Analysis of the Ecological Carrying Capacity of Fish Resources in Shengjin Lake, Anhui Province, China.

The carrying capacity of fish is related to the sustainability of fisheries' activities in water bodies. The fish carrying capacity of a water body is the maximum fish yield that can be carried by the natural bait organisms in the water body under the ideal natural conditions without feeding and fertilization. The evaluation of fish productivity is an important basis for rational stocking, rational fishing, and the scientific utilization of natural bait resources in a water area. This paper adopts the background data of the Shengjin Lake fishery ecosystem and uses the bait-based estimation method. The results show that (1) the annual yield of silver carp fed on phytoplankton is 1.5 million kg; (2) the annual yield of bighead carp fed on zooplankton is 1.295 million kg; (3) the annual yield of benthos is 310,000 kg; (4) the annual yield of organic detritus is 280,000 kg; and (5) as the coverage of water grass in Shengjin Lake is less than 10%, it should be protected and restored rather than used by fish. In general, the annual maximum carrying capacity of fish in Shengjin Lake is 3.385 million kg, except for water and grass.

Transition from horizontal expansion to vertical growth in the oyster prismatic layer.

Biomimetic materials inspired by biominerals have substantial applications in various fields. The prismatic layer of bivalve molluscs has extraordinary flexibility compared to inorganic CaCO3. Previous studies showed that in the early stage, minerals expanded horizontally and formed prism domains as a Voronoi division, while the evolution of the mature prisms were thermodynamically driven, which was similar to grain growth. However, it was unclear how the two processes were correlated during shell formation. In this study, we used scanning electronic microscopy and laser confocal scanning microscopy to look into the microstructure of the columnar prismatic layer in the pearl oyster Pinctada fucata. The Dirichlet centers of the growing domains in mature prisms were calculated, and the corresponding Voronoi division was reconstructed. It was found that the domain pattern did not fit the Voronoi division, indicating the driving forces of the mature prisms evolution and the initiation stage were different. During the transition from horizontal expansion to vertical growth, the minerals broke through the inner periostracum and squeezed out the organic materials to the inter-prism space. Re-arrangement of the organic framework pattern was driven by elastic relaxation at the vertices, indicating the transition process was thermodynamically driven. Our study provided insights into shell growth in bivalves and pave the way to synthesize three-dimensional material biomimetically.

Dual Roles of the Lysine-Rich Matrix Protein (KRMP)-3 in Shell Formation of Pearl Oyster, Pinctada fucata.

Matrix proteins play important roles in shell formation. Our group firstly isolated three cDNAs encoding lysine-rich matrix protein from Pinctada fucata in 2006. However, the functions of KRMPs are not fully understood. In addition, KRMPs contain two functional domains, the basic domain and the Gly/Tyr domain respectively. Based on the modular organization, the roles of their two domains were poorly characterized. Furthermore, KRMPs were then reported in other two species, P. maxima and P. margaritifera, which indicated that KRMPs might be very important for shell formation. In this study, the characterization and function of KRMP-3 and its two functional domains were studied in vitro through purification of recombinant glutathione S-transferase tagged KRMP-3 and two KRMP-3 deletion mutants. Western blot and immunofluorescence revealed that native KRMP-3 existed in the EDTA-insoluble matrix of the prismatic layer and was located in the organic sheet and the prismatic sheath. Recombinant KRMP-3 (rKRMP-3) bound tightly to chitin and this binding capacity was duo to the Gly/Tyr-rich region. rKRMP-3 inhibited the precipitation of CaCO3, affected the crystal morphology of calcite and inhibited the growth of aragonite in vitro, which was almost entirely attributed to the lysine-rich region. The results present direct evidence of the roles of KRMP-3 in shell biomineralization. The functional rBR region was found to participate in the growth control of crystals and the rGYR region was responsible to bind to chitin.

Amorphous calcium carbonate precipitation by cellular biomineralization in mantle cell cultures of Pinctada fucata.

The growth of molluscan shell crystals is generally thought to be initiated from the extrapallial fluid by matrix proteins, however, the cellular mechanisms of shell formation pathway remain unknown. Here, we first report amorphous calcium carbonate (ACC) precipitation by cellular biomineralization in primary mantle cell cultures of Pinctada fucata. Through real-time PCR and western blot analyses, we demonstrate that mantle cells retain the ability to synthesize and secrete ACCBP, Pif80 and nacrein in vitro. In addition, the cells also maintained high levels of alkaline phosphatase and carbonic anhydrase activity, enzymes responsible for shell formation. On the basis of polarized light microscopy and scanning electron microscopy, we observed intracellular crystals production by mantle cells in vitro. Fourier transform infrared spectroscopy and X-ray diffraction analyses revealed the crystals to be ACC, and de novo biomineralization was confirmed by following the incorporation of Sr into calcium carbonate. Our results demonstrate the ability of mantle cells to perform fundamental biomineralization processes via amorphous calcium carbonate, and these cells may be directly involved in pearl oyster shell formation.

Enzymatic synthesis of 2'-deoxyuridine by whole cell catalyst co-expressing uridine phosphorylase and thymidine phosphorylase through auto-induction system.

Genes encoding uridine phosphorylase (UP) and thymidine phosphorylase (TP) from Escherichia coli K12 were cloned and recombined respectively into plasmids pET-21a(+) and pET-28a(+). The recombinant plasmids BL21/pET21a-UP and BL21/pET28a-TP were co-transformed into E. coli BL21(DE3) to construct highly effective BTU strain (BL21/pET28a-TP, pET21a-UP) overexpressing both of UP and TP. BTU was cultivated in ZYM-Fe-5052 medium for 10 h and used as catalyst to synthesize 2'-deoxyuridine (dUR). It was found to increase the productivity of dUR by 8-9 fold when compared to wild E. coli K12 and E. coli BL21(DE3) strains. A series of experiments were carried out to find out the optimal conditions for synthesis of dUR. At 50°C, with 0.25‰ dry wt./v to catalyze the reaction of 2:1 ß-thymidine: uracil (60 mM ß-thymidine, 30 mM uracil), the conversion rate of dUR would reach 61.6% at 1 h, which was much higher than the rates obtained by BTU strain cultured in LB medium and induced by IPTG. This result proved co-expression and auto-induction were efficient methods in enhancing the expression quantity and activity of nucleoside phosphorylases, and they also had significant implications for large-scale industrial production of dUR and synthesis of other nucleoside derivatives.

A novel acidic matrix protein, PfN44, stabilizes magnesium calcite to inhibit the crystallization of aragonite.

Magnesium is widely used to control calcium carbonate deposition in the shell of pearl oysters. Matrix proteins in the shell are responsible for nucleation and growth of calcium carbonate crystals. However, there is no direct evidence supporting a connection between matrix proteins and magnesium. Here, we identified a novel acidic matrix protein named PfN44 that affected aragonite formation in the shell of the pearl oyster Pinctada fucata. Using immunogold labeling assays, we found PfN44 in both the nacreous and prismatic layers. In shell repair, PfN44 was repressed, whereas other matrix proteins were up-regulated. Disturbing the function of PfN44 by RNAi led to the deposition of porous nacreous tablets with overgrowth of crystals in the nacreous layer. By in vitro circular dichroism spectra and fluorescence quenching, we found that PfN44 bound to both calcium and magnesium with a stronger affinity for magnesium. During in vitro calcium carbonate crystallization and calcification of amorphous calcium carbonate, PfN44 regulated the magnesium content of crystalline carbonate polymorphs and stabilized magnesium calcite to inhibit aragonite deposition. Taken together, our results suggested that by stabilizing magnesium calcite to inhibit aragonite deposition, PfN44 participated in P. fucata shell formation. These observations extend our understanding of the connections between matrix proteins and magnesium.

Patterns of expression in the matrix proteins responsible for nucleation and growth of aragonite crystals in flat pearls of Pinctada fucata.

The initial growth of the nacreous layer is crucial for comprehending the formation of nacreous aragonite. A flat pearl method in the presence of the inner-shell film was conducted to evaluate the role of matrix proteins in the initial stages of nacre biomineralization in vivo. We examined the crystals deposited on a substrate and the expression patterns of the matrix proteins in the mantle facing the substrate. In this study, the aragonite crystals nucleated on the surface at 5 days in the inner-shell film system. In the film-free system, the calcite crystals nucleated at 5 days, a new organic film covered the calcite, and the aragonite nucleated at 10 days. This meant that the nacre lamellae appeared in the inner-shell film system 5 days earlier than that in the film-free system, timing that was consistent with the maximum level of matrix proteins during the first 20 days. In addition, matrix proteins (Nacrein, MSI60, N19, N16 and Pif80) had similar expression patterns in controlling the sequential morphologies of the nacre growth in the inner-film system, while these proteins in the film-free system also had similar patterns of expression. These results suggest that matrix proteins regulate aragonite nucleation and growth with the inner-shell film in vivo.

Structural characterization of amorphous calcium carbonate-binding protein: an insight into the mechanism of amorphous calcium carbonate formation.

ACC (amorphous calcium carbonate) plays an important role in biomineralization process for its function as a precursor for calcium carbonate biominerals. However, it is unclear how biomacromolecules regulate the formation of ACC precursor in vivo. In the present study, we used biochemical experiments coupled with bioinformatics approaches to explore the mechanisms of ACC formation controlled by ACCBP (ACC-binding protein). Size-exclusion chromatography, chemical cross-linking experiments and negative staining electron microscopy reveal that ACCBP is a decamer composed of two adjacent pentamers. Sequence analyses and fluorescence quenching results indicate that ACCBP contains two Ca²âº-binding sites. The results of in vitro crystallization experiments suggest that one Ca²âº-binding site is critical for ACC formation and the other site affects the ACC induction efficiency. Homology modelling demonstrates that the Ca²âº-binding sites of pentameric ACCBP are arranged in a 5-fold symmetry, which is the structural basis for ACC formation. To the best of our knowledge, this is the first report on the structural basis for protein-induced ACC formation and it will significantly improve our understanding of the amorphous precursor pathway.

Ubiquitylation functions in the calcium carbonate biomineralization in the extracellular matrix.

Mollusks shell formation is mediated by matrix proteins and many of these proteins have been identified and characterized. However, the mechanisms of protein control remain unknown. Here, we report the ubiquitylation of matrix proteins in the prismatic layer of the pearl oyster, Pinctada fucata. The presence of ubiquitylated proteins in the prismatic layer of the shell was detected with a combination of western blot and immunogold assays. The coupled ubiquitins were separated and identified by Edman degradation and liquid chromatography/mass spectrometry (LC/MS). Antibody injection in vivo resulted in large amounts of calcium carbonate randomly accumulating on the surface of the nacreous layer. These ubiquitylated proteins could bind to specific faces of calcite and aragonite, which are the two main mineral components of the shell. In the in vitro calcium carbonate crystallization assay, they could reduce the rate of calcium carbonate precipitation and induce the calcite formation. Furthermore, when the attached ubiquitins were removed, the functions of the EDTA-soluble matrix of the prismatic layer were changed. Their potency to inhibit precipitation of calcium carbonate was decreased and their influence on the morphology of calcium carbonate crystals was changed. Taken together, ubiquitylation is involved in shell formation. Although the ubiquitylation is supposed to be involved in every aspect of biophysical processes, our work connected the biomineralization-related proteins and the ubiquitylation mechanism in the extracellular matrix for the first time. This would promote our understanding of the shell biomineralization and the ubiquitylation processes.

Novel basic protein, PfN23, functions as key macromolecule during nacre formation.

The fine microstructure of nacre (mother of pearl) illustrates the beauty of nature. Proteins found in nacre were believed to be "natural hands" that control nacre formation. In the classical view of nacre formation, nucleation of the main minerals, calcium carbonate, is induced on and by the acidic proteins in nacre. However, the basic proteins were not expected to be components of nacre. Here, we reported that a novel basic protein, PfN23, was a key accelerator in the control over crystal growth in nacre. The expression profile, in situ immunostaining, and in vitro immunodetection assays showed that PfN23 was localized within calcium carbonate crystals in the nacre. Knocking down the expression of PfN23 in adults via double-stranded RNA injection led to a disordered nacre surface in adults. Blocking the translation of PfN23 in embryos using morpholino oligomers led to the arrest of larval development. The in vitro crystallization assay showed that PfN23 increases the rate of calcium carbonate deposition and induced the formation of aragonite crystals with characteristics close to nacre. In addition, we constructed the peptides and truncations of different regions of this protein and found that the positively charged C-terminal region was a key region for the function of PfN23 Taken together, the basic protein PfN23 may be a key accelerator in the control of crystal growth in nacre. This provides a valuable balance to the classic view that acidic proteins control calcium carbonate deposition in nacre.

The role of matrix proteins in the control of nacreous layer deposition during pearl formation.

To study the function of pearl oyster matrix proteins in nacreous layer biomineralization in vivo, we examined the deposition on pearl nuclei and the expression of matrix protein genes in the pearl sac during the early stage of pearl formation. We found that the process of pearl formation involves two consecutive stages: (i) irregular calcium carbonate (CaCO(3)) deposition on the bare nucleus and (ii) CaCO(3) deposition that becomes more and more regular until the mature nacreous layer has formed on the nucleus. The low-expression level of matrix proteins in the pearl sac during periods of irregular CaCO(3) deposition suggests that deposition may not be controlled by the organic matrix during this stage of the process. However, significant expression of matrix proteins in the pearl sac was detected by day 30-35 after implantation. On day 30, a thin layer of CaCO(3), which we believe was amorphous CaCO(3), covered large aragonites. By day 35, the nacreous layer had formed. The whole process is similar to that observed in shells, and the temporal expression of matrix protein genes indicated that their bioactivities were crucial for pearl development. Matrix proteins controlled the crystal phase, shape, size, nucleation and aggregation of CaCO(3) crystals.

Subcellular localization of N-deoxyribosyltransferase in Lactobacillus fermentum: cell surface association of an intracellular nucleotide metabolic enzyme.

N-deoxyribosyltransferases are essential enzymes in the nucleotide salvage pathway of lactobacilli. They catalyze the exchange between the purine or pyrimidine bases of 2'-deoxyribonucleosides and free pyrimidine or purine bases. In general, N-deoxyribosyltransferases are referred to as cytoplasmic enzymes, although there is no experimental evidence for this subcellular localization. In this work, the subcellular localization of N-deoxyribosyltransferase II (NTD) from Lactobacillus fermentum was examined by subcellular fractionation, transmission electron microscopy, and fluorescence microscopy. Our results indicate that L. fermentum NTD are distributed not only in the cytoplasm but also on the cell wall surface, and further studies showed that surface-attached NTD can be released into the culture broth and conventional buffers.

A possible mechanism for the formation of annual growth lines in bivalve shells.

We report a unique shell margin that differed from the usual shell structure of Pinctada fucata. We observed empty organic envelopes in the prismatic layer and the formation of the nacreous layer in the shell margin. All the characteristics of the growing margin indicated that the shell was growing rapidly. To explain this anomaly, we propose the concept of "jumping development". During jumping development, the center of growth in the bivalve shell jumps forward over a short time interval when the position of the mantle changes. Jumping development explains the unusual structure of the anomalous shell and the development of annual growth lines in typical shells. Annual growth lines are the result of a discontinuity in the shell microstructure induced by jumping development.

A new method to extract matrix proteins directly from the secretion of the mollusk mantle and the role of these proteins in shell biomineralization.

Considering the continuous and substantive secretory ability of the mantle in vitro, we report a new technique to produce shell-matrix proteins by inducing the mantle, after removal from the organism's body, to secrete soluble-matrix proteins into phosphate buffer. By this method, a large amount of matrix proteins could be obtained in 2 h. Experiments involving in vitro calcium carbonate crystallization and organic framework calcium carbonate crystallization indicated that these proteins retain high bioactivity and play key roles in shell biomineralization. Phosphate buffer-soluble proteins secreted by the margin of the mantles (MSPs) were used to reconstruct the stages in the growth of the prismatic layer of the decalcified organic frameworks. The MSPs were observed to aggregate calcites in vitro, and this ability enabled the mollusk to form big calcites in the prismatic layer. During shell biomineralization, an important stage after the self-assembly of the biomacromolecules and the formation of crystals is the assembly of the two parts to form a firm structure. Moreover, a new type of matrix protein, functioning as the binding factor between the crystals and the organic frameworks, was shown to exist in the phosphate buffer-soluble proteins secreted by the central part of mantles (CSPs). Nanoscale-sized bowl-like aragonites, with heights of ∼800 nm, were induced by CSPs in vitro. This method is a successful example of obtaining functional proteins through secretion by animal tissues.

Proteomics study of COI1-regulated proteins in Arabidopsis flower.

Jasmonates (JAs) are a new class of plant hormone that regulate expression of diverse genes to mediate various plant responses. The Arabidopsis F-box protein COI1 is required for plant defense and male fertility in JA signal pathway. To further investigate the regulatory role of COI1 in male fertility, we compared the proteomics profiles of Arabidopsis wild type (WT) flowers with coi1-1 mutant male-sterile flowers using two-dimensional difference gel electrophoresis coupled with matrix-assisted laser desoption/ionization-time-of-flight mass spectrometry. Sixteen proteins with potential function in specific biological processes such as metabolism processes and defense/stress responses were differentially expressed in WT and coi1-1 mutant flowers. Verification on a phi class glutathione transferase AtGSTF9, one out of these 16 identified proteins, revealed that the expression of AtGSTF9 was severely downregulated in flowers of coi1-1 mutant compared with that of WT. Further function analyses of these genes would provide new insights into the molecular basis of COI1-regulated male fertility.

Cloning and characterization of the activin like receptor 1 homolog (Pf-ALR1) in the pearl oyster, Pinctada fucata.

The signal transduction mechanisms in mollusks are still elusive since the genome information is incomplete and cell lines are not available. In previous study, we cloned a highly conserved Smad3 homolog (designated as Pf-Smad3) from the pearl oyster, Pinctada fucata. It seems that transforming growth factor beta (TGFbeta) signaling may play similar roles in the oyster as in vertebrate. Here we report a cDNA encoding an activin like receptor 1 homolog (designated as Pf-ALR1) of the oyster, another kind of TGFbeta superfamily member. Compared to the activin receptor-like kinases (ALK) in human, the amino acid sequence of Pf-ALR1 is more similar to that of ALK1, especially the L45 loop. Reverse transcription-polymerase chain reaction results indicate that Pf-ALR1 mRNA is expressed ubiquitously in the adult oyster. Thus, Pf-ALR1 may be important for many physiological processes in the oyster. To lay a basis for further investigation of the TGFbeta signal pathway functions in the oyster shell formation, in this report, the Pf-ALR1 mRNA expression in the oyster mantle was detected by in situ hybridization. The results show that Pf-ALR1 in the oyster mantle is mainly expressed at the inner epithelial cells of the outer fold and the outer epithelial cells of the middle fold, similarly as Pf-Smad3. The mRNA levels of Pf-ALR1 and Pf-Smad3 are all changed after shell notching. These results indicate that both Pf-ALR1 and Pf-Smad3 may take part in shell formation and repair. The results of drug treatment experiments with in-vitro cultured oyster mantle tissue cells demonstrate that the mRNA expression levels of Pf-Smad3, Pf-ALR1 and two oyster nuclear factor-kappaB (NF-kB) members can be adjusted and correlated. All our observations suggest that there should be similar TGFbeta signal pathways in the oyster and vertebrate. However, the potential functions of Pf-ALR1 and the relations of TGFbeta and NF-kB members in the oyster all need to be thoroughly investigated.

Calcineurin mediates the immune response of hemocytes through NF-kappaB signaling pathway in pearl oyster (Pinctada fucata).

Calcineurin (CN), a multifunctional protein, mediates the immune response through diverse signaling pathways in mammals, while the function of CN in the immune response of molluscan hemocytes still remains unclear. In the present study, we detected the distribution of CN in various tissues and the expression levels of Pf-CNA and Pf-CNB gene in hemocytes of Pinctada fucata. After the preparation of hemocyte monolayers, we checked the response of enzymatic activity of CN, the degradation level of IkappaBalpha, the activity of iNOS and the production of NO, and IL-2 to the challenge of lipopolysaccharide (LPS) and cyclosporin A (CsA). CN activity in hemocytes was very sensitive to both the stimulation of LPS and the inhibition of CsA. Most importantly, IkappaBalpha degradation in hemocytes was induced by LPS and attenuated by CsA. Consequently, the activity of iNOS was elevated and the production of NO was increased. Additionally, we found that the synthesis of IL-2 was increased by LPS but was apparently weakened by CsA. In vivo bacterial clearance experiments showed that CsA significantly decreased the ability of in vivo bacteria clearance in pearl oyster. All the results revealed, for the first time, that CN mediated the immune response of molluscan hemocytes via activating NF-kappaB signaling pathway.

Calcineurin plays an important role in the shell formation of pearl oyster (Pinctada fucata).

Calcineurin (CN) is a multifunctional protein involved in many important physiological processes in mammalians, but the function of CN in mollusks is still largely unknown. In the present study, through the shell regeneration system, the changes of enzymatic activity of CN were determined in the process of shell regeneration in pearl oyster Pinctada fucata. CN was activated immediately and continuously in the shell regeneration process. The speed of shell regeneration was measured and the ultrastructure of inner shell surface was observed by scanning electron microscopy after inhibiting CN by intramuscular injection of immunosuppresant cyclosporine A (CsA). The results showed that the speed of shell regeneration was delayed and the morphology of calcite and aragonite in the inner shell surface became abnormal when CN was inhibited by CsA. Meanwhile, RT-PCR analysis revealed that the expression of P. fucata BMP-2 in mantle tissue decreased with CsA injection. In vitro secretion level of proteoglycans (PGs) in primary cultures of mantle cells was also decreased when mantle cells were exposed to CsA. Taken together, our results, for the first time, show that CN is involved in the shell formation through regulating the expression of Pf-BMP-2 in mantle tissue, which controls the secretion of PGs/GAGs of the mantle epithelial cells.

Identification and comparison of amorphous calcium carbonate-binding protein and acetylcholine-binding protein in the abalone, Haliotis discus hannai.

Nacre has two different microarchitectures: columnar nacre and sheet nacre. We previously identified an important regulator of the morphology of sheet nacre tablets, which was named amorphous calcium carbonate-binding protein (pf-ACCBP). However, little is known about its counterpart in columnar nacre. Moreover, pf-ACCBP shares significant sequence similarity with a group of acetylcholine-binding proteins (AChBP) that participate in neuronal synapses transmission, but the relationships between the two proteins, which are homologous in sequences but disparate in function, have not been studied yet. Here, we identified an amorphous calcium carbonate-binding protein and an acetylcholine-binding protein in the abalone, Haliotis discus hannai, named hdh-ACCBP and hdh-AChBP, respectively. Studies of hdh-ACCBP indicated that it was a counterpart of pf-ACCBP in gastropods that might function similarly in columnar nacre formation and supersaturated extrapallial fluid. Analysis of hdh-AChBP showed that unlike previously identified AChBP, hdh-AChBP was not only expressed in the nervous system but could also be detected in non-nervous system cells, such as the goblet cells of the mantle pallial. Additionally, its expression patterns during embryo and larval development did not accord with ganglion development. These phenomena indicated that AChBP might play more general roles than just in neuronal synapses transmission. Comparison of hdh-ACCBP and hdh-AChBP revealed that they were quite different in their post-translational modification and oligomerization and that they were controlled under different transcriptional regulation systems, consequently obtaining disparate expression profiles. Our results also implied that ACCBP and AChBP might come from a common ancestor through gene duplication and divergence.

Culture of outer epithelial cells from mantle tissue to study shell matrix protein secretion for biomineralization.

Mantle tissue plays an important role in shell biomineralization by secreting matrix proteins for shell formation. However, the mechanism by which it regulates matrix protein secretion is poorly understood, largely because of the lack of cellular tools for in vitro study and techniques to evaluate matrix protein secretion. We have isolated the outer epithelial cells of the mantle of the pearl oyster, Pinctada fucata, and evaluated cellular metabolism by measuring the secretion of the matrix protein, nacrein. A novel sensitive sandwich enzyme-linked immunosorbent assay (ELISA) was established to quantify nacrein. Mantle explant culture was demonstrated to provide dissociated tissue cells with high viability. Single dissociated cell types from explant culture were separated by density in a discontinuous Percoll gradient. The outer epithelial cells were isolated from other cell types by their higher density and identified by immunolabeling and ultrastructure analysis. ELISA assays revealed that the outer epithelial cells retained the ability to secrete nacrein in vitro. Moreover, increased nacrein secretion resulted from an increased Ca(2+) concentration in the culture media of the outer epithelial cells, in a concentration-dependent manner. These results confirm that outer epithelial cell culture and the ELISA method are useful tools for studying the regulatory mechanisms of shell biomineralization.