified preparations of hirudin and pseudohirudin.

High antithrombin activity was maintained in the salivary gland secretion of starved medicinal leeches (Hirudo medicinalis) despite harvesting of the secretion twice in a month. However, after third or fourth harvesting every month antithrombin activity was no longer detectable. Moreover, in salivary gland secretion high antitrypsin activity was found but no antichymotrypsin activity. On the other hand, intestinal chyme of starved leeches exhibited high antichymotrypsin activity. Low antithrombin and antitrypsin activities were due to contamination by salivary gland secretion. There was a correlation between antichymotrypsin and antithrombin activities of hirudin preparations purified from bdellins and fractionated by isoelectric focusing. Pseudohirudin preparations exhibited neither antithrombin nor antichymotrypsin activities.

Animals↗
PubMedDate not supplied

[Neural control of somatic muscle function in the earthworm, Allobophora longa, and in the leech, Hirudo medicinalis].

Studies have been made on the electrical activity of the segmentary nerves and connectives of the abdominal nervous chain in the earthworm and leech. It was shown that the electrical activity of the isolated piece of the abdominal chain of the leech is manifested of periodic outbursts of impulsation. Presumably this central periodicity accounts for the discharge-like pattern of muscle rhythmic activity which was revealed in our earlier investigations. The electrical activity in the central nervous system of the earthworm depends on afferent influences which pass to the ganglia from the peripheral sensory nervous cells. Stimulation of the abdominal nervous chain did not result in extra discharges of muscle activity, but only affected some of the parameters of the latter.

Afferent Pathways↗
PubMed1983

Formation of the receptive fields of leech mechanosensory neurons during embryonic development.

We have charted the embryonic development of the epidermal receptive fields of the PV and PD mechanosensory neurons present in each segmental ganglion of the ventral nerve cord of the glossiphoniid leech Haementeria ghilianii. The receptive field of either of these neurons is established by several peripheral axons, each of which grows into its own skin territory, so that the receptive field is subdivided into a quiltwork of contiguous, mutually exclusive subfields of various sizes. The largest subfield develops first from the primary peripheral axon of the PD or PV neuron. The PD and PV primary axons grow directly to their respective territories and begin to innervate them at approximately the same time (see also Kuwada, J.Y., and A.P. Kramer (1983) J. Neurosci. 3: 2098-2111). The smaller secondary and minor subfields develop later from the secondary and intersegmental peripheral axons, respectively. These axons, too, grow directly to their appropriate skin territories. The arborizations of the peripheral axons expand until the adult receptive field pattern is established late in embryogenesis; they do not appear to initially overgrow and later trim down to the normal boundaries of the adult receptive field. The receptive field of the PV neuron develops from a stereotyped skeletal branching pattern of the peripheral axons that is elaborated in a regular way to form a grid-like pattern that matches the arrangement of muscle fibers in the body wall. Thus, this branching pattern may be the result of axon growth along prespecified pathways, perhaps delineated by the muscles. Throughout embryonic development, as in the adult leech, axon branches of neighboring homologous P neurons overlap considerably, but separate axon branches of the same neuron are virtually non-overlapping. These observations suggest that developing branches of a neuron are excluded from territory occupied by other branches of the same cell, in contrast to branches belonging to different neurons which can occupy the same territory.

Animals↗
PubMed1983

Sprouting and regeneration of sensory axons after destruction of ensheathing glial cells in the leech central nervous system.

To test the importance of glia during regeneration of mechanosensory neuron axons in the leech central nervous system, individual glial cells that ensheathed the axons were destroyed by intracellular protease injection. Recordings with intracellular microelectrodes showed that glial-desheathed axons re-established synaptic connections with their appropriate target cell, a motor neuron, as frequently and as selectively as control, glial-ensheathed axons. Intracellular staining with horseradish peroxidase showed that desheathed regenerating axons sprouted more than controls, and loss of the glial cell in some cases caused uninjured (intact) axons to sprout. Successful, accurate regeneration could occur whether axons grew along normal or along aberrant pathways. The distal stumps of some sensory axons severed from cell bodies and maintained in organ culture survived without their glial sheath for up to 3 weeks. These experiments show that, although loss of the glial cell affects sensory axon growth, the glial cell is not required for accurate axonal regeneration in the leech central nervous system.

Animals↗
PubMed1983

Embryonic development of the leech nervous system: primary axon outgrowth of identified neurons.

This paper describes the embryonic development of the leech nervous system and focuses on the differentiation of two identified pressure sensory (P) neurons, the PD and PV neurons. In the adult leech the P neurons have distinctive cell body locations in the central nervous system (CNS), different peripheral axon branching patterns, and different receptive field territories in the skin. The embryonic P neurons also have distinct and reproducible locations in the CNS and have been studied with recording and dye-filled microelectrodes from the time the first growth cones are projected from their somata. The peripheral axons of the P neurons are among the earliest peripheral axons to develop and may play an important role in the formation of peripheral nerves. The first or primary peripheral axons of the P neurons grow directly to their separate target territories. The specificity of the P neurons for their targets is probably not due to temporal differences in the outgrowth of their primary axons. Instead, the PD neuron seems to exhibit a preference early in embryogenesis for the target of its primary axon despite an apparent opportunity to occupy the target of the PV primary axon. It is hypothesized that the primary peripheral axons of the P neurons are among the first axons projected from the CNS and follow environmental cues to reach and innervate their target territories.

Animals↗
PubMed1984

[Secretion of the salivary glands of the medicinal leech Hirudo medicinalis inhibits ADP-induced aggregation of human platelets and platelet adhesion to a collagen-coated surface].

As was earlier demonstrated, secretion of medical leech exhibits high antithrombin activity of hirudin and inhibits contact activation of factor XII. Attempts were made to investigate the effects of the leech secretion on (i) the ADP-induced aggregation of platelets in suspension, registered by photometry according to Born, and (ii) platelet adhesion to the calf skin collagen-coated surface measured by scanning electron microscopy. The following results were obtained: 1) the secretion at doses of 0.13 vol % and 0.75 vol % inhibits the ADP-induced aggregation of gel-filtered human platelets by 50 and 97%, respectively; 2) the secretion inhibits the total adhesion and initial attachment of the gel-filtered platelets to the collagen substrate by 60-70% (dose 0.1-1.0 vol %) and 87-88% (dose 10 vol %); 3) platelet spreading on the collagen substrate and adhesion of the platelets from suspension to the upper surface of the spread platelets are inhibited by the secretion as well, but these effects are not statistically significant. The data obtained suggest that the secretion contains components which inhibit both the coagulating and platelet-vascular wall pathways of hemostasis.

Adenosine Diphosphate↗