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Immunocytochemical electron microscopic study and Western blot analysis of caldesmon and calponin in striated muscle of the fruit fly Drosophila melanogaster and in several muscle cell types of the earthworm Eisenia foetida.

Caldesmon and calponin are two proteins that are characteristic of vertebrate smooth muscle. In invertebrates, caldesmon has only been studied in some molluscan muscles, and no previous references to calponin have been found. The aim of this paper was to investigate the presence and distribution of caldesmon and calponin in several invertebrate muscle cell types, classified according to their ultrastructural pattern: transversely striated muscle (flight muscle from Drosophila melanogaster), obliquely striated muscle (muscular body wall and inner muscular layer of the pseudoheart from the earthworm Eisenia foetida), and a muscle of doubtful classification which seems to be intermediate between smooth muscle and obliquely striated muscle (outer muscular layer of the pseudoheart, from E. foetida), using electron microscopy immunocytochemistry and Western blot analysis. Immunoreactions to both caldesmon and calponin were observed in the outer muscular layer cells from the earthworm pseudoheart but neither in the transversely striated muscle of D. melanogaster nor in the obliquely striated muscle from the earthworm. Present findings suggest that caldesmon- and calponin-like proteins are also present in invertebrate muscle cells, but only in those that are ultrastructurally similar to the vertebrate smooth muscle cells. Since discrepancies in the classification of some invertebrate muscles are common in the literature, the use of distinctive markers, such as troponin, caldesmon and calponin may improve our understanding of the nature and properties of many invertebrate muscles showing an ultrastructural pattern that does not resemble any of the classic muscle types.

Animals↗

Some features of the submicroscopic morphology of synapses in frog and earthworm.

Electron micrographs are presented of synaptic regions encountered in sections of frog sympathetic ganglia and earthworm nerve cord neuropile. Pre- and postsynaptic neuronal elements each appear to have a membrane 70 to 100 A thick, separated from each other over the synaptic area by an intermembranal space 100 to 150 A across. A granular or vesicular component, here designated the synaptic vesicles, is encountered on the presynaptic side of the synapse and consists of numerous oval or spherical bodies 200 to 500 A in diameter, with dense circumferences and lighter centers. Synaptic vesicles are encountered in close relationship to the synaptic membrane. In the earthworm neuropile elongated vesicles are found extending through perforations or gaps in the presynaptic membrane, with portions of vesicles appearing in the intermembranal space. Mitochondria are encountered in the vicinity of the synapse, and in the frog, a submicroscopic filamentary component can be seen in the presynaptic member extending up to the region where the vesicles are found, but terminating short of the synapse itself.

Animals↗

Structural changes at cut ends of earthworm giant axons in the interval between dye barrier formation and neuritic outgrowth.

We describe structural changes at the cut ends of invertebrate myelinated earthworm giant axons beginning with the formation of a dye barrier (15 minutes posttransection or postcalcium addition) and ending with the formation of a neuritic outgrowth (2-10 days posttransection). The morphology of the cut end, and the location and morphological configuration of the dye barrier, were assessed by time-lapse confocal, fluorescence microscopy and by electron microscopy. During the interval from 15 to 35 minutes postcalcium addition, the dye barrier continuously migrated away from a cut axonal end; the dye barrier then remained stable for up to 5 hours. The size, packing density, and arrangement of membranous structures were correlated with changes in the dye barrier from 15 to 35 minutes postcalcium addition. During this interval, uptake of an externally placed hydrophilic dye by these membranous structures was also variable. After 35 minutes postcalcium addition, the membranous structures remained stable until they completely disappeared between 1 and 2 days posttransection. The disappearance of membranous structures always preceded neuritic outgrowth, which only arose from cut axonal ends. These results demonstrate that the dye barrier and associated membranous structures, which form after transection of earthworm giant axons, are very dynamic in the short term (35 minutes) with respect to their location and morphological configuration and suggest that axolemmal repair must be completed before neuritic outgrowth can occur.

Animals↗

Glial cells in the earthworm ventral nerve cord make an A-type nexus.

Nexuses have been demonstrated between glial cell processes in the earthworm ventral nerve cord. The nexuses have been demonstrated by freeze-cleavage to be A-type neuxuses, thus demonstrating for the first time the type of nexus present in the Annelida. If the nexal cleavage pattern has physiological significance then the results suggest that physiologically the nexuses of the earthworm may be similar to those of vertebrates.

Animals↗

Impulse conduction in the myelinated giant fibers of the earthworm. Structure and function of the dorsal nodes in the median giant fiber.

The dorsal openings in the myelin sheath of the median giant fiber (MGF) of the earthworm (Lumbricus terrestris L.) have been studied with electronmicroscopical and electrophysiological methods. The fine structure of the dorsal nodes (DN) resembles in many aspects the Ranvier nodes in vertebrate and crustacean nerve fibers. The nodal membrane directly faces the extracellular collagenous capsule of the ventral cord and displays a conspicuous electrondense undercoat. The myelin sheath of the paranode shows a characteristic differentiation into large desmosomal contracts. Recordings of the transmembrane and longitudinal surface currents along the dorsal side of the MGF during spike propagation support the view that an active inward current is restricted there to the DN. The inward current density in the DN reaches outstandingly high values similar to those measured in vertebrate nodes of Ranvier. The nodal activity can be blocked by application of tetrodotoxin and local anaesthetics. Local electrical stimulation of only one DN may suffice to elicit propagated actions potentials up and down the MGF. It is concluded that the dorsal nodes of the median giant fiber of the earthworm are highly specialized excitable structures mediating saltatory impulse conduction in these fibers.

Animals↗

Evaluation of three antidotes on arsenic toxicity in the common earthworm (Lumbricus terrestris).

The common earthworm (Lumbricus terrestris) is being evaluated in our laboratories as a substitute for mice in metal toxicity studies. These two disparate species have enzymes in common, such as catalase, superoxide dismutase and glutathione-S-transferase. Also, worms respond similarly to these rodents for selenium and nickel toxicity. Worms are less sensitive, however, to metal toxicity. In this study earthworms were challenged with three different arsenic compounds: arsenite, arsenate and the vesicant phenyldichloroarsine (PDA). The median lethal dose for each arsenic compound was determined. The order of toxicity of the arsenic compounds to the worms was PDA > arsenite > arsenate (24 h LD50 values were 189.5, 191.0 and 519.4 mumol kg-1, respectively). Individual mammalian dithiol antidotes, namely the sodium salt of 2,3-dimercapto-1-propanesulfonic acid (DMPS), meso-dimercaptosuccinic acid (DMSA) or 2,3-dimercapto-1-propanol (BAL), were injected into the worms 5 min after various doses of the arsenic compound were administered. The decreases in acute toxicity values were recorded. All three antidotes protected the worms against arsenic toxicity with varying degrees of effectiveness. The protective action for the inorganic arsenic compounds was in the order DMPS > DMSA > BAL. For the organic arsenical, PDA, the most effective antidote was BAL.

Animals↗

Diurnal rhythm and effect of temperature on oxygen consumption in earthworms, Amynthas gracilis and Pontoscolex corethrurus.

Different species of earthworms show distinct patterns of oxygen consumption pattern. Amynthas gracilis has a diurnal rhythm of oxygen consumption, consuming more oxygen at night, whether being incubated at 20, 25, or 30 degrees C. The higher oxygen consumption of A. gracilis is directly related to its behavior, as it shows higher activity at night. In contrast, Pontoscolex corethrurus showed no obvious diurnal rhythms of oxygen consumption or behavior. In addition, its oxygen demand is not related to temperature. A. gracilis has higher oxygen consumption than P. corethrurus at 20 and 30 degrees C. The difference in temperature adaptation of oxygen consumption between these two species may be a strategy by which earthworms adapt to different temperatures. This might explain why A. gracilis disperses on the soil surface at night after a rain, in contrast to P. corethrurus for which such behavior was not been observed.

Analysis of Variance↗

High-performance liquid chromatography/inductively coupled plasma mass spectrometry with iodine-specific detection for profiling the metabolites produced in the earthworm Eisenia veneta by exposure to 2-fluoro-4-iodoaniline.

High-performance liquid chromatography/inductively coupled plasma mass spectrometry (HPLC/ICPMS) provided a rapid and specific means for profiling the iodine-containing metabolites produced by the earthworm Eisenia veneta following exposure to 2-fluoro-4-iodoaniline. Profiles were obtained, using gradient reversed-phase HPLC, from extracts of whole earthworms and from coleomic fluid with as little as 25 ng/peak of iodine detected. The use of ICPMS in this way provides a convenient means of determining the metabolic fate of iodinated compounds without the need for radiolabelled compounds.

Aniline Compounds↗

Comparative toxicity and biochemical responses of certain pesticides to the mature earthworm Aporrectodea caliginosa under laboratory conditions.

This study was conducted to investigate the toxicity of aldicarb, cypermethrin, profenofos, chlorfluazuron, atrazine, and metalaxyl toward mature Aporrectodea caliginosa earthworms. The effects of the LC(25) values of these pesticides on the growth rate in relation to glucose, soluble protein, and activities of glutamic-oxaloacetic transaminase (GOT), glutamic-pyruvic transaminase (GPT), acid phosphatase (AcP), and alkaline phosphatase (AIP) were also studied. The results showed that aldicarb was the most toxic of the tested pesticides, followed in order by cypermethrin, profenofos, chlorfluazuron, atrazine, and metalaxyl. A reduction in growth rate was observed in all pesticide-treated worms, which was accompanied by a decrease in soluble protein and an increase in transaminases and phosphatases. Relationships between growth rate, protein content, transaminases, and phosphatases provided strong evidence for the involvement of pesticidal contamination in the biochemical changes in earthworms, which can be used as a bioindicator of soil contamination by pesticides.

Animals↗

Hemolytic function of opsonizing proteins of earthworm's coelomic fluid.

Synthetic 2-hydroxyethylmethacrylate copolymer particles (HEMA) can be opsonized in the coelomic fluid of Eisenia foetida earthworms. The incomplete coelomic fluid (i.e., the coelomic fluid after incubation with HEMA particles) exerts a lower level of hemolytic activity compared to complete coelomic fluid. The decreased hemolysis can be compensated by the addition of isolated opsonins. On the other hand, isolated opsonins do not possess direct hemolytic capacity. It can be suggested that at least one of the isolated opsonins is involved in the hemolytic process. These results support the hypothesized cooperation of humoral and cellular mechanisms in earthworm defence.

Animals↗

Autogeneic but not allogeneic earthworm effector coelomocytes kill the mammalian tumor cell target K562.

Earthworm coelomocytes have been used as effector cells against the human tumor target, K562. To first assess the viability of effectors, incorporation of [3H]-thymidine was tested and was higher in autogeneic (A<==A, self) than in allogeneic (A<==>B, nonself) coelomocytes. A<==>A showed significantly greater numbers in S, G2, or M phases than A<==>B coelomocytes. When A<==>A or A<==>B were cultured, no significant cell killing occurred in either, as measured in a 4-hr 51Cr release assay. A<==>A but not A<==>B killed K562 target cells. Cytotoxicity was dependent upon membrane binding between small, electron-dense coelomocytes and targets; it was enhanced by adding PHA. The heat labile supernatant from A<==>A but not from A<==>B killed K562 targets after cultivation for 10 min at 22 degrees C, but not immediately after washing. Recognition of, binding to, and killing of foreign cells in a natural killer cell-like reaction may reflect natural immunity in earthworms.

Animals↗

Cadmium disposition in the earthworm Eisenia fetida.

The disposition of cadmium was examined in the earthworm Eisenia fetida using two exposure media, filter paper, and artificial soil. Uptake and elimination rates were estimated to be 0.03039 and 0.00895 hr-1, respectively, for 1.25 micrograms Cd/cm2 filter paper exposure and 0.00512 and 0.00029 hr-1, respectively, for 10 micrograms Cd/g artificial soil exposure. The distribution of cadmium was examined using whole body autoradiography, tissue distribution, and subcellular localization. Cadmium tissue distribution was exposure-dependent, while cadmium subcellular localization apparently was not exposure-dependent. Acid-soluble thiol levels, examined as an index of cadmium exposure, were too variable in nonexposed earthworms to serve as an indicator of cadmium exposure.

Animals↗

PCBs increase molecular-related activities (lysozyme, antibacterial, hemolysis, proteases) but inhibit macrophage-related functions (phagocytosis, wound healing) in earthworms.

Both humoral and cellular immunodefense responses of the earthworms, Eisenia fetida andrei, Eisenia hortensis, and Lumbricus terrestris, have been compared after exposure to the PCB Aroclor 1254. Responses mediated by free factors, detected by in vitro assays for lysozyme, hemolysis, and proteases, were increased in both Eisenia. Antibacterial activity directed against pathogenic bacteria was increased in E.f. andrei. The resistance of L. terrestris against non-pathogenic bacteria was decreased, confirming that the bacteria were treated by different systems according to their pathogenicity. Nonspecific cellular functions, including phagocytosis and those related to wound healing, decreased dramatically in all earthworms.

Aeromonas hydrophila↗

Electron-microscopic observations of normal coelomocytes from the earthworm, Lumbricus terrestris.

Coelomocytes of the earthworm, Lumbricus terrestris, were studied by transmission electron microscopy. Four morphological cell types are distinguishable: lymphocytic coelomocytes, granulocytic coelomocytes, eleocytes (chloragogen cells), and inclusion-containing coelomocytes. Within these major categories, several distinct cell types differ and may represent developmental stages. The two types of lymphocytic coelomocytes are small with central nuclei and scanty cytoplasms. Two types of granulocytic coelomocytes differ greatly in shape and content; both have small dark-staining granules that resemble lysosomes. Electrocytes, derived from chloragogen tissue, contain a variety of granules, inclusions and vacuoles. Inclusion-containing coelomocytes appear as two types which may be immature and mature forms. Although these cells resemble those that have been referred to as erythroid cells in other invertebrates, the large inclusion bodies are apparently unrelated to hemoglobin; they can undergo morphologic transformation and be extruded by exocytosis. This information on lymphocytic, granulocytic and inclusion-containing coelomocytes is crucial to understanding more about cellular immunity in the earthworm.

Animals↗

The accumulation and intracellular compartmentation of cadmium, lead, zinc and calcium in two earthworm species (Dendrobaena rubida and Lumbricus rubellus) living in highly contaminated soil.

The earthworm Lumbricus rubellus contained more Ca and Zn, and less Pb and Cd, than Dendrobaena rubida living in the same contaminated disused-mine soil. Differences in the kinetics of Ca turnover may account for some of the inter-specific differences in heavy metal burdens, although the calciferous glands do not seem to be directly involved in heavy metal excretion. A comparison of the present findings with published data indicated that the concentration of soil Ca and the bioavailability of heavy metals, both factors being allied to soil pH, are important exogenous determinants of heavy metal accumulation by different earthworm populations. Electron microprobe X-ray analysis of air-dried smears of chloragogenous tissue showed that the metals were fairly specifically compartmentalized into two distinct organelles in both worms: Ca, Pb and Zn were found (associated with P) in the chloragosomes; Cd was found (with S and probably in stoichiometric association) in a more electron-lucent vesicular component, designated the 'cadmosome', but which may be identical with the debris vesicles which are characteristic inclusions in conventionally-fixed chloragocytes. The in vivo incorporation of Pb by the chloragosomes of D. rubida was accompanied by the loss of Ca, Zn and P.

Animals↗

Isolation of genomic DNA from the earthworm species Eisenia fetida.

Our interest in detecting genotoxic exposure in earthworms led us to isolate high quality DNA from the Eisenia fetida species. For that, we compared a modification of the conventional phenol-chloroform extraction procedure, usually referred to as the Maniatis procedure, to two commercially available kits reportedly eliminating multiple partitions in phenol and chloroform, namely the Qiagen and Nucleon protocols. From the 260 nm optical density values, the commercial kits extracts hinted toward higher DNA recovery with those procedures. However, the 260/280 nm ratios indicated that the quality of the DNA isolated with the modified Maniatis procedure was purer than that isolated with the commercial kits, the latter being most probably contaminated by proteins and/or RNA. The Maniatis procedure was slightly modified by the introduction of a potassium acetate step for protein precipitation and by shortening the proteinase K treatment from 12-18 h to only 2 h. The higher quality of the DNA isolated by phenol-chloroform extraction was confirmed by quantification with the fluorescent 3,5-diaminobenzoic acid assay. Preliminary results suggest that the modified Maniatis procedure herein described is not only applicable for DNA adducts studies using 32P-postlabelling techniques but is also suitable for DNA extraction from other earthworm species such as Lumbricus terrestris.

Animals↗

Cadmium-binding proteins induced in the earthworm.

Earthworms, Eisenia foetida, grown in composts of different cadmium concentrations accumulated cadmium in a dose-dependent manner and the cadmium was bound to three different molecular weight cadmium-binding proteins induced in the earthworm. The three proteins were stable to heat treatment and accompanied by a concomitant increase of absorbance at 254 nm and not at 280 nm. Each of the three proteins is a mixture of isoproteins; the molecular weight of one of the three proteins was estimated to be about 7,000 daltons, on the assumption that the cadmium-binding protein is similar in chromatographic properties to metallothionein.

Animals↗

Digestive enzymes of some earthworms.

4 species of tropical earthworms differed with regard to enzyme activity. The maximum activity of protease and of cellulase occurred in the posterior region of the gut of the earthworms. On the average Octochaetona surensis shows maximum activity and Drawida calebi shows minimum activity for all the enzymes studied.

Animals↗