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PubMed1996

Earthworm leukocytes that are not phagocytic and cross-react with several human epitopes can kill human tumor cell lines.

Earthworm coelomocytes (leukocytes) effect cytotoxicity at significantly high levels against the NK-sensitive, human tumor cell line, K562, and the NK-resistant targets (U937, BSM, CEM). By cytofluorimetric analyses using mouse anti-human monoclonal antibodies and by morphological evaluations, two types of coelomocytes were identified: (1) small (8-11 micron) electron-dense cells (SC): CD11a+, CD45RA+, CD45RO+, CDw49b+, CD54+, beta 2-m+ and Thy-1+; (2) large (12-15 micron) electron-lucent cells (LC) that are negative for these markers. Both cell types were negative for other CD and MHC class I and class II markers. SC were active during recognition, rapidly binding to targets; LC were phagocytic. Release of 51Cr revealed rapid, significant, and equal levels of killing at 4 degrees, 20 degrees, and 37 degrees C. We propose that primitive NK-like activity appeared early in evolution.

Animals↗
PubMed1977

Intra- and extraganglionic nerve endings formed by neurosecretory cells of the cerebral ganglion of the earthworm (Lumbricus terrestris L.).

In the cerebral ( = supraesophageal, suprapharyngeal) ganglion of the earthworm, a number of neurosecretory Gomori-positive perikarya are bipolar; others are unipolar, or multipolar. Some of the neurosecretory cell processes project centrally into a fibrous zone; peripheral processes enter small nerves which leave the dorsocaudal aspect of the ganglion. In the central fibrous zone, the neurosecretory fibers form varicose Gomoripositive terminals. Here, also zinc-iodine-osmium (ZIO)-positive fibers and monoamine fluorescent fibers are found. With the electron microscope, nerve terminals containing synaptic vesicles and either large neurosecretory "peptidergic" granular vesicles (diameter more than 1500 A), or smaller granular vesicles (diameter about 1300 A, or 900 A) are observed. These axon endings mainly form axo-dendritic synapses. "Peptidergic" profiles are both pre- and postsynaptic. Some of the extraganglionic "peptideric" fibers appear to terminate around vessels, but most of them form terminals on the visceral muscle cells which surround the ganglion. We think that the central neurosecretory processes communicate with the fibers of the synaptic zone of the ganglion. The peripheral neurosecretory "peptidergic" fibers are supposed to form a primitive neurohemal area and/or to function as vasomotor nerves. The fibers innervating the visceral muscle cells may represent vegetative nerves.

Animals↗
PubMed1979

Elemental analysis of histochemically defined cells in the earthworm Lumbricus terrestris.

A method for preparation of biological specimens for electron probe X-ray microanalysis is described, that aims at retaining the original elemental distribution within the tissue at the cellular level. The tissue is without any chemical fixation, quench-frozen, and 16-micron sections are prepared with a conventional cryomicrotome, transferred to a carbon specimen holder and freeze-dried. Adjacent serial sections, collected on glass slides and stained with various histological procedures, are used to correlate the data obtained by X-ray microanalysis with other histochemical information on the same cell or tissue. To demonstrate the possibilities of the method, sections of the earthworm Lumbricus terrestris were analyzed. In the chloragogenous cells, high concentrations of Ca, Zn and P were found. The inner and outer muscle layer show slightly different properties, both with regard to elemental composition and to myofibrillar ATPase activity.

Animals↗
PubMed1989

The effect of lead incorporation on the elemental composition of earthworm (Annelida, Oligochaeta) chloragosome granules.

The elemental compositions of chloragosome 'granules' in the earthworm Lumbricus rubellus living in non-polluted (Dinas Powys) and heavily Pb-polluted (Wemyss) soils were determined by fully quantitative electron probe X-ray microanalysis. P, Ca, S and Zn were the major elemental components of the chloragosomes. High Pb concentrations were found in chloragosomes of Wemyss animals; Pb was not detected in chloragosomes of Dinas Powys animals. Partial correlation and regression analysis indicated that the in vivo accumulation of Pb by chloragosomes was accompanied by diminished chloragosomal Ca concentrations. Pb is bound by P-containing ligand(s) in the chloragosome matrix. The sequestration of Pb by chloragosomes results in the detoxification of the metal by accumulative immobilization.

Animals↗
PubMed1989

Zinc sequestration by earthworm (Annelida: Oligochaeta) chloragocytes. An in vivo investigation using fully quantitative electron probe X-ray micro-analysis.

The elemental compositions of chloragosome "granules" in the earthworm Lumbricus rubellus living in non-polluted and heavily Zn-polluted soils were determined by fully quantitative electron probe X-ray microanalysis. P, Ca, S and Zn were the major elemental components of the chloragosomes. The in vivo accumulation of Zn by the chloragosomes was accompanied by diminished chloragosomal Ca concentrations. Zn was apparently bound by at least two ligand pools (Pool 1 = uncharacterised; Pool 2 = P-containing ligands, binding approximately 45% and 55% of the Zn, respectively) in the "control" chloragosomes. In Zn-contaminated chloragosomes, most (approximately 70%) was bound by P-containing ligand(s) but some (less than 1%) was also bound by S-containing ligands. It is suggested that the sequestration of Zn in chloragosomes results in the detoxification of the metal by accumulative immobilisation.

Animals↗
PubMed1978

Evidence of the presence of extraperoxisomal catalase in chloragogen cells of the earthworm, Lumbricus terrestris L.

The DAB reactivity of the midintestine of the earthworm, consisting of epithelial layer, muscle layer, and chloragogen tissue, was examined electron microscopically. Besides the mitochondrial membranes of the examined cell types and the hemoglobin content of the blood vessels and chloragogen cells, a considerable DAB reactivity was found in the whole cytosol of the chloragocytes. The DAB reaction of the cytosol was more intensive when incubation medium for catalase, less intensive when incubation medium for peroxidase, was used and did not occur when H2O2 was omitted. Cytosol of the chloragogen cells was isolated and preliminary assay of catalase and peroxidase activities was made. Cytosol samples showed moderate peroxidase activity, but catalase activity measured by the decomposition of hydrogen peroxide showed a very high rate. Catalase and peroxidase activities of the cytosol were heat-sensitive and might have been inhibited by azide and cyanide, respectively. Results prove the assumption that the intensive DAB reactivity of the chloragocyte cytosol is caused by its extraperoxisomal catalase content.

Animals↗
PubMed1982

Electrical coupling and dye transfer between axon segments in the medium giant axon of the earthworm.

Electrical coupling between axon segments has been studied in the medium giant axon, which had been partially isolated from the ventral nerve cord of the earthworm. Evidence has been obtained that in addition to the coupling structures in the septum there are diffusion- and current-pathways through the pseudo-myelin which seem to be more permeable to fluorescein than the channels in the septum. These findings offer an explanation for the discrepancy of the experimentally determined specific resistance of the septum and the expected values based on nexus density and channel size in the septum.

Animals↗
PubMed1978

Cytochemical observations of coelomocytes from the earthworm, Lumbricus terrestris.

Coelomocytes of the earthworm, Lumbricus terrestris, were stained by cytochemical techniques to determine the biochemical composition of the seven different cell types and subtypes. The enzymes acid phosphatase and beta-glucuronidase are present in all types of coelomocytes, but are especially abundant in basophils and neutrophils; the differences in enzyme amounts correlate well with the differences in phagocytic activity of the various cell types. No peroxidase is present. The cytoplasmic basophilia of basophils is due primarily to ribonucleic acid. Basophils also contain large deposits of glycogen, with neutrophils and chloragogen cells containing somewhat lesser amounts. The predominant granules of the two types of acidophils and of granulocytes are composed of a basic protein and a neutral mucopolysaccharide or glycoprotein. A second granule population, present in low numbers in acidophils and granulocytes, but in larger numbers in basophils and neutrophils, is small in size and lipid-positive and may, in part, represent lysosomes. Lipid is especially abundant in the vesicles and granules of the two types of chloragogen cells. Some granules of chloragogen cells also contain ferrous and ferric iron and a substance with pseudoperoxidase activity. The cytoplasm contains protein, glycogen, and a neutral mucopolysaccharide. In addition, acid mucopolysaccharides are variably present in the cytoplasm of chloragogen cells, the only coelomocytes to contain this class of substances.

Acid Phosphatase↗
PubMed1979

The septum of the lateral axon of the earthworm: a thin section and freeze-fracture study.

Septa occur between the axonal segments in the lateral giant septate axon of the nerve cord of the earthworm. This septum is demonstrated here to be permeable to fluorescein and to exhibit a negligible time delay for impulse transmission. Periodic anastomoses between the two lateral axons of the nerve cord are revealed by fluorescein. The permeability of the septum is correlated with the demonstration that nexuses occur along the septum. In thin sections, the nexuses may appear as long septilaminar or pentalaminar membrane appositions, but most frequently appear as a series of short or punctate membrane appositions. In freeze-fracture replicas, the nexuses appear as particles 10-12 nm in diameter on the PF face and as pits on the EF face. The particles and pits are arranged in plaques, in anastomosing strands, or most frequently in small plaques with strands of particles or pits emerging from the periphery. In addition to the nexuses, a junction characterized by the presence of 31 nm diameter hemispherical densities on the cytoplasmic surfaces of the septal membranes is revealed in thin sections. The densities are paired on the adjacent septal membranes, and most frequently are shown by optical diffraction to be arranged on the membrane surfaces in hexagonal or rhomboidal lattices with a centre-to-centre spacing of 34.8 nm. In freeze-fracture replicas, an array of particles and pits with a similar lattice symmetry and spacing to the arrays of hemispherical densities is demonstrated.

Action Potentials↗
PubMed1977

Earthworm coelomocytes in vitro.

Contamination and low viability of earthworm coelemocytes in tissue culture have delayed in vitro studies. Using penicillin, streptomycin, tetracycline and Amphotericin B, Lumbricus terrestis coelomocytes were maintained viable and uncontaminated for 10 days at 15degreesC in medium L-15 supplemented with 5 to 10% fetal bovine serum. The coelomocytes survived for at least 10 days with 85% viability as assessed by trypan blue exclusion assays and phagocytosis of heat-killed yeast. Studies on the thymidine uptake, however, were negative. With the involvement of coelomocytes in tissue graft rejection, in vitro techniques can now be applied to study their capacity in the immune response.

Amphotericin B↗
PubMed1997

Lesion of leukocytes, erythrocytes, and mesothelial cells by the coelomic fluid of Eisenia foetida earthworms.

Coelomic fluid of Eisenia foetida earthworms is known to exert strong proteolytic, hemolytic, bacteriostatic, and cytolytic properties. Ultrastructural observations revealed that coelomic fluid causes multiple ruptures and defects in the erythrocyte membrane as well as in the membrane of murine peritoneal leukocytes. Incubation of peritoneal cells in coelomic fluid resulted in a disorganization of the macrophage surface microvilli, changes in the organization of cytoplasmic organelles and disruption and degranulation of mast cells. Severe mesothelial damage was observed after intraperitoneal administration of the coelomic fluid.

Animals↗
PubMed2001

Effect of earthworm (G-90) extract on formation and lysis of clots originated from venous blood of dogs with cardiopathies and with malignant tumors.

The stability of homeostasis is important to keep a balance between coagulation and fibrinolysis. A disorder of homeostasis leads to different physiological changes and causes different diseases such as cardiopathies and malignant tumors. Cardiopathies is characterized by a hypercoagulation. In the malignant tumors, besides the hypercoagulation due to plasminogen activators (PA) formed inside the tumor, a disorder of homeostasis leads also to acceleration of the fibrinolysis. The variety of internal and external factors in both cases determine the deviation of time for the clots formation, as well as the lyses of blood and fibrin clots. In this study the venous blood as well as the blood and the fibrin clots, derived from healthy dogs, the dogs with cardiopathies and with malignant tumors, were examined for the time of coagulation and fibrinolysis by adding different substances. In these experiments we used a glycolipoprotein extract from earthworm tissue homogenate (G-90) and the proteolytic enzymes P I and P II, isolated from G-90. The efficacy of the tested substances was comparable with the clinically administered anticoagulants. The most significant differences in clotting time among the three tested groups of dogs were obtained by application of the original G-90. The results suggest a possibility that G-90, along with the fibrinolytic enzymes and other biologically active factors, also contains a factor that decelerates the formation of clot in a specific medium, such as the blood from the dogs with malignant tumors.

Animals↗
PubMed2003

A systematic approach for optimisation of supercritical-fluid extraction of polycyclic aromatic hydrocarbons from earthworms.

A systematic approach using a mathematical model as an alternative to time-consuming empirical optimisation of a supercritical-fluid extraction (SFE) procedure is presented. The model was applied to the extraction of 15 polycyclic aromatic hydrocarbons (PAH). The selected fat-containing matrix is the earthworm used in ecotoxicological absorption studies. Settings for optimal recovery were established for the important parameters (temperature, pressure, amount of trapping sorbent, flow, and dynamic extraction time) using a D-optimal experimental design (including quadratic terms and interactions). The recoveries were modelled using a two sigmoid-model with parameters for each of the individual PAH. The main objective was to optimise the conditions for 15 PAH congeners by maximisation of the lowest recovery. The results show that for some parameters, e.g. the amount of sorbent material, optimal conditions are identical for all PAH. For other parameters, e.g. extraction time, the optimum is PAH dependent. The advantage of this optimisation procedure is that, within three days of analysis (73 experiments), optimised extraction conditions for the extraction of the set of 15 PAH were found but also optimum conditions for specific subsets can be extracted from the collected data for specific subsets.

Aluminum Oxide↗
PubMed2003

Assessing the impact of triazine herbicides on organophosphate insecticide toxicity to the earthworm Eisenia fetida.

A standard Organization for Economic Cooperation and Development (OECD) filter paper test was used to assess the acute toxicity of chlorpyrifos, atrazine, cyanazine, and simazine to the earthworm Eisenia fetida. Acute toxicity of chlorpyrifos was also determined in combination with the three-triazine herbicides. Surprisingly, atrazine and cyanazine caused mortality at concentrations lower than chlorpyrifos. Atrazine and cyanazine also increased the toxicity of chlorpyrifos 7.9- and 2.2-fold, respectively. However, simazine caused no toxicity to the worms and did not affect chlorpyrifos toxicity in binary mixture experiments. Possible mechanisms for the greater-than-additive toxicity for the binary combinations of atrazine and cyanazine with chlorpyrifos were investigated, including changes in uptake and biotransformation rates of chlorpyrifos in the presence of atrazine. Uptake of chlorpyrifos into the worms decreased slightly when atrazine was present in the system, therefore eliminating increased uptake as a possible explanation for the increased toxicity. Body residue analysis of worms indicated increased metabolite formation, suggesting the greater-than-additive response may be due to increased biotransformation to more toxic oxon metabolites.

Animals↗
PubMed2006

Evidence for proteins involved in prophenoloxidase cascade Eisenia fetida earthworms.

The prophenoloxidase cascade represents one of the most important defense mechanisms in many invertebrates. Following the recognition of microbial saccharides by pattern recognition molecules, proteinases cleave inactive prophenoloxidase to its active form, phenoloxidase. Phenoloxidase is a key enzyme responsible for the catalysis of the melanization reaction. Final product melanin is involved in wound healing and immune responses. Prophenoloxidase cascade has been widely described in arthropods; data in other invertebrate groups are less frequent. Here we show detectable phenoloxidase activity in 90-kDa fraction of the coelomic fluid of earthworms Eisenia fetida. Amino acid sequencing of peptides from the active fraction revealed a partial homology with invertebrate phenoloxidases and hemocyanins. Moreover, the level of phenoloxidase activity is lower and the activation slower as compared to other invertebrates.

Animals↗
PubMed2003

Hox genes from the earthworm Perionyx excavatus.

The Hox genes of the oligochaete, Perionyx excavatus, were surveyed using PCR and phylogenetic analysis. We were able to identify 11 different Hox gene fragments. Comparative and phylogenetic analyses revealed that this oligochaete would have at least five Hox genes of the anterior group, including three copies of labial-type, five of the central group and one of the posterior group. This is the first report regarding sequence information and phylogenetic analysis of Hox genes in the earthworm.

Animals↗
PubMed2005

Neurochemical characterization of nervous elements innervating the body wall of earthworms (Lumbricus, Eisenia): immunohistochemical and pharmacological studies.

The distribution and chemical neuroanatomy of nervous elements and certain pharmacological-physiological characteristics of the innervation of the body wall in earthworms are described. Solitary sensory bipolar cells can be found among the epithelial cells. These bipolar cells contain serotonin, tyrosine hydroxylase, histamine, gamma-amino-butyric acid (GABA), Eisenia tetradecapeptide, proctolin or rhodopsin in various combinations. In the body wall, the plexus sub-muscularis is composed of nerve fibres only, whereas the plexus sub-epithelialis and muscularis also contain solitary nerve cells. These cells display histamine, GABA or neuropeptide Y immunoreactivity. The fibres of the three plexuses are reactive to serotonin, histamine, Eisenia tetradecapeptide, proctolin, GABA and neuropeptide Y antibodies. FMRFamide-immunoreactive fibres of the plexus muscularis originate from the central nervous system, whereas axons containing the other studied molecules are derived from both peripheral and central structures. High pressure liquid chromatography assays have revealed serotonin, dopamine and histamine in the body wall. Contractions of the body wall musculature can be elicited with serotonin and FMRFamide. Serotonin-evoked contractions are suppressed by the application of GABA. Serotonin acts both directly on the muscle cell receptors and indirectly through initiating transmitter release from the nervous elements, whereas the FMRFamide-induced contractions seem to be mediated through the muscle cell receptors only. The pharmacological profiles of the serotonin and GABA receptors resemble those of the vertebrate 5-HT(3) and GABA(B) receptor types. Our findings indicate that both the sensory and efferent system of the annelid body wall operate by means of a variety of neuroactive compounds, suggesting a complex role of signalling systems in the regulation of this organ.

Animals↗
PubMed1999

Effect of high-level oxygen exposure on the peroxidase activity and the neuromelanin-like pigment content of the nerve net in the earthworm, Lumbricus terrestris.

Histochemical examination of 1-microm tissue sections from the dorsal nerve plexus of the earthworm, Lumbricus terrestris, reveals multiple brown intraneuronal granules. These granules contain material morphologically and histochemically consistent with neuromelanin. When viewed with transmission electron microscopy, these were seen as single membrane-enclosed biphasic granules with diameters of 370-730 nm. Exposure of L. terrestris to high-level environmental oxygen resulted in an increase in the number of neuromelanin-like pigment granules within the neurons of the circular muscle layer. As measured by ortho-phenylenediamine hydrochloride, the endogenous peroxidase activity of extracts from worms incubated in high-level environmental oxygen was 51% more than controls. The endogenous peroxidase activity was localized in situ with 3,3-diaminobenzidine (DAB) and was found to increase in and around the neuromelanin-like pigment-containing neurons within the circular muscle layer. These studies suggest that the nerve net of L. terrestris may serve as a model to study the role of neuromelanin production in oxidative stress and its relationship to endogenous peroxidases.

Animals↗