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PubMed2001

Effects of norepinephrine and epinephrine on resting membrane potential in body wall muscle cells of Lumbricus terrestris earthworm.

Norepinephrine and to a lesser extent epinephrine increased the resting membrane potential of earthworm body wall muscle cells. Ouabain, phentolamine, propranolol, and replacement of Ca(2+) with Mg(2+) in the incubation medium abolished this effect. External 3'5'-cAMP in high concentration, dibutyryl cAMP, and dibutyryl cGMP did not induced hyperpolarization of muscle cell membranes. It was concluded that norepinephrine and epinephrine increased the resting membrane potential in earthworm body wall muscle cells via activation of Na(+), K(+) pump. This process involves Ca(2+) and sarcolemmal adrenergic structures, similar to alpha-adrenoceptors in vertebrate, but not the cyclic nucleotide systems.

Adrenergic alpha-Agonists↗
PubMed2001

Effect of cholinergic agonists on resting membrane potential of earthworm body wall muscle cells.

Carbachol in a concentration of 5x10(-8) mol/liter does not hyperpolarize, and in a concentration of 5x10(-6) mol/liter depolarizes the membrane of somatic muscle cell in earthworm. d-tubocurarine, alpha-bungarotoxin, atropine, and hexamethonium added to the incubation medium did not abolish the carbachol-induced decrease in resting membrane potential. Each of these drugs alone had no effect on resting membrane potential in muscle cells. Presumably, the acetylcholine-sensitive receptor-channel membrane complex in earthworm muscle cell differs from acetylcholine receptor in skeletal muscle fibers and peripheral neurons of vertebrates.

Acetylcholine↗
PubMed2002

Calcium mechanism of norepinephrine activation of ionic pump in somatic cells of lumbricus terrestris earthworm muscle wall.

Blockade of norepinephrine-induced hyperpolarization in earthworm muscle cells was observed in a calcium-free medium, after substitution of Ca(2+)with Mn(2+), and in the presence of verapamil or chlorpromazine to the incubation saline. Changes in Ca(2+)concentration in the saline and addition of caffeine had no effect on the resting potential of muscle cells. It was hypothesized that signal transduction from norepinephrine-activated membrane adrenoceptors to the ionic pump in earthworm muscle cells depends on influx of extracellular Ca(2+)and subsequent involvement of Ca(2+)-accepting proteins similar to calmodulin in vertebrates.

Adrenergic alpha-Agonists↗
PubMed2003

Purification, characterization and crystallization of a group of earthworm fibrinolytic enzymes from Eisenia fetida.

Seven fibrinolytic enzymes were purified from the earthworm Eisenia fetida. The molecular weights of the enzymes were 24663, 29516, 29690, 24201, 24170, 23028 and 29595, and the respective isoelectric points were 3.46, 3.5, 3.5, 3.68, 3.62, 3.94 and 3.46. All the proteases showed different fibrinolytic activity on fibrin plates. Studies on substrate specificity and inhibition indicated that they belonged to different types of serine proteases. N-Terminal sequencing indicated their high homology to those from the earthworm Lumbricus rubellus. All the enzymes have been crystallized.

Amino Acid Sequence↗
PubMed2003

An antimicrobial peptide of the earthworm Pheretima tschiliensis: cDNA cloning, expression and immunolocalization.

A cDNA encoding a putative antimicrobial peptide (named PP-1) was obtained using a rapid amplification of cDNA ends from the Asian earthworm, Pheretima tschiliensis. PP-1 showed 77.6% homology with the antimicrobial peptide lumbricin I isolated from the earthworm Lumbricus rubellus. PP-1 lacked an obvious signal peptide sequence. RT-PCR analysis demonstrated that this gene was expressed mainly in the body wall. PP-1 was expressed in Escherichia coli as a fusion protein with a maltoze-binding protein. A polyclonal antiserum was raised in mice using this recombinant fusion protein as antigen. Immunohistochemical studies showed that PP-1 was only in the mucus of the epidermis.

Amino Acid Sequence↗
PubMed2003

Two populations of miniature excitatory synaptic ionic currents in somatic muscle cells of Lumbricus terrestris earthworm body wall.

Three types of miniature excitatory synaptic currents were recorded in the same synaptic region of earthworm muscle cells: monoexponential (tau=1.2 msec) and biexponential (tau(1)=1.2 and tau(2)=8.0 msec). It was hypothesized that earthworm muscle cells contain at least two populations of acetylcholine-sensitive ionic channels, which do not belong to classical nicotinic and muscarinic acetylcholine receptors.

Animals↗
PubMed2004

Earthworms and litter distribution affect plant-defensive chemistry.

Studies on plant-defensive chemistry have mainly focused on plants in direct interaction with aboveground and occasionally belowground herbivores and pathogens. Here we investigate whether decomposers and the spatial distribution of organic residues in soil affect plant-defensive chemistry. Litter concentrated in a patch (vs. homogeneously mixed into the soil) led to an increase in the aucubin content in shoots of Plantago lanceolata. Earthworms increased total phytosterol content of shoots, but only when the litter was mixed homogeneously into the soil. The phytosterol content increased and aphid reproduction decreased with increasing N concentration of the shoots. This study documents for the first time that earthworms and the spatial distribution of litter may change plant-defensive chemistry against herbivores.

Animals↗
PubMed2006

Earthworm cytochrome P450 determination and application as a biomarker for diagnosing PAH exposure.

We developed a new microsome purification method and used the Omura and Sato method to measure the total content of cytochrome (Cyt) P450 in earthworm (Eisenia fetida) microsomes. In method development, two different pretreatments, i.e. solubilization or manual separation were used to purify worm microsomes. Solubilization was more effective than manual separation and difference spectra showed a peak at 450 +/- 1 nm in microsomes received solubilization pretreatment. We conducted a 48 h contact test by exposing worms on pyrene (Py) and benzo[a]pyrene (BaP) spiked filter paper. A dose-response relationship was established between total P450 content and the concentration of Py or BaP ranging from 10(-6) mg mL(-1) to 10(-2) mg mL(-1). Results show that total Cyt P450 content in earthworms is a promising biomarker for diagnosing PAHs exposure at sublethal dose ranges.

Animals↗
PubMed1979

Glutathione S-transferases in earthworms (Lumbricidae).

Glutathione S-transferase activity (EC 2.5.1.18) was demonstrated in six species of earthworms of the family Lumbricidae: Eisenia foetida, Lumbricus terrestris, Lumbricus rebellus, Allolobophora longa, Allolobophora caliginosa and Allolobophora chlorotica. Considerable activity was obtained with 1-chlorl-2,4-dinitrobenzene and low activity with 3,4-dichloro-1-nitrobenzene, but no enzymic reaction was detectable with sulphobromophthalein 1,2-epoxy-3-(p-nitrophenoxy)propane of trans-4-phenylbut-3-en-2-one as substrates. Enzyme prepartations from L. rubellus and A. longa were the most active, whereas A. chlorotica gave the lowest activity. The ratio of the activities obtained with 1-chloro-2,4-dinitrobenzene and 3,4-cichloro-1-nitrobenzene was very different in the various species, but no phylogenetic pattern was evident. Isoelectric focusing gave rise to various activity peaks as measured with 1-chloro-2,4-dinitrobenzene as a substrate, and the activity profiles of the species examined appeared to follow a taxonomic pattern. The activity of Allolobophora had the highest peak in the alkaline region, whereas that of Lumbricus had the highest peak in the acid region. Eisenia showed a very complex activity profile, with the highest peak ne pH 7. As determined by an enzymic assay, all the species contained glutathione, on an average about 0.5 mumol/g wet wt. Conjugation with glutathione catalysed by glutathione S-transferases may consequently be an important detoxification mechanism in earthworms.

Animals↗
PubMed2000

Physical mapping of rDNA genes, (TTAGGG)n telomeric sequence and other karyological features in two earthworms of the family Lumbricidae (Annelida: Oligochaeta).

A cytogenetical study was carried out on the chromosomes and nuclear DNA amounts of the terrestrial earthworms Octodrilus complanatus and Eisenia foetida (Annelida: Oligochaeta: Lumbricidae). Chromosomes were studied using Giemsa staining, banding methods and fluorescent in situ hybridization (FISH) with two repetitive DNA probes [rDNA and (TTAGGG)n]. rDNA FISH and silver staining consistently identified one chromosome pair per spread in both species. The telomeric sequence (TTAGGG)n hybridized with termini of all the chromosomes in both earthworms. Flow cytometry DNA assays showed that O. complanatus and E. foetida had different nuclear DNA contents (2C value=1.72 and=1.40 pg, respectively) but very similar base composition in their genomes.

Animals↗
PubMed1997

Scanning (atomic) force microscopy imaging of earthworm haemoglobin calibrated with spherical colloidal gold particles.

Scanning (atomic) force microscopy (SFM) permits high-resolution imaging of a biological specimen in physiological solutions. Untreated extracellular haemoglobin molecules of the common North American earthworm. Lumbricus terrestris, were imaged in NH4Ac solution using calibrated SFM. Individual molecules and their top and side views were clearly identified and were comparable with the images of the same molecule obtained by scanning transmission electron microscopy (STEM). A central depression, the presumed mouth of the hole, was detected. We analysed 75 individual molecules for their lateral dimensions. Compression varied for different molecules, presumably because of the variation of the interaction between the SFM tip and the protein molecule. Two effective heights which correspond to the heights of the points of the haemoglobin molecules first and last touched by the tip, h1 and h2, respectively, were measured for each protein and ranged between 1.58 and 16.2 nm for h1 and 1.23 and 13.6 nm for h2. The apparent diameter was measured and ranged from 44.9 to 86.6 nm (63.2 +/- 10.5 nm, n = 75), which is about twice the diameter of the molecule reported by STEM for the top view orientation. The higher the measured effective heights, the worse was the tip convolution effect. In order to determine the tip parameters (semivertical angle, curvature of radius and the cut-off height) and to calibrate images of earthworm haemoglobin molecules, spherical gold particles were scanned as standards. The tip sectional radii at distances of h1 and h2 above the tip apex were subtracted from the apparent diameter of the protein. The calibrated lateral dimension was 29.1 +/- 3.85 nm, which is close to the reported scanning transmission electron microscopy data 30.0 +/- 0.8 nm. The results presented here demonstrate that the calibration approach of imaging gold particles is practical and relatively accurate. Calibrated SFM imaging can be applied to the study of other biomacromolecules.

Animals↗
PubMed1999

Biochemical characteristics of Eiseniapore, a pore-forming protein in the coelomic fluid of earthworms.

The cytolytic protein Eiseniapore (38 kDa) from coelomic fluid of the earthworm Eisenia fetida functionally requires sphingomyelin as revealed by using mammalian erythrocytes and phospholipid vesicles. The effects of ions, glycoproteins and phospholipids were investigated for the two-step Eiseniapore action mode, binding and pore formation in different assays. Eiseniapore lysis is activated by thiol groups but inhibited by metal ions. Eiseniapore binding to target membranes is inhibited by Eiseniapore-regulating factor, vitronectin, heparin and lysophosphatidylcholine. Ca2+ and Mg2+ were found to be not necessary for membrane binding or lytic activity. Sphingomyelin was essential for Eiseniapore-induced leakage of liposomes. We describe a cytolytic protein/toxin in Eiseniapore which differs from the established classification; it can be activated by thiol groups and is inhibited by sphingomyelin. Electron microscopy of erythrocyte membranes confirmed ring-shaped structures (pores) with a central channel with outer (10 nm) and inner (3 nm) diameters as shown previously [Lange, S., Nüssler, F., Kauschke, E., Lutsch, G., Cooper, E.L. & Herrmann, A. (1997) J. Biol. Chem. 272, 20 884-20 892] using artificial membranes. Functional evidence of pore formation by Eiseniapore was revealed as protection of lysis by carbohydrates occurred at an effective diameter above 3 nm. From these results, we suggest a plausible explanation for the mechanism by which components of the earthworm's immune system destroy non-self components.

Animals↗
PubMed1997

Interaction of earthworm hemolysin with lipid membranes requires sphingolipids.

Lytic activity in the coelomic fluid of earthworm (Eisenia fetida fetida) has been ascribed to eiseniapore, a hemolytic protein of 38 kDa. Since receptors for eiseniapore on target cell membranes are not known, we used lipid vesicles of various composition to determine whether specific lipids may serve as receptors. Lytic activity of eiseniapore was probed by the relief of fluorescence dequenching from the fluorophore 8-aminonaphthalene-1,3, 6-trisulfonic acid originally incorporated into the vesicle lumen as a complex with p-xylene-bis-pyridinium bromide. Hemolysin binds to and disturbs the lipid bilayer only when distinct sphingolipids consisting of a hydrophilic head group as phosphorylcholine or galactosyl as well as the ceramide backbone, e.g. sphingomyelin, are present. Cholesterol enhances eiseniapore lytic activity toward sphingomyelin-containing vesicles probably due to interaction with sphingomyelin. Leakage of vesicles was most efficient when the lipid composition resembled that of the outer leaflet of human erythrocytes. Presumably, an oligomeric protein pore formed by six monomers is responsible for leakage of sphingomyelin-containing vesicles. The secondary structure of eiseniapore did not change upon binding to lipid membranes. The lytic activity of eiseniapore was completely abolished after its denaturation or after preincubation with polyclonal antibodies. Our results suggest that the presence of specific sphingolipids is sufficient to mediate lytic activity of eiseniapore. This action contributes to our understanding of earthworm immune responses.

Animals↗
PubMed1998

Novel galactose-binding proteins in Annelida. Characterization of 29-kDa tandem repeat-type lectins from the earthworm Lumbricus terrestris.

Novel type lectins were found in the phylum Annelida, i.e. in the earthworm, tubifex, leech, and lugworm. The lectins (29-31 kDa) were extracted from the worms without the use of detergent and purified by affinity chromatography on asialofetuin-agarose. On the basis of the partial primary structures of the earthworm Lumbricus terrestris 29-kDa lectin (EW29), degenerate primers were synthesized for use in the reverse transcriptase-polymerase chain reaction. An amplified 155-base pair fragment was used to screen a cDNA library. Four types of full-length clones were obtained, all of which encoded 260 amino acids, but which were found to differ at 29 nucleotide positions. Since three of them resulted in non-silent substitutions, EW29 mRNA was considered to be a mixture of at least three distinct polynucleotides encoding the following proteins: Ala44-Gln197-Ile213 (clone 5), Gly44-Gln197-Val213 (clone 7), and Ala44-His197-Ile213 (clones 8 and 9; different at the nucleotide level, but encoding an identical polypeptide). Genomic polymerase chain reaction using DNA from a single worm revealed that the single worm already had four sets of cDNAs. The EW29 protein showed two features. First, the lectin was composed of two homologous domains (14,500 Da) showing 27% identity with each other. When each of the domains was separately expressed in Escherichia coli, the C-terminal domain was found to bind to asialofetuin-agarose as strongly as the whole protein, whereas the N-terminal domain did not bind and only retardation was observed. EW29 was found to exist as a monomer under non-denaturing conditions. It had significant hemagglutinating activity, which was inhibited by a wide range of galactose-containing saccharides. Second, EW29 contained multiple short conserved motifs, "Gly-X-X-X-Gln-X-Trp." Similar motifs have been found in many carbohydrate-recognizing proteins from an extensive variety of organisms, e.g. plant lectin ricin B-chain and Clostridium botulinum 33-kDa hemagglutinin. Therefore, these carbohydrate-recognition proteins appear to form a protein superfamily.

Amino Acid Sequence↗
PubMed1998

Identification and cloning of a glucan- and lipopolysaccharide-binding protein from Eisenia foetida earthworm involved in the activation of prophenoloxidase cascade.

Coelomic fluid of Eisenia foetida earthworms contains a 42-kDa protein named coelomic cytolytic factor 1 (CCF-1) that was described previously to be involved in cytolytic, opsonizing, and hemolytic properties of the coelomic fluid. Cloning and sequencing of CCF-1 reveal significant homology with the putative catalytic region of beta-1,3- and beta-1,3-1,4-glucanases. CCF-1 also displays homology with coagulation factor G from Limulus polyphemus and with Gram-negative bacteria-binding protein of Bombyx mori silkworm, two proteins involved in invertebrate defense mechanisms. We show that CCF-1 efficiently binds both beta-1,3-glucan and lipopolysaccharide. Moreover, CCF-1 participates in the activation of prophenoloxidase cascade via recognition of yeast and Gram-negative bacteria cell wall components. These results suggest that the 42-kDa CCF-1 protein of E. foetida coelomic fluid likely plays a role in the protection of earthworms against microbes.

Acute-Phase Proteins↗
PubMed2001

Metal ion trafficking in earthworms. Identification of a cadmium-specific metallothionein.

Exposure to cadmium poses a considerable risk to human health and environmental safety. Earthworms reside in the most contaminated sites on earth, displaying a phenomenal tolerance to toxic heavy metals. They exhibit a distinct metabolic pathway that allows the bio-accumulation of cadmium to yield body burdens in excess of 1/1000th of total dry body weight, a most impressive figure by any standard. However, the precise molecular mechanism underlying this phenomenon remains to be unraveled. This study meets this challenge by fully characterizing the major metal-binding protein in earthworms, namely the two isoforms of metallothionein. Chemical analysis of recombinant protein showed that although both isoforms bind equimolar amounts of cadmium (6 mol), wMT-2 is more stable during proton competition. Furthermore, isoform-specific transcript analysis demonstrated that only wMT-2 is responsive to cadmium in a dose and temporal manner. The specific sequestration of cadmium to wMT-2 protein was confirmed in situ using polyclonal antisera. The latter also provided the means for mapping the cellular and intracellular distribution of metallothionein, thus yielding a holistic insight into its involvement in cadmium transit during absorption, storage, and excretion. The structure-function relationship of wMT-2 and its role in cadmium detoxification through sequestration and compartmentalization is discussed.

Amino Acid Sequence↗
PubMed2002

Immunohistochemical localization of nNOS in the skin and nerve fibers of the earthworm Lumbricus terrestris L. (Annelida Oligochaeta).

The epidermis of the earthworm Lumbricus terrestris is a multifunctional tissue. It is composed of supporting, mucous, neuroendocrine-like, sensory and basal cells. NO is considered to be a molecule that regulates numerous functional activities (also in non-neuronal cells) in vertebrates. In the earthworm epidermis, we found neuronal NO synthase immunopositivity in orthochromatic and metachromatic mucous cells, neuroendocrine-like cells and in epidermal and subepidermal nerve fibers and striated muscle fibers. It is suggested that NO has a multitude of biological actions, affecting functional activities of the epidermis such as tissue homeostasis, control of secretion, proliferation, respiration, defense, water-salt balance, as well as regulation of tonus in vascular and striated muscles.

Animals↗
PubMed2001

Evidence for perforin-like activity associated with earthworm leukocytes.

Earthworm (Eisenia fetida) coelomic fluid contains several leukocytes (coelomocytes): basophils, acidophils and neutrophils as well as chloragocytes. Small coelomocytes and coelomocyte lysate are cytotoxic for the tumor cell target K562. The expression of a lytic factor was investigated by immunocytochemistry using light and transmission electron microscopy. A rat-anti-mouse-perforin-mAb labeled mainly small coelomocytes (nearly 20%) as visualized by light microscopy. TEM analysis using immunogold showed a homogenous labeling in the cytoplasm of small coelomocytes. The highest number of immunogold particles was estimated in coelomocytes with many small cytoplasmic granules. Coelomocytes with large lysosomal granules were also labeled but less intensely. No antibody binding was observed for chloragocytes either in light or electron microscopy. This suggests that the perforin-like activity is associated with only one cell type and that chloragocytes are responsible for other lytic activities. MALDI-MS revealed calreticulin usually associated with perforin in mammalian cells that mediate lysis (e.g. NK, CTL). Together, results strongly suggest the presence of putative perforin in earthworms. This in turn supports the hypothesis that perforin is a conserved component important in immune defense during evolution.

Journal Article↗