A possible neuroendocrine system in polynoid polychaetes.
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Animals↗
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Collagenous and other organizations in mature annelid cuticle and epidermis.
The mature annelid cuticle contains orthogonally oriented collagen in a matrix capped superficially by a dense epicuticle with external corpuscles. The underlying epidermis is a simple columnar epithelium with two major cell types, mucous-secreting cells which secrete through channels in the cuticle to the exterior of the worm, and "supportive" cells which presumably produce and increase the cuticle by secreting into it. The structures of supportive cells, previously interpreted as specialized for establishing interfibrillar collagen order, are revealed by glutaraldehyde fixation as common cellular components without the qualities deemed useful to align collagen. Cell processes which penetrate and sometimes pass completely through the cuticle are not stable, not in geometric order, and lack cilia-like structure. Cilia, unlike the ubiquitous cellular processes, are highly restricted to regions of the epidermis with specialized functions. Cellular control, or other control, of collagen fibrillogenesis remains unestablished.
Animals↗
Collagen deposition on a preformed grid.
Appearance of collagen fibrils in the cuticle was seen by electron microscopy to be preceded by formation of a finely filamentous matrix material. At first, the fine filaments of the matrix are unorganized. However, signs of orthogonal ordering soon appear in the most superficial portion of the cuticle, and subsequently appear more basally and closer to the underlying epidermis. Meanwhile, fibrils of different staining properties and identifiable as collagen begin to be deposited in the superficial portion of the cuticle, the same region which first showed organized fine filaments. Then, like the fine filaments before them, the collagen fibrils polymerize more basally. Collagen appears to polymerize on the preformed skeleton of fine filaments as though the fine filaments lagen fibrils seems to require direct cellular intervention but occur first in that portion of the cuticle which is furthest away from the underlying epidermis. The fine filaments may be self ordering, extracellular macromolecules which in turn determine the polymerization of collagen fibrils.
Animals↗
On the neurosecretory system of Dendrobaena atheca Cernosvitov.
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Isolate from the annelid, Reteterebella queenslandia (Australia), active against Ehrlich ascites tumor.
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Determining the purity of samples from natural products by coupling HPLC and CCD spectrometry.
Isolation and identification of natural products is a very important and active research field. However, establishing the purity of the samples during the isolation process is quite difficult, especially when the retention times are similar for two desired components in HPLC. Although some technologies, e.g. MS and NMR, offer effective ways of obtaining purity information about the samples, the expensive instrumentation required or the off-line nature of coupling (generally speaking) make purity analysis somewhat inconvenient. In this paper, an on-line analytical system coupling HPLC and a CCD spectrometer for determination of purity for each eluate was developed in a thin layer spectrometric cell. The effectiveness of the system was demonstrated by differentiating Tanshinone I, Tanshinone IIA, and their mixture. The time-resolved UV-Vis spectra promptly revealed significant differences between the three samples while conventional single wavelength detection (CSWLD) could not. The system was then used to distinguish two steroid compounds which behaved as a single component in CSWLD. The compounds were isolated from a Chinese marine invertebrate animal, a marine annelid, Arenicola cristata, referred to here as Stimpson. The method reported here provided an efficient, convenient, fast, and inexpensive approach holding promise for on-line determination of the purity of samples isolated from natural products.
Abietanes↗
Effects of MAP kinase pathway and other factors on meiosis of Urechis unicinctus eggs.
The eggs of Urechis unicinctus Von Drasche, an echiuroid, are arrested at P-I stage in meiosis. The meiosis is reinitiated by fertilization. Immunoblotting analysis using anti-ERK2 and anti-phospho-MAPK antibodies revealed a 44 kDa MAP kinase species that was constantly expressed in U. unicinctus eggs, quickly phosphorylated after fertilization, and dephosphorylated slowly before the completion of meiosis I. Phosphorylation of the protein was not depressed by protein synthesis inhibitor Cycloheximide (CHX), but was depressed by the MEK1 inhibitor PD98059. Under PD98059 treatment, polar body extrusion was suppressed and the function of centrosome and spindle was abnormal though GVBD was not affected, indicating that MAP kinase cascade was important for meiotic division of U. unicinctus eggs. Other discovery includes: A23187 and OA could parthenogenetically activate U. unicinctus eggs and phosphorylated 44 kDa MAP kinase species, indicating that the effect of fertilization on reinitiating meiosis and phosphorylation of 44 kDa MAP kinase specie is mediated by raising intracellular free calcium and by phosphorylation of some proteins, and that phosphotase(s) sensitive to OA is responsible for arresting U. unicinctus eggs in prophase I. diC8, an activator of PKC, accelerated the process of U. unicinctus egg meiotic division after fertilization and accelerated the dephosphorylation of 44 kDa MAP kinase specie, which implied that the acceleration effect of PKC on meiotic division was mediated by inactivation of MAP kinase cascade. Elevating cAMP/PKA level in U. unicinctus eggs had no effect on meiotic division of the eggs.
1-Methyl-3-isobutylxanthine↗
Differing mechanisms of cAMP- versus seawater-induced oocyte maturation in marine nemertean worms II. The roles of tyrosine kinases and phosphatases.
Instead of blocking oocyte maturation as it does in most animals, cAMP causes oocytes of marine nemertean worms to initiate maturation (=germinal vesicle breakdown, "GVBD"). To characterize cAMP-induced GVBD in nemerteans, inhibitors of tyrosine kinase signaling were tested on Cerebratulus sp. oocytes that had been incubated in cAMP-elevating drugs versus seawater (SW) alone. Such tests yielded similar results for Src-like tyrosine kinase blockers, as the inhibitors prevented mitogen-activated protein kinase (MAPK) activation without stopping either GVBD or maturation-promoting factor (MPF) activation in both SW and cAMP-elevating treatments. Alternatively, genistein, a general tyrosine kinase antagonist, and piceatannol, an inhibitor of the tyrosine kinase Syk, reduced GVBD and MAPK/MPF activities in SW-, but not cAMP-induced maturation. Similarly, inhibitors of the human epidermal growth factor receptor-2 (HER-2) tyrosine kinase prevented GVBD and MAPK/MPF activations in oocytes treated with SW, but not with cAMP-elevating drugs. Antagonists of either protein tyrosine phosphatases (PTPs) or the dual-specificity phosphatase Cdc25 also reduced GVBD and MAPK/MPF activities in SW-treated oocytes without generally affecting cAMP-induced maturation. Collectively, these data suggest cAMP triggers GVBD via pathways that do not require MAPK activation or several components of tyrosine kinase signaling. In addition, such differences in tyrosine kinase cascades, coupled with the dissimilar patterns of Ser/Thr kinase signaling described in the accompanying study, indicate that nemertean oocytes are capable of utilizing multiple mechanisms to activate MPF during GVBD.
Animals↗
Differing mechanisms of cAMP- versus seawater-induced oocyte maturation in marine nemertean worms I. The roles of serine/threonine kinases and phosphatases.
Unlike in most animals, oocytes of marine nemertean worms initiate maturation (=germinal vesicle breakdown, GVBD) following an increase, rather than a decrease, in intraoocytic cAMP. To analyze how serine/threonine (Ser/Thr) kinase cascades involving mitogen-activated protein kinase (MAPK), maturation-promoting factor (MPF), cAMP-dependent protein kinase (PKA), and phosphatidylinositol 3-kinase (PI3K) regulate nemertean GVBD, oocytes of Cerebratulus sp. were treated with pharmacological modulators and stimulated with cAMP-elevating drugs or seawater (SW) alone. Both cAMP elevators and SW triggered GVBD while activating MAPK, its target p90Rsk, and MPF. Similarly, neither cAMP- nor SW-induced GVBD was affected by several Ser/Thr phosphatase inhibitors, and both stimuli apparently accelerated GVBD via a MAPK-independent, PI3K-dependent mechanism. However, inhibitors of Raf-1, a kinase that activates MAPK kinase, blocked GVBD and MAPK activation during SW-, but not cAMP-induced maturation. In addition, MPF blockers more effectively reduced GVBD and MAPK activity in SW versus in cAMP-elevating treatments. Moreover, the two maturation-inducing stimuli yielded disparate patterns of PKA-related MAPK activations and phosphorylations of putative PKA substrates. Collectively, such findings suggest that in maturing oocytes of Cerebratulus sp., Ser/Thr kinase cascades differ during cAMP- versus SW-induced GVBD in several ways, including MAPK activation modes, MPF-feedback loops, and PKA-related signaling pathways. Additional differences in cAMP- versus SW-induced oocyte maturation are also described in the accompanying study that deals with the roles of tyrosine kinase signaling during GVBD.
Androstadienes↗
Characterization of carbonic anhydrases from Riftia pachyptila, a symbiotic invertebrate from deep-sea hydrothermal vents.
The symbiotic hydrothermal vent tubeworm Riftia pachyptila needs to supply its internal bacterial symbionts with carbon dioxide, their inorganic carbon source. Our aim in this study was to characterize the carbonic anhydrase (CA) involved in CO(2) transport and conversion at various steps in the plume and the symbiotic tissue, the trophosome. A complete 1209 kb cDNA has been sequenced from the trophosome and identified as a putative alpha-CA based on BLAST analysis and the similarities of total deduced amino-acid sequence with those from the GenBank database. In the plume, the putative CA sequence obtained from cDNA library screening was 90% identical to the trophosome CA, except in the first 77 nucleotides downstream from the initiation site identified on trophosome CA. A phylogenetic analysis showed that the annelidan Riftia CA (CARp) emerges clustered with invertebrate CAs, the arthropodan Drosophila CA and the cnidarian Anthopleura CA. This invertebrate cluster appeared as a sister group of the cluster comprising mitochondrial and cytosolic isoforms in vertebrates: CAV, CAI II and III, and CAVII. However, amino acid sequence alignment showed that Riftia CA was closer to cytosolic CA than to mitochondrial CA. Combined biochemical approaches revealed two cytosolic CAs with different molecular weights and pI's in the plume and the trophosome, and the occurrence of a membrane-bound CA isoform in addition to the cytosolic one in the trophosome. The physiologic roles of cytosolic CA in both tissues and supplementary membrane-bound CA isoform in the trophosome in the optimization of CO(2) transport and conversion are discussed.
Amino Acid Sequence↗
Two distinct heme distal site states define Cerebratulus lacteus mini-hemoglobin oxygen affinity.
The nerve tissue hemoglobin of Cerebratulus lacteus (CerHb) is the smallest naturally occurring known hemoglobin. Stabilization of the diatomic bound species (e.g., O(2)) is achieved through a network of hydrogen bonds based on three key residues TyrB10, GlnE7, and ThrE11. The first two residues are typically associated in hemoglobins with enhanced O(2) affinity, related to hydrogen bond stabilization of the heme-bound O(2) resulting in a decrease of the ligand dissociation rates. In contrast to the above observations, the affinity of CerHb for O(2) is only moderate, and the rate of O(2) dissociation is unexpectedly high. To gain insight on the diverse molecular mechanisms controlling ligand affinities, we have analyzed w.t. CerHb and its ThrE11-->Val mutant by means of joint molecular dynamics and quantum mechanics simulation techniques, complementing recent site-directed mutagenesis experiments. Our results suggest that the observed O(2) dissociation rates can only be explained through a dynamic equilibrium between high and low affinity states of the w.t. CerHb heme distal site.
Amino Acid Substitution↗
Silica sand as an artificial control sediment in a 20 day Neanthes arenaceodentata toxicity test.
The use of artificial control sediments in toxicity tests offers several advantages over field-collected control sediments, especially for laboratory-cultured organisms such as Neanthes arenaceodentata. Ten side-by-side trials were conducted using a field-collected sediment from West Beach, Whidbey Island, Washington State, USA and an artificial sediment composed of a silica quartz sand commonly used for masonry applications. After a 20-d exposure to sediment and clean overlying seawater, a similar biological response (as measured by the final average individual dry weight of Neanthes) was observed. The average individual dry weight was 14.77 +/- 3.83 and 14.70 +/- 3.57 mg/worm for the artificial and field-collected negative controls, respectively.
Animals↗
Identification of the subunit loci in the extracellular multisubunit hemoglobin from annelid Perinereis aibuhitensis.
Giant hemoglobin (Hb) from Perinereis aibuhitensis is made of several types of protein components including single-chain globin (a), disulfide-bridged globin trimer (A-b-B), disulfide-bridged dimers of nonglobin chain (or linkers; L1-L1, L2-L2, and L1-L2), and oligomers of L1-L2 [(L1-L2)n]. The intact form of this giant Hb is a two-tiered hexagonal structure composed of 12 identical units, or so-called submultiples (six submultiples to a tier). To obtain a view of the three-dimensional architectural arrangement of these components in the intact form, we identified the subunit loci by using two mutually complementary chemical modifications and a colloidal gold labeling technique. Using the chemical modifications, we discovered that (i) linkers L1-L2 and L2-L2 were located at the exterior of the Hb, (ii) linker L1-L1 and globin a were buried in the interior, and (iii) linker (L1-L2)n and globin trimer A-b-B were located at both exterior and interior loci. The labeling with an L2-specific colloidal gold revealed the predominant loci of L2 at the outer and inner boundaries between neighboring submultiples in a hexagonal form. By combining these results with those from our previous reports [S. Ebina, K. Matsubara, K. Nagayama, M. Yamaki, and T. Gotoh (1995) Proc. Natl. Acad. Sci. USA 92, 7367-7371; K. Matsubara, M. Yamaki, Nagayama, H. Ishii, K. Imai, T. Gotoh, and S. Ebina (1996), in press], we deduced the following conclusions concerning the Hb architecture. The L1-L1 chains perhaps together with (L1-L2)n chains form a scaffold on which submultiples assemble into a two-tiered hexagonal arrangement, probably by connecting the carbohydrates in globin a. The L1-L2 and L2-L2 chains reinforce the connections of the submultiples by binding carbohydrates, perhaps those carbohydrates in globin A. We proposed to call this type of non-protein-dependent structural level as seen in such a carbohydrate-glued protein aggregate "protein-plus structure."
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Characterization of a novel cDNA sequence encoding invertebrate tachykinin-related peptides isolated from the echiuroid worm, Urechis unicinctus.
Tachykinin is one of the most well-known bioactive peptides found in vertebrates, and tachykinin-related peptides have also been isolated from various invertebrate species. Urechistachykinin I (Leu-Arg-Gln-Ser-Gln-Phe-Val-Gly-Ser-Arg-NH(2)) and II (Ala-Ala-Gly-Met-Gly-Phe-Phe-Gly-Ala-Arg-NH(2)) were purified from the ventral nerve cords of echiuroid worm, Urechis unicinctus. In the present study, we described the characterization of a novel cDNA encoding the urechistachykinin precursor. Amino acid sequence analysis of the deduced polypeptide revealed that the urechistachykinin precursor included seven structurally related peptides, unlike mammalian tachykinin precursors which encode only one or two tachykinin peptides. This is the first identification of an invertebrate tachykinin-related peptide cDNA.
Amino Acid Sequence↗
Protein and gene structure of a chlorocruorin chain of Eudistylia vancouverii.
The polychaete annelid, Eudistylia vancouverii, contains as oxygen carrier a hexagonal bilayer (HBL) chlorocruorin. One of the globin chains, chain a1, has 142 amino acids (Mr 16,054.99) and its sequence deviates strongly from other nonvertebrate globin sequences. Unprecedented, it displays a Phe at the distal position E7 as well as at position B10, creating a very hydrophobic heme pocket probably responsible for the low oxygen affinity of the native molecule. Phylogenetic analysis of annelid globin chains clearly proves that globin chain a1 belongs to type I of globin chains having a pattern of 3 cysteine residues essential for the aggregation into a HBL structure. The gene coding for globin chain a1 is interrupted by 2 introns at the conserved positions B12.2 and G7.0. Based on protein and gene structure it can therefore be concluded that the globin chains of chlorocruorins are not fundamentally different from other annelid globin chains.
Amino Acid Sequence↗
Cloning and expression of a lombricine kinase from an echiuroid worm: insights into structural correlates of substrate specificity.
Phosphagen kinases constitute a large family of enzymes catalyzing the reversible phosphorylation of guanidino acceptor compounds. These guanidino substrates differ substantially in size and chemical properties. In spite of the appearance of X-ray crystal structures for two members of this family, creatine kinase (CK) and arginine kinase (AK), the structural correlates of substrate specificity remain to be fully elucidated. We have determined the cDNA and deduced amino acid sequences for lombricine (guanidinethylphosphoserine) kinase (LK) from the echiuroid worm Urechis caupo and expressed the cDNA in Escherichia coli. The recombinant protein was purified by affinity chromatography and showed high capacity for phosphorylation of lombricine. Phosphagen kinases consist of a small, N-terminal domain and a much larger domain connected by a linker sequence. A key event in catalysis in CK and AK, and certainly all other phosphagen kinases, is a large conformational change involving involving a rotation of the two domains and the movement of two highly conserved flexible loops (one located in the small domain; the other located in the large domain of these enzymes) which clamp down on the substrates. Multiple sequence alignments of Urechis LK with the only other LK sequence available and CK, AK and glycocyamine kinase sequences, confirm the importance of the small flexible loop located in the N-terminal domain of phosphagen kinases as one component of the structural determinants of guanidine specificity. The role of the other flexible loop in the large domain in terms of substrate specificity remains questionable.
Amino Acid Sequence↗
Regulation of maternal messenger RNA translation during oogenesis and embryogenesis in Urechis caupo.
We have tested the hypothesis that the selective translation of mRNAs in oocytes and embryos is controlled by when during oogenesis individual maternal mRNAs are synthesized. In an earlier paper we described the isolation of cDNA probes to 21 maternal mRNAs which accumulate with different patterns during oogenesis in Urechis caupo (E. T. Rosenthal and F. H. Wilt, 1986, Dev. Biol. 117, 55-62). Many of these probes have now been used to analyze the translation of the maternal mRNAs in growing oocytes, full-grown oocytes, and embryos. The translation of all of the mRNAs studied changes dramatically when full-grown Urechis oocytes are fertilized. Our data do not support the idea that the translation of the different maternal mRNAs depends on when they were synthesized during oogenesis. This strongly suggests that, at some level, the ability of the cell's translational machinery to distinguish between different maternal mRNAs must reside directly, or indirectly, in the mRNA sequences. We have also found that in Urechis, as in Xenopus, the selective translation of maternal mRNAs is correlated with their selective adenylation. Consequently, we have sequenced the 3' ends of 16 translationally controlled maternal mRNAs in an effort to detect consensus sequences that might regulate adenylation. No such consensus sequences have been found, suggesting that the mRNA characteristics involved in the regulation of polyadenylation may reside in a wide variety of sequences or in more subtle features, such as secondary structure.
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