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PubMed1999

Mechanism of the kainate-induced intracellular acidification in leech Retzius neurons.

We examined the effect of the glutamatergic agonist kainate on the membrane potential, the intracellular Na+ concentration ([Na+]i), the intracellular-free Ca2+ concentration, and on the intracellular pH of Retzius neurons of the medicinal leech, Hirudo medicinalis, in order to investigate the mechanism responsible for the intracellular acidification caused by glutamatergic stimulation. The recordings were made with Na+- and pH-sensitive microelectrodes and iontophoretically injected Fura-2. Bath application of kainate evoked a marked membrane depolarization, a [Na+]i increase, and an intracellular acidification. The intracellular acidification was unaffected by reversal of the electromotive force for H+, suggesting that an influx of H+ from the interstitial space does not contribute to the acidification. While the Ca2+ channel blockers La3+ and Co2+ had no effect on the kainate-induced intracellular acidification, suggesting that a Ca2+ influx via voltage-dependent Ca2+ channels was not relevant, the acidification was reduced in Ca2+-free saline solution. In Na+-free saline solution the kainate-induced intracellular acidification was absent, suggesting the involvement of Na+ influx in generating the acidification. When injected iontophoretically Na+ induced an intracellular acidification but Li+, K+, Rb+ or Cs+ did not. Furthermore, a [Na+]i increase induced by blocking the Na+/K+ pump also led to an intracellular acidification. We conclude that the [Na+]i increase is the crucial event underlying the kainate-induced intracellular acidification. Possible mechanisms linking the [Na+]i increase to the intracellular acidification are discussed.

Acids↗

PubMed1978

Isolation of a collagen fraction from the body-wall glycoproteins of the leech (Hirudo medicinalis), and characterization of its carbohydrate--amino acid portion.

Fractionation of the leech (Hirudo medicinalis) body-wall glycoproteins yielded a collagen fraction containing only D-glucose and D-galactose as its carbohydrate constituents. Digestion of the collagen with trypsin and pronase, and alkaline degradation of the resulting glycopeptides, gave a product that contained a disaccharide linked to hydroxylysine. Mild, acid hydrolysis of the N-acetylated glycopeptides yielded a disaccharide consisting of a D-glucose and a D-galactose residue. Various chemical and enzymic reactions of the disaccharide, the glycosyloxylysine, and the glycopeptide fraction indicated that the disaccharide is 2-O-alpha-D-glucopyranosyl-D-galactose, and that this is beta-glycosidically linked to O-5 of the hydroxylysine residue in the collagen.

Animals↗

PubMed1998

Structural analysis of leech galactocerebrosides using 1D and 2D NMR spectroscopy, gas chromatography-mass spectrometry, and FAB mass spectrometry.

Cerebrosides were isolated from the leech species, Hirudo medicinalis, and purified to homogeneity by silicic acid chromatography, followed by preparative thin-layer chromatography. Their structure was determined by spectroscopic and chemical methods. 1D and 2D 1H NMR spectroscopy, DQF-COSY and HMQC indicated that the head group consists of a single galactose residue in the beta configuration. The galacto configuration was determined by the characteristic chemical shift, the spin-spin splitting and the multiplicity of the characteristic resonance of its equatorial H-4 proton, as well as by the splittings of the other ring protons. GC, GC-MS and fast-atom-bombardment mass spectrometry studies indicated that C24:0 and C22:0 are the major saturated fatty acid species. Unsaturated fatty acids present were C25:2, C27:2, C27:3, C28:3, C29:3, C30:3, C33:3. GC-MS indicated the presence of hydroxylated C27:2 and one other unidentified hydroxylated fatty acid. The cerebroside contained an unusual polyunsaturated sphingosine analogue, namely 2-amino-1,3-dihydroxydocsatriene.

Animals↗

PubMed1989

Animal and vegetal teloplasms mix in the early embryo of the leech, Helobdella triserialis.

In embryos of the glossiphoniid leech, Helobdella triserialis, as in many annelids, cytoplasmic reorganization prior to first cleavage generates distinct animal and vegetal domains of yolk-deficient cytoplasm, called teloplasm. Both domains are sequestered to the D' macromere, progenitor of the definitive segmental tissues, during the first three rounds of cell division. And it has been believed that during the fourth round of cell division, the obliquely equatorial cleavage of macromere D' cleanly segregates animal teloplasm into an ectodermal precursor, cell DNOPQ, and vegetal teloplasm into a mesodermal precursor, cell DM. But here we report a hitherto unobserved cytoplasmic rearrangement between the second and the fourth divisions that seems to mix the animal and vegetal domains of teloplasm. The newly observed rearrangement consists of the movement of vegetal teloplasm toward the animal pole of cell D' between the second and the fourth cell divisions. Animal and vegetal teloplasms form a single pool of teloplasm in cell D' which is then divided between DM and DNOPQ at the fourth division. The movement of teloplasm was inferred by examination of embryos fixed and sectioned between the second and the fourth rounds of cleavage and was confirmed in living embryos microinjected with rhodamine 123, a fluorescent mitochondrial stain.

Animals↗

PubMed2001

Anticoagulants and inhibitors of platelet aggregation derived from leeches.

Increased life expectancy is associated with aging populations in the developed countries, and we can expect an increased incidence of cardiovascular and inflammatory diseases and cancers. A priority for medical research is to reduce such morbidity. Leeches have been demonstrated to be a useful source of drugs to treat cardiovascular diseases, as they have evolved highly specific mechanisms to feed on their hosts by blocking blood coagulation. Powerful molecules acting at different points in the coagulation cascade or in the inhibition of platelet aggregation have been purified from these animals. Moreover, clinical trials confirm their potential to treat cardiovascular diseases.

Animals↗

PubMed1990

Quaternary structure of the giant extracellular hemoglobin of the leech Macrobdella decora.

The molecular dimensions of the extracellular hemoglobin of the leech Macrobdella decora, determined by scanning transmission electron microscopy were 29.8 nm x 19.5 nm (diameter x height) for negatively stained specimens. Measurements of molecular mass (Mm) of unstained specimens with the microscope gave Mm = 3560 +/- 160 kDa. Small-angle X-ray scattering measurements gave a diameter of 28.0(+/- 0.5) nm, radius of gyration 10.5(+/- 0.2) nm and volume 7500(+/- 300) nm3. The hemoglobin had no carbohydrate and its iron content was found to be 0.23(+/- 0.02)% (w/w), corresponding to a minimum Mm of 24,000(+/- 1300) kDa. SDS/polyacrylamide gel electrophoresis of the unreduced hemoglobin showed that it consisted of three subunits, which have apparent Mm values of 12 (1), 25 (2) and 29 kDa (3). The reduced hemoglobin consisted of four subunits, I (12 kDa), II (14 kDa), III (26 kDa) and IV (30 kDa). Subunit 1 corresponded to subunit I, subunit 2 to subunits III and IV and subunit 3 to subunit II. Partial N-terminal sequences were obtained for subunit 1, the two chains of subunit 2 and one of the two chains of subunit 3, suggesting that the hemoglobin consists of at least five different polypeptide chains. The percentage fraction of the three unreduced subunits was determined by densitometry of SDS/polyacrylamide gel patterns and quantitative determination of Coomassie R-250 dye bound to the individual bands in reduced and unreduced patterns to be, monomer (subunit I) : non-reducible subunit (subunit 2) : reducible dimer (subunit 3) = 0.35 : 0.29 : 0.35 (S.D. = +/- 0.05). This corresponded to a stoichiometry of 74 +/- 11 : 37 +/- 5 : 38 +/- 6, assuming the molecular masses to be 17 kDa, 30 kDa and 34 kDa, taking into account the anomalously high mobility of annelid globins in SDS-containing gels. The stoichiometry calculated from the amino acid compositions of the hemoglobin and the three subunits was 82 +/- 12 : 29 +/- 4 : 40 +/- 8. Gel filtration of the hemoglobin at pH 9.8, at neutral pH subsequent to dissociation at pH 4 and at neutral pH in the presence of urea and Gu.HCl provided no evidence for the existence of a putative 1/12 of the whole molecule (Mm approx. 300 kDa). Furthermore, the largest subunits obtained had Mm of 60 to 100 kDa and had a much decreased content of subunit 2, suggesting that the hemoglobin was not a simple multimeric protein. Three-dimensional reconstruction from microscope images provided a model of Macrobdella hemoglobin that is very similar to the reconstruction of Lumbricus hemoglobin: the radial mass distribution curves are virtually superimposable.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗

PubMed1998

Morphological organization of neuropile glial cells in the central nervous system of the medicinal leech (Hirudo medicinalis).

Neuropile glial (NPG) cells in the central nervous system of the medicinal leech, Hirudo medicinalis, were studied by histological, histochemical and immunocytochemical techniques. The NPG cells are often surrounded by electron-dense microglial cells. The central cytoplasm of NPG cells shows a significant zonation. The zone around the nucleus contains mitochondria, glycogen and vesicles. The cytoplasm also contains many ribosomes, a few dictyosomes and distinct inclusions up to 2 microns in diameter. A second zone around the perinuclear region is marked by the occurrence of bundles of intermediate filaments that correspond in thickness to glial filaments of vertebrates. We found a positive reaction with polyclonal antibodies against human glial fibrillary acidic protein (GFAP), and the areas of intense fluorescence correspond to the regions where intermediate filaments were found to be abundant. The peripheral zone contains numerous membrane stacks that could not be contrasted by lanthane nitrate or tannic acid. Therefore, the membrane stacks could be part of an extensive smooth endoplasmic reticulum, which is characteristic of cells with active lipid metabolism.

Animals↗

PubMed1996

Penile replantation using the leech Hirudo medicinalis.

A 37-year-old chronic schizophrenic man underwent penile replantation after complete autoamputation using a kitchen knife. We report the first case of using medicinal leeches to salvage a penile replant after the development of postoperative venous congestion.

Adult↗

PubMed1999

A stable prostacyclin-like substance produced by the medicinal leech Hirudo medicinalis.

The medicinal leech Hirudo medicinalis produces a low-molecular mass compound with properties similar to those of prostacyclin. It extracted with organic solvent, had affinity to 6-keto-PGF1alpha antibodies, inhibited human platelet aggregation induced in vitro by thrombin (by 50% at 4 pg/ml), and caused hypotension and secretion of plasminogen (t-PA) into the blood stream of rats. A main distinction from prostacyclin is stability of the substance due to covalent binding with the polypeptide chain of destabilase. Because of the high aggregability of destabilase, the molecules of the protein-lipid complex are organized into micelles that can change their spatial orientation depending on the nature of the solvent. Incorporation of hirudin and blood plasma kallikrein inhibitor into the micelle structure causes the formation of liposomes (with a molecular mass of the structural monomer 25 kDa). This complex with polypeptides provides not only stability but also rapid transmembrane penetration. The pure prostacyclin-like substance has a molecular mass of 391 Da and can be produced on destruction of the destabilase polypeptide chain.

Animals↗

PubMed2003

NTP, the photoproduct of nifedipine, activates caffeine-sensitive ion channels in leech neurons.

Leech P neurons possess caffeine-sensitive ion channels in intracellular Ca(2+) stores and in the plasma membrane. The following results indicate that these channels are also activated by 2,6-dimethyl-4-(2-nitrosophenyl)-3,5-pyridinedicarboxylic acid dimethyl ester (NTP), the photoproduct of the L-type Ca(2+) channel-blocker nifedipine: (1) Just like caffeine, NTP evoked Ca(2+) influx and intracellular Ca(2+) release, as well as the influx of various other divalent cations and that of Na(+). (2) In the presence of high NTP or caffeine concentrations the plasma membrane channels close, suggesting desensitization of the channel-activating mechanism. (3) Depending on the concentration, NTP and caffeine induce cross-desensitization or act additively. (4) NTP was effective in the same neurons as caffeine (P, N, Leydig, 101), and it was ineffective in neurons in which caffeine was also ineffective (AP, T, L, 8, AE). (5) In Retzius neurons, NTP and caffeine evoked intracellular Ca(2+) release but no Ca(2+) influx. Despite these parallels, the effects of NTP and caffeine were not identical, which may be due to differences in the mechanisms of channel activation or desensitization and/or to substance-specific side effects. The caffeine-sensitive ion channels were activated by NTP concentrations > or =10 microM, which is almost three orders of magnitude smaller than the threshold concentration of caffeine.

Animals↗

PubMed2001

Nonassociative learning in the leech Hirudo medicinalis.

In the present study we examined nonassociative learning of the induction of swimming which was evoked by weak electrical stimulation in the leech Hirudo medicinalis. The behavioural response to stimuli applied repeatedly to the body wall at an inter-trial interval (ITI) of 1 min decreased, eventually ceased, and then recovered spontaneously. More rapid reduction of the behavioural response occurred in repeated training sessions. This decrement of response conformed to the operational definition of habituation. Moreover, a noxious stimulus (i.e. brushing on the skin) facilitated the decremented response (dishabituation). In addition, we compared response decrement in naive animals with decrement in dishabituated and in sensitized animals. The analysis of the best fitting functions of the habituation, the habituation of dishabituation and the habituation of sensitization revealed interesting differences in these processes.

Animals↗

PubMed1999

Differential glycosylation and proteolytical processing of LeechCAM in central and peripheral leech neurons.

LeechCAM is a recently described member of the Ig-superfamily which has five Ig-domains, two FNIII-domains, a transmembrane domain, and a cytoplasmic domain. Phylogenetic analysis indicated that LeechCAM is the leech homolog of apCAM, FasII, and vertebrate NCAM. Using a leechCAM-specific monoclonal antibody we show by immunoblot analysis and by Triton X-114 phase separation experiments that in addition to existing in a transmembrane version LeechCAM is likely to be proteolytically cleaved into a secreted form without the transmembrane domain and the intracellular tail. Furthermore, by immunoprecipitation we demonstrate that LeechCAM is glycosylated with the Laz2-369 glycoepitope, an epitope that has been specifically implicated in regulation of axonal outgrowth and synapse formation.

Animals↗

PubMed1996

Structural characterization of osmoregulator peptides from the brain of the leech Theromyzon tessulatum: IPEPYVWD and IPEPYVWD-amide.

Neurons immunoreactive to an antiserum (a-OT) directed specifically against the C-terminal part (prolyl-leucyl-glycinamide) of vertebrate oxytocin (OT) were detected in the brain of the leech Theromyzon tessulatum. With high pressure gel permeation chromatography followed by reversed-phase HPLC on brain extracts, evidence was given of the presence of three peptides (P1, P2, P3) immunoreactive to a-OT. Results of injection experiments in T. tessulatum and of titrations of each peptide at the different physiological stages of the animals which showed a peak in peptide P1 amount at stage 3B, indicated that P1 is the active OT-like peptide. Using three steps of reversed-phase HPLC, Edman degradation and electrospray mass spectrometry, two sequences for P1 (IPEPYVWD and IPEPYVWD-amide) were found. These peptides differ from peptides to the oxytocin/vasopressin family and are unique in the animal kingdom. Confirmation of their action on the hydric balance and their distribution in the CNS were presented.

Animals↗

PubMed2000

Molecular crosstalk in host-parasite relationships: schistosome- and leech-host interactions.

The host-parasite relationship is based on subtle interplay between parasite survival strategies and host defense mechanisms. In this context, parasites often use the same or similar immune signaling molecules and/or molecular mimicry to escape host immunosurveillance. Both processes represent an adaptive strategy to ensure host immunocompatibility. This bidirectional communication between parasites and their hosts includes the renin-angiotensin, opioid and opiate systems. Here, Michel Salzet, André Capron and George Stefano review recent work on the interaction of common signaling mechanisms in schistosomes, leeches and their host.

Angiotensins↗

PubMed2001

Frequency coding of positional information by an identified neuron, the AP cell, in the leech, Whitmania pigra.

The synaptic connections from pressure-sensitive receptors (P cells) to identified neurons of unknown function (known as anterior pagoda or AP cells) were used to study the way in which leeches process information about the position of a mechanical stimulus on its skin. We elicited spikes in P cells by injecting current intracellularly while recording from AP neurons. The postsynaptic responses consisted of an increase in impulse frequency. We show here that the AP neuron can encode positional information in terms of the frequency of its action potentials. Thus, the AP neuron can serve as an indicator of integrative mechanisms used in the processing of sensory information that is important for the behavior of the animal.

Action Potentials↗

PubMed1997

A leech homolog of twist: evidence for its inheritance as a maternal mRNA.

In the development of leeches such as Helobdella robusta, mesodermal and ectodermal fates segregate to cells DM and DNOPQ, respectively, at fourth cleavage. As one step in identifying genes that may act in mesoderm determination, we have cloned the H. robusta homolog to the Drosophila gene twist. This homolog, designated Hro-twi, exhibits high (> 90%) amino acid identity with other twist-class genes within its basic-helix loop-helix (b-HLH) DNA binding motif and dimerization domain. Like twist, Hro-twi contains CAX-rich stretches: three stretches 5' to the b-HLH and one located 3' of the b-HLH motif. RT-PCR analysis suggests that Hro-twi is present throughout development, beginning as a maternal transcript in the oocyte.

Amino Acid Sequence↗

PubMed1990

Isolation and purification of novel hirudins from the leech Hirudinaria manillensis by high-performance liquid chromatography.

The isolation and purification of novel hirudins from a crude extract of the leech Hirudinaria manillensis and their analytical characterization are reported. Initial purification by gel permeation chromatography on Sephadex G50 and anion-exchange chromatography on Q Sepharose fast-flow removed most contaminants and yielded a highly active extract. Two isohirudins (designated hirudin P6 and P18) were isolated and purified by successive reversed-phase high-performance liquid chromatography on silica-based stationary phases and anion-exchange chromatography on Mono Q. The final products were characterized by reversed-phase high-performance liquid chromatography, 252Cf plasma desorption time-of-flight mass spectrometry and capillary zone electrophoresis. The molecular masses determined by 252Cf plasma desorption mass spectrometry were 7416 dalton for hirudin P6 and 7199 dalton for hirudin P18.

Amino Acids↗

PubMed2006

[An unusual cause of hemoptysis: a leech].

We report an exceptional case of hemoptysis observed in a 38-year-old woman with an uneventful past history. Bronchial fibroscopy demonstrated the presence of a leech attached to the laryngeal orifice. This condition is common in endemic areas but is underdiagnosed. The association of hemoptysis, voice alterations, and difficult respiration are suggestive signs. Extraction should be achieved rapidly to avoid death due to acute respiratory failure.

Adult↗