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At least 973 records · Page 54Linked to original sources
PubMed2001

Stochastic WNT signaling between nonequivalent cells regulates adhesion but not fate in the two-cell leech embryo.

BACKGROUND: In the leech Helobdella robusta, an annelid worm, the early pattern of cell divisions is stereotyped. The unequal first cleavage yields cells AB and CD, which differ in size, cytoplasmic inheritance, normal fate, and developmental potential. RESULTS: Here we report a dynamic and transcription-independent pattern of WNT signaling in the two-cell stage of H. robusta. Surprisingly, HRO-WNT-A is first expressed in a stochastic manner, such that either AB or CD secretes the protein in each embryo. This stochastic phase is followed by a deterministic phase during which first AB, then CD expresses HRO-WNT-A. When contact between the cells is reduced or eliminated, both AB and CD express HRO-WNT-A simultaneously. Finally, bathing embryos in anti-HRO-WNT-A antibody during first cleavage reduces the adhesion between cells AB and CD. CONCLUSIONS: Our findings show that the stochastic phase of HRO-WNT-A signaling in the two-cell stage of Helobdella is negatively regulated by cell-cell contact and that this early signaling affects cell adhesion without affecting cell fate. We speculate that the primordial function of wnt class genes may have been to regulate cell-cell adhesion and that the nuclear signaling components of the wnt pathway arose later in association with the evolution of diverse cell types.

Animals↗
PubMed2000

RNAi of the receptor tyrosine phosphatase HmLAR2 in a single cell of an intact leech embryo leads to growth-cone collapse.

Receptor protein tyrosine phosphatases (RPTPs) are important for growth-cone migration [1-5], but their specific roles have yet to be defined. Previously, we showed that the growth cones of the Comb cell, an embryonic cell in the leech, express high levels of an RPTP called HmLAR2 [6,7]. Here, we report the use of RNA interference (RNAi) to block expression of HmLAR2 in individual Comb cells in the developing embryo. HmLAR2 mRNA levels were reduced in the soma, processes and growth cones of Comb cells injected with double-stranded RNA (dsRNA) for HmLAR2, but no decrease was detected when control dsRNAs were injected. Consistent with this observation, the level of phosphotyrosine increased significantly in the growth cones of Comb cells injected with HmLAR2 dsRNA. Within 24 hours, the growth cones of treated cells showed a distinct collapsed phenotype, with sharp reductions in lamellipodial surface area and in numbers of filopodia. These experiments indicate a key role for LAR-like RPTPs in maintaining the integrity of the growth cone.

Animals↗
PubMed1999

Presence of invertebrate dystrophin-like products in obliquely striated muscle of the leech, Pontobdella muricata (Annelida, Hirudinea).

Dystrophin is a 427-kDa cytoskeletal protein, which occurs in scant amounts in vertebrate muscle and nerve cells. No previous references to dystrophin or associated proteins in invertebrates at the protein level have been found, while two recent studies investigated the presence of genes encoding proteins homologous to dystrophin in sea urchin and other invertebrates such as Drosophila melanogaster. In this study, the possible presence and distribution of dystrophin-like proteins were studied in different invertebrate muscle cell types and species through Western blot analysis and light and electron microscope immunohistochemistry using a panel of antibodies whose specificities have been determined in vertebrates. Crude protein extracts of leech Pontobdella muricata were analysed by Western blotting. The revealed protein band, with 140 kDa molecular weight, was related to dystrophin, utrophin or dystrophin-related protein-2 (DRP2) according to the specificities of the antibodies used to detect them. The immunofluorescence study showed positive immunoreactions in obliquely striated muscle of this hyrudinean. The immunoelectron microscopy study confirmed specific immunogold labelling beneath the sarcolemma of muscle cells. We thus assume that this protein is an invertebrate dystrophin-like product that is referred to as IDLp140. The potential functions of this invertebrate dystrophin-like protein in invertebrate muscles are discussed relative to previous data in vertebrate tissues.

Animals↗
PubMed2001

Proteinase inhibitors from the medicinal leech Hirudo medicinalis.

The medicinal leech Hirudo medicinalis produces various types of proteinase inhibitors: bdellins (inhibitors of trypsin, plasmin, and acrosin), hirustasin (inhibitor of tissue kallikrein, trypsin, alpha-chymotrypsin, and granulocyte cathepsin G), tryptase inhibitor, eglins (inhibitors of alpha-chymotrypsin, subtilisin, and chymasin and the granulocyte proteinases elastase and cathepsin G), inhibitor of factor Xa, hirudin (thrombin inhibitor), inhibitor of carboxypeptidase, and inhibitor of complement component C1s. This review summarizes data on their primary and tertiary structures, action mechanisms, and biological activities.

Animals↗
PubMed2003

The neurite-stimulating influence of components of medicinal leech salivary gland secretions in organotypic culture of spinal ganglia.

The effects of components from medicinal leech (Hirudo medicinalis) salivary gland secretions and the therapeutic agent Piyavit on the growth of chick embryo neurites in organotypic culture were studied. Native destabilase and bdellin A at concentrations of 0.01, 0.02, 0.05, and 0.1 ng/ml, bdellin B at a concentration of 0.05 ng/ml, and eglin at a concentration of 0.1 ng/ml had neurite-stimulating activity, evident on the third day of organotypic culture of spinal ganglia. The stimulatory activity of destabilase was lost after revere-phase chromatography. The neurite-stimulating activity of the extract of the therapeutic agent Piyavit (200 ng/ml) in organotypic ganglion culture appeared to result from the neurite-stimulating salivary gland components within this agent, suggesting that Piyavit could be used for the treatment of neurodegenerative disorders.

Animals↗
PubMed1989

Function of identified interneurons in the leech elucidated using neural networks trained by back-propagation.

Mechanical stimulation of the body surface of the leech causes a localized withdrawal from dorsal, ventral and lateral stimuli. The pathways from sensory to motor neurons in the reflex include at least one interneuron. We have identified a subset of interneurons contributing to the reflex by intracellular recording, and our analysis of interneuron input and output connections suggests a network in which most interneurons respond to more than one sensory input, most have effects on all motor neurons and in which each form of the behaviour is produced by appropriate and inappropriate effects of many interneurons. To determine whether interneurons of this type can account for the behaviour, or whether additional types are required, model networks were trained by back-propagation to reproduce the physiologically determined input-output function of the reflex. Quantitative comparisons of model and actual connection strengths show that model interneurons are similar to real ones. Consequently, the identified subset of interneurons could control local bending as part of a distributed processing network in which each form of the behaviour is produced by the appropriate and inappropriate effects of many interneurons.

Animals↗
PubMed1998

A neuronal network for computing population vectors in the leech.

The correlation of neuronal activity with sensory input and behavioural output has revealed that information is often encoded in the activity of many neurons across a population, that is, a neural population code is used. The possible algorithms that downstream networks use to read out this population code have been studied by manipulating the activity of a few neurons in a population. We have used this approach to study population coding in a small network underlying the leech local bend, a body bend directed away from a touch stimulus. Because of the small size of this network we are able to monitor and manipulate the complete set of sensory inputs to the network. We show here that the population vector formed by the spike counts of the active mechanosensory neurons is well correlated with bend direction. A model based on the known connectivity of the identified neurons in the local bend network can account for our experimental results, and is suitable for reading out the neural population vector. Thus, for the first time to our knowledge, it is possible to link a proposed algorithm for neural population coding with synaptic and network mechanisms in an experimental system.

Action Potentials↗
PubMed1990

Peripheral organs control central neurogenesis in the leech.

Interactions between developing nerve centres and peripheral targets are known to affect neuronal survival and thus regulate the adult number of neurons in many systems. Here we provide evidence that peripheral tissues can also influence cell numbers by stimulating the production of neurons. In the leech Hirudo medicinalis, there is a population of several hundred neurons that is found only in the two segmental ganglia that innervate the genitalia and which seem to be added gradually during post-embryonic maturation. By monitoring 5-bromo-2'-deoxyuridine incorporation immunohistochemically, we have now determined that these neurons are actually born late in embryogenesis, well after all other central neurons are born and after efferent and afferent projections are established between these ganglia and the periphery. Ablation of the male genitalia early in embryogenesis, or evulsion of the nerves that connect them to the ganglia, prevent the birth of these neurons. However, they fail to appear ectopically when male genitalia are transplanted to other segments, despite innervation by local ganglia. We conclude that the generation of the late-appearing neurons depends on a highly localized signal produced by the male genitalia, to which only the ganglia that normally innervate these organs have the capacity to respond.

Animals↗
PubMed1990

Intrinsic segmental identity of segmental founder cells of the leech embryo.

Segmentation occurs in several animal phyla, and the cellular mechanisms generating this structural periodicity vary considerably. In the leech, an annelid worm, segmental founder cells arise through a fixed cell lineage (Fig. 1), and come together in a longitudinally repeating array through a stereotyped pattern of morphogenesis. In this paper we demonstrate that founder cells forced to differentiate in a foreign segmental environment give rise to their normal, segment-specific clones of neuronal descendants, even in segments in which those neuronal phenotypes would not normally be observed. These findings indicate that the individual founder cells possess segmental identity at or shortly after the time of their birth, and further suggest that such identities are established by a mechanism in which the parent stem cell 'counts' mitotic cycles.

Animals↗
PubMed1984

Kinetics of the inhibition of human leucocyte elastase by eglin from the leech Hirudo medicinalis.

The rate constants for the inhibition of human leucocyte elastase by eglin from the leech Hirudo medicinalis were determined by using a pre-steady-state kinetic approach. kon and koff for complex-formation and dissociation were 1 X 10(6)M-1 X S-1 and 8 X 10(-4)S-1 respectively. Ki was calculated as the ratio koff/kon = 8 X 10(-10)M, the binding of eglin to elastase was reversible and the inhibition mechanism was of the fully competitive type. The mechanistic properties of the system and the biological significance of the rate constants are discussed.

Animals↗
PubMed1992

Site-directed mutagenesis of the leech-derived factor Xa inhibitor antistasin. Probing of the reactive site.

Antistasin (ATS) is a leech-derived 119-amino-acid protein which exhibits potent and highly selective inhibition of coagulation Factor Xa. It inhibits Factor Xa according to a common mechanism of serine-proteinase inhibitors in which a conformationally rigid substrate-like reactive site is presented to the enzyme. In this study a recombinant version of ATS was expressed and purified utilizing a yeast expression system in order to probe the reactive site P1 (Arg-34) and P1' (Val-35) residues by site-directed mutagenesis. The results demonstrate the requirement for a positively charged residue in the P1 position of ATS, with an arginine residue preferred over a lysine, yielding K1 values of 61 pM and 1.28 nM respectively. Mutation of the P1 arginine residue to the non-polar amino acid leucine abolished its inhibitory potency toward Factor Xa. The role of the C-terminal domain of ATS, which shares significant amino acid sequence identity with the N-terminal domain, was investigated by creating a second reactive site in the corresponding position of the C-terminal domain. The inhibitory activity of this mutant demonstrated that the C-terminal domain of ATS is not folded into the proper conformation necessary to create a functional inhibitory domain.

Amino Acid Sequence↗
PubMed1995

Solubilization, molecular forms, purification and substrate specificity of two acetylcholinesterases in the medicinal leech (Hirudo medicinalis).

Two acetylcholinesterases (AChE) differing in substrate and inhibitor specificities have been characterized in the medical leech (Hirudo medicinalis). A 'spontaneously-soluble' portion of AChE activity (SS-AChE) was recovered from haemolymph and from tissues dilacerated in low-salt buffer. A second portion of AChE activity was obtained after extraction of tissues in low-salt buffer alone or containing 1% Triton X-100 [detergent-soluble (DS-) AChE). Both enzymes were purified to homogeneity by affinity chromatography on edrophonium- and concanavalin A-Sepharose columns. Denaturing SDS/PAGE under reducing conditions gave one band at 30 kDa for purified SS-AChE and 66 kDa for DS-AChE. Sephadex G-200 chromatography indicated a molecular mass of 66 kDa for native SS-AChE and of 130 kDa for DS-AChE. SS-AChE showed a single peak sedimenting at 5.0 S in sucrose gradients with or without Triton X-100, suggesting that it was a hydrophylic monomer (G1). DS-AChE sedimented as a single 6.1-6.5 S peak in the presence of Triton X-100 and aggregated in the absence of detergent. A treatment with phosphatidylinositol-specific phospholipase C suppressed aggregation and gave a 7 S peak. DS-AChE was thus an amphiphilic glycolipid-anchored dimer. Substrate specificities were studied using p-nitrophenyl esters (acetate, propionate and butyrate) and corresponding thiocholine esters as substrates. SS-AChE displayed only limited variations in Km values with charged and uncharged substrates, suggesting a reduced influence of electrostatic interactions in the enzyme substrate affinity. By contrast, DS-AChE displayed higher Km values with uncharged than with charged substrates. SS-AChE was more sensitive to eserine and di-isopropyl fluorophosphate (IC50 5 x 10(-8) and 10(-8) M respectively) than DS-AChE (5 x 10(-7) and 5 x 10(-5) M.

Acetylcholinesterase↗
PubMed2001

Stretch-activated cation channels of leech neurons exhibit two activity modes.

Single-channel recordings were used to characterize two activity modes of stretch activated channels (SACs) in identified neurons of the leech. Clear-cut differences in the activity pattern of SACs from freshly desheathed cell bodies and from cultured AP cells were observed. SACs of inside-out patches, made by 'gentle' sealing and excised from cell bodies of freshly desheathed ganglia exhibited spike-like (SL) activity, with a mean channel open time (MCOT) shorter than 10 ms. Fitting of dwell open-time distributions revealed time constants shorter than 2 and 10 ms. This activity was characterized by a chord conductance of about 115 pS. SACs from cultured cells often displayed activity just after excision. MCOT exceeded 200 ms and the time constants of open-time interval distributions were longer than 10 and 100 ms. Furthermore, this activity pattern was characterized by both sub- (about 80 and 40 pS) and super-conductance (150 pS) levels, hence denoted as multiconductance (MC) mode. The percentage of open time spent at the main subconductance level (80 pS) was significantly higher in patches isolated from growth cones than in those from cell bodies of cultured neurons. The two activity modes (SL and MC) should belong to the same channel because both modes have a common main conductance value and exhibit outward rectification, stretch sensitivity and blockage by Gd3+ and gentamicin. Cytochalasin D applied to the cytoplasmic side induced activation of SACs or increased their ongoing activity. Thus, the observed differences in the expression of the two activity modes of SACs might be associated with different arrangements of the cortical cytoskeleton.

Animals↗
PubMed1998

Identification of Man alpha1-3Man alpha1-2Man and Man-linked phosphate on O-mannosylated recombinant leech-derived tryptase inhibitor produced by Saccharomyces cerevisiae and determination of the solution conformation of the mannosylated polypeptide.

The production of recombinant leech-derived tryptase inhibitor (rLDTI) by two different strains of Saccharomyces cerevisiae resulted in the secretion of non-glycosylated and glycosylated rLTDI. Monosaccharide analysis and a-mannosidase treatment demonstrated that glycosylated rLDTI was exclusively alpha-mannosylated. A trypsin digest of reduced and S-carboxymethylated glycosylated rLDTI was separated on a reverse-phase HPLC column. Glycopeptides identified by a combination of matrix-assisted laser desorption mass spectrometry, amino acid sequence analysis, and monosaccharide analysis revealed the presence of different glycoforms. It was found that Ser24, Ser33 and Ser36 were partially glycosylated with a single mannose residue, whereas Thr42 in glycosylated rLDTI from both strains was fully occupied with manno-oligosaccharides with a degree of polymerization ranging over 1-3 and 1-13 depending on the yeast strain. In phosphorylated rLDTI a single phosphate group was predominantly located at the innermost Man residue of units of mannobiose, mannotriose, mannotetraose and mannopentaose at Thr42. Oligosaccharides released by alkaline treatment were reduced by sodium borohydride and separated by high-pH anion-exchange chromatography on a CarboPac MA1 column, and analyzed by one- and two-dimensional 1H-NMR spectroscopy. Besides the major oligosaccharide Man alpha1-2Man-ol, the (for yeast protein O-glycosylation) unusual Man alpha1-3Man alpha1-2Man-ol was determined. The solution conformation of glycosylated rLDTI was investigated by two-dimensional NMR spectroscopy. Structure calculations by means of distance geometry showed that glycosylated rLDTI is compactly folded and contained small secondary structure elements. Analysis of the chemical shifts showed that amino acids Val32-Ser33, Ser36-Ser39 and Thr42 were affected by the O-mannosylation. In addition, changes in chemical shift were observed within the beta-hairpin peptide regions Val13-Ser16 and Gly18-Tyr21 attributed to direct interactions of the mannose residue at Ser36. Furthermore, the protein-linked oligosaccharides were spatially grouped in a position opposite of the canonical binding loop.

Amino Acid Sequence↗
PubMedDate not supplied

Holomeric vs. meromeric segmentation: a tale of centipedes, leeches, and rhombomeres.

Explaining the origin and evolution of segmentation is central to understanding the body plan of major animal groups such as arthropods, annelids, and vertebrates. One major shortcoming of current views on segmentation is the failure to recognize the existence of two layers of segmentation. I distinguish here holomeric segmentation, involving the whole body axis (or the whole axis of an appendage) and producing "true" segments (eosegments); and meromeric segmentation, producing merosegments within one or more eosegment(s). In terms of developmental mechanisms, meromeric segmentation is probably the same as compartmentalization. This process follows two rules: (1) merosegments are formed from a stereotyped pattern of subdivisions, where only the merosegments in contact to the anterior or posterior boundary of the eosegment are allowed to divide; (2) contiguous eosegments undergoing meromeric segmentation generate merosegments according to identical lineage patterns apart from possible lineage truncation in one or a few terminal eosegments. The segmentation model proposed in this paper is mainly supported by evidence from comparative morphology, but it is compatible with known cellular and developmental mechanisms. The development of vertebrate rhombomeres, the annulation of leeches, the subdivision of the distal part of insect antenna into flagellomeres and the segmentation of centipedes are interpreted here in terms of meromeric segmentation. Some of these phenomena, like centipede segmentation, have thus far defied all attempts at an explanation, both in mechanistic (developmental) and phylogenetic terms. The model presented in this paper suggests a rich research agenda at all levels, from molecular and genetic to morphological and phylogenetic.

Animals↗
PubMedDate not supplied

A redescription of Trypanosoma cotti Brumpt and Lebailly, 1904 and its development in the leech, Calliobdella punctata.

Trypanosoma cotti Brumpt and Lebailly, 1904 is redescribed from Brumpt's blood slides prepared from the sea scorpion (Enophrys bubalis). Developmental stages occur in the intestine of the marine leech Calliobdella punctata, which acts as the vector. Division occurs only in the amastigote stage which subsequently changes into a sphaeromastigote and epimastigote forms. Yellow pigment granules characterize all developmental stages except slender epimastigotes. No metatrypanosomes were observed.

Animals↗
PubMed2002

Calcium influx into dendrites of the leech Retzius neuron evoked by 5-hydroxytryptamine.

5-Hydroxytryptamine (5-HT) is a ubiquitous neurotransmitter and neuromodulator that affects neural circuits and behaviours in vertebrates and invertebrates. In the present study, we have investigated 5-HT-induced Ca(2+) transients in subcellular compartments of Retzius neurons in the leech central nervous system using confocal laser scanning microscopy, and studied the effect of 5-HT on the electrical coupling between the Retzius neurons. Bath application of 5-HT (50mM) induced a Ca(2+) transient in axon, dendrites and cell body of the Retzius neuron. This Ca(2+) transient was significantly faster and larger in dendrites than in axon and cell body, and was half-maximal at a 5-HT concentration of 5-12mM. The Ca(2+) transient was suppressed in the absence of extracellular Ca(2+) and by methysergide (100mM), a non-specific antagonist of metabotropic 5-HT receptors, and was strongly reduced by bath application of the Ca(2+) channel blocker Co(2+) (2mM). Injection of the non-hydrolysable GTP analogue GTPgammaS increased and prolonged the dendritic 5-HT-induced Ca(2+) transient. The non-selective protein kinase inhibitor H7 (100mM) and the adenylate cyclase inhibitor SQ22536 (500 mM) did not affect the Ca(2+) transient, and the membrane-permeable cAMP analogue dibutyryl-cAMP (500 mM) did not mimic the effect of 5-HT application. 5-HT reduced the apparent electrical coupling between the two Retzius neurons, whereas suppression of the Ca(2+) influx by removal of external Ca(2+) improved the transmission of action potentials at the electrical synapses which are located between the dendrites of the adjacent Retzius neurons. The results indicate that 5-HT induces a Ca(2+) influx through calcium channels located primarily in the dendrites, and presumably activated by a G protein-coupled 5-HT receptor. The dendritic Ca(2+) increase appears to modulate the excitability of, and the synchronization between, the two Retzius neurons.

Animals↗