Further data on expression of acetylcholinesterase during oogenesis and embryonic development of Ciona intestinalis and Ascidia malaca (Ascidiacea,Urochordata): a biochemical and histochemical study (original) (raw)
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Caryologia -Firenze-
We have used unfertilized eggs, embryos and larvae of the sea squirt Ciona intestinalis (Ascidia- cea, Urochordata) to study the effects of tributyltin (TBT) on acetylcholine esterase (AChE) activity. Our results showed an increase of AChE enzyme activity levels after TBT exposure in both unfertilized eggs and embryos. The effects of TBT were more pronounced during the gastrula stage, while no differences between exposed larvae and controls were observed. A mechanism of TBT action on enzyme activity during ascidian embryonic development and the possibility of employing AChE in C. intestinalis as a biomarker of TBT exposure are suggested and discussed.
Journal of Experimental Zoology, 1983
Acetylcholinesterase (AChE) activity has been studied in Artemia salina embryos and larvae by quantitative, histochemical, and electrophoretic methods. An AChE activity is present in dehydrated encysted gastrulae and remains low in rehydrated developing embryos, showing at hatching an increase that becomes faster during the nauplius stages (twice the gastrula activity at the nauplius I1 stage), and continues linearly in the first metanauplius stages. The histochemical staining, faint and diffuse in dehydrated gastrulae, in more advanced embryos becomes localized in cell membranes and stronger in differentiating tissues; most AChE activity is localized in the nervous and muscular cells, and a fainter one in the differentiating metanauplius segments. Dehydrated cysts show one electrophoretic AChE band, that is no longer detectable in rehydrated embryos and larvae, while two other bands appear, that migrate more slowly; in advanced larval stages, the more cathodic band is predominant. In dehydrated cysts, isoelectric focusing separates three AChE bands with isoelectric points (IP) at pH 5.0, 5.7, and 5.9; in hydrated embryos and larvae, the band at pH 5.0 is absent and another is seen at pH 5.5. Eserine, DFP, and BW284c51 inhibit the enzyme activity strongly and cause paralysis of larvae; also tubocurarine blocks the motility. These results are discussed in relation to the differentiation of the neuromuscular system and to the development of motility. As a hypothesis, in early embryonic stages, before any neuromuscular differentiation is evident, a cholinergic-like system might regulate non-neural cell activities and interactions, associated with embryogenetic events, This work is dedicated to our master, Professor Antonio Min-Address reprint requests to Dr. M Raineri, Istituto di Anato ganti.
Purification and characterization of an ascidian larval acerylcholinesterase
Biochimica et biophysica acta, 1980
Larval acetylcholinesterase (acetylcholine acetylhydrolase, EC 3.1.1.7) of the ascidian Ciona intestinalis (L.) was purified by a two-step affinity chromatography procedure. Concavanalin A-Sepharose chromatography in batches provided the initial purification and was followed by chromatography on columns to which competitive inhibitors of acetylcholinesterase had been attached. The most efficient of these used m-carboxyphenylmethylammonium iodide coupled to Sepharose 4B via a hydrophobic 6-carbon spacer. In combination with the concanavalin A-Sepharose step, this affinity resin yielded recoveries of 30-39% with specific activities ranging from 580-730 units/mg protein, a total purification of 5000-7000-fold. Analysis of this product by polycrylamide gel electrophoresis in the presence of SDS and beta-mercaptoethanol revealed a single major polypeptide of M(r) 65 000-70 000. This protein was identified as the basic catalytic subunit of acetylcholinesterase by its coelectrophoresis wit...
Acetylcholine synthesis and possible functions during sea urchin development
European journal of histochemistry : EJH
Cholinergic neurotransmitter system molecules were found to play a role during fertilisation and early cell cycles of a large number of invertebrate and vertebrate organisms. In this study, we investigated the presence and possible function of choline acetyltransferase (ChAT, the biosynthetic enzyme of acetylcholine) in gametes of the sea urchin, Paracentrotus lividus, through localisation and functional studies. ChAT-like molecules were detected in oocytes, mature eggs and zygotes with indirect immunofluorescence methods. Positive immunoreactivity was found in the ovarian egg cytoplasm and surface as well as at the zygote surface. This suggests the eggs' capacity to autonomously synthesise acetylcholine (ACh), the signal molecule of the cholinergic system. Acetylcholinesterase (AChE, the lytic enzyme of acetylcholine) was also found in ovarian eggs, with a similar distribution; however, it disappeared after fertilisation. Ultrastructural ChAT localisation in sperms, which was c...
Characterization and evaluation of cholinesterase activity in the cockle Cerastoderma glaucum
Aquatic Biology, 2011
The aim of the present study was to partially characterize the biochemical properties of cholinesterase (ChE) activity in the tissues of the cockle Cerastoderma glaucum, using different substrates and sample volumes, and to evaluate ChE response following caging exposure in a fishing harbour. Comparative analysis of the Michaelis-Menten kinetics in the studied tissues of C. glaucum indicated that ChE activity, using acetylthiocholine as substrate, displayed a tissuedependent response, with activity levels in the following order of magnitude: whole animal > rest of animal (adductor and retractor muscles, mantle and foot) > visceral mass > gills. The transfer of C. glaucum to the Téboulba fishing harbour caused a time-dependent reduction in acetylcholinesterase activity.
Biological Bulletin, 1996
This study constitutes the first report of a biochemical characterization, involving both substrates and inhibitors and electrophoretic analysis, of a cholinesterase (ChE) from a polychaete annelid (Nereis diversicolor). The ChE of N. diversicolor appears to be an acetylcholinesterase (AChE); i.e., it hydrolyzes acetylthiocholine iodide at a higher rate than other substrates and is inhibited by eserine but not by iso-OMPA. The ChEs of Eisenia,fetida are different from that of N. diversicolor and include at least two types of PrChEs. The AChE activity is located principally in the anterior region of the worm (head) in N. diversicolor, whereas the ChE activity of E. fetida is located throughout the body. The electrophoretic characterization of N. diversicolor and E. ,fetida ChEs showed,
Neurogenesis and occurrence of cholinesterase in eggs of Triatoma infestans
Comparative Biochemistry and Physiology Part C: Comparative Pharmacology, 1980
1. Eggs of T. infestans incubated at 30~C and 50~i:, r.h. hatched in 15 days. 2. The neurogenesis of T. i~!festans embryo was studied. The neuroblasts appeared on the third day and the nerve cells on the fourth day. 3. The ontogenesis of cholinesterases was investigated. ActiviD was first detected on the fourth day coincidentally with the nerve cells appearance. 4. The cholinesterases were characterized as butyrylcholinesterase and acetylcholinesterase. 5. The oxygen analogs of several phosphorothionates insecticides were found to be potent inhibitors of the embryonic acetylcholinesterase.
Molecular and Biochemical Parasitology, 2003
We describe the molecular cloning, expression and biochemical characterisation of recombinant forms of two secreted acetylcholinesterases from adult Dictyocaulus viviparus. The two variants (designated Dv-ACE-1 and Dv-ACE-2) were 613 and 615 amino acids long and showed 94.7% identity to one another. The highest level of identity to other cholinesterases was with ACE-2 of Caenorhabditis elegans. Dv-ACE-1 and Dv-ACE-2 showed 48.0 and 47.7% identity to C. elegans ACE-2 over 577 amino acids, respectively. The primary structure of both enzymes showed conservation of the catalytic triad and of a tryptophan residue known to be critical for the choline-binding site, but differed in the number of potential glycosylation sites and at one amino acid in the peripheral anionic site. Southern blotting and PCR experiments indicated that the genes encoding these enzymes are distinct. When expressed in Pichia pastoris, the enzymes were active, but differed subtly in their biochemical characteristics. Both enzymes exhibited a preference for acetylcholine as substrate, but differed in the extent of excess substrate inhibition and in their optimal pH for activity. The lack of an obvious carboxy-terminal membrane anchor and the presence of an insertion at the molecular surface were other features which, thus far, appear to be characteristic of parasite secreted acetylcholinesterases.