Identification of low molecular weight molecules as new components of the nacre organic matrix

Nacre of Pinctada margaritifera displays a number of interesting biological activities on bone, mainly concentrated in a water-soluble organic matrix representing 0.24% of the nacre weight. Dialysis of that matrix through 8 kDa and 1 kDa cut-off membranes showed that 60% of it is made of small molecules of molecular masses below 1 kDa. Reversed-phase high-performance liquid chromatography of the small molecule fractions and subsequent electrospray ionization mass spectrometric analysis of 19 fractions thereof indicated the presence of at least 110 different molecules, in the range 100 Da–700 Da. Evidence for aggregate-forming properties of the small molecules was given. Amino acid analysis revealed that most of the small molecules were not peptides and tandem mass spectrometric gas-phase fragmentations clearly indicated a structural relationship between several molecules. Intriguingly, differences of a single Dalton between mono-charged ions peaks were observed. Further, approximately 40 analytes could be arranged in a ladder-like manner with mass spaces of 57 Da. Some of the water-soluble peptide sequences obtained after MS/MS fragmentation revealed that the 57 Da shift corresponds to the repetition of glycine residues. Furthermore, the exchange of glycine against alanine explains the 14 Da shift observed between some peptides. These data show for the first time that small molecules, especially peptides, are prevalent components of nacre. The molecular species described in this report might have a functional role in nacre.     

Comparative proteomic analysis revealed the metabolic mechanism of excessive exopolysaccharide synthesis by Bacillus mucilaginosus under CaCO3 addition

Abstract

The metabolic mechanism of excessive exopolysaccharide (BMPS) synthesis by Bacillus mucilaginosus CGMCC5766 under CaCO₃ addition was investigated. Under CaCO₃ (5?g/L), the maximum BMPS concentration reached 28.4?g/L, which was 11.2 folds higher than that of the control. Proteomics was then used to analyze the proteins with substantial differences expressed by B. mucilaginosus with and without CaCO₃ addition. The proteomic results revealed that the enzymes related to the central metabolic pathway, amino acid biosynthesis, and nucleotide metabolism were depressed. By contrast, the UDP–glucose pyrophosphorylase involved in BMPS biosynthesis was overexpressed and converted metabolic flux from the biomass accumulation to the biosynthesis of BMPS. This research provides a new and widened perspective into understanding the mechanism of BMPS biosynthesis and applying theoretical and practical significance for the improvement of BMPS production from B. mucilaginosus.     

²⁶Mg labeling of the sea urchin regenerating spine: Insights into echinoderm biomineralization process

This paper reports the results of the first dynamic labeling experiment with regenerating spines of sea urchins Paracentrotus lividus using the stable isotope ²⁶Mg and NanoSIMS high-resolution isotopic imaging, which provide a direct information about the growth process. Growing spines were labeled twice (for 72 and 24 h, respectively) by increasing the abundance of ²⁶Mg in seawater. The incorporation of ²⁶Mg into the growing spines was subsequently imaged with the NanoSIMS ion microprobe. Stereom trabeculae initially grow as conical micro-spines, which form within less than 1 day. These micro-spines fuse together by lateral outgrowths and form a thin, open meshwork (inner stereom), which is subsequently reinforced by addition of layered thickening deposits (outer stereom). The (longitudinal) growth rate of the inner stereom is ca. 125 μm/day. A single (ca. 1 μm) thickening layer in the stereom trabeculae is deposited during 24 h. The thickening process is contemporaneous with the formation micro-spines and involves both longitudinal trabeculae and transverse bridges to a similar degree. Furthermore, the skeleton-forming cells remain active in the previously formed open stereom for at least 10 days, and do not migrate upwards until the end of the thickening process. The experimental capability presented here provides a new way to obtain detailed information about the skeleton formation of a multitude of marine, calcite producing organisms.     

Long-term effects of induced mineral accretion on growth, survival and corallite properties of Porites cylindrica Dana

The mineral accretion technology involves the accumulation of dissolved mineral ions within the vicinity of underwater electrodes and their deposition via electrochemical processes onto the positive electrode forming a natural substrate. Branches of Porites cylindrica were reared under the presence of mineral accretion (treated nubbins), without mineral accretion (untreated), and under undisturbed (control) conditions for 6 months (mineral accretion phase). The electricity for the treated nubbins was cut off after the sixth month, and all remaining nubbins were allowed to grow for another 6 months (post mineral accretion phase). The longitudinal growth rate of the treated nubbins was relatively high during the mineral accretion phase then dropped during the post mineral accretion phase. Statistical analysis revealed longitudinal growth to be significant over time, but the significant differences between treatments lay only between the first until the second bi-monthly period (from January to May 2000) and the rest of the observation period (June to January 2001). This indicates that growth enhancement occurred only during the early stages of the mineral accretion phase. There were also significant differences in girth growth between phases and between treatments as well as a significant phase by treatment interaction again indicating that significant differences between treatments occurred only during a certain phase. In terms of overall survival, the treated nubbins fared better than the untreated nubbins. The corals in all treatments showed the same trend in corallite size and density after 1 year. Corallite sizes increased from the tip towards the base of the transplant. The reverse trend occurred with corallite density which decreased from the tip towards the base. Phenotypic alteration of the corallites (decrease in size and increase in density from the middle region of the nubbin towards the base) had occurred in the treated nubbins while they were exposed to mineral accretion. This effect disappeared, however, after 6 months. It appears, therefore, that enhancement of growth took place only during active mineral accretion, though there were positive, longer-term effects on survival.     

Regulation of CaCO(3) formation in coccolithophores

Coccolithophores impact the ocean carbon cycle principally through the generation of CO₂ during CaCO₃ production. Coccolithophore biomineralization has been examined most extensively in Pleurochrysis carterae and Emiliania huxleyi both of which produce mineralized scales—coccoliths—composed of elaborate calcite crystals attached to an underlying organic base plate. Calcification of preformed base plates is mediated by acidic polysaccharides and occurs in Golgi-derived structures known as mineralizing vesicles. In Pleurochrysis a high capacity calcium-binding polysaccharide PS2 is required for efficient nucleation of calcitic protocrystals. A galacturonomannan PS3 is required for the growth and transformation of the protocrystals into a massive double disc of calcite. The genes that regulate expression of the glycans have not yet been identified. In addition to the coccolith-bearing diploid phases, Pleurochrysis and Emiliania possess both haploid and diploid non-calcifying stages, which are self-perpetuating via binary fission. One non-calcifying Pleurochrysis phase fails to synthesis PS2 and spontaneously reverts to the mineralizing morphotype in laboratory cultures. As yet, there is little information on environmental factors that effect the expression or silencing of calcifying genes or favor the growth of calcifying over non-calcifying phases. These issues will need extensive investigation, if we are to appreciate the role of coccolithophores in the regulation of atmospheric CO₂ levels.     

Growth and survival of coral transplants with and without electrochemical deposition of CaCO3

This study aims to investigate experimentally the effect of electrochemical deposition of CaCO₃ on linear and girth growth, survival and skeletal structure of Porites cylindrica Dana. Transplanted coral nubbins were subjected to up to 18 V and 4.16 A of direct current underwater to induce the precipitation of dissolved minerals. Naturally growing colonies showed a significant increase in percentage longitudinal growth over the treated and untreated corals. Survival followed a similar trend as the growth rate. Lowest survival rates were found in the untreated nubbins. Phenotypic alterations were observed in the treated nubbins where the basal corallites decreased in size with a concomitant increase in their number per unit area. This was probably due to increased mineral concentration (such as Ca²⁺, Na⁻, Mg²⁺, CO₃²⁻, Cl⁻, OH⁻ and HCO₃⁻) at the basal region of the nubbins. These alterations were accompanied by a significant increase in girth growth rates of the treated nubbins at their basal regions. The abundance of mineral ions at the basal region thus appeared to be utilized by the numerous small polyps for a lateral increase in size of the nubbins instead of a longitudinal increase.     

Quantifying CaCO3 Microprecipitates Within Developing Surface Mats of Marine Stromatolites Using GIS and Digital Image Analysis

The unique geochemical coupling of organic molecules and mineral CaCO₃ provides a fluorescence signature detectable using conventional confocal scanning laser microscopy (CSLM). The surface microbial mats of open-water marine stromatolites (Bahamas) exist in a continuum of states ranging from a Type 1 (i.e., nonlithifying) to Type 2 (i.e., lithified micritic laminae present) to Type 3 (i.e., fused grain layer). An approach was developed here, that utilizes geographical information systems (GIS) and digital image analysis, coupled with CSLM to estimate concentrations of calcium carbonate precipitates in developing marine stromatolites. We propose that the area occupied by particles within each image can be used to estimate concentrations of precipitates. Fluorescent polymeric microbeads and bacteria were used to calibrate the approach. We used this approach to demonstrate that CaCO₃ precipitates in lithifying layers were quantifiable and significantly different (p < 0.0001) from those in nonlithifying layers. The approach provided a useful tool for the unambiguous assessment of relative changes in microbial precipitates occurring over small ( μ m to mm) spatial scales, and that characterize the formation of lithified layers (micritic laminae) in open-water marine stromatolites.     

Coccolithophore (–CaCO3) flux in the Sea of Okhotsk: seasonality, settling and alteration processes

Coccolithophore fluxes were determined in the Sea of Okhotsk using samples from a 1 year experiment (12 August 1990 to 12 August 1991) with sediment traps at 258 and 1061 m depth. A special study was made on Coccolithus pelagicus, using fragmentation and the degree of etching, as indicators of transport mechanisms. A Corrosion Index for C. pelagicus is developed. The coccolithophore flux pattern at 258 m depth was characterised by a strong seasonality, with flux peaks during autumn 1990 (late November to early December) and spring 1991 (March). The assemblage consisted almost entirely of the two species C. pelagicus and Emiliania huxleyi. During autumn, coccolithophore transportation to 258 m depth mainly occurred within cylindrical fecal pellets and marine snow aggregates of silicoflagellates, and through agglutination on tintinnids. Grazing caused severe fragmentation of coccoliths and disintegration of coccospheres. Marine snow aggregates contained many intact coccospheres of C. pelagicus. During spring, coccolithophores were probably removed from the euphotic zone by the ballast effect of sinking diatoms. The coccolithophore flux peak in spring occurred immediately after the ice had retreated from the trap station, and the trapped assemblage included coccoliths of subtropical species. These features indicate drifting from an ice-free location to the south or east.The coccolith and coccosphere flux at 1061 m was respectively 7 and 12 times lower than at 258 m depth, and maximum fluxes were recorded 2 months later. Increasing carbonate dissolution from 258 to 1061 m depth is expressed in the coccolithophore–CaCO₃ flux reduction of 82%, and in the increasing percentage of etched coccoliths of Coccolithus pelagicus from 32 to >90%.     

Calcareous impact on arbuscular mycorrhizal fungus development and on lipid peroxidation in monoxenic roots

The present work underlined the negative effects of increasing CaCO₃ concentrations (5, 10 and 20 mM) both on the chicory root growth and the arbuscular mycorrhizal fungus (AMF) Glomus irregulare development in monoxenic system. CaCO₃ was found to reduce drastically the main stages of G. irregulare life cycle (spore germination, germinative hyphae elongation, root colonization, extraradical hyphae development and sporulation) but not to inhibit it completely. The root colonization drop was confirmed by the decrease in the arbuscular mycorrhizal fungal marker C16:1ω5 amounts in the mycorrhizal chicory roots grown in the presence of CaCO₃. Oxidative damage evaluated by lipid peroxidation increase measured by (i) malondialdehyde (MDA) production and (ii) the antioxidant enzyme peroxidase (POD) activities, was highlighted in chicory roots grown in the presence of CaCO₃. However, MDA formation was significantly higher in non-mycorrhizal roots as compared to mycorrhizal ones. This study pointed out the ability of arbuscular mycorrhizal symbiosis to enhance plant tolerance to high levels of CaCO₃ by preventing lipid peroxidation and so less cell membrane damage.     

Conditioning film and initial biofilm formation on electrochemical CaCO3 deposition on a metallic net in the marine environment

Electrochemical deposition of minerals is a unique technology for artificial reef constructions, relying on calcium carbonate (CaCO₃) build-up over metallic structures through electrolysis of seawater. The present study traces the first 72 h following electric current termination on bacterial biofilm build-up on a metallic net covered with CaCO₃. 16S rRNA clone libraries indicated a dynamic succession. Proteobacteria and Bacteroidetes were evident at all sampling times while Cyanobacteria appeared only within the first 8 h. A significant increase in total organic carbon (TOC) and total protein was observed after 48 h with a significant correlation (R ² = 0.74), indicating TOC is a good tool for characterizing initial biofilm formation. 18S rRNA gene sequences obtained 72 h following current termination indicated a significant presence of Cnidarians (51%). Understanding the dynamics among primary bacterial settlers is important because they play a crucial role in driving the colonization of sessile invertebrate communities on artificial, as well as natural surfaces.     

Optimization of conditions for the production of lignocellulolytic enzymes by Sphingobacterium sp. ksn-11 utilizing agro-wastes under submerged condition

Abstract

The present work was aimed at studying the production of lignocellulolytic enzymes, namely cellulase, xylanase, pectinase, mannanase, and laccase by a newly isolated bacterium Sphingobacterium sp. ksn-11, utilizing various agro-residues as a substrate under submerged conditions. The production of lignocellulolytic enzymes was found to be maximum at the loading of 10%(w/v) agro-residues. The enzyme secretion was enhanced by two-fold at 2?mM CaCO_3, optimum pH 7, and temperature 40°. The Field Emission Gun-Scanning Electron Microscope (FEG-SEM) results have shown the degradative effect of lignocellulases; cellulase, xylanase, mannanase, pectinase, and laccase on corn husk with 3.55?U/ml, 79.22?U/ml, 12.43?U/ml, 64.66?U/ml, and 21.12?U/ml of activity, respectively. The hydrolyzed corn husk found to be good adsorbent for polyphenols released during hydrolysis of corn husk providing suitable conditions for stability of lignocellulases. Sphingobacterium sp. ksn is proved to be a promising candidate for lignocellulolytic enzymes in view of demand for enzymes in the biofuel industry.     

Germination response of Arabian desert species to gibberellic acid and potassium nitrate seed treatment

Abstract

Desert plant species commonly use seed dormancy to prevent germination during unfavorable environmental conditions and thus increase the probability of seedling survival. Seed dormancy presents a challenge for restoration ecology, particularly in desert species for which our knowledge of dormancy regulation is limited. In the present study the effect of gibberellic acid (GA₃) and potassium nitrate (KNO₃) on seed dormancy release was investigated on eight Arabian desert species. Both treatments significantly enhanced the germination of most species tested. GA₃ was more effective than KNO₃ in enhancing germination percentage, reducing mean germination time and synchronizing the germination in most of the studied species. Light requirement during germination was species-specific, but in general the presence of light promoted germination more effectively when combined with KNO₃ and GA₃. The wide variation in dormancy and germination requirements among the tested species is indicative of distinct germination niches, which might assist their co-existence in similar habitat/environmental conditions. Seed pre-treatments that optimize germination in this habitat must therefore be assessed for individual species to improve the outcomes of ecological restoration.     

Yield of submerged paddy and uptake of Zn,P and N as affected by liming and Zn fertilizers

The effects of CaCO₃, Zn sources and levels on the yield of submerged paddy and uptake of Zn, P and N to paddy were studied in green-house at Haryana Agricultural University, Hissar. Powdered CaCO₃ was mixed at 0,4 and 8 per cent and Zn was added at 0,5 and 10 ppm through ZnSO₄.7H₂O, ZnO and Zn EDTA separately. Dry weight at tillering and heading and grain and straw at maturity decreased significantly with 4 and 8 per cent CaCO₃ in comparison to the control. Increasing Zn application increased the dry weight and grain yield. Zn EDTA gave highest yield of paddy followed by ZnSO₄.7H₂O and ZnO.Increasing the application of CaCO₃ from 0–8 per cent decreased the concentration and uptake of Zn and increasing Zn application from 0–10 ppm increased concentration and uptake of Zn in paddy at tillering, heading and maturity. Zn EDTA gave the highest concentration and uptake of Zn followed by ZnSO₄.7H₂O and ZnO. There was interaction between Zn sources and CaCO₃.The concentration and uptake of N and P in paddy dry matter at tillering and heading and straw and grain at maturity decreased as compared to control with increasing CaCO₃ addition. The concentration and uptake of N increased and that of P decreased in paddy dry matter straw and grain with increasing Zn application. The highest concentration of N was observed with ZnO, followed by ZnSO₄.7H₂O and Zn EDTA. But highest uptake of N was observed with Zn EDTA followed by ZnSO₄.7H₂O and ZnO. As regards concentration and uptake of P, it was highest with ZnO followed by ZnSO₄.7H₂O and Zn EDTA.     

Conformation of the c552:aa3 electron transfer complex in Paracoccus denitrificans studied by EPR on oriented samples

The EPR spectral parameters of aa(3) oxidase and cyt c(552) from Paracoccus denitrificans were studied in purified oxidase and enriched cyt c(552). The orientation of the g-tensors of hemes a and c(552) were determined on partially ordered membranes, enriched cyt c(552) and a c(552):aa(3) subcomplex. The known correlation of g-tensor to molecular axes in histidine/methionine ligated hemes permits us to position cyt c(552) with respect to the parent membrane. Taken together with previous data on the interaction surface between aa(3) oxidase and cyt c(552), these results allow us to arrive at a single conformation for the c(552):aa(3) electron transfer complex.     

The role of long-chain fatty-acid-CoA ligase 3 in vitamin D3 and androgen control of prostate cancer LNCaP cell growth

The antiproliferative effect of 1alpha,25(OH)(2)D(3) on human prostate cancer cells is well known, but the mechanism is still not fully understood, especially its androgen-dependent action. Based on cDNA microarray results, we found that long-chain fatty-acid-CoA ligase 3 (FACL3/ACS3) might play an important role in vitamin D(3) and androgen regulation of LNCaP cell growth. The expression of FACL3/ACS3 was found to be significantly upregulated by 1alpha,25(OH)(2)D(3) and the regulation was shown to be time-dependent, with the maximal regulation over 3.5-fold at 96h. FACL3/ACS3 was a dominant isoform of FACL/ACS expressed in LNCaP cells as indicated by measuring the relative expression of each isoform. 1alpha,25(OH)(2)D(3) had no significant effect on the expression of FACL1(FACL2), FACL4 and FACL6 except for its downregulation of FACL5 at 24 and 48h by around twofold. The upregulation of FACL3/ACS3 expression by 1alpha,25(OH)(2)D(3) was accompanied with increased activity of FACL/ACS as demonstrated by enzyme activity assay using a (14)C-labeled substrate preferential for FACL3/ACS3. The growth inhibitory effect of 1alpha,25(OH)(2)D(3) on LNCaP cells was significantly attenuated by FACL3/ACS3 activity inhibitor. Androgen withdrawal (DCC-serum), in the presence of antiandrogen Casodex or in AR-negative prostate cancer cells (PC3 and DU145), vitamin D(3) failed to regulate FACL3/ACS3 expression. The upregulation of FACL3/ACS3 expression by vitamin D(3) was recovered by the addition of DHT in DCC-serum medium. Western blot analysis showed that the expression of androgen receptor (AR) protein was consistent with vitamin D(3) regulation of FACL3/ACS3 expression. Taken together, the data suggest that the upregulation of FACL3/ACS3 expression by vitamin D(3) is through an androgen/AR-mediated pathway and might be one of the contributions of the vitamin D(3) antiproliferative effect in prostate cancer LNCaP cells.     

1,25-Dihydroxyvitamin D3 induces Ca-mediated apoptosis in adipocytes via activation of calpain and caspase-12

Induction of apoptotic cell death is emerging as a promising strategy for prevention and treatment of obesity because removing of adipocytes via apoptosis may result in reducing body fat and a long-lasting maintenance of weight loss. However, the mechanisms controlling adipocyte apoptosis are unknown and even the ability of adipocytes to undergo apoptosis has not been conclusively demonstrated. We have shown previously that the specific Ca²⁺ signal, sustained increase in intracellular Ca²⁺, triggers apoptotic cell death via activation of Ca²⁺-dependent proteases and that the apoptosis-inducing effect of the hormone 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) is mediated through Ca²⁺ signaling. Here, we report that 1,25(OH)₂D₃ induces apoptosis in mature mouse 3T3-L1 adipocytes via activation of Ca²⁺-dependent calpain and Ca²⁺/calpain-dependent caspase-12. Treatment of adipocytes with 1,25(OH)₂D₃ induced, in concentration- and time-dependent fashion, a sustained increase in the basal level of intracellular Ca²⁺. The increase in Ca²⁺ was associated with induction of apoptosis and activation of μ-calpain and caspase-12. Our results demonstrate that Ca²⁺-mediated apoptosis can be induced in mature adipocytes and that the apoptotic molecular targets activated by 1,25(OH)₂D₃ in these cells are Ca²⁺-dependent calpain and caspase-12. These findings provide rationale for evaluating the role of vitamin D in prevention and treatment of obesity.     

1α,25(OH)2D3 is an autocrine regulator of extracellular matrix turnover and growth factor release via ERp60 activated matrix vesicle metalloproteinases

Growth plate chondrocytes produce proteoglycan-rich type II collagen extracellular matrix (ECM). During cell maturation and hypertrophy, ECM is reorganized via a process regulated by 1α,25(OH)₂D₃ and involving matrix metalloproteinases (MMPs), including MMP-3 and MMP-2. 1α,25(OH)₂D₃ regulates MMP incorporation into matrix vesicles (MVs), where they are stored until released. Like plasma membranes (PM), MVs contain the 1α,25(OH)₂D₃-binding protein ERp60, phospholipase A₂ (PLA₂), and caveolin-1, but appear to lack nuclear Vitamin D receptors (VDRs). Chondrocytes produce 1α,25(OH)₂D₃ (10⁻⁸ M), which binds ERp60, activating PLA₂, and resulting lysophospholipids lead to MV membrane disorganization, releasing active MMPs. MV MMP-3 activates TGF-β1 stored in the ECM as large latent TGF-β1 complexes, consisting of latent TGF-β1 binding protein, latency associated peptide, and latent TGF-β1. Others have shown that MMP-2 specifically activates TGF-β2. TGF-β1 regulates 1α,25(OH)₂D₃-production, providing a mechanism for local control of growth factor activation. 1α,25(OH)₂D₃ activates PKCα in the PM via ERp60-signaling through PLA₂, lysophospholipid production, and PLCβ. It also regulates distribution of phospholipids and PKC isoforms between MVs and PMs, enriching the MVs in PKCζ. Direct activation of MMP-3 in MVs requires ERp60. However, when MVs are treated with 1α,25(OH)₂D₃, PKCζ activity is decreased and PKCα is unaffected, suggesting a more complex feedback mechanism, potentially involving MV lipid signaling.     

Synthesis of 2α-substituted-14-epi-previtamin D3 and its genomic activity

We synthesized and isolated 2α-substituted analogs of 14-epi-previtamin D₃ after thermal isomerization at 80 °C for the first time. The VDR binding affinity and transactivation activity of osteocalcin promoter in HOS cells were evaluated, and the 2α-methyl-substituted analog was found to have greater genomic activity than 14-epi-previtamin D₃.     

Orchestin, a calcium-binding phosphoprotein, is a matrix component of two successive transitory calcified biomineralizations cyclically elaborated by a terrestrial crustacean

Orchestia cavimana is a crustacean that cyclically replaces its calcified cuticle during molting cycles in order to grow. Its terrestrial way of life requires storage of calcium during each premolt period, as calcareous concretions, in tubular diverticula of the midgut. During the postmolt period the stored calcium is reabsorbed and is translocated through the storage organ epithelium as calcified small spherules. In a previous study, we sequenced and characterized a remarkable component of the organic matrix of the premolt storage structures, Orchestin, which is a calcium-binding phosphoprotein. In this paper, we analyzed the spatiotemporal expression of the orchestin gene by Northern blotting and in situ hybridization, and its translated product by immunocytochemistry. We found evidence that the gene and the protein are expressed specifically during premolt in the storage organs. More interestingly, we demonstrated that the protein is synthesized also during the postmolt period, as a component of the organic matrix of the calcium resorption spherules. Thus, Orchestin is a matrix component that is synthesized by the same cells to contribute alternately to the elaboration of two different calcifications. These results, in addition to the physical and chemical features of the protein, suggest that Orchestin is probably a key molecule in the calcium carbonate precipitation process leading to the cyclic elaboration of two transitory calcified mineralizations by the crustacean Orchestia.