Organic compounds in the extrapalial fluid and haemolymph of Anodonta cygnea (L.) with emphasis on the seasonal biomineralization process

Bivalve mollusks, such as the freshwater mussel Anodonta cygnea, show seasonal changes in calcification. This cycle of calcification must either be a cause or a consequence for seasonal fluctuations in the organic composition of the animal's fluids, haemolymph and extrapallial fluid, the liquid media for biomineralization. We monitored the fluids of A. cygnea, throughout a 1-year cycle, for the presence of organic constituents, known to be important for biomineralization, such as proteins, glycosaminoglycans (GAGs) and hexosamines. Proteins were subjected to further study, namely through the total amino acid determination and fraction separation by agarose gel electrophoresis. GAG levels were fairly constant throughout the year, with a maximum concentration in July and a minimum in January, a feature also detected for glucosamine, although with higher fluctuations. Proteins showed highly increased concentrations during June and July, both in total amounts and individual fractions. All fractions showed similar trends throughout the year, with lowest general levels in October, the starting month of a period when some fractions were not detectable at all. All fractions ended this low period in May, when a sometimes-important increase could be detected. As to the total amino acid composition of the fluids, the general trend followed that of proteins, except for ornithine (Orn), a non-proteic amino acid. The overall fluctuations detected in the biological fluids of A. cygnea suggest that the main variation related to the calcification cycle must be quantitative, since no different compounds appear in specific periods, to achieve also specific results.     

Intracellular targeting of Gag proteins of the Drosophila telomeric retrotransposons

Drosophila has two non-long-terminal-repeat (non-LTR) retrotransposons that are unique because they have a defined role in chromosome maintenance. These elements, HeT-A and TART, extend chromosome ends by successive transpositions, producing long arrays of head-to-tail repeat sequences. These arrays appear to be analogous to the arrays produced by telomerase on chromosomes of other organisms. While other non-LTR retrotransposons transpose to many chromosomal sites, HeT-A and TART transpose only to chromosome ends. Although HeT-A and TART belong to different subfamilies of non-LTR retrotransposons, they encode very similar Gag proteins, which suggests that Gag proteins are involved in their unique transposition targeting. We have recently shown that both Gags localize efficiently to nuclei where HeT-A Gag forms structures associated with telomeres. TART Gag does not associate with telomeres unless HeT-A Gag is present, suggesting a symbiotic relationship in which HeT-A Gag provides telomeric targeting. We now report studies to identify amino acid regions responsible for different aspects of the intracellular targeting of these proteins. Green fluorescent protein-tagged deletion derivatives were expressed in cultured Drosophila cells. The intracellular localization of these proteins shows the following. (i) Several regions that direct subcellular localizations or cluster formation are found in both Gags and are located in equivalent regions of the two proteins. (ii) Regions important for telomere association are present only in HeT-A Gag. These are present at several places in the protein, are not redundant, and cannot be complemented in trans. (iii) Regions containing zinc knuckle and major homology region motifs, characteristic of retroviral Gags, are involved in protein-protein interactions of the telomeric Gags, as they are in retroviral Gags.     

Correlation between the morpho-cytohistochemistry of the outer mantle epithelium of Anodonta cygnea with seasonal variations and following pollutant exposure

Seasonal and experimental conditions induce morphological and cytochemical variations in the outer mantle epithelium (OME) of the freshwater bivalve Anodonta cygnea, probably influencing the shell calcification mechanism. In this study, OME samples were taken from untreated animals in autumn, winter, spring and summer as well as from animals exposed to divalent metals (cadmium, chromium, lead, copper and zinc) and pesticides (setoxidim and dimethoate) and observed by light microscopy. The present results showed that OME cells have larger cell volumes and increased amounts of secreted macromolecules during spring and summer than in autumn and winter. This correlates with higher shell calcification rates in spring and summer and lower shell calcification rates in autumn and winter. The experiments showed that incubation with pollutants for 8 months dramatically reduced the cellular volume so that the density of cytoplasmic material appeared higher that in the control samples. The pronounced changes in OME cells suggest a significant decrease in secretory activity following exposure to toxic agents and this has implications for the shell calcification process.     

Structural evidence that MOAP1 and PEG10 are derived from retrovirus/retrotransposon Gag proteins

The Gag proteins of retroviruses play an essential role in virus particle assembly by forming a protein shell or capsid and thus generating the virion compartment. A variety of human proteins have now been identified with structural similarity to one or more of the major Gag domains. These human proteins are thought to have been evolved or “domesticated” from ancient integrations due to retroviral infections or retrotransposons. Here, we report that X-ray crystal structures of stably folded domains of MOAP1 (modulator of apoptosis 1) and PEG10 (paternally expressed gene 10) are highly similar to the C-terminal capsid (CA) domains of cognate Gag proteins. The structures confirm classification of MOAP1 and PEG10 as domesticated Gags, and suggest that these proteins may have preserved some of the key interactions that facilitated assembly of their ancestral Gags into capsids.     

Defect of human immunodeficiency virus type 2 Gag assembly in Saccharomyces cerevisiae

We have previously shown that the expression of human immunodeficiency virus type 1 (HIV-1) Gag protein in Saccharomyces cerevisiae spheroplasts produces Gag virus-like particles (VLPs) at the plasma membrane, indicating that yeast has all the host factors necessary for HIV-1 Gag assembly. Here we expand the study by using diverse primate lentiviral Gags and show that yeast does not support the production of HIV-2 or simian immunodeficiency virus SIVmac Gag VLPs but allows the production of SIVagm and SIVmnd Gag VLPs. Particle budding was observed at the surfaces of cells expressing SIVagm and SIVmnd Gags, but cells expressing HIV-2 and SIVmac Gags showed only membrane-ruffling structures, although they were accompanied with electron-dense submembrane layers, suggesting arrest at an early stage of particle budding. Comparison of HIV-1 and HIV-2 Gag expression revealed broadly equivalent levels of intracellular Gag expression and Gag N-terminal myristoylation in yeast. Both Gags showed the same membrane-binding ability and were incorporated into lipid raft fractions at a physiological concentration of salt. HIV-2 Gag, however, failed to form a high-order multimer and easily dissociated from the membrane, phenomena which were not observed in higher eukaryotic cells. A series of chimeric Gags between HIV-1 and HIV-2 and Gag mutants with amino acid substitutions revealed that a defined region in helix 2 of HIV-2 MA (located on the membrane-binding surface of MA) affects higher-order Gag assembly and particle production in yeast. Together, these data suggest that yeast may lack a host factor(s) for HIV-2 and SIVmac Gag assembly.     

The action of Cd, Cu, Cr, Zn, and Pb on fluid composition of Anodonta cygnea (L.): organic components

The heavy metals, Cd, Cu, Cr, Zn, and Pb, were used to incubate healthy specimens of the freshwater mussel species, Anodonta cygnea. Afterwards, their biological fluids, either haemolymph or extrapallial fluid were analyzed for the presence of several organic constituents, known to be important for biomineralization, such as proteins, glycosaminoglycans (GAGs) and glucosamine. Proteins were subjected to further study, namely through the total amino acid determination after acid hydrolysis. The most disturbing pollutants tested seem to be Pb, Zn, and Cr, which caused highly decreased overall compositions, namely with respect to protein, and glucosamine, in comparison to the control group. This suggests that this group contributes to a decrease of the metabolic activity, and thus mineralization, in the exposed animals.     

Paracellular pathway in the shell epithelium of Anodonta cygnea

Ultrastructural study of cell-cell connections in the outer mantle epithelium (OME) on high-pressure-frozen specimens revealed zonula adherens, septate junctions and gap junctions in Anodonta cygnea. In order to evaluate the permeability of the paracellular pathway, the OME was incubated under gradients of lanthanum and calcium. After lanthanum incubation (4 mM) from the basal side, the septate junctions were penetrated completely by this tracer. When applied from the apical side, lanthanum deposits were located similarly over the entire length of the septate junctions up to the first dilatations of the intercellular space. Calcium deposits were also present in paracellular areas only when OME had been incubated simultaneously with calcium (6 mM) and lanthanum (4 mM) gradients. Lanthanum and calcium deposits were detected with ESI (Electron Spectroscopic Imaging) and identified with EELS (Electron Energy Loss Spectroscopy). On the other hand, electrophysiological observations showed a 48% reduction of conductance when the OME was bathed on both sides with solutions containing lanthanum (4 mM) and calcium (6 mM), compared to bathing with lanthanum-free solution (control). The conductance reduction was 52% when calcium was removed from the control solution. Supported by morphological and physiological evidence, it appears that, under in vivo conditions, calcium ions may diffuse paracellularly from the haemolymph towards the extrapallial fluid and vice-versa across the septate junctions in the OME of A. cygnea. Permeability of the septate junctions depended proportionally on the calcium concentration in fluids.     

Correlation between the morpho-cytohistochemistry of the outer mantle epithelium of Anodonta cygnea with seasonal variations and following pollutant exposure

Seasonal and experimental conditions induce morphological and cytochemical variations in the outer mantle epithelium (OME) of the freshwater bivalve Anodonta cygnea, probably influencing the shell calcification mechanism. In this study, OME samples were taken from untreated animals in autumn, winter, spring and summer as well as from animals exposed to divalent metals (cadmium, chromium, lead, copper and zinc) and pesticides (setoxidim and dimethoate) and observed by light microscopy. The present results showed that OME cells have larger cell volumes and increased amounts of secreted macromolecules during spring and summer than in autumn and winter. This correlates with higher shell calcification rates in spring and summer and lower shell calcification rates in autumn and winter. The experiments showed that incubation with pollutants for 8 months dramatically reduced the cellular volume so that the density of cytoplasmic material appeared higher that in the control samples. The pronounced changes in OME cells suggest a significant decrease in secretory activity following exposure to toxic agents and this has implications for the shell calcification process.     

Effects of starvation on body composition and cold tolerance in the collembolan Orchesella cincta and the isopod Porcellio scaber

The effects of long-term starvation on the body composition of the isopod Porcellio scaber (Latreille) and the collembolan Orchesella cincta (L.) were studied, by determining the body composition in starved and fed animals. A period under summer conditions (19 degrees C, 75% RH and L/D 16/8 photoperiod), was followed by a period under winter conditions (5 degrees C, 75% RH and LD 6/18 photoperiod). O. cincta was held under summer conditions for 3weeks, during which its protein and lipid content decreased, while its water content increased. In P. scaber, the same occurred during the 6weeks they were kept under summer conditions. During subsequent weeks under winter conditions, changes in cold tolerance of the animals were investigated. Cold tolerance and haemolymph osmolality were measured once a week. Starved animals had lower cold tolerance than fed ones. For P. scaber a decreased haemolymph osmolality was found in starved animals compared to fed ones. This is assumed to be caused by a combination of the consumption of carbohydrates out of the haemolymph and of protein reserves and the accumulation of body water. O. cincta appeared to be capable of osmoregulation, as haemolymph osmolality did not differ between starved and fed animals, despite differences in body water content. Decreased cold tolerance in starved animals of both species may be caused by increased water content or, more probably, by the decrease in reserves needed to produce cryoprotective substances.     

A Bivalve Biomineralization Toolbox

Mollusc shells are a result of the deposition of crystalline and amorphous calcite catalyzed by enzymes and shell matrix proteins (SMP). Developing a detailed understanding of bivalve mollusc biomineralization pathways is complicated not only by the multiplicity of shell forms and microstructures in this class, but also by the evolution of associated proteins by domain co-option and domain shuffling. In spite of this, a minimal biomineralization toolbox comprising proteins and protein domains critical for shell production across species has been identified. Using a matched pair design to reduce experimental noise from inter-individual variation, combined with damage-repair experiments and a database of biomineralization SMPs derived from published works, proteins were identified that are likely to be involved in shell calcification. Eighteen new, shared proteins likely to be involved in the processes related to the calcification of shells were identified by the analysis of genes expressed during repair in Crassostrea gigas, Mytilus edulis, and Pecten maximus. Genes involved in ion transport were also identified as potentially involved in calcification either via the maintenance of cell acid–base balance or transport of critical ions to the extrapallial space, the site of shell assembly. These data expand the number of candidate biomineralization proteins in bivalve molluscs for future functional studies and define a minimal functional protein domain set required to produce solid microstructures from soluble calcium carbonate. This is important for understanding molluscan shell evolution, the likely impacts of environmental change on biomineralization processes, materials science, and biomimicry research.     

Microclimate and variations in haemolymph composition in the freezing-tolerant New Zealand alpine weta Hemideina maori Hutton (Orthoptera: Stenopelmatidae)

The microclimate in the habitat of the New Zealand alpine weta Hemideina maori is very variable with winter temperatures down to −6 °C under the rocks where the insects are found. Subfreezing temperatures may in winter prevail for up to 17 days but diurnal cycles of freezing and thawing are common, as is also the case in summer. Rates of temperature change can be very high and up to −7.20 °C/h. During winter, humidity was high for extended periods ranging from 70% to 100% relative humidity (RH). In the summer, humidity ranged from 30% RH during the day to 100% RH at night. The supercooling point of the haemolymph was approximately −8 °C year round, caused by a heat labile substance. The supercooling point of the haemolymph of an insect of the same genus, Hemideina femorata not regularly exposed to subfreezing temperatures, was ca. −16.5 °C. Thermal hysteresis was not detected in the haemolymph of H. maori. Haemolymph osmolality varied from 380 mOsm (summer) to 700 mOsm (winter). Body water content was ca. 75% all year round. Total concentrations of sodium, potassium and chloride in haemolymph varied from 170 mM (winter) to 250 mM (summer). The total concentration of free amino acids varied from 58 mM (summer) to 263 mM (winter). This variation was mostly due to proline which varied from ca. 15 mM (summer) to ca. 100 mM (winter). The freeze-tolerant weta H. maori is exposed to a highly variable and cold environment all year round and several properties of its haemolymph composition can be attributed to these climatic conditions, e.g. the presence of ice-nucleating agents and an increase in the concentration of proline during cold hardening in the autumn. Accepted: 22 February 1999     

Human Immunodeficiency Virus Type 1 Gag Polyprotein Multimerization Requires the Nucleocapsid Domain and RNA and Is Promoted by the Capsid-Dimer Interface and the Basic Region of Matrix Protein

The human immunodeficiency virus type 1 (HIV-1) Gag polyprotein directs the formation of virions from productively infected cells. Many gag mutations disrupt virion assembly, but little is known about the biochemical effects of many of these mutations. Protein-protein interactions among Gag monomers are believed to be necessary for virion assembly, and data suggest that RNA may modify protein-protein interactions or even serve as a bridge linking Gag polyprotein monomers. To evaluate the primary sequence requirements for HIV-1 Gag homomeric interactions, a panel of HIV-1 Gag deletion mutants was expressed in bacteria and evaluated for the ability to associate with full-length Gag in vitro. The nucleocapsid protein, the major RNA-binding domain of Gag, exhibited activity comparable to that of the complete polyprotein. In the absence of the nucleocapsid protein, relatively weak activity was observed that was dependent upon both the capsid-dimer interface and basic residues within the matrix domain. The relevance of the in vitro findings was confirmed with an assay in which nonmyristylated mutant Gags were assessed for the ability to be incorporated into virions produced by wild-type Gag expressed in trans. Evidence of the importance of RNA for Gag-Gag interaction was provided by the demonstration that RNase impairs the Gag-Gag interaction and that HIV-1 Gag interacts efficiently with Gags encoded by distantly related retroviruses and with structurally unrelated RNA-binding proteins. These results are consistent with models in which Gag multimerization involves indirect contacts via an RNA bridge as well as direct protein-protein interactions.     

Redundant roles for nucleocapsid and matrix RNA-binding sequences in human immunodeficiency virus type 1 assembly

RNA appears to be required for the assembly of retroviruses. This is likely due to binding of RNA by multiple Gags, which in turn organizes and stabilizes the Gag-Gag interactions that form the virion. While the nucleocapsid (NC) domain is the most conspicuous RNA-binding region of the human immunodeficiency virus type 1 (HIV-1) Gag polyprotein, we have previously shown that NC is not strictly required for efficient particle production. To determine if an RNA requirement for HIV-1 assembly exists, we analyzed virions produced by an NC deletion mutant for the presence of RNA. The results revealed that virions without NC still contained significant amounts of RNA. Since these packaged RNAs are probably incorporated by other RNA-binding sequences in Gag, an RNA-binding site in the matrix protein (MA) of Gag was mutated. While this mutation did not interfere with HIV-1 replication, a construct with both MA and NC mutations (MX/NX) failed to produce particles. The MX/NX mutant was rescued in trans by coassembly with several forms of Gag: wild-type Gag, either of the single-mutant Gags, or Gag truncations that contain MA or NC sequences. Addition of basic sequences to the MX/NX mutant partially restored particle production, consistent with a requirement for Gag-RNA binding in addition to Gag-Gag interactions. Together, these results support an RNA-binding requirement for Gag assembly, which relies on binding of RNA by MA or NC sequences to condense, organize, and stabilize the HIV-1 Gag-Gag interactions that form the virion.     

In vivo homodimerisation of HTLV-1 Gag and MA gives clues to the retroviral capsid and TM envelope protein arrangement

During retroviral particle formation, the capsid precursors (Gag) associate with the cell membrane via their matrix (MA) domain to form viral assembling particles. After budding, Gag and its proteolytically matured MA, form a shell in the released immature and mature particles, respectively. Although the arrangement of Gag domains in vitro and their radial organisation in retroviral particles have been extensively studied, little is known concerning Gag inter-subunit interactions in authentic retroviruses. We report that human T-cell leukemia virus type 1 Gag homodimerises in the cell via a disulphide bonding at cysteine 61 in the MA domain. Most Gags are homodimeric after budding and MAs are also dimeric in mature authentic virions. Molecular modelling of the MA domain indicates that non-covalent interactions at the MA dimer interface may also be important for Gag (and MA) dimerisation. In addition, all amino acids previously reported to be involved in MA-transmembrane (TM) interactions are located on the MA face opposite to the dimer interface. The model reveals that homodimerisation is compatible with a hexameric network of Gag and MA dimers that look like the hexameric networks observed for other retroviruses. These data, together with previous studies, lead us to propose a supra-molecular arrangement model in which the transmembrane glycoproteins of the virion envelope are anchored in a hexameric cage hole formed by the MA.     

Seasonal variations of pH, pCO2, pO2, HCO3- and Ca2+ in the haemolymph: implications on the calcification physiology in Anodonta cygnea

A study about the relationship between the physical–chemical parameters and the calcium carbonate balance between the haemolymph fluid and mantle calcareous structures was carried out in Anodonta cygnea. An intense peak of HCO₃ ⁻ and a highest pH in December–January months may be understood as a preparation period for creating alkaline conditions. An intense pH decrease from January to February in parallel with the HCO₃ ⁻ reduction seems to indicate the beginning process of carbonate precipitation. On the other hand, the following calcium and HCO₃ ⁻ increases in February–May associated with a continuous and gradual pH fall profile may infer two combined aspects: calcium and HCO₃ ⁻ absorption from external environment and a simultaneous intense calcium carbonate deposition in the haemolymph. So, the pCO₂ peak in this period reflects a subsequent result on equilibrium balance between HCO₃ ⁻ absorption and deposition. The only significant pO₂ increase in the next period, from February to June, is related with an energetic increase to support the metabolic activity favouring the posterior intense pCO₂ peaks. The extended time of CO₂ production in the haemolymph from May to November should induce an increased metabolic acidosis with subsequent intense formation of both HCO₃ ⁻ and Ca²⁺ ions in the same period. This seems to result from CaCO₃ deposits dissolution in the haemolymph, the most direct calcareous source. Additionally, the later increase of metabolic succinic acid during autumn may greatly potentiate this acidosis increasing the dissolution process. Consequently, the pH profile present two simultaneous alkaline peaks in July and October, probably due to a strong HCO₃ ⁻ release from the CaCO₃ dissolution. So, the present seasonal results indicate that in the freshwater bivalve A. cygnea, the low metabolism with higher pH from the early winter is the main cause which may favour a calcareous precipitation, while the high metabolism with lower pH from the early summer may function as an inductor of calcareous dissolution in the haemolymph.     

Recovery by the Norway lobster Nephrops norvegicus (L.) from the physiological stresses of trawling: Influence of season and live-storage position

Live Norway lobsters (Nephrops norvegicus L.) were trawled at depths of 30 to 55 m off the coast of Jutland (Denmark) in late winter (March) and in summer (August) in 2006. Water temperatures at the bottom and surface of the sea were 7 °C and 2 °C during the winter, and 12 °C and 21 °C in the summer, respectively. The recovery of specific physiological and metabolic variables from the intense stresses associated with capture (trawling and air-exposure during sorting) was followed in seawater at 5 °C in winter or 18 °C in summer. Recovery was compared in lobsters held individually in two different live-storage positions, either resting vertically on the tail or sitting horizontally. In winter, many animals were alive when brought on board and approximately 86% were still alive at the end of experimentation (96 h). In summer very few animals were alive when brought on board and, of these, approximately 95% were dead at 24 h. When compared with values measured in laboratory controls, the stresses of capture elicited very high haemolymph lactate contents in both seasons, although levels recovered within 24 h. Trawling also caused very high haemolymph glucose concentrations, which differed with season. In winter, haemolymph glucose was elevated for 24 h to levels significantly higher than in summer. In summer, glucose had returned to control levels by 4 h. At 4 h after trawling, haemolymph O₂ status was not markedly influenced in either season, but there were significant disturbances of acid-base status. In winter, a potential metabolic lactic acidosis was compensated by a marked respiratory alkalosis, with significantly increased haemolymph pH and decreased CO₂ total content and partial pressure. These effects disappeared gradually over 96 h. Summer lobsters showed combined metabolic and respiratory acidosis at 4 h, although this had recovered to control values in the small number of survivors sampled at 24 h. The capture stresses elicited very high haemolymph crustacean hyperglycaemic hormone (CHH) titres, significantly higher in summer than in winter. In winter, CHH titre had declined significantly at 24 h, whereas it exhibited a further significant increase at 24 h in summer. Live-storage position had no significant effect on survival or recovery from capture stresses in either season. The results demonstrate that Nephrops were much more stressed by trawling at high summer temperatures and had difficulty recovering from this, with pronounced negative effects on their survival, irrespective of their live-storage position.     

One‐year experiment on the physiological response of the Mediterranean crustose coralline alga,Lithophyllum cabiochae,to elevated pCO2 and temperature

The response of respiration, photosynthesis, and calcification to elevated pCO₂ and temperature was investigated in isolation and in combination in the Mediterranean crustose coralline alga Lithophyllum cabiochae. Algae were maintained in aquaria during 1 year at near‐ambient conditions of irradiance, at ambient or elevated temperature (+3°C), and at ambient (ca. 400 μatm) or elevated pCO₂ (ca. 700 μatm). Respiration, photosynthesis, and net calcification showed a strong seasonal pattern following the seasonal variations of temperature and irradiance, with higher rates in summer than in winter. Respiration was unaffected by pCO₂ but showed a general trend of increase at elevated temperature at all seasons, except in summer under elevated pCO₂. Conversely, photosynthesis was strongly affected by pCO₂ with a decline under elevated pCO₂ in summer, autumn, and winter. In particular, photosynthetic efficiency was reduced under elevated pCO₂. Net calcification showed different responses depending on the season. In summer, net calcification increased with rising temperature under ambient pCO₂ but decreased with rising temperature under elevated pCO₂. Surprisingly, the highest rates in summer were found under elevated pCO₂ and ambient temperature. In autumn, winter, and spring, net calcification exhibited a positive or no response at elevated temperature but was unaffected by pCO₂. The rate of calcification of L. cabiochae was thus maintained or even enhanced under increased pCO₂. However, there is likely a trade‐off with other physiological processes. For example, photosynthesis declines in response to increased pCO₂ under ambient irradiance. The present study reports only on the physiological response of healthy specimens to ocean warming and acidification, however, these environmental changes may affect the vulnerability of coralline algae to other stresses such as pathogens and necroses that can cause major dissolution, which would have critical consequence for the sustainability of coralligenous habitats and the budgets of carbon and calcium carbonate in coastal Mediterranean ecosystems.     

喀斯特高原深水水库-万峰湖富营养化特征分析

以贵州喀斯特高原深水水库-万峰湖为例,研究了其夏季和冬季湖沼学和富营养化特征。结果表明:冬季和夏季湖沼学及富营养化特征区别明显,夏季水温,pH值,溶解氧,电导率等指标在10 m和30 m分别出现明显的分界点,而冬季仅在30 m出现分界点,表明夏季属于双分层型,冬季属于单分层型。表层和底层的水温变化幅度较小,底层温度较高,在10℃以上;底层全年处于厌氧状态,钙离子浓度在0.192~0.235 mg/L 之间,镁离子的浓度在0.149~0.196 mg/L 之间,但是垂直和季节变化较小。总磷浓度在0~40 m较低,在0.02~0.04 mg/L之间,底层达0.794 mg/L;总氮浓度在0~40 m维持在2.18~3.54 mg/L,夏季略高于冬季,总氮浓度随深度逐渐降低。根据单因子指数判断,万峰湖大多时期属于中富营养状态。万峰湖不同季节的分层特征,反映出了喀斯特高原深水水库湖沼学变化特征,水体分层影响着水环境因子的变化和水体富营养化程度。     

厚壳贻贝whirlin类贝壳基质蛋白的重组表达与功能分析

贝壳历来是生物工程和材料学研究的重要对象。贝壳中的贝壳基质蛋白质在贝壳的形成与发育过程中具有重要的调控作用。Whirlin类蛋白质(Whirlin-like protein,WLP)是一种从厚壳贻贝(Mytilus coruscus)中鉴定的新型贝壳基质蛋白质。序列分析结果显示,该蛋白质含有PDZ(postsynaptic density/Discs large/Zonula occludens)结构域,而该结构域对贝壳生物矿化的影响目前尚无报道。为深入了解WLP在贝壳形成中对碳酸钙晶体的影响,在序列分析基础上,采用密码子优化结合原核重组表达,获得其重组表达产物后,开展了重组WLP对碳酸钙晶体形貌及晶型的影响研究,结晶速度抑制以及碳酸钙晶体结合分析。分析结果表明,重组WLP能诱导文石型碳酸钙晶体的形貌和方解石型碳酸钙晶体的晶型发生改变;同时重组WLP对碳酸钙晶体具有结合作用,且能抑制碳酸钙晶体的结晶速度。上述结果表明,WLP对贝壳的形成及发育具有重要影响,并可能在贝壳肌棱柱层的形成中发挥了重要作用。