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PubMed2003

Gene fishing: the use of a simple protocol to isolate multiple homeodomain classes from diverse invertebrate taxa.

Comparison of relevant gene sequence and functional data is central to understanding the evolution of metazoan development. The conservation of portions of regulatory genes, such as homeoboxes, allows for the design of PCR-based sequence isolation and amplification strategies. Here we describe a simple protocol that uses a degenerate primer pair to isolate a variety of homeobox-containing genes from diverse metazoan taxa. In a nonexhaustive survey, we have isolated 28 gene sequence fragments from 15 taxa, representing eight invertebrate phyla (Mollusca, Echiura, Annelida, Platyhelminth, Acoela, Ctenophora, Cnidaria, and Porifera). Based on BLAST and parsimony analyses, these gene fragments affiliate with several gene groups (PAIRED-like, HOX, and ParaHOX) and several single genes, including pancreas/duodenum homeoboxes (Pdx), empty spiracles (ems/Emx), gastrulation brain homeoboxes (Gbx), hematopoietically expressed homeoboxes (HEX), brain specific homeobox (bsh/BarH1/BarH2), NK-1 (NK-1/s59/slouch), and ladybird (Lbl/Lbe/Lbx). In several cases, these fragments represent the first reported orthologue for the phylum or superphyletic group (i.e., Lophotrochozoa).

Amino Acid Sequence↗
PubMed2005

Nervous and muscle system development in Phascolion strombus (Sipuncula).

Recent interpretations of developmental gene expression patterns propose that the last common metazoan ancestor was segmented, although most animal phyla show no obvious signs of segmentation. Developmental studies of non-model system trochozoan taxa may shed light on this hypothesis by assessing possible cryptic segmentation patterns. In this paper, we present the first immunocytochemical data on the ontogeny of the nervous system and the musculature in the sipunculan Phascolion strombus. Myogenesis of the first anlagen of the body wall ring muscles occurs synchronously and not subsequently from anterior to posterior as in segmented spiralian taxa (i.e. annelids). The number of ring muscles remains constant during the initial stages of body axis elongation. In the anterior-posteriorly elongated larva, newly formed ring muscles originate along the entire body axis between existing myocytes, indicating that repeated muscle bands do not form from a posterior growth zone. During neurogenesis, the Phascolion larva expresses a non-metameric, paired, ventral nerve cord that fuses in the mid-body region in the late-stage elongated larva. Contrary to other trochozoans, Phascolion lacks any larval serotonergic structures. However, two to three FMRFamide-positive cells are found in the apical organ. In addition, late larvae show commissure-like neurones interconnecting the two ventral nerve cords, while early juveniles exhibit a third, medially placed FMRFamidergic ventral nerve. Although we did not find any indications for cryptic segmentation, certain neuro-developmental traits in Phascolion resemble the conditions found in polychaetes (including echiurans) and myzostomids and support a close relationship of Sipuncula and Annelida.

Animals↗
PubMed2006

Evidence for the presence of thyroid stimulating hormone, thyroglobulin and their receptors in Eisenia fetida: a multilevel hormonal interface between the nervous system and the peripheral tissues.

The present study describes the localization and distribution of thyroid-stimulating hormone (TSH), thyroglobulin (TGB) and their receptors in Eisenia fetida (Annelida, Oligochaeta) as revealed by immunohistological methods. Immunopositive neuronal and non-neuronal cells are present in both the central nervous system and some peripheral organs (e.g. foregut and coelomocytes). TSH- and TGB-immunopositive neurons in the various ganglia of the central nervous system are differentially distributed. Most of the immunoreactive cells are found in the suboesophageal ganglion. The stained cells also differ in their shapes (round, oval, pear-shaped) and sizes (small, 12-25 microm; medium, 20-35 microm; large, 30-50 microm). In all ganglia of the central nervous system, TSH-positive neurons additionally show gamma aminobutyric acid (GABA) immunopositivity. Non-neuronal cells also take part in hormone secretion and transport. Elongated TSH-positive cells have been detected in the capsule of the central ganglia and bear granules or vacuoles in areas lacking neurons. Many of capillaries show immunoreactivity for all four tested antibodies in the entire central nervous system and foregut. Among the coelomocytes, granulocytes and eleocytes stain for TSH and its receptor and for TGB but not for thyroid hormone receptor. Most of the granulocytes are large (25-50 microm) but a population of small cells (10-25 microm) are also immunoreactive. None of the coelomocytes stain for GABA. We therefore suggest that the members of this hormone system can modify both metabolism and immune functions in Eisenia. Coelomocytes might be able to secrete, transport and eliminate hormones in this system.

Animals↗
PubMed1983

Bodian's silver method reveals molecular variation in the evolution of neurofilament proteins.

The recent demonstration that Bodian's silver method specifically stains mammalian neurofilament subunits (NFs), but not other intermediate filament proteins (IFs), provides a specific marker for the identification of neurofilament polypeptides. We have applied the Bodian stain to SDS-PAGE separated polypeptides in nervous tissues from 9 species, representing neuronal evolution in 4 major phyla: chordata, mollusca, arthropoda and annelida. Every species tested except the arthropod showed intense silver staining of a set of polypeptides, each subsequently identified as NFs by immunomethods. These results demonstrate that the affinity of NFs for Bodian's silver stain is conserved during the evolution of nervous systems in a diverse spectrum of animals. Further, considerable variation in the molecular weight of NF subunits was found among the 6 vertebrates studied. This variation suggests that the molecular weight of NFs has not been conserved during evolution, a quality which appears to be unusual for a structural protein.

Animals↗
PubMed1995

Isolation and structural characterization of enkephalins in the brain of the rhynchobdellid leech Theromyzon tessulatum.

This paper reports the purification of four peptides related to enkephalins from the brain of the leech Theromyzon tessulatum. After reverse-phase HPLC purification, the sequence of the enkephalins (YGGFM, YGGFL, FM, FL) was established by a combination of automated Edman degradation, electrospray mass spectrometry measurement, and co-elution experiments in reverse-phase HPLC with synthetic peptides. ELISA titrations performed on each purified peptide indicated that the major amount was borne by the leucine-enkephalin. The ratio of leucine-enkephalin and methionine-enkephalin of 2:1 is in line with previous immunocytochemical data obtained on T. tessulatum brains. The presence of enkephalins in T. tessulatum, an animal belonging to the oldest group of coelomate metazoans (the Annelida) establishes the very ancient phylogenetic origin of opioids and their conservation in the course of evolution.

Amino Acid Sequence↗
PubMed1990

Polypeptides related to mammalian procholecystokinin in the brain of an invertebrate, a marine worm, Nereis diversicolor: evidence from in ovo translation of mRNA.

Total mRNA extracted from brain of a sea worm Nereis diversicolor (Annelida, Polychaeta) were injected into Xenopus oocytes. The in ovo-translated polypeptides were analyzed through electrophoresis of immunoprecipitated products or Western blotting techniques. Some of these polypeptides cross-reacted antibodies elaborated against three mammalian (human and rat) procholecystokinin (pro-CCK) sequences. Polypeptides of 15, 64, and 70 kDa were determined, but two families of less obvious products, whose molecular masses were between 27-34 kDa and 46-60 kDa, appeared. Furthermore, the Western blotting technique also showed a cross-reaction between some of the polypeptides (64 and 70 kDa) extracted from brains and the antibodies used. From this work, evidence is given of the presence of epitopes shared by cellular polypeptides extracted from the brain, in ovo-translated products and mammalian pro-CCK. Furthermore, these data suggest the existence of a CCK precursor in the brain of N. diversicolor.

Animals↗
PubMed1991

Cells showing immunoreactivity for calcitonin or calcitonin gene-related peptide (CGRP) in the central nervous system of some invertebrates.

In the central nervous system of some species of several invertebrate phyla, including land planarians (Platyhelminthes), ribbon worms (Nemertina), slugs (Mollusca), polychaetes, earthworms and leeches (Annelida), pill bugs (Arthropoda), and beard worms (Pogonophora), salmon calcitonin-immunoreactive cells and rat calcitonin gene-related peptide (CGRP)-immunoreactive cells were found by immunohistochemistry. These immunoreactive cells were located in the region surrounding the neuropile, although the sizes of the cells varied according to species. Some of them were round or polygonal and regarded as apolar nerve cells because of their lack of cytoplasmic processes, whereas others were spindle-shaped or elongated, being comparable with unipolar nerve cells because of extension of their cytoplasmic processes in the direction of the neuropile. In some cases, it was noted that the cytoplasmic processes had complicated branches or formed loop-like structures at their ends. These observations suggest that a calcitonin-like or CGRP-like substance is extensively present in invertebrates as well as vertebrates.

Animals↗
PubMed1992

Some polypeptides in the nervous system of the marine worm, Nereis diversicolor, are related to the sodium influx stimulating peptide of the pulmonate freshwater snail, Lymnaea stagnalis.

Total mRNA, extracted from brain of the marine worm, Nereis diversicolor (Annelida, Polychaeta), was translated either in vitro using a rabbit reticulocyte lysate or in ovo (Xenopus laevis oocyte). The synthesized polypeptides were analyzed by electrophoresis and Western blotting techniques using polyclonal antisera raised against three peptides: sodium influx stimulating peptide (SISP) sequences 10-19 and 67-76 and a monoclonal antibody raised against purified native SISP (1-77) of Lymnaea stagnalis. Among the products translated in vitro, three polypeptides of 80, 72, and 64 kDa were recognized by the anti-SISP (10-19) polyclonal antiserum and by the monoclonal antiserum, but not by anti-SISP (67-76). Some of the in ovo translated products showed almost identical immunoreactivity to both the anti-SISP (10-19) and the monoclonal antibody. These polypeptides have molecular masses of 80, 72, and 43 kDa. No polypeptides were recognized by anti-SISP (67-76). Western blotting analysis of brain extracts revealed a number of proteins that reacted with the antiserum raised against SISP (10-19) and the monoclonal antiserum. Several perikarya of brain ganglionic nuclei and ventral nerve cord were immunoreactive to anti-SISP (10-19). The monoclonal antiserum gave similar results, although with a less intense immunoreaction. The infracerebral region was also stained, suggesting that the immunoreactive material is released as a true neurohormone into the hemolymph. The largest polypeptides, in particular those translated from brain mRNA, could be neuropeptide precursors containing a SISP-related sequence.

Animals↗
PubMed1993

Calibrating the bootstrap test of monophyly.

It has been suggested that the bootstrap test of monophyly is too conservative, i.e. the test rejects the hypothesis of monophyly when it is true far too often. Here, a method called the iterated bootstrap is described which estimates by randomization the probabilities associated with rejecting the hypothesis of monophyly when it is true and accepting the hypothesis of monophyly when it is false. Using this method, the bootstrap test can be calibrated by taking account of the errors associated with the hypothesis test. This method is applied to the high-level phylogeny of the Platyhelminthes using 18S rRNA sequences. The analysis suggests that the data cannot unequivocally resolve the placement of the Platyhelminthes with respect to the Annelida and Insecta.

Animals↗
PubMed1978

Anatomical and ultrastructural study of the pharyngeal bulb in Protodrilus (Polychaeta, Archiannelida) II. The stomodeal and its cuticle.

The epidermal and stomodeal cuticles of Protodrilus are described then compared. The thin epidermal cuticle, the thickness of which is about the same over all the body, is characterized both by the absence of fibrils in its deepest part and by the extension of epidermal microvilli above the cuticle. The stomodeal cuticle, the thickness of which is as variable as that of the epithelium, presents two layers of fibrils comparable to the collagen fibrils described in the cuticle of other Annelida, as well as a relatively diversified supramicrovillous coating. The anterior cuticular thickening or grating plate, is characterized by the length of the epithelial microvilli, the thickness of the cuticular matrix and the superficial cuticular zone with supramicrovillous denticles supported by an axis of fibrous bundles. In the stomodeal cuticle, the fibrillar material seems to give to the cuticle best resistance to deformation during the pharyngeal bulb contraction, while an especially elaborated supramicrovillous coating is found in regions most exposed to friction. These features contrast with the relative simplicity of the epidermal cuticle.

Animals↗
PubMed1995

Identification of a cytolytic protein in the coelomic fluid of Eisenia foetida earthworms.

Total coelomic fluid of earthworms Eisenia foetida (Oligochaeta, Annelida) is capable of lysing different mammalian tumor cell lines. This cytolytic activity is different from tumor necrosis factor (TNF)-mediated lysis and is not due to proteolysis. Total coelomic fluid was subjected to ion-exchange chromatography separation and a fraction with prominent cytolytic activity was used to elicit monoclonal antibodies that were screened for their capacity to neutralize the cytolytic effect of total coelomic fluid. One of the prepared neutralizing IgG antibodies was used for the immunoaffinity purification of a cytolytic factor from total coelomic fluid. SDS-PAGE and Western blot analyses revealed a protein band with an apparent molecular weight of 42 kDa. This cytolytic protein (termed CCF-1 or coelomic cytolytic factor 1) can be adsorbed on the surface of opsonized particles and may be involved in opsonizing and hemolytic effects of coelomic fluid.

Animals↗
PubMed1996

Electrospray ionization mass spectrometric composition of the 400 kDa hemoglobin from the pogonophoran Oligobrachia mashikoi and the primary structures of three major globin chains.

Maximum entropy analysis of the electrospray ionization mass spectra of the native, carbamidomethylated, reduced and reduced and carbamidomethylated forms of the extracellular ca. 400 kDa hemoglobin of the pogonophoran Oligobrachia mashikoi has shown it to consist of eight globin chains: (a1-a5), 14861.1, 14937.1, 15040.7, 15070.6 and 15310.6 Da and b-dl, 15173.2, 15605.1 and 14775.4 Da, respectively. Although chains a1-a5 are monomeric, chains b + c form a disulfide-bonded dimer of 30776.8 Da and chains b + c + d1 form a disulfide-bonded trimer of 45551.9 Da. The major chains a5, b and c were separated by reverse-phase chromatography, and their cDNA's amplified by PCR using redundant oligomers based on their N-terminal amino-acid sequences. The complete amino-acid sequences of chains a5 (142 residues), b (140 residues) and c (147 residues) were derived from protein and cDNA sequencing and represent the first pogonophoran globin sequences. They have a high percent identity (35-52%) with the globin chains of the approximately 3500 kDa hexagonal bilayer hemoglobins from the annelids Lumbricus and Tylorrhynchus and the vestimentiferan Lamellibrachia, suggesting a very close relationship among the phyla Annelida, Pogonophora and Vestimentifera. Two free cysteine residues (Cys-73 and Cys-83), which we proposed to be the most probable candidates for the sulfide-binding sites in the Lamellibrachia chains (Suzuki, T., Takagi, T. and Ohta, S. (1990) Biochem. J. 266, 221-225), are also conserved in three chains (Cys-73 for chains b and c, and Cys-83 for chain a5) of Oligobrachia hemoglobin, in agreement with the probable role of the hemoglobin in the binding and transport of sulfide to the symbiotic bacteria which provide the metabolic fuel in the two phyla.

Amino Acid Sequence↗
PubMed1994

Effects of cadmium, copper, lead and zinc on growth, reproduction and survival of the earthworm Eisenia fetida (Savigny): Assessing the environmental impact of point-source metal contamination in terrestrial ecosystems.

The earthworm Eisenia fetida (Annelida: Oligochaeta) was exposed to a geometric series of concentrations of cadmium, copper, lead and zinc in artificial soil using the OECD recommended protocol. Mortality, growth and cocoon production were measured over 56 days to determine LC50 and EC50 values. No observed effect concentrations (NOECs) were also estimated. Furthermore, the percentage of viable cocoons and number of juveniles emerging per cocoon was recorded. Cocoon production was more sensitive than mortality for all the metals, particularly cadmium and copper for which NOEC reproduction values were an order of magnitude lower than those for NOEC mortality. However, there was no significant effect of metals on the viability of cocoons. The weights of earthworms declined in all treatments (including the controls) during the experiment. This was probably due to the lack of suitable food in the OECD standard soil medium used. It was concluded that future experiments should include animal manure in the test medium. The LC50, EC50 and NOEC values determined in this study were compared with concentrations of metals in soils in the vicinity of a smelting works at Avonmouth, southwest England. The 14-day LC50 for zinc in Eisenia fetida was exceeded in soils covering an area of 75 km2 around the works, compared to 4.2 km2 for copper and 4.7 km2 for lead. Soil values for cadmium did not exceed the LC50 value anywhere in the region. Similar estimates of relative effects on reproduction confirmed that zinc is most likely to be responsible for the absence of earthworms from sites close to the Avonmouth works. However, the OECD standard test overestimated the potential effects of metals on populations, since earthworms can be found as close as 1 km from the smelting works. The discrepancy between test and field observations was probably due to the greater availability of the metals in the artificial soil.

Journal Article↗
PubMed1978

Intestinal arginase in vertebrates and invertebrates.

1. Arginase was found to be present in the intestine in all species of Annelida, Arthropoda and Chordata studied. 2. The activity of intestinal arginase differs from species to species, the differences reaching two orders of magnitude (100 x). 3. The highest activity of intestinal arginase was observed in the rodents (mouse, rat, hamster). 4. In animals in which the enzyme activity was high or moderately high, arginase activity showed topographical differentiation along the long axis of the intestine.

Animals↗
PubMed1988

Collagen cross-linking: distribution of hydroxypyridinium cross-links among invertebrate phyla and tissues.

1. Using a specific HPLC assay, a wide variety of marine invertebrate connective tissues was screened for the 3-hydroxypyridinium amino acids that are prominent intermolecular cross-linking residues in the collagens of many vertebrate connective tissues. 2. One or both of the two structural forms that exist, hydroxylysyl pyridinoline (HP) and lysyl pyridinoline (LP), was found in organisms from the following phyla: coelenterata, Annelida, Echinodermata, Mollusca and Arthropoda. 3. Neither amino acid was found in tissues from representative species of Porifera and Chordata. 4. Of special note was an unusually high ratio of LP to HP in Limulus polyphemus gill cartilage.

Animals↗
PubMed1995

Alterations in polyamine levels of nematode, earthworm, leech and planarian during regeneration, temperature and osmotic stresses.

Free-living nematodes, Caenorhabditis elegans and Dorylaimus fodori, contain putrescine and spermidine. Putrescine, spermidine and spermine occur in the parasitic Nematoda, Ascaris suum, Anisakis simplex and Dirofilaria immitis. Earthworms, Eisenia foetida, Tubifex hattai and Pheretima communissima and the leech, Hirudo nipponia (belonging to Annelida) and the planarian, Dugesia japonica (belonging to Platyhelminthes) contain homospermidine and spermine in addition to putrescine and spermidine. Regenerated heads of E. foetida and D. japonica are rich in putrescine indicating the stimulation of its synthesis during regeneration. Putrescine and spermidine levels temporarily increase after heat shock in C. elegans, E. foetida and D. japonica and cold shock and hypertonic osmotic shock treatments in D. japonica.

Animals↗
PubMed2004

The complete amino acid sequences of four globins from the land leech Haemadipsa zeylanica var. japonica.

The amino acid sequences of four globins from the land leech, Haemadipsa zeylanica var. japonica, were determined using nucleotide sequencing and protein sequencing. The mature globin-molecules were composed of 146 amino acid residues for M-1 globin, 156 for M-2 globin, 143 for D-1 globin, and 149 for D-2 globin. Alignment of the four kinds of globins by Clustal X revealed 22 invariant amino acids. The four globins were 26-33% identical. A striking feature of amino acid alteration was: the replacement of the E7 distal-His of D-1 globin by phenylalanine because histidine is conserved among the rest of the globins of H. zeylanica, those of other representative species (Lumbricus and Tylorrhynchus) of Annelida and most other hemoglobins. A phylogenetic tree constructed of 18 globin structures including two species of leeches, H. zeylanica (a land leech) and Macrobdella decora (a freshwater leech), T. heterochaetus (a representative species of polychaetes), L. terrestris (a representative species of oligochaetes), and human alpha and beta globins strongly indicated that the leech globins first separated from globin lineage of annelids.

Amino Acid Sequence↗
PubMed2005

A beta-galactose-specific lectin isolated from the marine worm Chaetopterus variopedatus possesses anti-HIV-1 activity.

A 30 kDa beta-galactose-specific lectin named CVL was isolated from the polychaete marine worm Chaetopterus variopedatus (Annelida) and its anti-HIV-1 activity in vitro was determined. Results showed that CVL inhibited cytopathic effect induced by HIV-1 and the production of viral p24 antigen. The EC(50) values were 0.0043 and 0.057 microM, respectively. Time-of-addition analysis of anti-HIV-1 activity indicated its action was at the early stage of virus replication. CVL could blocked the cell-to-cell fusion process of HIV infected and uninfected cells with an EC(50) of 0.073 microM. The inhibition of HIV-1 entry into host cells was demonstrated by using fluorescence-based real-time quantify PCR. At CVL concentration of 0.33 microM and 0.07 microM, 86% and 21% virus attachment were blocked, respectively. The anti-HIV-1 action of CVL might relate to blockade of HIV-1 entry into cells.

Animals↗