Calcium-rich Environments Research Articles

Quantification of Low Amounts of Zoledronic Acid by HPLC-ESI-MS Analysis: Method Development and Validation.

Zoledronic acid (ZA) is used in the treatment of various bone pathologies, but it forms complexes with calcium ions present in body fluids, decreasing ZA bioavailability. Thereby, the study first describes the identification of ZA-calcium complexes that form in calcium-rich environments, in order to establish the bioavailable ZA concentration. Then, a new method for quantification of low ZA amounts in milieus that mimics in vivo conditions by using simulated body fluid and calcium sulfate hemihydrate was described. Almost all analytical methods of ZA quantification described in the literature require compound derivatization. At very low concentrations, derivatization is prone to analyte loss, therefore compromising the analytical results. In our study, we avoided ZA derivatization by using a high-performance liquid chromatography and electrospray ionization mass spectrometry (HPLC-ESI-MS) system, conducting the investigation based on the fragmentation mass extracted ion chromatograms specific to the ZA protonated form. The method was validated by selectivity, precision, accuracy, linearity, signal to noise ratio, and limit of detection and limit of quantification calculation. Experimentally, this method can detect ranges of 0.1–0.5 ng/mL and precisely quantify ZA concentrations as low as 0.1 ng/mL. This method could provide the basis for quantifying low amounts of ZA in the blood during long-term administration.

GUT BACTERIAL DIVERSITY BETWEEN POLYGRID LAND SNAILS REFLECT ENVIRONMENTAL DIFFERENCES

GUT BACTERIAL DIVERSITY BETWEEN POLYGRID LAND SNAILS REFLECT ENVIRONMENTAL DIFFERENCES

The influence of calcium-rich environments in siliceous industrial residues on the hydration reaction of cementitious mixtures

The surface reactivity of waste glass powder (WGP) and sugarcane bagasse ash (SCBA) was evaluated in the partial replacement of cement using simple experimental procedures. As a blend, WGP and SCBA showed an excellent dosage at 5 wt % of each, and the availability and reactivity of silanol groups seem to have been the most important aspect in allowing the growth of calcium silicate hydrate (CSH) onto the quartz surface. XRD and SEM analyses have indicated that WGP and SCBA did not dissolve in the reaction and may have worked as a platform for the conversion of Ca(OH)2 into CSH. In addition, amorphous WGP was converted into quartz phase during hydration step. Unlike SCBA, whose surface is formed by “pure” silanol groups, WGP may possess one unit of CaO6 for each three groups of SiO4 in its structure and this may have enhanced the ability of the WGP surface to convert Ca(OH)2 into CSH. BET and BJH sorption methods showed that SCBA presented higher specific area and pore volume. However, XRF analysis showed that WGP possesses an important participation of calcium, which may have contributed for the enhancement of its acid-base features. Separately, both waste materials were able to replace at least 10 wt % of cement without compromising its compressive strength. In general, the results indicated the potential of those waste to be used in cement replacement and increase the raw material options in the cement production process.

The Transformation of Two-Line Ferrihydrite into Crystalline Products: Effect of pH and Media (Sulfate versus Nitrate)

Two-line ferrihydrite (Fh), ubiquitous in soils, groundwater, and aquatic sediments, may serve as an important sink for sequestering trace metals, metalloids, and organic matter via adsorption/coprecipitation due to its high surface area and reactivity. Although considerable attention has been paid to the transformation process of this thermodynamically metastable solid, little is known about the transformation products, the crystallization rates, or the transformation routes of two-line Fh in sulfate- and calcium-rich environments. This work systematically investigates the transformation of 2-line ferrihydrite produced by using different neutralization reagents (CaO vs NaOH) at different pHs (4 and 8), temperatures (25 °C, 40 °C, and 80 °C), and media (sulfate vs nitrate). X-ray diffraction, Raman, Fourier transform infrared spectroscopy, and chemical extraction were employed to characterize the transformed solids as well as the crystallization rate of 2-line Fh. The results show that the crystallization...

Structural and Mechanical Effects of Calcium on the Lipid Bilayer

Calcium is a ubiquitous divalent cation involved in fundamental biochemical and biophysical processes. Its integral roles in cell signaling, protein-mediated fusion, and action potential propagation have been well studied with a variety of techniques. Calcium is also commonly maintained at strong asymmetric concentration gradients, which often span several orders of magnitude, across mammalian cell membranes in proper physiological conditions. Yet, there has been a comparative lack of investigation of the effect of divalent cations on lipid bilayer structure and mechanical properties. Of the few studies done, experimental methods are limited to undefined resolutions and indirect spectroscopic or fluorescent techniques. This is in part due to the inherent difficulty in preparing well aligned samples with ions for high resolution experiments; common multilayer preparation techniques are subject to miscibility limits and sample segregation. Computationally, molecular dynamics simulations have predicted a calcium “condensing” effect that is associated with bilayer thickening and increased hydrocarbon chain order. However, these methods are limited by still-developing approaches to modeling divalent cation structure and interactions. Here, we report lamellar x-ray diffraction measurements of lipid bilayer thickness in partially charged multilayers containing calcium. Additionally, we report an apparent calcium-induced mechanical softening effect in giant unilamellar vesicles (GUVs) as measured by micropipette aspiration. This effect is marked by a measurable reduction in the elastic stretching coefficient of GUVs in calcium-rich environments. Both the thickening and mechanical softening effects may be important for proteins and molecules that couple directly to the lipid bilayer in physiological calcium concentrations. Future investigations involving PG-containing membranes and bacterial protoplasts may help lead to a better understanding of how invading bacteria thrive in calcium-rich environments, or if calcium-rich membrane domains have some non-trivial influence on the mechanisms of antibiotics and/or host defense antimicrobial peptides.

Adaptive molecular evolution of the two-pore channel 1 gene TPC1 in the karst-adapted genus Primulina (Gesneriaceae).

Limestone karst areas possess high floral diversity and endemism. The genus Primulina, which contributes to the unique calcicole flora, has high species richness and exhibit specific soil-based habitat associations that are mainly distributed on calcareous karst soils. The adaptive molecular evolutionary mechanism of the genus to karst calcium-rich environments is still not well understood. The Ca2+-permeable channel TPC1 was used in this study to test whether its gene is involved in the local adaptation of Primulina to karst high-calcium soil environments. Specific amplification and sequencing primers were designed and used to amplify the full-length coding sequences of TPC1 from cDNA of 76 Primulina species. The sequence alignment without recombination and the corresponding reconstructed phylogeny tree were used in molecular evolutionary analyses at the nucleic acid level and amino acid level, respectively. Finally, the identified sites under positive selection were labelled on the predicted secondary structure of TPC1. Seventy-six full-length coding sequences of Primulina TPC1 were obtained. The length of the sequences varied between 2220 and 2286 bp and the insertion/deletion was located at the 5' end of the sequences. No signal of substitution saturation was detected in the sequences, while significant recombination breakpoints were detected. The molecular evolutionary analyses showed that TPC1 was dominated by purifying selection and the selective pressures were not significantly different among species lineages. However, significant signals of positive selection were detected at both TPC1 codon level and amino acid level, and five sites under positive selective pressure were identified by at least three different methods. The Ca2+-permeable channel TPC1 may be involved in the local adaptation of Primulina to karst Ca2+-rich environments. Different species lineages suffered similar selective pressure associated with calcium in karst environments, and episodic diversifying selection at a few sites may play a major role in the molecular evolution of Primulina TPC1.

Efficient Degumming of Rice Bran Oil by Immobilized PLA1 from Thermomyces lanuginosus.

Phospholipase A1 (PLA1) immobilized in calcium alginate can effectively overcome the mass transfer resistance at the lipid-water interface making more room for the enzyme to separate itself from the products of reaction and to bind with the next available molecule at the interface. The reaction of an immobilized PLA1 hydrolase from Thermomyces lanuginosus was comparatively faster than of its free form. The rate of phospholipid hydrolysis by PLA1 was studied in calcium-rich and calcium-depleted environments; and the extent of phosphorus removed from the crude rice bran oil as well as the amount of free fatty acids produced during the reaction were used as indices for analysing the rate of enzymatic hydrolysis under standard conditions of pH, temperature, time of incubation and agitation. The immobilized PLA1 was found to be superior in removing phosphorus in the presence of 10 mM bivalent calcium ions in a solution. As compared to a maximum of 72.52% phosphorus removed by 0.01 kg of free enzyme per kg of oil, the same amount of immobilized PLA1 removed phosphorus from oil by 94.12% under the same experimental conditions (pH=6, 60 °C, 1-hour incubation). Both the free PLA1 and its immobilized form had shown extended rates of hydrolysis in a calcium-rich environment. The mass fractions of free fatty acids produced by the free enzyme and by its immobilized form were 14.9 and 14.16%, respectively, under the above experimental conditions. The removal of phosphorus from oil was accompanied by a significant reduction in colour and restoration of iodine value to the desired level.

Doubling the cross-linking interface of a rationally designed beta roll peptide for calcium-dependent proteinaceous hydrogel formation.

We have rationally engineered a stimulus-responsive cross-linking domain based on a repeats-in-toxin (RTX) peptide to enable calcium-dependent formation of supramolecular hydrogel networks. The peptide isolated from the RTX domain is intrinsically disordered in the absence of calcium. In calcium rich environments, the peptide binds Ca(2+) ions and folds into a beta roll (β-roll) secondary structure composed to two parallel β-sheet faces. Previously, we mutated one of the faces to contain solvent exposed leucine side chains which are localized only in the calcium-bound β-roll conformation. We demonstrated the ability of this mutant peptide to self-assemble into hydrogels in the presence of calcium with the aid of additional peptide-based cross-linking moieties. Here, we have expanded this approach by engineering both β-roll faces to contain leucine residues, thereby doubling the cross-linking interface for each monomeric building block. These leucine rich surfaces impart a hydrophobic driving force for self-assembly. Extensive characterization was performed on this double-faced mutant to ensure the retention of calcium affinity and subsequent structural rearrangement similar to that of the wild type domain. We genetically fused an α-helical leucine zipper capable of forming tetrameric coiled-coil bundles to the peptide and the resulting chimeric protein self-assembles into a hydrogel only in calcium rich environments. Since this new mutant peptide enables cross-linking on both surfaces simultaneously, a higher oligomerization state was achieved. To further investigate the cross-linking capability, we constructed concatemers of the β-roll with maltose binding protein (MBP), a monomeric globular protein, without the leucine zipper domains. These concatemers show a similar sol-gel transition in response to calcium. Several biophysical techniques were used to probe the structural and mechanical properties of the mutant β-roll domain and the resulting supramolecular networks including circular dichroism, fluorescence resonance energy transfer, bis-ANS binding, and microrheology. These results demonstrate that the engineered β-roll peptides can mediate calcium-dependent cross-linking for protein hydrogel formation without the need for any other cross-linking moieties.

Physiological and genomic features of highly alkaliphilic hydrogen-utilizing Betaproteobacteria from a continental serpentinizing site.

Serpentinization, or the aqueous alteration of ultramafic rocks, results in challenging environments for life in continental sites due to the combination of extremely high pH, low salinity and lack of obvious electron acceptors and carbon sources. Nevertheless, certain Betaproteobacteria have been frequently observed in such environments. Here we describe physiological and genomic features of three related Betaproteobacterial strains isolated from highly alkaline (pH 11.6) serpentinizing springs at The Cedars, California. All three strains are obligate alkaliphiles with an optimum for growth at pH 11 and are capable of autotrophic growth with hydrogen, calcium carbonate and oxygen. The three strains exhibit differences, however, regarding the utilization of organic carbon and electron acceptors. Their global distribution and physiological, genomic and transcriptomic characteristics indicate that the strains are adapted to the alkaline and calcium-rich environments represented by the terrestrial serpentinizing ecosystems. We propose placing these strains in a new genus ‘Serpentinomonas’.

Arsenic mobility controlled by solid calcium arsenates: A case study in Mexico showcasing a potentially widespread environmental problem

An As-contaminated perched aquifer under an urban area affected by mining was studied over a year to determine the contamination source species and the mechanism of As mobilization. Results show that the dissolution of calcium arsenates in residues disposed on an inactive smelter has caused high levels of As pollution in the adjoining downgradient 6-km perched aquifer, reaching up to 158 mg/L of dissolved As, and releasing a total of ca. 7.5 tons of As in a year. Furthermore, free calcium ion availability was found to control As mobility in the aquifer through the diagenetic precipitation of calcium arsenates (Ca5H2(AsO4)4·cH2O) preventing further mobilization of As. Results shown here represent a model for understanding a highly underreported mechanism of retention of arsenate species likely to dominate in calcium-rich environments, such as those in calcareous sediments and soils, where the commonly reported mechanism of adsorption to iron(III) oxyhydroxides is not the dominant process.

Engineering of an Environmentally Responsive Beta Roll Peptide for Use As a Calcium-Dependent Cross-Linking Domain for Peptide Hydrogel Formation

We have created a set of rationally designed peptides that form calcium-dependent hydrogels based on the beta roll peptide domain. In the absence of calcium, the beta roll domain is intrinsically disordered. Upon the addition of calcium, the peptide forms a beta helix secondary structure. We have designed two variations of our beta roll domain. First, we have mutated one face of the beta roll domain to contain leucine residues so that the calcium-dependent structural formation leads to dimerization through hydrophobic interactions. Second, an α-helical leucine zipper domain is appended to the engineered beta roll domain as an additional means of forming intermolecular cross-links. This full peptide construct forms a hydrogel only in calcium-rich environments. The resulting structural and mechanical properties of the supramolecular assemblies are compared with the wild-type domain using several biophysical techniques including circular dichroism, FRET, bis-ANS binding and microrheology. The calcium responsiveness and rheological properties of the leucine beta roll containing construct confirm the potential of this allosterically regulated scaffold to serve as a cross-linking domain for stimulus-responsive biomaterials development.

A Novel “Clip-and-link” Activity of Repeat in Toxin (RTX) Proteins from Gram-negative Pathogens: COVALENT PROTEIN CROSS-LINKING BY AN ASP-LYS ISOPEPTIDE BOND UPON CALCIUM-DEPENDENT PROCESSING AT AN ASP-PRO BOND

Clinical isolates of Neisseria meningitidis produce a repeat in toxin (RTX) protein, FrpC, of unknown biological activity. Here we show that physiological concentrations of calcium ions induce a novel type of autocatalytic cleavage of the peptide bond between residues Asp(414) and Pro(415) of FrpC that is insensitive to inhibitors of serine, cysteine, aspartate, and metalloproteases. Moreover, as a result of processing, the newly generated amino-terminal fragment of FrpC can be covalently linked to another protein molecule by a novel type of Asp-Lys isopeptide bond that forms between the carboxyl group of its carboxyl-terminal Asp(414) residue and the epsilon-amino group of an internal lysine of another FrpC molecule. Point substitutions of negatively charged residues possibly involved in calcium binding (D499K, D510A, D521K, and E532A) dramatically reduced the self-processing activity of FrpC. The segment necessary and sufficient for FrpC processing was localized by deletion mutagenesis within residues 400-657, and sequences homologous to this segment were identified in several other RTX proteins. The same type of calcium-dependent processing and cross-linking activity was observed also for the purified ApxIVA protein of Actinobacillus pleuropneumoniae. These results define a protein cleavage and cross-linking module of a new class of RTX proteins of Gram-negative pathogens of man, animals, and plants. In the calcium-rich environments colonized by these bacteria this novel activity is likely to be of biological importance.

Timing of the Calcium Intake and Effect of Calcium Deficiency on Behaviour and Egg Laying in Captive Great Tits, Parus major

The calcium demand of egg-laying birds is much higher than in other vertebrates during reproduction. We showed elsewhere that a low level of calcium availability can greatly affect the eggshell quality and reproduction of free-living passerines. However, there are few data on calcium demand and calcium intake in relation to egg laying and behaviour and egg-laying performance under conditions of calcium shortage in nondomesticated birds. We examined these aspects in an experiment with captive great tits, Parus major, on a diet deficient in calcium, with or without snail shells as an additional calcium source. More than 90% of the calcium intake for egg production took place during the egg-laying period. Females ingested about 1.7 times as much calcium as they deposited in eggshells. Removing the snail shells after the first egg resulted in eggshell defects and interruptions of laying after 1-3 d. Females without snail shells doubled their searching effort and started to burrow in the soil and to eat sand, small stones, and their own eggs. Most calcium was consumed in the evening, probably to supplement the calcium available from the medullary bone with an additional calcium source in the gut during eggshell formation. The results demonstrated that eggshell formation requires accurate timing of the calcium intake and that obtaining sufficient calcium is time-consuming, even in calcium-rich environments. These factors pertaining to calcium intake greatly affect the ability of birds to collect sufficient calcium for eggshell formation in calcium-poor areas.

Environmental calcium and hypocalcin activity in the Stannius corpuscles of the channel catfish, Ictalurus punctatus (Rafinesque)

Stannius corpuscle homogenates from the channel catfish, Ictalurus punctatus (Rafinesque), produced a significant hypocalcemia in male killifish, Fundulus heteroclitus L., adapted to artificial calcium-deficient sea water. Homogentes from catfish adapted to calcium enriched fresh water had higher activities than those from calfish adapted to low calcium fresh water. It is suggested here that in the calcium rich environments, the glands contained more hypocalcin, the active hypocalcemic principle(s) in Stannius corpuscles of fishes (Pang et al., 1974).