软体动物的一氧化氮及其合酶的研究进展

一氧化氮作为一种重要的信息分子,参与调节软体动物的嗅觉、运动、取食、机体防御及学习行为。本文从生理、生化、形态定位以及信号转导几方面综述了有关软体动物一氧化氮及其合酶的最新研究进展。     

Topology of the 10 subunits within the Decamer of KLH, the hemocyanin of the marine gastropod Megathura crenulata

Immunoelectron microscopy has been performed using negatively stained immune complexes of keyhole limpet hemocyanin isoform 1 (KLH1) decamers and a functional unit-specific monoclonal antibody anti-KLH1-c1. The antibody links hemocyanin molecules at both the collar and the collarless edge of the decamer, indicating a peripheral localization of functional units c. In isoform 2 (KLH2) the positions of functional units c have been identified with the peanut agglutinin (PNA), which has previously been shown to exclusively bind to KLH2-c. Ferritin linked to PNA was used to visualize labeled molecules electron microscopically. The pattern of labeling also indicates a peripheral localization of the c functional units. The data presented in this paper support only one of two possible models for the subunit orientation within the hemocyanin decamer.     

The central nervous system of Bulla gouldiana: peptide localization

In the present study, we describe the structure of the central nervous system (CNS) of the marine gastropod Bulla gouldiana, and compare it with the structure of the CNS of the related mollusc, Aplysia californica. In addition, we performed an immunohistochemical analysis of a series of peptides, and the synaptic vesicle protein, synapsin I, in the central nervous system of B. gouldiana. The most common peptide in the B. gouldiana nervous system is the molluscan cardioexcitatory peptide (FMRFamide), which is present in a significant proportion of B. gouldiana neurons. A smaller number of neurons exhibit immunoreactivity to antisera raised against the calcitonin gene related peptide, vasopressin, vasoactive intestinal peptide, cholecystokinin, galanin and enkephalin. In some instances there is colocalization of two or more peptides. Very few neurons or axons exhibit synapsin I-like immunoreactivity. The patterns of immunoreactivity to these antisera is quite similar to the patterns that have been described in other gastropods, including Lymnaea stagnalis and Aplysia californica. These observations emphasize the importance of FMRFamide-like compounds in phylogenetically old nervous systems and indicate that compounds similar to mammalian peptides are present in the gastropod. Thus, the production of a wide variety of peptide molecules and their use in neuronal function appears to be a highly conserved phylogenetic process.     

Astaxanthin binding and structural stability of the apple snail carotenoprotein ovorubin

Ovorubin (OR) is the major perivitellin of the eggs of Pomacea canaliculata. The astaxanthin (ASX) binding and structural stability of OR were investigated by fluorescence spectroscopy and circular dichroism (CD). The apo-OR (without astaxanthin) shows a single, high affinity binding site for ASX (K(D)=0.5 microM). The quenching of tryptophan fluorescence by ASX indicates that about 22% are near the carotenoid-binding site in a non-polar environment, as indicated by tryptophan resonance energy transfer to the ligand. Secondary structure (alpha+beta) was virtually not affected by cofactor removal. Holo-OR shows unusually high thermal stability. The removal of ASX does not affect the thermal or chemical stability of the quaternary structure. In conclusion, although subtle changes were observed, ASX is not essential for OR stability, unlike most invertebrate carotenoproteins. This supports the idea that OR plays an important physiological role in the storage, transport and protection of carotenoids during snail embryogenesis.     

Hsp70 in the atrial neuroendocrine units of the snail, Achatina fulica

Heat shock proteins (Hsps) are evolutionary conserved peptides well known as molecular chaperones and stress proteins. Elevated levels of extracellular Hsps in blood plasma have been observed during the stress responses and some diseases. Information on the cellular sources of extracellular Hsps and mechanisms regulating their release is still scanty. Here we showed the presence and localization of Hsp70 in the neuroendocrine system in the atrium of the snail, Achatina fulica. The occurrence of the peptide in snail atrium lysate was detected by Western blot analysis. Immunoperoxidase and immunogold staining demonstrated that Hsp70-immunoreactivity is mainly confined to the peculiar atrial neuroendocrine units which are formed by nerve fibers tightly contacted with large granular cells. Immunolabelling intensity differed in morphologically distinct types of secretory granules in the granular cells. The pictures of exocytosis of Hsp70-immunolabeled granules from the granular cells were observed. In nerve bundles, axon profiles with Hsp70-immunoreactive and those with non-immunoreactive neurosecretory granules were found. In addition, Hsp70-like material was also revealed in the granules of glia-interstitial cells that accompanied nerve fibers. Our findings provide an immuno-morphological basis for a role of Hsp70 in the functioning of the neuroendocrine system in the snail heart, and show that the atrial granular cells are a probable source of extracellular Hsp70 in the snail hemolymph.     

Hexamethonium sensitivity of the swim musculature of the pteropod mollusc, <Emphasis Type="Italic">Clione limacina</Emphasis>

Swimming in reduced electrophysiological preparations of the pteropod mollusc, Clione limacina, was blocked by bath application of hexamethonium even though pattern generator activity continued with this treatment. Neuromuscular recordings indicated that hexamethonium blocked synaptic input from Pd-3 and Pd-4 motoneurons to slow-twitch muscle cells, while connections from Pd-1A and Pd-2A motoneurons to fast-twitch muscle cells were variable in their response to hexamethonium—synaptic inputs were suppressed in most cases and occasionally blocked, but the latter only with high concentrations and long incubations. Acutely dissociated wing muscle cells showed a concentration-dependency in the percentage of contracted cells with bath application of acetylcholine, and this contractile activity was blocked in preparations that were first bathed in hexamethonium. Intracellular recordings from dissociated slow-twitch muscle cells showed conductance-increase depolarizations of approximately 20 mV following 1 s pressure ejections of 10⁻⁴ M acetylcholine from micropipettes placed immediately adjacent to the muscle cells. These responses were blocked when hexamethonium was bath applied prior to the pressure-applied acetylcholine. The results suggest the Pd-3/Pd-4 motoneuron to slow-twitch muscle cell junctions are cholinergic with nicotinic-like receptors, while the Pd-1A/Pd-2A to fast-twitch muscle cell connections are likely cholinergic, but with a different receptor type.     

Modelling the ontogeny of ectotherms exhibiting indeterminate growth

Numerous growth functions exist to describe the ontogeny of animals. Such functions (e.g., von Bertalanffy's equation, thermal-unit growth coefficient) are currently applied to ectotherms even though they fail to provide analytical expressions that adapt to a wide range of fluctuating temperatures. The underlying mechanisms responsible for the ontogeny of ectotherms exhibiting indeterminate growth have not yet been summarised in terms of a simple but meaningful mathematical equation. Here, a growth function is developed, with parameters having physical or biological interpretation that accommodates indeterminate growth under fluctuating temperatures assuming the latter vary seasonally. The equation is derived as a special case of von Bertalanffy's equation providing realistic growth trajectories throughout the ontogeny of several groups of ectotherms (R²>0.90). The results suggest that the effect of temperature on growth trajectory supersedes that of reproduction in an environment with fluctuating temperature. Furthermore, values of the allometric weight exponent (0<b<0.75) indicate that the rules of body surface and body weight do not apply under certain circumstances. Finally, the growth function circumvents problems associated with models based on thermodynamic and chemical kinetic principles (e.g., inability to predict growth of organisms in which ontogeny exceeds 3 months) and on rule of thermal summation (e.g., reliable only in a certain range of temperature). The growth function can handle a wide range of temperature fluctuations, encompass life stages and apply to key organisms in ecology, fisheries and agriculture.     

Stable isotope records (O, C) of Jurassic aragonitic shells from England and NW Poland: palaeoecologic and environmental implications

Shells of fully marine Middle to Upper Jurassic molluscs from England and north-western Poland were analysed with respect to their stable isotope (δ¹⁸O, δ¹³C) compositions, and palaeoecological and environmental life conditions of these molluscs were inferred from them. Light microscopical and SEM inspection and an analysis of the minor element content (Fe, Mn, Mg, Sr) suggest rather unaltered isotope signals. The δ¹⁸O and δ¹³C values show a characteristic distribution among three groups of co-occurring organisms. Benthic (adult) bivalves generally preserved higher δ¹⁸O and δ¹³C values than ammonites, whereas planktic bivalve larvae tend to possess the lowest δ¹⁸O but higher δ¹³C than adult bivalves. As this distribution pattern is found in numerous horizons and sections of Bathonian to Kimmeridgian age in NW Poland and England, it is thought to reflect real palaeoenvironmental parameters. All observations can be incorporated in a single model that assumes (i) seasonally induced temperature stratification of the water column, (ii) a correlation between phytoplankton blooms and reproduction season of planktic-planktotrophic bivalves, and (iii) insignificant vital effects with respect to the δ¹³C in bivalves, but strong biological control in ammonites. In addition, the δ¹⁸O evolution suggests that the Late Bajocian to Middle/Late Bathonian and Early Oxfordian to Late Kimmeridgian were considerably warmer than the latest Bathonian to Late Callovian time interval. The oxygen isotopic records from other European regions indicate a similar pattern of long-term palaeotemperature evolution. The comparatively high water temperatures during the Callovian to Oxfordian of the Isle of Skye (NW Scotland) are enigmatic, however. In the Early Oxfordian, sea surface and bottom temperatures began to rise in continental Europe and England. These changes coincide with a south-westward drift of the West European crustal plate, but a causal relationship remains to be demonstrated.     

Hyperosmotic volume regulation in the gills of the ribbed mussel, Geukensia demissa: rapid accumulation of betaine and alanine

The content of betaine and alanine in gills of the ribbed mussel Geukensia demissa increases rapidly following transfer of the tissues from 250 to 1000 mOsm seawater (SW). Increases in alanine, proline and glycine account for most of the increase in the amino acid pool. The betaine content increases from 45 to 150 μmol/g dry weight within 12 h. Transfer of isolated gills from 250 to 1000 mOsm SW results in a temporary cessation of all ciliary activity. Within 20–40 min following transfer, ciliary activity has recovered. Recovery of ciliary activity precedes recovery of tissue hydration. The uric acid content of gills is unchanged by exposure to hyperosmotic media, suggesting that uric acid is not a store of nitrogen for alanine synthesis from pyruvate. In other organisms, the accumulation of betaine in response to hyperosmotic stress is a slow (days to weeks) process that probably involves changes in gene expression. The rapid, large increases in betaine reported here suggest that gene expression is not a factor in volume recovery by euryhaline bivalve tissues exposed to acute hyperosmotic stress.     

Partial characterisation of high-molecular weight glycoconjugates in the trail mucus of the freshwater pond snail Lymnaea stagnalis

We have studied the glycoconjugates in trail mucus of the pond snail Lymnaea stagnalis. The mucus was dissolved with 6 M guanidinium hydrochloride (GuHCl) and the major component was comprised of very high-M_r glycoconjugates that were eluted in the void volume of a Sepharose CL-4B gel-filtration column. This high-M_r material was pooled and thereafter subjected to density gradient centrifugation first in 4 M GuHCl/CsCl and subsequently 0.2 M GuHCl/CsCl to further remove non-glycosylated proteins and DNA. The harvested glycoconjugate pool chromatographed in the void volume of Sepharose CL-2B. However, reduction of disulfide bonds lowered the molecular size of approximately 80% of the void material yielding a major fragment and some minor smaller fragments in gel chromatography. The reduced glycoconjugates were digested with papain and yielded high molecular weight, proteinase-resistant glycopeptides. This fragmentation pattern is similar to that found for oligomeric gel-forming mucins in mammals and the amino acid composition (60% Ser/Thr) and sugar analysis of the glycopeptides is consistent with mucin-like molecules, there being no significant amounts of xylose or uronic acids. The residual 20% of the preparation, which apparently resisted reduction and protease digestion, had a similar amino acid composition to the bulk, but was somewhat different in sugar composition, containing some xylose and a significant amount of glucuronic acid. The two groups of molecules had very different morphologies in the electron microscope. Taken together, these data suggest that trail mucus is a complex mixture of at least two families of protein-glycoconjugate molecules based upon the gel-forming mucin and proteoglycan families, though we cannot rule out that polysaccharides may also be present.