Effect of dietary phosphate on intestinal calcium and phosphate absorption

Intestinal Ca and P absorption was investigated on rachitic chicks raised on diets with a 1% Ca and 0.3% or 1% P contents. 45Ca and 32P absorption was determined by the technique of the isolated gut sac in vivo. In addition, 32P transport was also measured by the everted gut sac procedure in vitro. Treatment with vit. D3 during 7 days increased the 45Ca absorption in animals fed diets containing 0.3% or 1% P. 32P absorption showed an increase after 2 days of treatment and a decrease afterwards. The reduction of 32P absorption was larger in animals fed diet with 1% P. Study of 32P transport with the everted gut sac technique showed an increase after vit. D3 and a loss of intracellular P, regardless the duration of treatment.     

Precipitation of nucleotides by calcium phosphate

Earlier it has been shown that nucleic acids of high molecular weight can be introduced into cells by coprecipitation with calcium phosphate. We have studied the requirements for calcium phosphate coprecipitation of shorter nucleotides. The degree of coprecipitation of dodecanucleotides lacking terminal phosphate varied between 25 and 72%. Tetramers with a 5′-monophosphate were coprecipitated to 29–87% by calcium phosphate. A high content of guanosine residues and an increased number of terminal phosphate groups increased the degree of coprecipitation of nucleotides. The trinucleotide pppA2′p5′A2′p5′A was effectively precipitated by calcium phosphate but the monophosphate and the core structure were not.     

A comparison of calcium phosphate coprecipitation and electroporation

Plasmid-based transfection assays provide a rapid means to measure homologous and nonhomologous recombination in mammalian cells. Often it is of interest to examine the stimulation of recombination by DNA damage induced by radiation, genotoxic chemicals, or nucleases. Transfection is frequently performed by using calcium phosphate coprecipitation (CPP), because this method is well suited for handling large sample sets, and it does not require expensive reagents or equipment. Alternative transfection methods include lipofection, microinjection, and electroporation. Since DNA strand breaks are known to stimulate both homologous and nonhomologous recombination, the induction of nonspecific damage during transfection would increase background recombination levels and thereby reduce the sensitivity of assays designed to detect the stimulation of recombination by experimentally induced DNA damage. In this article, we compare the stimulatory effects of nuclease-induced double-strand breaks (DSBs) on homologous and nonhomologous recombination for molecules transfected by CPP and by electroporation. Although electroporation yielded fewer transfectants, both nonhomologous and homologous recombination were stimulated by nuclease-induced DSBs to a greater degree than with CPP. Ionizing radiation is an effective agent for inducing DNA strand breaks, but previous studies using CPP generally showed little or no stimulation of homologous recombination among plasmids damaged with ionizing radiation. By contrast, we found clear dose-dependent enhancement of recombination with irradiated plasmids transfected using electroporation. Thus, electroporation provides a higher signal-to-noise ratio for transfection-based studies of damage-induced recombination, possibly reflecting less nonspecific damage to plasmid DNA during transfection of mammalian cells.     

Ex vivo detection of calcium phosphate and calcium carbonate in rat blood serum

The total calcium (tCa) in blood serum comprises free Ca²⁺ ions (fCa), protein-bound calcium (prCa), and complexed calcium by small anions (cCa). The cCa fraction, in addition to fCa, has been indicated to have some physiological activity. However, there is little evidence for the structure of its constituents. Here we report an ex vivo detection of the cCa constituents by synchrotron X-ray absorption near-edge structure spectroscopy. We collected the data directly on rat blood serum and, by making use of the reference samples, derived a spectrum that exhibits the features of cCa constituents. Among the features are those of the complexes of calcium phosphate and calcium carbonate. The detected complexes in the cCa fraction are mainly Ca(η²-HPO₄)(H₂O)₄ and Ca(η¹-HCO₃)(H₂O)₅⁺, in which HPO₄^2? and HCO₃^? serve as bidentate and unidentate ligands, respectively. The remained H₂O molecules on the coordination sphere of Ca²⁺ enable these complexes to behave partially like aquated Ca²⁺ ions in protein-binding. Besides, as the dominant part of prCa, albumin-bound calcium (albCa) exhibits a spectrum that closely resembles that of fCa, indicating weak interactions between the protein carboxyl groups and calcium. The weak-bound cCa and albCa, along with fCa and the relevant anions, compose a local chemical system that could play a role in maintaining the calcium level in blood.     

Transfection of adherent and suspended cells by calcium phosphate

DNA-calcium phosphate coprecipitates have been used for 30 years as an efficient method to introduce genetic material into cells. The method involves simple solutions that can be prepared or purchased by the experimentalist. All the numerous variations of the protocol found in the literature are based on the same principle--a spontaneous precipitation that occurs in supersaturated solutions. When DNA is present during this process, it is readily incorporated into the forming calcium phosphate precipitate. Although a wide range of conditions will lead to precipitates, high transfection efficiencies are only obtained within a narrow range of optimized parameters that assure certain properties of the precipitate. This paper describes several physico-chemical parameters that are critical to adapt the method to a particular cell line and/or cultivation condition. Examples of protocols that were established and tested within the authors' laboratory are presented. The article also emphasizes differences between transfections of adherent and suspended cells.     

Binding of calcium and phosphate ions to dentin phosphophoryn

The concomitant binding of calcium and inorganic phosphate ions by the highly phosphorylated rat dentin phosphophoryn (HP) was measured in the pH range of 7.4-8.5 by an ultrafiltration procedure. HP binds almost exclusively the triply charged PO4(3-) ion, and for each PO4(3-) ion bound, the protein binds about 1.5 additional Ca2+ ions. Therefore, the protein-mineral ion complex can be described as a protein with two different ligands, Ca2+ ions and calcium phosphate clusters having a stoichiometry of about Ca1.5PO4. Empirically the binding of calcium and phosphate can best be described as a function of a neutral ion activity product in which 2.5-10% of the phosphate is HPO4(2-). The stoichiometry of the bound clusters is similar to that of amorphous calcium phosphate, and it is clear that the protein does not sequester crystal embryos of octacalcium phosphate or hydroxyapatite. The protein-mineral ion complex is amorphous by electron diffraction analysis and does not catalyze the formation of a crystalline phase when aged in contact with its solution. About 15% of the bound phosphate is buried in protected domains, and it is stable with respect to dissociation for extended periods in phosphate-free calcium buffers. The buried mineral maintains the protein in an aggregated state even at calcium ion concentrations which are too low for the aggregation of unmineralized HP. In vivo HP should be ineffective in the nucleation of a crystalline mineral phase, if it is secreted in a mineralized aggregated state similar to casein and the bivalve phosphoprotein.     

Design and manufacture of combinatorial calcium phosphate bone scaffolds

It is well known that pore design is an important determinant of both the quantity and distribution of regenerated bone in artificial bone tissue scaffolds. A requisite feature is that scaffolds must contain pore interconnections on the order of 100-1000 μm (termed macroporosity). Within this range, there is not a definitive optimal interconnection size. Recent results suggest that pore interconnections permeating the scaffold build material on the order of 2-20 μm (termed microporosity) drive bone growth into the macropore space at a faster rate and also provide a new space for bone growth, proliferating throughout the interconnected microporous network. The effects of microstructural features on bone growth has yet to be fully understood. This work presents the manufacture and characterization of novel combinatorial test scaffolds, scaffolds that test multiple microporosity and macroporosity designs within a single scaffold. Scaffolds such as this can efficiently evaluate multiple mechanical designs, with the advantage of having the designs colocated within a single defect site and therefore less susceptible to experimental variation. This paper provides the manufacturing platform, manufacturing control method, and demonstrates the manufacturing capabilities with three representative scaffolds.     

Chemotherapy drug delivery from calcium phosphate nanoparticles

Calcium phosphate nanoparticles (nanoCaP) conjugated with cis-diamminedichloroplatinum (CDDP, cisplatin) were prepared through the electrostatic binding of an aquated species of cisplatin to the nanoCaP in a chloride-free solution. The agglomeration of the nanoCaP that typically occurs during synthesis of CaP was controlled through the addition of DARVAN 811 immediately after precipitation and before drug conjugation. In vitro drug release studies were completed and showed a sustained release of CDDP from the nanoconjugates over time. The cytotoxicity of the nanoCaP/CDDP was compared to that of the free drug in an in vitro cell proliferation assay using the CDDP resistant A2780cis human ovarian cancer cell line. The CDDP released from the nanoconjugates was equally effective as the free drug against the A2780cis cell line. Direct addition cytotoxicity studies revealed that the sterically-stabilized, negatively-charged drug nanoconjugates are unable to overcome drug resistance and had an increased IC50 value as compared to the free drug.     

Caseins are cross-linked through their ester phosphate groups by colloidal calcium phosphate

Artificial casein micelles were prepared by adding 30 mM calcium, 22 mM phosphate and 10 mM citrate to sodium caseinate solutions, and the content of the casein aggregates cross-linked by colloidal calcium phosphate was determined by high-performance gel chromatography on a TSK-GEL G4000SW column in the presence of 6 M urea. The content of the casein aggregates cross-linked by colloidal calcium phosphate in artificial whole casein micelles was 48% of total casein, and their relative casein composition determined by high-performance ion-exchange chromatography was 53.1% for alpha s1-casein, 15.8% for alpha s2-casein, 31.1% for beta-casein and 0% for kappa-casein. The order of cross-linking by colloidal calcium phosphate agreed with that of the ester phosphate content of casein constituents. The content of the casein aggregates cross-linked by colloidal calcium phosphate was higher in alpha s1-kappa-casein micelles than in beta-kappa-casein micelles. kappa- and gamma-caseins and dephosphorylated alpha s1-casein were not cross-linked by colloidal calcium phosphate. Although kappa-casein was not cross-linked, chemically phosphorylated kappa-casein, of which the average phosphate content was 8.5 per molecule, was cross-linked. It is concluded that caseins are cross-linked through their ester phosphate groups by colloidal calcium phosphate.     

Transport mechanism for calcium and phosphate in ram spermatozoa

Calcium uptake into ejaculated ram spermatozoa is highly enhanced by the addition of extracellular phosphate. Under identical conditions, extracellular calcium stimulates the uptake of phosphate by the cells. Both calcium and phosphate uptake are comparably inhibited by the sulfhydryl reagent mersalyl. The I50 was found to be 6.36 and 10.14 nmol mersalyl per mg protein for phosphate and calcium uptake, respectively. Calcium uptake is inhibited by mersalyl whether phosphate is present or not. Extracellular fructose causes a 5-fold increase in calcium uptake. When fructose and phosphate are present in the cell's medium, there is an additive effect, which indicates that two independent systems are involved in calcium transport into the cell. Ruthenium red, which blocks Ca2+ transport into the mitochondria, causes 70% and 95% inhibition of calcium uptake in the absence or in the presence of fructose, respectively. Ruthenium red does not affect phosphate uptake unless calcium was present in the incubation medium. The stimulatory effect of fructose upon calcium uptake can be mimicked by L-lactate and can be inhibited by the glycolytic inhibitor 2-deoxyglucose. Fructose and L-lactate stimulate mitochondrial respiration in a comparable way. Oligomycin, which inhibits mitochondrial ATP synthesis, does not inhibit Ca2+ uptake. This indicates that ATP is not involved in the mechanism by which mitochondrial respiration stimulates Ca2+ uptake. The calcium channel blocker, verapamil, inhibits Ca2+ uptake in the presence or absence of extracellular phosphate. The phosphate-dependent calcium transport mechanism is more sensitive to verapamil than is the phosphate-independent transporter. In summary, the data indicate that the plasma membrane of mammalian spermatozoa contains a calcium/phosphate symporter, a phosphate-independent calcium carrier and a calcium-independent phosphate carrier.