Environmental controls on the production of calcium carbonate by earthworms

Lumbricus terrestris earthworms produce calcium carbonate (CaCO3) granules with unknown physiological function. To investigate carbon sequestration potential, the influence of temperature and CO2 concentration ([CO2]) on CaCO3 production was investigated using three soils, five temperatures(3-20 C) and four atmospheric [CO2] (439-3793 ppm). Granule production rates differed between soils, but could not be related to any soil characteristics measured. Production rates increased with temperature, probably because of higher metabolic rate, and with soil CO2 concentration. Implications for carbon sequestration are discussed. CaCO3 production in earthworms is probably related to pH regulation of blood and tissue fluid in the high CO2 environment of the soil.

Synaptic vesicle glycoprotein 2A modulates vesicular release and calcium channel function at peripheral sympathetic synapses

Synaptic vesicle glycoprotein (SV)2A is a transmembrane protein found in secretory vesicles and is critical for Ca2+-dependent exocytosis in central neurons, although its mechanism of action remains uncertain. Previous studies have proposed, variously, a role of SV2 in the maintenance and formation of the readily releasable pool (RRP) or in the regulation of Ca2+ responsiveness of primed vesicles. Such previous studies have typically used genetic approaches to ablate SV2 levels; here, we used a strategy involving small interference RNA (siRNA) injection to knockdown solely presynaptic SV2A levels in rat superior cervical ganglion (SCG) neuron synapses. Moreover, we investigated the effects of SV2A knockdown on voltage-dependent Ca2+ channel (VDCC) function in SCG neurons. Thus, we extended the studies of SV2A mechanisms by investigating the effects on vesicular transmitter release and VDCC function in peripheral sympathetic neurons. We first demonstrated an siRNA-mediated SV2A knockdown. We showed that this SV2A knockdown markedly affected presynaptic function, causing an attenuated RRP size, increased paired-pulse depression and delayed RRP recovery after stimulus-dependent depletion. We further demonstrated that the SV2A–siRNA-mediated effects on vesicular release were accompanied by a reduction in VDCC current density in isolated SCG neurons. Together, our data showed that SV2A is required for correct transmitter release at sympathetic neurons. Mechanistically, we demonstrated that presynaptic SV2A: (i) acted to direct normal synaptic transmission by maintaining RRP size, (ii) had a facilitatory role in recovery from synaptic depression, and that (iii) SV2A deficits were associated with aberrant Ca2+ current density, which may contribute to the secretory phenotype in sympathetic peripheral neurons.

Biomineralisation by earthworms: an investigation into the stability and distribution of amorphous calcium carbonate

Background Many biominerals form from amorphous calcium carbonate (ACC), but this phase is highly unstable when synthesised in its pure form inorganically. Several species of earthworm secrete calcium carbonate granules which contain highly stable ACC. We analysed the milky fluid from which granules form and solid granules for amino acid (by liquid chromatography) and functional group (by Fourier transform infrared (FTIR) spectroscopy) compositions. Granule elemental composition was determined using inductively coupled plasma-optical emission spectroscopy (ICP-OES) and electron microprobe analysis (EMPA). Mass of ACC present in solid granules was quantified using FTIR and compared to granule elemental and amino acid compositions. Bulk analysis of granules was of powdered bulk material. Spatially resolved analysis was of thin sections of granules using synchrotron-based μ-FTIR and EMPA electron microprobe analysis. Results The milky fluid from which granules form is amino acid-rich (≤ 136 ± 3 nmol mg−1 (n = 3; ± std dev) per individual amino acid); the CaCO3 phase present is ACC. Even four years after production, granules contain ACC. No correlation exists between mass of ACC present and granule elemental composition. Granule amino acid concentrations correlate well with ACC content (r ≥ 0.7, p ≤ 0.05) consistent with a role for amino acids (or the proteins they make up) in ACC stabilisation. Intra-granule variation in ACC (RSD = 16%) and amino acid concentration (RSD = 22–35%) was high for granules produced by the same earthworm. Maps of ACC distribution produced using synchrotron-based μ-FTIR mapping of granule thin sections and the relative intensity of the ν2: ν4 peak ratio, cluster analysis and component regression using ACC and calcite standards showed similar spatial distributions of likely ACC-rich and calcite-rich areas. We could not identify organic peaks in the μ-FTIR spectra and thus could not determine whether ACC-rich domains also had relatively high amino acid concentrations. No correlation exists between ACC distribution and elemental concentrations determined by EMPA. Conclusions ACC present in earthworm CaCO3 granules is highly stable. Our results suggest a role for amino acids (or proteins) in this stability. We see no evidence for stabilisation of ACC by incorporation of inorganic components.

Calcium-mediated binding of DNA to 1,2-distearoyl-sn-glycero-3-phosphocholine-containing mixed lipid monolayers

The calcium-mediated interaction of DNA with monolayers of the non-toxic, zwitterionic phospholipid, 1,2-distearoyl-sn-glycero-3-phosphocholine when mixed with 50 mol% of a second lipid, either the zwitteronic 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine or neutral cholesterol was investigated using a combination of surface pressure-area isotherms, Brewster angle microscopy, external reflectance Fourier transform infrared spectroscopy and specular neutron reflectivity in combination with contrast variation. When calcium and DNA were both present in the aqueous subphase, changes were observed in the compression isotherms as well as the surface morphologies of the mixed lipid monolayers. In the presence of calcium and DNA, specular neutron reflectivity showed that directly underneath the head groups of the lipids comprising the monolayers, DNA occupied a layer comprising approximately 13 and 18% v/v DNA for the 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine and cholesterol-containing monolayers, respectively. The volume of the corresponding layer for 1,2-distearoyl-sn-glycero-3-phosphocholine only containing monolayers was ∼15% v/v DNA. Furthermore regardless of the presence and nature of the second lipid and the surface pressure of the monolayer, the specular neutron reflectivity experiments showed that the DNA-containing layer was 20–27 Å thick, suggesting the presence of a well-hydrated layer of double-stranded DNA. External reflectance Fourier transform infrared studies confirmed the presence of double stranded DNA, and indicated that the strands are in the B-form conformation. The results shed light on the interaction between lipids and nucleic acid cargo as well as the role of a second lipid in lipid-based carriers for drug delivery.

Production of acid whey hydrolysates applying an integrative process: effect of calcium on process performance

High ionic calcium concentration and the absence of caseinmacropeptides (CMP) in acid whey could influence the production of angiotensin-I-converting enzyme (ACE)-inhibitory hydrolysate and its bioactivity through the application of the integrative process. Therefore, the aim of the present study was to produce a hydrolysate from acid whey applying the integrative process. Process performance was evaluated based on protein adsorption capacity and conversion in relation to ACE-inhibitory activity (ACEi%) and ionic calcium concentration. Hydrolysates with high potency of their biological activity were produced (IC50 = 206-353 μg mL-1). High ionic calcium concentration in acid whey contributed to ACE-inhibitory activity. However, low β-lactoglobulin adsorption and conversion was observed. Optimisation of the resin volume increased the adsorption of β-lactoglobulin significantly but with lower selectivity. The changes in conversion value were not significant even at higher concentration of enzyme. Several ACE inhibitors derived from β-lactoglobulin that were identified before in sweet whey hydrolysates such as, IIAEKT, IIAE, IVTQ, LIVTQ, LIVTQT, LDAQ and LIVT were found. New peptides such as, SNICNI and ECCHGD derived from α-lactalbumin and BSA respectively were identified.

Calcium Flux in Neuroblastoma Cells Is a Coupling Mechanism between Non-Genomic and Genomic Modes of Estrogens

Estrogens have been demonstrated to rapidly modulate calcium levels in a variety of cell types. However, the significance of estrogen-mediated calcium flux in neuronal cells is largely unknown. The relative importance of intra- and extracellular sources of calcium in estrogenic effects on neurons is also not well understood. Previously, we have demonstrated that membrane-limited estrogens, such as E-BSA given before an administration of a 2-hour pulse of 17beta-estradiol (E(2)), can potentiate the transcription mediated by E(2) from a consensus estrogen response element (ERE)-driven reporter gene. Inhibitors to signal transduction cascades given along with E-BSA or E(2) demonstrated that calcium flux is important for E-BSA-mediated potentiation of transcription in a transiently transfected neuroblastoma cell line. In this report, we have used inhibitors to different voltage-gated calcium channels (VGCCs) and to intracellular store receptors along with E-BSA in the first pulse or with E(2) in the second pulse to investigate the relative importance of these channels to estrogen-mediated transcription. Neither L- nor P-type VGCCs seem to play a role in estrogen action in these cells; while N-type VGCCs are important in both the non-genomic and genomic modes of estrogen action. Specific inhibitors also showed that the ryanodine receptor and the inositol trisphosphate receptor are important to E-BSA-mediated transcriptional potentiation. This report provides evidence that while intracellular stores of calcium are required to couple non-genomic actions of estrogen initiated at the membrane to transcription in the nucleus, extracellular sources of calcium are also important in both non-genomic and genomic actions of estrogens. Copyright (c) 2005 S. Karger AG, Basel.

Microbial culture and checkerboard DNA-DNA hybridization assessment of bacteria in root canals of primary teeth pre- and post-endodontic therapy with a calcium hydroxide/chlorhexidine paste

Aim. To investigate the root canal microbiota of primary teeth with apical periodontitis and the in vivo antimicrobial effects of a calcium hydroxide/chlorhexidine paste used as root canal dressing. Design. Baseline samples were collected from 30 root canals of primary teeth with apical periodontitis. Then, the root canals were filled with a calcium hydroxide paste containing 1% chlorhexidine for 14 days and the second bacteriologic samples were taken prior to root canal filling. Samples were submitted to microbiologic culture procedure to detect root canal bacteria and processed for checkerboard DNA-DNA hybridization. Results. Baseline microbial culture revealed high prevalence and cfu number of anaerobic, black-pigmented bacteroides, Streptococcus, and aerobic microorganisms. Following root canal dressing, the overall number of cfu was dramatically diminished compared to initial contamination (P < 0.05), although prevalence did not change (P > 0.05). Of 35 probes used for checkerboard DNA-DNA hybridization, 31 (88.57%) were present at baseline, and following root canal dressing, the number of positive probes reduced to 13 (37.14%). Similarly, the number of bacterial cells diminished folowing application of calcium hydroxide/chlorhexidine root canal dressing (P = 0.006). Conclusion. Apical periodontitis is caused by a polymicrobial infection, and a calcium hydroxide/chlorhexidine paste is effective in reducing the number of bacteria inside root canals when applied as a root canal dressing.

Calcium Hydroxide Promotes Cementogenesis and Induces Cementoblastic Differentiation of Mesenchymal Periodontal Ligament Cells in a CEMP1-and ERK-Dependent Manner

Periodontal tissue engineering is a complex process requiring the regeneration of bone, cementum, and periodontal ligament (PDL). Since cementum regeneration is poorly understood, we used a dog model of dental pulpal necrosis and in vitro cellular wounding and mineralization assays to determine the mechanism of action of calcium hydroxide, Ca(OH)(2), in cementogenesis. Laser capture microdissection (LCM) followed by qRT-PCR were used to assay responses of periapical tissues to Ca(OH)(2) treatment. Additionally, viability, proliferation, migration, and mineralization responses of human mesenchymal PDL cells to Ca(OH)(2) were assayed. Finally, biochemical inhibitors and siRNA were used to investigate Ca(OH)(2)-mediated signaling in PDL cell differentiation. In vivo, Ca(OH)(2)-treated teeth formed a neocementum in a STRO-1- and cementum protein-1 (CEMP1)-positive cellular environment. LCM-harvested tissues adjacent to the neocementum exhibited higher mRNA levels for CEMP1, integrin-binding sialoprotein, and Runx2 than central PDL cells. In vitro, Ca(OH)(2) and CEMP1 promoted STRO-1-positive cell proliferation, migration, and wound closure. Ca(OH)(2) stimulated expression of the cementum-specific proteins CEMP1 and PTPLA/CAP in an ERK-dependent manner. Lastly, Ca(OH)(2) stimulated mineralization by CEMP1-positive cells. Blocking CEMP1 and ERK function abolished Ca(OH)(2)-induced mineralization, confirming a role for CEMP1 and ERK in the process. Ca(OH)(2) promotes cementogenesis and recruits STRO-1-positive mesenchymal PDL cells to undergo cementoblastic differentiation and mineralization via a CEMP1- and ERK-dependent pathway.

Effects of the Association between a Calcium Hydroxide Paste and 0.4% Chlorhexidine on the Development of the Osteogenic Phenotype In Vitro

The present study aimed to evaluate whether the association between a calcium hydroxide paste (Calen paste) and 0.4% chlorhexidine (CHX) affects the development of the osteogenic phenotype in vitro. With rat calvarial osteogenic cell cultures, the following parameters were assayed: cell morphology and viability, alkaline phosphatase activity, total protein content, bone sialoprotein immunolocalization, and mineralized nodule formation. Comparisons were carried out by using the nonparametric Kruskal-Wallis test (level of significance, 5%). The results showed that the association between Calen paste and 0.4% CHX did not affect the development of the osteogenic phenotype. No significant changes were observed in terms of cell shape, cell viability, alkaline phosphatase activity, and the total amount of bone-like nodule formation among control, Calen, or Calen + CHX groups. The strategy to combine Ca(OH)(2) and CHX to promote a desirable synergistic antibacterial effect during endodontic treatment in vivo might not significantly affect osteoblastic cell biology. (J Endod 2008;34:1485-1489)

Effect of a calcium hydroxide-based paste associated to chlorhexidine on RAW 264.7 macrophage cell line culture

Objective. The objective of this study was to evaluate the effect of a calcium hydroxide Ca(OH)(2)-based paste (Calen) associated or not to 0.4% chlorhexidine digluconate (CHX) on RAW 264.7 macrophage cell line culture. Study design. The cell viability (MTT assay), immunostimulating properties (NO dosage), and anti-inflammatory properties (NO, TNF-alpha, and IL-1 alpha dosage) were evaluated after cell exposure to the materials. Data were analyzed statistically by Kruskal-Wallis test at 5% significance level. Results. There was low immunostimulating activity of the Calen paste associated or not to 0.4% CHX in the different materials` concentrations evaluated (P > .05). Anti-inflammatory activity with inhibition of NO and cytokine (TNF-alpha and IL1-alpha) release was observed only with Ca(OH)(2) + CHX at the highest concentration (25 mu g/mL). Conclusion. As the Calen paste associated to 0.4% CHX did not alter cell viability or the immunostimulating and anti-inflammatory properties, the addition of CHX brought no benefits to the Ca(OH)(2)-based paste with regard to the tested parameters. (Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2008;106:e44-e51)

Influence of Post and Resin Cement on Stress Distribution of Maxillary Central Incisors Restored with Direct Resin Composite

The current study evaluated the influence of two endodontic post systems and the elastic modulus and film thickness of resin cement on stress distribution in a maxillary central incisor (MCI) restored with direct resin composite using finite element analysis (FEA). A three-dimensional model of an MCI with a coronary fracture and supporting structures was performed. A static chewing pressure of 2.16 N/mm(2) was applied to two areas on the palatal surface of the composite restoration. Zirconia ceramic (ZC) and glass fiber (GF) posts were considered. The stress distribution was analyzed in the post, dentin and cement layer when ZC and GF posts were fixed to the root canals using resin cements of different elastic moduli (7.0 and 18.6 GPa) and different layer thicknesses (70 and 200 mu m). The different post materials presented a significant influence on stress distribution with lesser stress concentration when using the GF post. The higher elastic modulus cement created higher stress levels within itself. The cement thicknesses did not present significant changes.

Bond Strength of Fiber Posts in Different Root Thirds Using Resin Cement

Purpose: The aim of the study was to assess the in vitro bond strength (BS) of glass fiber posts (GF) and carbon fiber posts (CF) in the cervical, middle, and apical thirds of root canals cemented with RelyX-Unicem (RX) and Cement-Post (CP). Materials and Methods: Forty maxillary canines were divided into 4 groups (n = 10) according to the cement and post used: group 1: GF and RX; group 2: CF and RX; group 3: GF and CP; group 4: CF and CP. The push-out test was applied in the cervical, middle and apical thirds of each specimen to assess bond strength of the cement/post complex to the root canal wall. The data obtained were submitted to ANOVA (Bonferroni test, p < 0.05), and fracture analysis was done with SEM. Results: The GF posts presented the best results when cemented with RX and with CF (p < 0.05). RX presented the highest BS values for both GF and CF (p < 0.05). For all the groups, BS was higher in the cervical third, followed by the middle and apical thirds. Fracture analysis showed a predominance of cohesive fracture of posts for RX, and a predominance of adhesive fracture between dentin/cement, and mixed failure mode for CP. Conclusion: GF posts cemented with RX presented the highest BS values in all root thirds.