Architecture of the organic matrix in the sternal CaCO3 deposits of Porcellio scaber (Crustacea, Isopoda)

Before the molt terrestrial isopods resorb calcium from the posterior cuticle and store it in large deposits within the first four anterior sternites. In Porcellio scaber the deposits consist of three structurally distinct layers consisting of amorphous CaCO3 (ACC) and an organic matrix that consists of concentric and radial elements. It is thought that the organic matrix plays a role in the structural organization of deposits and in the stabilization of ACC, which is unstable in vitro. In this paper, we present a thorough analysis of the ultrastructure of the organic matrix in the CaCO3 deposits using high-resolution field-emission scanning electron microscopy. The spherules and the homogeneous layer contain an elaborate organic matrix with similar structural organization consisting of concentric reticules and radial strands. The decalcification experiments reveal an inhomogeneous solubility of ACC within the spherules probably caused by variations in the stabilizing properties of matrix components. The transition between the three layers can be explained by changes in the number of spherule nucleation sites.     

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.     

A novel highly acidic polysaccharide with inhibitory activity on calcification from the calcified scale "coccolith" of a coccolithophorid alga, Pleurochrysis haptonemofera

Coccolith, a calcified scale with species-specific fine structure produced by marine unicellular coccolithophorid algae, consists of calcium carbonate (CaCO(3)) crystals and a small amount of organic matrices. A novel polysaccharide named coccolith matrix acidic polysaccharide (CMAP) was isolated from the coccolith of a coccolithophorid alga, Pleurochrysis haptonemofera. The structure of CMAP was determined by chemical analysis and NMR spectroscopy including COSY, TOCSY, HMQC, and HMBC to be a polysaccharide composed of the following unit: -->4) l-iduronic acid (alpha1-->2) meso-tartaric acid (3-->1) glyoxylic acid (1-->. It has four carboxyl groups per a disaccharide unit as observed in another polysaccharide PS-2 characterized previously in Pleurochrysis carterae. CMAP showed a strong inhibitory activity on CaCO(3) precipitation. These results suggest that CMAP serves as a regulator in the calcification of the coccolith.     

Effects of ammonia and phosphate limitation on the activated sludge treatment of calcium-containing chemical waste

Laboratory-scale biotreaters were used to study the effects of NH(3)-N and PO(4)-P nutrients on the activated sludge treatment of a chemical waste containing soluble calcium (1300 mg/L). Units receiving high or low levels of NH(3)-N and PO(4)-P were similar in their ability to remove organic compounds from the waste. Adaptation of sludges to low PO(4)-P levels (<0.1 mg/L effluent) resulted in a marked accumulation of CaCO(3) in the biosolids, whereas those receiving high PO(4)-P (2-4 mg/L effluent) had little CaCO(3). Microscopic observations of CaCO(3) containing sludges showed substantial amounts of CaCO(3) crystals imbedded in the biomass. These flocs also appeared to be enriched with nonfilamentous bacterial species in contrast to flocs devoid of CaCO(3) which had a floc structure of filamentous and nonfilamentous organisms. Scanning electron micrographs of flocs grown under low NH(3)-N showed a microbial fibrillar network of exocellular material interconnecting cells in the floc matrix. The sludges adapted to low NH(3)-N also produced higher amounts of extractable polysaccharide. CaCO(3) containing biosolids were more dense, larger, and settled better (low SVI, high ISV) than flocs devoid of the precipitates. It is not known from our experiments whether PO(4)-P or some inorganic or organic polymer produced by the floc bacteria are involved in inhibiting CaCO(3) precipitation in the activated sludge treatment of calcium-containing wastes.     

Abalone nacre insoluble matrix induces growth of flat and oriented aragonite crystals

Mollusc shell formation takes place in a preformed extracellular matrix, composed of insoluble chitin, coated with proteins and dissolved macromolecules. The water-soluble matrix is known to have a strong influence on the growth of CaCO(3), whereas the role of the insoluble matrix on mineralization is unclear. Therefore, we mineralized the EDTA (ethylenediaminetetraacetic acid) insoluble organic matrix of abalone nacre with a modified double-diffusion set-up, where the diffusing solutions were constantly renewed. Control experiments were performed with cellulose and chitosan foils. The mineralized matrices/foils were analyzed with SEM. We show that the insoluble matrix of abalone nacre induces the growth of flat and roughly polygonal CaCO(3) crystals. In some of the experiments with the insoluble matrix, the growth of three-dimensional parallel sheets of densely packed platelets inside the insoluble matrix was observed. XRD on these samples revealed that they consist of oriented aragonite.     

Effects of pig manure and wheat straw on growth of mung bean seedlings grown in aluminium toxicity soil

Crop production in red soil areas may be limited by Al toxicity. A possible alternative to ameliorate Al toxicity is the application of such organic manure as crop straw and animal manure. A pot experiment was conducted to investigate the effects of organic materials on the alleviation of Al toxicity in acid red soil. Ground wheat straw, pig manure or CaCO3 were mixed with the soil and incubated, at 85% of water holding capacity and 25 degrees C, for 8 weeks. After the incubation, 14 seedlings of mung bean (Phaseolus aures Roxb) were allowed to grow for 12 days. Results showed that application of organic material or CaCO3 increased soil pH and decreased soil monomeric inorganic Al concentrations. Growth of mung bean seedling was improved sustantially by the application of organic material or CaCO3. Pig manure or wheat straw was more effective in ameliorating Al toxicity than was CaCO3. Mung bean plants receiving pig manure or wheat straw contained relatively high concentrations of P, Ca and K in their leaves. It is suggested that the beneficial effect of organic manure on mung bean is likely due to decreasing concentrations of monomeric inorganic Al concentrations in soil solution and improvement of mineral nutrition.     

The human pancreatic stone protein

Chronic calcifying pancreatitis (CCP) is characterized by the presence of stones in pancreatic ducts. Calcium carbonate (CaCO3) is the main constituent of stones, to which is associated an organic matrix consisting primarily of one protein of Mr 14,000, the pancreatic stone protein or PSP. PSP is not present as such in pancreatic juice, but in polymorphic forms with higher molecular weights. These secretory forms (PSP S2-5, Mr 16-19,000) are synthesized in the acinar cells of the pancreas and secreted along the same secretory pathway as the exocrine enzymes. The heterogeneity of the forms of higher Mr (PSP S2-5) is probably due to different glycosylation patterns. PSP and PSP S1 are generated by the cleavage of an Arg-Ile bond in the N-terminal part of PSP S2-5. The N-terminal sequence of PSP (40 amino acids) is identical to that of PSP S1, whose complete sequence (133 amino acids) has been determined. Yet, the two proteins differ by their pI. Pancreatic juice is normally supersaturated in CaCO3, suggesting the presence of a stabilizer preventing CaCO3 precipitation. The PSP S could play that role, since an activity inhibiting the nucleation and growth in vitro of CaCO3 crystals was found in pancreatic juice, associated with these proteins. Moreover, PSP S concentration was significantly lower in the pancreatic juice of patients with CCP than in control patients. Proteins homologous to PSP S were also found in the dog, rat, swine, monkey and ox. They constitute a new family of pancreatic secretory proteins, whose biological role would be to maintain pancreatic juice in a stable state towards CaCO3.     

Design strategies of sea urchin teeth: structure,composition and micromechanical relations to function.

The teeth of sea urchins comprise a variety of different structural entities, all of which are composed of magnesium-bearing calcite together with a small amount of organic material. The teeth are worn down continuously, but in such a way that they remain sharp and functional. Here we describe aspects of the structural, compositional and micromechanical properties of the teeth of Paracentrotus lividus using scanning electron microscopy, infrared spectrometry, atomic absorption. X-ray diffraction and microindentation. The S-shaped single crystalline calcitic fibres are one of the main structural elements of the tooth. They extend from the stone part to the keel. The diameter of the fibres increases gradually from less than 1 micron at the stone tip to about 20 microns at the keel end, while their MgCO3 contents decrease from about 13 mol% to about 4.5 mol%. Each fibre is coated by a thin organic sheath and surrounded by polycrystalline calcitic discs containing as much as 35 mol% MgCO3. This structure constitutes a unique kind of gradient fibre-reinforced ceramic matrix composite, whose microhardness and toughness decrease gradually from the stone part to the keel. Primary plates are also important structural elements of the tooth. Each primary plate has a very unusual sandwich-like structure with a calcitic envelope surrounding a thin apparently amorphous CaCO3 layer. This central layer, together with the primary plate/disc interface, improves the toughness of this zone by stopping and blunting cracks. The self-sharpening function of the teeth is believed to result from the combination of the geometrical shape of the main structural elements and their spatial arrangement, the interfacial strength between structural elements, and the hardness gradient extending from the working stone part to the surrounding zones. The sea urchin tooth structure possesses an array of interesting functional design features, some of which may possibly be applicable to materials science.     

Interfacial molecular recognition between polysaccharides and calcium carbonate during crystallization

Based on the basic principles of biomineralization, using beta-cyclodextrin, hepatin and soluble starch as organic matrices, employing the biomimetic method, the crystallization of CaCO(3) was studied by Fourier transform infrared spectroscopy (FT-IR), X-ray powder diffraction analysis (XRD), scanning electronic microscopy (SEM), thermal gravity-differential thermal analysis and conductivity analysis. The results show that polysaccharides have an effect on the crystallization of CaCO(3). CaCO(3) crystals also have an effect on polysaccharides. The possible mechanism of this interaction is discussed.     

The carbonic anhydrase domain protein nacrein is expressed in the epithelial cells of the mantle and acts as a negative regulator in calcification in the mollusc Pinctada fucata

Signals and organic matrix proteins secreted from the mantle are critical for the development of shells in molluscs. Nacrein, which is composed of a carbonic anhydrase domain and a Gly-X-Asn repeat domain, is one of the organic matrix proteins that accumulates in shells. In situ hybridization revealed that nacrein was expressed in the outer epithelial cells of the mantle of the pearl oyster Pinctada fucata. The recombinant nacrein protein inhibited the precipitation of calcium carbonate from a saturated solution containing CaCl2 and NaHCO3, indicating that it can act as a negative regulator for calcification in the shells of molluscs. Because deletion of the Gly-X-Asn repeat domain of nacrein had a significant effect on the ability of nacrein to inhibit the precipitation of calcium carbonate, it is conceivable that the repeat domain has a primary role in the inhibitory function of nacrein in shell formation. Together these studies suggest that nacrein functions as a negative regulator in calcification in the extrapallial space between the shell and the mantle by inhibiting the precipitation of CaCO3.     

Formation of transient amorphous calcium carbonate precursor in quail eggshell mineralization: an in vitro study

To understand the mechanism of quail eggshell biomineralization, we have performed two CaCO(3) precipitation experiments. In the reprecipitation experiments, supersaturated Ca(HCO(3))(2) was prepared by bubbling CO(2) through a slurry of biogenic CaCO(3) obtained from bleach-treated eggshell followed by filtration to obtain a clear solution for crystallization experiments. The nucleated crystals were collected at various time intervals and analyzed. In the second experiment, the extracted SOM from the bleach-treated eggshell was added to the supersaturated clear solution of Ca(HCO(3))(2) solution obtained by bubbling CO(2) gas through a slurry of synthetic CaCO(3) followed by filtration. The crystals/precipitates collected at various time intervals were analyzed. Both experiments showed that amorphous CaCO(3) (ACC) was precipitated in the early stages, which then transformed to the most stable crystalline calcite phase. Amino acid analysis of the soluble organic matrixes (SOM) indicated the presence of high amounts of Glx and Asx amino acids. Ovomucoid--an acidic glycoprotein, and lysozyme--a basic protein, are the two major components along with a few low molecular weight peptides present in the SOM of quail eggshell matrix. Both ovomucoid and lysozyme did not induce precipitation of the ACC phase in in vitro conditions, while the fraction containing low molecular weight peptides induced the precipitation of ACC, suggesting that the latter play an important role in the eggshell biomineralization. Thus, organisms can produce inorganic minerals which assume nonequilibrium morphologies and intricate architecture by precipitating transient ACC, which then transformed into the crystalline phase. Altogether, these observations further demonstrate that this strategy may be common in both vertebrate and invertebrate mineralized structures.     

Organization and mode of secretion of the granular prismatic microstructure of Entodesma navicula (Bivalvia: Mollusca)

The term homogeneous has been applied to molluscan microstructures that lack a readily discernible microstructure and as a result, it has become rather a ‘dustbin’ term, covering a multitude of unrelated finely crystalline textures. Here we investigate in detail the outer ‘homogeneous’ layer of the lyonsiid bivalve Entodesma navicula. The apparently equigranular crystals (up to 10 µm) are, in fact, short prisms which grow in a dense organic matrix with their c‐axes and fibre axes coincident, perpendicular to the growth surface. These prisms are distinct from the aragonitic prisms grown by other bivalves in both their morphology and their mode of growth and so we propose the term granular prismatic microstructure. The organic content of granular prisms (7.4%) is the highest yet recorded for any molluscan microstructure and it is apparent that the short prisms have grown within a gel‐filled space. Although this high organic content is likely to make the microstructure metabolically expensive to produce, it has the benefit of making the valves very flexible. This may be advantageous in the intertidal zone inhabited by E. navicula by allowing a tight seal between the valves.     

Nautilin-63, a novel acidic glycoprotein from the shell nacre of Nautilus macromphalus

In molluscs, and more generally in metazoan organisms, the production of a calcified skeleton is a complex molecular process that is regulated by the secretion of an extracellular organic matrix. This matrix constitutes a cohesive and functional macromolecular assemblage, containing mainly proteins, glycoproteins and polysaccharides that, together, control the biomineral formation. These macromolecules interact with the extruded precursor mineral ions, mainly calcium and bicarbonate, to form complex organo-mineral composites of well-defined microstructures. For several reasons related to its remarkable mechanical properties and to its high value in jewelry, nacre is by far the most studied molluscan shell microstructure and constitutes a key model in biomineralization research. To understand the molecular mechanism that controls the formation of the shell nacreous layer, we have investigated the biochemistry of Nautilin-63, one of the main nacre matrix proteins of the cephalopod Nautilus macromphalus. After purification of Nautilin-63 by preparative electrophoresis, we demonstrate that this soluble protein is glycine-aspartate-rich, that it is highly glycosylated, that its sugar moieties are acidic, and that it is able to bind chitin in vitro. Interestingly, Nautilin-63 strongly interacts with the morphology of CaCO(3) crystals precipitated in vitro but, unexpectedly, it exhibits an extremely weak ability to inhibit in vitro the precipitation of CaCO(3) . The partial resolution of its amino acid sequence by de novo sequencing of its tryptic peptides indicates that Nautilin-63 exhibits short collagenous-like domains. Owing to specific polyclonal antibodies raised against the purified protein, Nautilin-63 was immunolocalized mainly in the intertabular nacre matrix. In conclusion, Nautilin-63 exhibits 'hybrid' biochemical properties that are found both in the soluble and insoluble proteins, rendering it difficult to classify according to the standard view on nacre proteins. DATABASE: The protein sequences of N63 appear on the UniProt Knowledgebase under accession number P86702.     

几种生物CaCO_3霰石结晶的取向性

ＣａＣＯ３结晶广泛分布于生物界,其主要结晶形式为方解石、霰石及球霰石。用Ｘ－射线衍射法对三角帆蚌及合浦珍珠母贝的珍珠层、墨鱼骨和大黄鱼耳石的ＣａＣＯ３结晶进行测定,发现各样品均有一定取向性,以三角帆蚌和合浦珍珠母贝珍珠层的取向性为最强,墨鱼骨的取向性次之,大黄鱼耳石的取向性最小,以上材料粉末样的衍射分析表明,各样品对应ｄ值间差异极小,均为Ｘ射线衍射卡（５—０４５３）所表征的ＣａＣＯ３霰石结构。     

Proteins of calcified endoskeleton: II partial amino acid sequences of endoskeletal proteins and the characterization of proteinaceous organic matrix of spicules from the alcyonarian, Synularia polydactyla

Calcified organic substances in the skeleton contain a protein-polysaccharide complex taking a key role in the regulation of bio-calcification. However, information concerning the matrix proteins in alcyonarian and their effect on calcification process is still unknown. For this reason, we have studied the organic matrix of endoskeletal spicules from the alcyonarian coral, Synularia polydactyla, to analyze the proteins with their sequences and investigate the functional properties by a molecular approach. The separated spicules from the colony were identified by scanning electron microscope (SEM). The soluble organic matrix comprised 0.04% of spicule weight. By recording decline of pH in the experimental design, the inhibitory effect of the matrix on CaCO3 precipitation was revealed. Prior to electrophoresis, our analysis of proteins extracted from the soluble organic matrix of the spicules revealed an abundance of proteins in molecular weight. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the preparations showed seven bands of proteins with an apparent molecular mass of 109, 83, 70, 63, 41, 30 and 22 kDa. The proteins were electrophoresed on Tricine-SDS-PAGE after electro-elution treatment, and then transferred to polyvinylidene difluoride (PVDF) membranes and their N-termini were sequenced. Two bands of proteins of about 70 and 63 kDa successfully underwent N-terminal amino acid sequencing. For the detection of calcium binding proteins, a Ca2+ overlay analysis was conducted on the extract by 45Ca autoradiography. The 109 and 63 kDa calcium binding proteins were found to be radioactive. Periodic acid schiff staining indicated that 83 and 63 kDa proteins were glycosylated. An assay for carbonic anhydrase, which is thought to play an important role in the process of calcification revealed low level of the activity. These findings suggest that the endoskeletal spicules of alcyonarian corals have protein-rich organic matrices, which might be related to the calcification process.     

Oxidation of elemental sulfur by bacteria and fungi in soil

Laboratory experiments were used to determine the effects of antibiotics, organic C and CaCO3 amendments of sterile reinoculated soil on S0 oxidation by bacteria and fungi. The rate of S0 oxidation in soil with nystatin added was higher than in soil amended with penicillin + streptomycin. This tells us that bacteria were more efficient than fungi in the S0 oxidation process. It was demonstrated that neutrophilic chemolithotrophs were more efficient in this process than heterotrophs. Glucose introduced to the soil had a negative effect and CaCO3 had a positive effect on S0 oxidation. In soil enriched with glucose the number of chemolithotrophs was very low in comparison with extremely numerous heterotrophic bacteria and fungi. It suggests that the role of heterotrophs in S0 oxidation could be important in habitats rich in organic C, e.g. rhizosphere. In soil containing S0, qualitative changes of fungal communities to genera with higher S0 oxidation ability was also noted. In the presented paper, after comparison of the own results with the data of others concerning the natural soils, the role of various microbial groups in S0 oxidation process in soils is discussed.     

Characterization and variations of organic parameters in teleost fish endolymph during day-night cycle, starvation and stress conditions

The aim of the present work was to examine the modifications of the organic composition of fish endolymph under environmental conditions (day-night cycle, starvation and Cl2-stress) known to modify otolith growth. Endolymph electrophoretic patterns were compared. An antibody raised against the trout otolith organic matrix allowed examining the variations of organic matrix precursors in the endolymph under the above conditions. Western blot analysis showed bands around 60-80 kDa. A 50% decrease of immunolabelling was observed during the night whereas increases were seen after starvation (factor 3) or stress (factor 2) suggesting that these variations could be related to the organic matrix deposit. A factor retarding in vitro CaCO3 crystallization (FRC) was shown to co-precipitate with endolymph proteins and its apparent molecular mass (determined by measuring the activity after electro elution of gel electrophoresis) was estimated around 20 kDa. The FRC activity was stable during day-night cycle whereas it decreased by 70% and nearly 100% under starvation and stress respectively. These results suggest that the FRC, although retarding in vitro crystallization, plays a major role in the process of otolith calcification and that the decreases measured after starvation and stress are responsible for the decreases of the otolith growth. The variations of these two parameters (precursors and FRC) could contribute for the changes in the microstructure of the otolith.     

几种生物CaCO3霰石结晶的取向性

CaCO3结晶广泛分布于生物界,其主要结晶形式为方解石、霰石及球霰石。用X－射线衍射法对三角帆蚌及合浦珍珠母贝的珍珠层、墨鱼骨和大黄鱼耳石的CaCO3结晶进行测定,发现各样品均有一定取向性,以三角帆蚌和合浦珍珠母贝珍珠层的取向性为最强,墨鱼骨的取向性次之,大黄鱼耳石的取向性最小,以上材料粉末样的衍射分析表明,各样品对应d值间差异极小,均为X射线衍射卡（5－0453）所表征的CaCO3霰石结构。     

Gene targeting reveals the role of Oc90 as the essential organizer of the otoconial organic matrix

A critical part of the functional development of our peripheral balance system is the embryonic formation of otoconia, composite crystals that overlie and provide optimal stimulus input to the sensory epithelium of the gravity receptor in the inner ear. To date neither the functions of otoconial proteins nor the processes of crystal formation are clearly defined. Using gene targeting and protein analysis strategies, we demonstrate that the predominant mammalian otoconin, otoconin-90/95 (Oc90), is essential for formation of the organic matrix of otoconia by specifically recruiting other matrix components, which includes otolin, a novel mammalian otoconin that we identified to be in wildtype murine otoconia. We show that this matrix controls otoconia growth and morphology by embedding the crystallites during seeding and growth. During otoconia development, the organic matrix forms prior to CaCO3 deposition and provides optimal calcification efficiency. Histological and ultrastructural examinations show normal inner ear epithelial morphology but reduced acellular matrices, including otoconial, cupular and tectorial membranes, in Oc90 null mice, likely due to an absence of Oc90 and a profound reduction of otolin. Our data demonstrate the critical roles of otoconins in otoconia seeding, growth and anchoring and suggest mechanistic similarities and differences between otoconia and bone calcification.     

Structural and chemical characterization of inorganic deposits in calcified human mitral valve

X-ray diffraction, i.r. absorption, and chemical analyses have been carried out on the mineral deposits of calcified human mitral valves and glutaraldehyde-preserved porcine aortic grafts. The mineral deposits isolated from highly calcified mitral valves and porcine aortic grafts are constituted of type B-carbonate apatite. Magnesium substituted beta-tricalcium phosphate is present, together with an apatitic phase similar to dahllite, in the ashes of poorly calcified mitral valves. The contraction of the unit cell of beta-tricalcium phosphate due to magnesium incorporation is compared with the variation of the lattice constants of synthetic beta-tricalcium phosphate at different degree of magnesium substitution for calcium. The results reveal the important role of magnesium on the calcification of human valves. In fact, the apatitic phase deposited at the beginning of the calcification process, when there is a high magnesium content, converts completely into beta-tricalcium phosphate by heat treatment at 1,000 degrees C. On the other hand, when the calcification becomes massive, magnesium content appears highly reduced, and the deposited apatitic phase is characterized by a high thermal stability.