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Neuronal control of the cardiac responses to osmotic stress in the gastropod limpet Patella caerulea.

Mediterranean limpets Patella caerulea were exposed to different salinity conditions and treated with drugs interfering with neuronal control of heartbeat. Heart rate was monitored using a non-invasive method. Limpets were superfused with control (33 g l(-1)), hyposaline (0 and 10 g l(-1)) or hypersaline (56 and 66 g l(-1)) artificial seawater. Under osmotic stress the limpets showed an initial increase of heart rate, followed by acardia, particularly under hyposalinity. The tachycardia observed after exposure to 56 g l(-1) was abolished in the animals injected with a selective sodium channel blocker, tetrodotoxin (TTX), or with a serotoninergic antagonist, methysergide. Injection of TTX also partly prevented the acardia occurring at 0 g l(-1). The acardia was completely prevented after injection with atropine and benzoquinonium, two selective cholinergic antagonists. These findings indicate that cardiac responses of P. caerulea to variations in external salinity are regulated by an extrinsic neuronal control involving the serotoninergic and the cholinergic systems in the tachycardic and acardic responses, respectively.

Adaptation, Physiological↗

Development of dorsoventral polarity and mesentoblast determination in Patella vulgata.

In Patella vulgata the 32-cell stage represents a pause in the mitotic activity prior to the differentiation of the mesentoblasts mother cell 3D. At the onset of this stage, the embryo is radially symmetrical. Nevertheless, the plane of bilateral symmetry is indicated as it passes through the macromeres forming the vegetal cross-furrow. From the early beginning of the 32-cell stage, all four macromeres introdude far into the interior and tough the centrally radiating cells of the first quartet of micromeres. The two cross-furrow forming macromeres (3B and 3D) intrude the farthest and come into contact with the greatest number of micromeres. Finally, the contacts are extended significantly and maintained with only one of these macromeres. From that moment, this cell can be called the macromere 3D and the dorsoventral axis is determined. The evolution of the internal cell contacts between the micromeres of the first quartet and the macromeres indicates an essential role of the former in the determination of one of the latter as the masentoblasts mother cell, and thus in the determination of dorsoventral polarity.

Animals↗

Cell lineage of the prototroch of Patella vulgata (Gastropoda, Mollusca).

The trochophore larva of the archaeogastropod mollusc Patella vulgata has a well-developed locomotory organ, the ciliated prototroch. This structure is formed from specific founder cells, the trochoblasts. Two methods were employed to determine the composition and cell lineage of the prototroch. Fluorescent cell-lineage tracer injection in trochoblasts and trochoblast founder cells was used to show how the various trochoblasts became incorporated into the prototroch. Scanning electron microscopy was used to study both differentiation, more specifically ciliation, of trochoblasts and localization of trochoblasts in the prototroch. The results obtained with both methods are in accordance with each other. During early development all trochoblasts involved in prototroch formation become cell cycle-arrested and develop cilia. Subsequently, the trochoblasts shift in position to form a circular prototroch and a number of trochoblasts deciliate. As a result of these processes the mature prototroch consists of a number of heavily ciliated cells as well as a number of deciliated cells. Comparison of prototrochs from a number of spiralian species shows that this structure is very conserved during evolution. The significance of this is discussed.

Animals↗

Follicle cell regulation of amino acid uptake and polyphosphoinositide metabolism in oocytes of the limpet Patella vulgata.

We investigated the role of follicle cells during meiosis reinitiation in the oocytes of Patella vulgata. Germinal vesicle breakdown, chromosome condensation, and metaphase-1 spindle formation were artificially induced by treating oocytes with 10 mM NH4Cl. By following the accumulation of 32PO4 and [3H]inositol in polyphosphoinositides (PPI) and by measuring chemical amounts of these lipids, we found that the presence of follicle cells caused PPI turnover to remain at a low level in the immature oocyte. On the other hand, the presence of these cells was necessary for maximal activation of the metabolism of these lipids during maturation. We also examined the nutritive role of follicle cells by studying the transport of leucine and found that the presence of these cells was necessary for optimal transport of this amino acid. We conclude that follicle cells can exert either a stimulatory or an inhibitory effect on the oocyte metabolism according to its state of maturity.

Animals↗

Expression of Patella vulgata orthologs of engrailed and dpp-BMP2/4 in adjacent domains during molluscan shell development suggests a conserved compartment boundary mechanism.

The engrailed gene is well known from its role in segmentation and central nervous system development in a variety of species. In molluscs, however, engrailed is involved in shell formation. So far, it seemed that engrailed had been co-opted uniquely for this particular process in molluscs. Here, we show that, in the gastropod mollusc Patella vulgata, an engrailed ortholog is expressed in the edge of the embryonic shell and in the anlage of the apical sensory organ. Surprisingly, a dpp-BMP2/4 ortholog is expressed in cells of the ectoderm surrounding, but not overlapping, the engrailed-expressing shell-forming cells. It is also expressed in the anlage of the eyes. Earlier it was shown that a compartment boundary exists between the cells of the embryonic shell and the adjacent ectoderm. We conclude that engrailed and dpp are most likely involved in setting up a compartment boundary between these cells, very similar to the situation in, for example, the developing wing imaginal disc in Drosophila. We suggest that engrailed became involved in shell formation because of its ancestral role, which is to set up compartment boundaries between embryonic domains.

Amino Acid Sequence↗

Protein synthesis controls cyclin stability in metaphase I-arrested oocytes of Patella vulgata.

The metaphasic block of Patella vulgata oocytes depends on protein synthesis as an emetine treatment triggers metaphase/anaphase transition and leads to the sequential disappearance of cyclin A and B. Both cyclins are stable in metaphase-arrested oocytes which indicates that inhibition of protein synthesis activates cyclin proteolysis. The use of extracts prepared from metaphase-arrested oocytes and from emetine-treated oocytes fully confirms these in vivo findings. Considering previous observations about the regulation of protein synthesis throughout the cell cycle, we propose the involvement of a transient inhibition of translation in the activation of cyclin proteolysis and in exit from the M-phase arrest.

Animals↗

Putative sense organs on the pallial tentacles of the limpet, Patella vulgata (L.).

The structure and ultrastructure of ciliary tufts on the pallial tentacles of the limpet Patella vulgata (L.) are described. The tip of each tentacle is covered by a dense crown of tufts and additional tufts can be seen scattered evenly across the surface of each tentacle. The cilia are nonmotile and nerve fibres run from the base of the ciliated cells suggesting a sensory function. Comparisons are made with ciliary tufts found in a Pacific species of limpet, Acmaea scutum, and other molluscan sensory structures.

Animals↗

Basal bodies in the odontoblasts of the limpet, Patella coerulea L. (Gastropoda).

The odontoblasts in the long radular gland of Patella coerulea L. are arranged in a terminal position; therefore newly formed teeth already have an upright position. The long and slender odontoblasts have only one to three lengthy and ramifying apical microvilli. Between these pinnate microvilli a fine filamentous material appears which probably corresponds to chitin microfibrils. Therefore, the pattern of chitin microfibrils seems to depend on the arrangement of odontoblasts' microvilli. For the first time, basal bodies were found in the apical part of odontoblasts which led to the assumption that the radular gland originally might have been a mucous gland, the secretion of which was transported by cilia.

Animals↗

Physico-chemical properties and rheological behaviour of Patella caerula paramyosin.

Paramyosin is a muscle protein which is characteristic of all invertebrates but which is not present in vertebrate muscles. Given the functional importance of paramyosin, the purpose of this paper was to study the physico-chemical properties, including the amino acid composition and rheological behaviour, of purified paramyosin and to investigate its mode of interaction with myosin. Paramyosin was purified from the limpet (Patella caerula) by an ethanol precipitation step. It was soluble at ionic strengths below 0.05 M NaCl and its maximum solubility at neutral pH occurred at approximately 0.4 M NaCl. At this high ionic strength, the pH dependence of solubility was such that paramyosin passed quickly into solution when pH exceeded pH 5, the transitional pH value. By using an immunological method, it was shown that interactions between paramyosin and myosin occurred, even in the presence of actin. The molecular assembly of both proteins was probably specified by hydrophobic interactions, as well as by interactions enhanced by divalent cations. The changes in the dynamic shear storage modulus (G') started between 40 degrees C and 50 degrees C, and reached a maximum at about 75 degrees C.

Actins↗

Differences in the rehabilitation period following two methods of anterior cruciate ligament replacement: semitendinosus/gracilis tendon vs. ligamentum patellae.

This study compared patient outcome during the early rehabilitation phase following two different methods of anterior cruciate ligament (ACL) reconstruction: ligamentum patellae (LP) and semitendinosus/gracilis tendon (SG) based reconstruction. The study included 50 consecutive patients treated by each method, examined 6 weeks and 3, 6 and 12 months after surgery. Patients in the SG group showed significantly better Lysholm scores at 6 and 12 months, Tegner Activity Scale scores at 3 months, and pain profile assessments at 6 weeks and 3 months than those in the LP group. Significant advantages were observed in LP group in the Overall Knee Score at 6 weeks and in range-of-motion at 6 weeks and 3 and 6 months post-surgery. Stability tests revealed no significant differences between patients in the two groups. SG-based reconstruction of the ACL thus demonstrates advantages over LP-based reconstruction regarding pain and function, while LP-based reconstruction was associated with an earlier return of motion.

Adolescent↗

Abnormal bone scans of the tibial tunnel 2 years after patella ligament anterior cruciate ligament reconstruction: correlation with tunnel enlargement and tibial graft length.

Tibial bone tunnels were examined with bone scans 2 years after patella ligament ACL reconstruction in 68 patients. At 2 years, scan uptake at the tibial tunnel was increased in 29% of patients. Marked increase of scintigraphic uptake was associated with tibial tunnel enlargement of more than 35% and a graft length in the tibial tunnel over 14 mm. Scan uptake was correlated to tunnel enlargement (r = 0.64, P < 0.01) and tunnel enlargement was correlated to graft length inside the tibial tunnel (r = 0.59 P < 0.001). No correlation was found between scan uptake or tunnel enlargement and anterior laxity, sagittal tunnel position and subjective outcome. Scintigraphy indicates the enlarged tibial tunnels are filled with remodelling bone. Tibial fixation location influences ligament healing inside the tunnel: Return of osseous homeostasis at the tibial tunnel can take more than 2 years when fixation is more than 14 mm below the joint.

Adolescent↗

Evaluation of tibial bone-tunnel changes with X-ray and computed tomography after ACL reconstruction using a bone-patella tendon-bone autograft.

Thirteen patients who underwent anterior cruciate ligament (ACL) reconstruction with bone-patella tendon-bone autografts (BTB) using interference screws were the subjects of this study. We analysed the relationship between bone-tunnel changes and clinical results 2 years after ACL reconstruction. To investigate changes, X-ray images were used to evaluate bone-tunnel enlargement, and computed tomography (CT) was used to evaluate the sclerotic area around the bone tunnel. The KT-2000 was used to measure the discrepancy of tibial anterior displacement between the affected and nonaffected sides (DTAD). There was no correlation between bone-tunnel enlargement and DTAD. On the other hand, in the CT evaluation, there was a significant correlation between the sclerotic area and DTAD. Our results suggest that it is more significant to analyse the area of sclerotic change than bone-tunnel enlargement for clinical evaluation. We cannot evaluate bone-tunnel changes correctly with two-dimensional X-rays and cannot analyse the sclerotic area using X-rays. Therefore, we recommend that CT, with which it is possible to analyse the sclerotic area, be used to evaluate bone-tunnel changes and clinical results.

Adult↗

Distalization of the patella during tibial callus distraction.

Consecutive distalization of the patella is described in two patients undergoing segmental transportation after high tibial corticotomy. Revision surgery with loosening and proximal reattachment of a portion of the patellar ligament bridging the callus distraction zone could re-establish the correct patellar position. Despite excellent callus formation after tibial corticotomy just below the tibial tuberositas, this procedure should be performed more distally as the fibers of the patellar tendon spread laterally and distally.

Adult↗

Novel and conserved roles for orthodenticle/ otx and orthopedia/ otp orthologs in the gastropod mollusc Patella vulgata.

The orthodenticle/ otx and orthopedia/ otp classes of homeobox gene families have been identified in all three major classes of bilaterians: deuterostomes, lophotrochozoans, and ecdysozoans. Otx genes have been studied extensively and play a role in the development of anterior neural structures. Otp genes have been found to be involved in nervous system development in mouse and Drosophila. To date, no members of these genes are known in molluscs. We cloned orthologs of orthodenticle/ otx and orthopedia/ otpfrom the gastropod Patella vulgata, and designated them Pv-otx and Pv-otprespectively. Our analysis of the spatio-temporal expression pattern of otx and otp orthologs during P. vulgata embryogenesis leads to the following conclusions. First, Pv-otx is expressed in and around the stomodaeum and our analysis thus supports the previously suggested conservation of the protostome and deuterostome larval mouth regions. Second, we find that Pv-otp is involved in the development of the larval apical sensory organ, suggesting a conserved role for this gene family in nervous system development. A similar conserved role in nervous system development has been proposed for orthodenticle/otx genes and we suggest that part of the cells expressing Pv-otx are involved in the development of the anterior nervous system. Last, we postulate that otx genes were ancestrally involved in the development of ciliary bands in bilaterians.

Amino Acid Sequence↗

Expression patterns of fork head and goosecoid homologues in the mollusc Patella vulgata supports the ancestry of the anterior mesendoderm across Bilateria.

We have characterised orthologues of the genes fork head and goosecoid in the gastropod Patella vulgata. In this species, the anterior-posterior (AP) axis is determined just before gastrulation, and leads to the specification of two mesodermal components on each side of the presumptive endoderm, one anterior (ectomesoderm), and one posterior (endomesoderm). Both fork head and goosecoid are expressed from the time the AP axis is specified, up to the end of gastrulation. fork head mRNA is detected in the whole endoderm, as well as in the anterior mesoderm, whereas goosecoid is only expressed anteriorly, in the three germ layers. The two genes are thus coexpressed in the anterior mesoderm, suggesting the latter's homology with vertebrate prechordal mesoderm. In addition, since prechordal plate is known to belong to an anterior, so called "head organiser", and since its inductive role is dependent on the function of the vertebrate fork head and goosecoid orthologues, we further suggest that the anterior mesoderm may also have a role in anterior inductive patterning in Spiralia. Finally, we propose that a mode of axial development involving two organisers, one anterior and one posterior, is ancestral to the Bilateria, and that both organisers evolved from the single head organiser of a putative hydra-like ancestor.

Animals↗

Spatio-temporal expression of a gene encoding a putative RNA-binding protein during the early larval development of the mollusc Patella vulgata.

In the mollusc Patella vulgata a cDNA clone named Esther 32 (E32) was found to be expressed in a specific spatio-temporal pattern. DNA sequence analysis showed that E32 represents a putative RNA-binding protein containing a KH domain. In early trochophore larvae, expression of E32 was found in all cells except the already differentiated trochoblasts and the apical tuft cells. Later on in development, expression was also abolished in the presumptive shell gland and restricted to specific areas, among which were the head and foot anlage. This suggests that E32 is involved in maintaining cells in an undifferentiated state via a post-transcriptional mechanism.

Amino Acid Sequence↗

Metal concentrations in the radula of the common limpet, Patella vulgata L., from 10 sites in the UK.

Metal (Al, As, Ca, Cd, Cu, Fe, K, Mg, Mn, Na, Pb, Zn) levels in the feeding organ or radula of the common limpet Patella vulgata L. were surveyed in 10 populations over a approximately 150-km stretch of coastline in north-east England. The most northern population was at Beadnell in Northumberland and the most southern was at Port Mulgrave in North Yorkshire; sites included unspoilt bays and areas heavily affected by industrial contamination such as the River Tees estuary. We hypothesized that the radula might be used as an indicator of environmental contamination. There were significant differences between the sites in the ratio of radula length to shell length. Limpets from Whitburn had the smallest radula fraction (mean = 1.665), while those from Port Mulgrave the largest (mean = 1.998). Such variation is common in the literature and we detected no correlate and propose no cause. Iron was clearly the dominant metal in the radulae, with an overall of mean of 1.46% of radular weight, though this is rather low in comparison to values in the literature. Iron is naturally secreted into the developing radula as a putative hardening agent. The next most abundant metals, in descending order, were Na (at approximately 2000-8000 microg g(-1)), K, Mg, Ca (approximately 1000-1500 microg g(-1)), Zn, Cu, Al, Pb (approximately 7-75 microg g(-1)), Mn, As, Cd (approximately 0-1 microg g(-1)). All but Al and Cd showed significant differences between the sites, but not in any consistent or convincing geographic manner. Nevertheless, the variations in metal levels between sites (e.g. Fe > 72%, Cu and Zn > 10-fold) suggest an environmental cause, but we are unable to offer any responsible factor, for example, there appeared little effect of the River Tees estuary. Cadmium is at a relatively low level in the radula in comparison to published data on pedal mucus and the flesh, but Pb is relatively high in pedal mucus and the radula and this might suggest that the radula is a detoxification route for Pb. Although the relationship between radula metal content and environmental metal content is unknown, the radula is constantly replaced and so may yet have the potential to be of use as a bioindicator, integrating metal exposure over much shorter periods than whole body burdens.

Animals↗

Microinjection of lectins, hyaluronidase, and hyaluronate fragments interferes with cleavage delay and mesoderm induction in embryos of Patella vulgata.

In embryos of Patella vulgata at the 32-cell stage, one of the four vegetally located macromeres makes contacts with overlying animal micromeres. As a result, this macromere (designated 3D) divides significantly later than the other macromeres and forms the mesodermal stem cell 4d. Shortly before and during this interaction two types of extracellular matrix are present: a basal lamina-like layer on the tips of the micromeres and a loose fibrillar meshwork in the blastocoel. In this paper we examine the role of the matrix in cleavage delay and mesoderm determination. The microinjection of extracellular matrix-binding lectins, or of hyaluronidase, or of decasaccharide fragments of hyaluronate into the blastocoel results in embryos in which either no or two macromeres are delayed in cleavage and are presumably determined as mesodermal stem cells. We suggest that the fibrillar meshwork is needed for macromere elongation toward the micromeres and that the basal lamina-like layer is involved in the determination process itself.

Animals↗