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Worm Breeder's Gazette,
1995]
Cone snails are predatory snails. They harpoon their prey, which may be small fish, marine worms, or other snails, then inject a paralyzing venom through the hollow harpoon (Olivera et al, Science 249: 257). The venom is a complex mixture of tiny peptide toxins, different in each species. Several of these toxins have been shown to act on cell-surface molecules important for nervous system function such as the N-type Ca++ channel or the voltage-gated Na+ channel (Gray et al, Ann Rev Biochem 57: 665).
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Worm Breeder's Gazette,
1996]
There is precious little in the literature regarding the regenerative ability of nematodes (Poinar, 1988). The received wisdom has been that the determinate cleavage in these pseudocoelomates precludes any possibility of epimorphic regeneration (K.A. Wright pers. comm., 1988). Although Filipjev (1921) stated that regeneration is "completely absent" in nematodes, other reports by Micoletzky and Kreis (1930) and Allgen (1959) present another picture which seemingly contradicts such a blanket generalization. Last year it was reported that a gastrotrich (undescribed species of Turbanella) of the genera Macrodasyidae, a primitive sister group of the Nematoda, restored epidermis with complete wound closure following transection; restructuring of Y organ and intestine; and caudal adhesive tubes forming anew (Manylov, 1995). This is the first report of regeneration in this group. In another recent paper from the former Soviet Union, Voronov and Panchin (1995a) report that a nematode, of the order Enoplida (E.brevis), has a process of gastrulation which contradicts the patterns of cleavage formerly ascribed to the Enoplida (Malakhov,1994). They observed that up to the sixteenth cell stage cleavage is usually (though this can vary) equal and synchronous, producing blastomeres of equal appearance; elsewhere they observe that the primordia which gives rise to all the endoderm can be derived from either the anterior or posterior at the two-cell stage (Voronov and Panchin,1995b). This variability, they offer, makes the Enoplida different from other nematodes studied. Malakhov believes that this variability "can even engender the idea that the cleavage among members of marine Enoplida is indeterminate, but this is not so." (p.166). However, the cleavage of the Enoplida may be indeterminate enough to allow for the regenerative phenomena recently witnessed in a gastrotrich. In sum, Enoplid cleavage patterns would appear to be similar to the more primitive patterns seen in the Macrodasyidae, which is consistent with the notion that equal cleavage is ancestral and determination of early blastomere fate derived (Baguna and Boyer,1990). Also, it should be remembered that in addition to the single species reported by Micoletzky and Kreis, all nine of the species which Allgen found evidence of regeneration were marine Enoplids. Allgen,C.A.(1959)Free living marine nematodes. Further Zool. Results Swed. Antarct. Exp. 1901-03 vol.5 no.2: 1-293. Baguna,J.,B.C.Boyer(1990)Descriptive and experimental embryology of the Turbellaria: Present knowledge, open questions and future trends. In Marthy, H.(ed), Experimental Embryology in Aquatic Plants and Animals. NATO ASI 195; 95-128. Filipjev,I.N.(1921)Free living nematodes in the vicinity of Sevastapol. (in Russian), Akad. Nauk SSSR. Trudy osob. zool. lab. ser 2 41: 351-614. Malakhov,V.V.(1994)Nematodes: Structure, Development, Classification and Phylogeny. Smithsonian Inst. Press. Manylov,O.G.(1995)Regeneration in Gastrotricha - I. Light microscopical observations on the regeneration in Turbanella sp. . Acta Zool. 76:1-6. Micoletzky,H.,H.A.Kreis(1930)Freilbende marine Nematoden von den sunda-Inseln. Dansk natur. Foren. Vid. medd. Bd 87: 243-339. Poinar,G.O.(1992)Immune responses and wound repair. In Diseases of Nematodes. vol 1, p.133-40, CRC Press, Boca Raton, Florida. Voronov,D.A.,Y.V.Panchin(1995a)The early-stage of the cleavage in the free-living marine nematode Enoplus brevis (Enoplida, Enoplidae) in the normal and experimental conditions. Zool. Zhurn. 74(6): 31-38. Voronov,D.A.,Y.V.Panchin(1995b)Gastrulation in the free-living marine nematode Enoplus brevis and the localization of endodermal material at the stage of 2 blastomeres in the nematodes of the order Enoplida. Zool. Zhurn. 74(10): 10-18.
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Worm Breeder's Gazette,
1996]
Protein phosphatases (PP) are significant regulatory enzymes of all eukaryotes. They must be present in C.elegans as witnessed by DNA-deduced PP-like amino acid sequences unraveledin the framework of the genom project. About half of them belong to Ser/Thr PP, while the rest is Tyr PP. For the assay of the main Ser/Thr PP classes, namely PP1, PP2A, PP2B and PP2C Cohen and coworkers suggested a straightforward and relatively simple procedure (1) that was adopted for the analysis of the worm. Rabbit muscle phosphorylase a phosphorylated by phospho- rylase kinase, rabbit muscle inhibitor-1 and casein from bovine milk phosphorylated by bovine heart cAMP-dependent protein kinase were used as substrates. C.elegans Var. Bristol (N2) wild type strain were cultured, harvested, rinsed with the extraction buffer, frozen in liquid nitrogen and homogenized in four volumes of the ice cold extraction buffer (50 mM Tris-HCl pH=7.4; 0.1 mM EDTA; 10 mM DTT; 0.5% Triton X-100; 2mM PMSF; 5mM benzamidine and 1 mM o-phenantroline).The homogenates were cleared by cent- rifugation at 14000 g, 4 oC for 10 min and than were kept frozen in liquid nitrogen till the assays. Rabbit skeletal muscle inhibitor-2 (unphosphorylated) was used as a specific inhibitor ofPP1 with 32P-phosphorylase a substrate in the presence ofEDTA,a metal ion chelator(Fig.1).The titration curve reveals that about 75%of the total activity was supplied by PP1 and 5 nM of inhibitor-2 caused about 50% inhibition. Okadaic acid, a potent marine toxin and tumor promoter, was used to separate PP1 and PP2A activities with 32P-phosphorylase a substrate in the pre- sence of EDTA (Fig.2). The titration curve with okadaic acid is bi- phasic, in the first step about 30% of the total activity is inhibited with an IC50 of ~0.04 nM and the rest of activity was inhibited in a second step with an IC50 of ~70 nM. Complete inhibition was achieved by 1 micromol/liter okadaic acid concentration. It is known that PP2A is more sensitive to okadaic acid than PP1, thus the first step of the titration can be explained by the presence of PP2A while the second step can be attributed to PP1 inhibition. For the detection of PP2B we used 32P-inhibitor-1 substrate and Ca-calmodulin as an activator. Due to the interference of prote- ases the 32Pi liberated in the assays was estimated after ammonium molybdate extraction(2). About 50 nmol substrate was converted in 1 min by the extract. ~70% of the activity was inhibited by 4 mM EDTA or 1.5 mM trifluoperazine and ~30% was inhibited by 2 nM okadaic acid (not shown). Thus 70% of the total activity was due to PP2B and 30% to PP2A. PP2C was assayed with 32P-casein substrate in the presence of Mg2+-activator. ~10%of the activity was inhibited by inhibitor-2 (PP1) ~40% of the activity was inhibited by 2 nM okadaic acid (PP2A) and ~50% was stimulated by Mg2+ (PP2C). Half maximal activation was achieved at 2 mM Mg2+ concentration, maximal activation was measured at 20 mM (Fig.3.). Acknowledgements.This work was supported by the grants OTKA 6005 and 12840. (1) Cohen, P., Klump, S., Schelling, D.L. (1989) FEBS Lett. 250, 596-600. (2) Shenolikar, S., Ingebritsen, T.S. (1984) Meth. Enyzmol. 107, 103-129.
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Worm Breeder's Gazette,
1996]
To better understand the evolution of cell-specification mechanisms we study development in other nematode species. One of them is Cephalobus spec., an apparently more primitive representative of rhabditid nematodes (Skiba and Schierenberg, 1992, Dev. Biol. 151:597). Cephalobus lacks autofluorescence and birefringence of the gut granules as found in C.elegans. In order to analyse development of the intestine we therefore take advantage of 1.) its tissue-specific endocytotic activity leading to accumulation of fluorescently labelled transferrin (Bossinger et al., 1996, Roux's Arch. Dev. Biol. 205: 494) and 2.) the mAb 1CB4 which specifically recognizes gut cells in C.elegans (Okamoto and Thomson, 1985, J. Neurosci. 5: 643) and Cephalobus. In the last WBG we reported that gut differentiation in Cephalobus does not depend on an induction by P2 but may depend on AB. Here we present additional data leading to a modified interpretation. When AB was extruded at the 2-cell-stage, 87% (35/40) of the P1-derived partial embryos displayed gut differentiation while 13% (5/40) did not. When AB was extruded at the 3-cell stage 45% (15/33) of the partial embryos showed gut differentiation while 55% (18/33) did not (or very faintly). From this we conclude that in Cephalobus gut differentiation can take place without inductions from either P2 or AB (descendants). In another set of experiments we extruded P1 from 2-cell stages. Much to our surprise, 62% (20/32) of the emerging AB-derived embryos developed strong gut differentiation although lineage analysis shows that normally the gut in Cephalobus comes exclusively from descendants of the E- cell as in C. elegans. To exclude a potential confusion of AB/P1 (despite their size differences), we ascertained the typical different early lineage patterns in the non-extruded and the extruded cell. We found that in 65% (17/26) of the embryos AB was still able to produce gut-like cells when EMS and P2 were extruded in late 3-cell stages. In 35% (6/17) of these we even observed an overexpression of the gut markers in the AB lineage with 40-60 cells being recognized by the transferrin assay and nicely outlined by the antibody. These cells were considerably smaller than the normal 20-24 gut cells in untreated Cephalobus embryos, suggesting one or two additional rounds of cell division. Our results indicate that in Cephalobus both of the first two blastomeres carry the potential to develop gut and that an inhibitory interaction between AB and EMS (or their descendants) is necessarty to restrict the gut fate to the E-lineage. They also show that the relatively few divisions that normally take place in the gut lineage are not a prerequisite for proper differentiation. They can be interpreted as an early example of two cells competing for a primary fate as observed later in equivalence groups. Preliminary observations suggest that in manipulated embryos after the 8-AB cell stage some AB descendants acquire a slower cell-cycle rhythm and give rise to descendants with gut characteristics. This pattern is reminiscent of early embryogenesis in Enoplus brevis, a marine nematode in which a visible soma/germline differentiation is absent. In addition, other nematode species have been described in which gut is derived from the AB blastomere (V.V. Malakhov, 1994, Nematodes, Smithsonian Institution Press).