Magnesium and strontium interaction with carbonate-containing hydroxyapatite in aqueous medium

Crystallographic and spectroscopic studies were performed on the solid products obtained from magnesium and strontium interaction with carbonate-containing hydroxyapatite under aqueous conditions employing a cyclic pH variation technique. Low concentrations of magnesium and strontium make easier the transformation of carbonate-containing hydroxyapatite into β-tricalcium phosphate by heat treatment. At high concentration magnesium stabilizes the β-tricalcium phosphate crystal structure while the apatite crystal structure is maintained when the Ca/Sr molar ratio decreases. It is suggested that these ions are important in determining the structure of the calcium phosphate deposits in biological tissues during the physiological and pathological local pH variations.     

The high-affinity binding site for manganese on the oxidizing side of Photosystem II

Electron donation to Photosystem II (PS II) by diphenylcarbazide (DPC) is interrupted by the presence of endogenous Mn in PS II particles. Removal of this Mn by Tris treatment greatly stimulates the electron transport with DPC as donor. Binding of low concentration of exogenous Mn(II) to Tris-treated PS II particles inhibits DPC photooxidation competitively with DPC. This phenomenon was used to locate a highly specific Mn(II) binding site on the oxidizing side of Photosystem II with dissociation constant about 0.15 μM. The binding of Mn(II) is electrostatic in nature. Its affinity depends not only on the ionic strength, but also on the anion species of the salt in the medium. The effectiveness in decreasing the affinity follows the order F⁻ > SO²⁻₄ > CH₃COO⁻ > CI⁻ > Br⁻ > NO₃⁻. This observation is interpreted as follows: smaller ions, like F⁻, CH₃COO⁻, and larger ions, like SO²⁻₄, have inhibitory effects on Mn(II) binding, whereas ions with optimal size, like Cl⁻, Br⁻ and NO₃⁻, can stabilize the binding, resembling the anion requirement for reactivation of Cl⁻-depleted chloroplasts. We suggest that the binding site for Mn(II) we observed is the site for the endogenous Mn in the O₂-evolving complex of PS II. This site remains after Tris treatment, which removes all the endogenous Mn as well as the three extrinsic proteins, indicating that it is on the intrinsic component(s) of PS II reaction centers. Furthermore, the Cl⁻ requirement for O₂ evolution may be attributed, at least partly to its stabilizing effect on Mn binding.     

Synthesis and in vitro behavior of β-TCP zirconia/polymeric biocomposites for bio-applications

Novel biocomposites were fabricated by impregnating β-tricalcium phosphate (β-TCP)/zirconia particles into the polymers matrix. The composite materials were characterized using thermo-gravimetric analysis (TGA), X-ray diffraction (XRD), Fourier Transform Infrared (FT-IR) analyzes and Scanning Electron Microscopy (SEM). The results confirmed the conversion of hydroxyapatite (HA) to β-TCP at a sintering temperature of 1150 °C with or without zirconia powder. The in vitro behavior was assessed via measurement of calcium and phosphorus ions in SBF (simulated body fluid). FT-IR and SEM of the composites were performed pre and post immersion in SBF. The results prove that the bone like apatite layer formation was enhanced on the β-TCP-Z20/polymeric composite surface more than that on the β-TCP-Z10/polymeric composite. Therefore, the data confirmed that zirconia plays an important role in the enhancement of the apatite formation. The conclusions proved that the β-TCP-Z20/polymeric biocomposites, containing 20% of zirconia, are promising for bone remodeling applications.     

On the molecular mechanisms of the acid-induced dissociation of hydroxy-apatite in water

The enamel/saliva interface is mimicked by the comparably much simpler model of (001) surfaces of hydroxy-apatite ( Ca₁₀(PO₄)₆(OH)₂ ) in contact with aqueous solution. At neutral pH, the dissociation of ions is penalized by more than 150 kJ mol^-1 giving rise to very stable apatite-water interfaces. This picture changes drastically with decreasing pH, as the protonation of phosphate and hydroxide ions lowers the free energy of calcium ions dissociation. Our simulations suggest the mechanism of acid-induced apatite decomposition to i) require a considerable degree of protonation of the apatite surface. The first ion dissociation step ii) involves calcium ions which electrostatic binding has been locally destabilized through phosphate and hydroxide protonation. The depletion of calcium ions embedding the anions then allows iii) the dissociation of the anionic species. Along this line, the protective role of fluoride in caries prevention is related to the stabilization of the calcium triangles embedding the OH^-/F^- ions.     

Fructosan metabolism in Cichorium intybus roots

Incorporation of [¹⁴C]sucrose into difructosyl glucose (F₂G), trifructosyl glucose (F₃G) and tetrafructosyl glucose (F₄G) in the presence of various nucleoside triphosphates revealed that formation of F₄G and F₃G is retarded in the presence of ATP, and formation of F₃G and F₂G is significantly enhanced in the presence of CTP, whereas UTP has no effect on the synthesis of these oligosaccharides. Different fructosyl transferases seem to be responsible for the different fructosylation steps and self transfer seems to be the major pathway for fructosan synthesis. Utilization of added glucose, which is formed by sucrose sucrose fructosyl transferase action in vivo, is completely inhibited in acetate buffer whereas in phosphate, citrate and citrate-phosphate buffers glucose is actively utilized. In the presence of fluoride ions both glucose utilization and its conversion to CO₂ is inhibited by ca 50%. CO₂ production from [¹⁴C]glucose is completely inhibited in acetate ions. No evidence for the incorporation of ¹⁴C from [¹⁴C]glucose into [¹⁴C]sucrose is observed. The ratio of bound fructose to bound glucose is the same in the entire length of the root indicating that there is no preferential zone for fructosan synthesis.     

Uptake,incorporation and calcium-ionophore-stimulated mobilization of arachidonic,eicosapentaenoic and docosahexaenoic acids by leucocytes of the rainbow trout,Salmo gairdneri

Rainbow trout leucocytes contain high levels of neutral lipid (about 70% of total lipid on a wt% basis) consisting of mostly triacylglycerol, free sterols and sterol esters (25%, 15% and 52% of neutral lipid, respectively). The phospholipids, separated by thin-layer chromatography, consisted predominantly of phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine, each present at about 30% of the total phospholipid. Radiolabelling of the leucocytes for 1 h with 1 μCi (approx. 6 μM) [1−¹⁴C]20:4(n−6), [1−¹⁴C]20:5(n−3) or [1−¹⁴C]22:6(n−3) each gave similar uptake values (approx. 1 · 10⁵ cpm/10⁷ leucocytes). The incorporation into total phospholipids was highest for 22:6(n−3) and lowest for 20:4(n−6). A higher percentage of radiolabel from [1−¹⁴C]22:6(n − 3) was found incorporated into phosphatidylcholine and phosphatidylethanolamine as compared to that from [1−¹⁴C]20:4(n − 6) and [1−^14C]20:5(n−3), while the reverse situation was found with phosphatidylinositol and phosphatidylserine. The relative rates of incorporation into the different phospholipid classes for all three fatty acids were in the order phosphatidylinositol > sphingomyelin > diphosphatidylglycerol > phosphatidylcholine > phosphatidylethanolamine > phosphatidylserine. Calcium ionophore-challenge did not significantly alter the pattern of phospholipid radiolabel. Ionophore-challenge released large amounts of radiolabel, much of which was recovered after high-performance liquid chromatographic separation as free fatty acid/monohydroxy fatty acids, although only approx. 0.3% was recovered in leukotriene B₄ and leukotriene B₅ for the [1−¹⁴C]20:4(n−6) and [1−¹⁴C]20:5(n−3) labelled leucocytes, respectively. Other lipoxygenase products were also radiolabelled and tentatively identified as 20-carboxy-LTB₄, 20-hydroxy-LTB₄, 6-trans-LTB₄, 6-trans-12-epi-LTB₄, 6-trans-8-cis-12-epi-LTB₄ and the corresponding LTB₅ structures. No ‘6-series’ leukotrienes were produced from [1−¹⁴C]22:6(n−3), nor was there any evidence for the synthesis of ‘5-series’ leukotrienes via retroconversion of 22:6(n−3) to 20:5(n−3). This latter finding shows that, despite the preponderance of 22:6(n−3) in the membranes of trout leucocytes, this fatty acid is not a substrate for leukotriene generation.     

Fluoride inhibition of yeast enolase: Crystal structure of the enolase–Mg2+–F−–Pi complex at 2.6 Å resolution

Enolase in the presence of its physiological cofactor Mg²⁺ is inhibited by fluoride and phosphate ions in a strongly cooperative manner (Nowak, T, Maurer, P. Biochemistry 20:6901, 1981). The structure of the quaternary complex yeast enolase–Mg²⁺–F^?–P_i has been determined by X-ray diffraction and refined to an R = 16.9% for those data with F/σ(F) ≥ 3 to 2.6 Å resolution with a good geometry of the model. The movable loops of Pro-35-Ala-45, Val-153-Phe-lo9, and Asp-255-Asn-266 are in the closed conformation found previously in the precatalytic substrate–enzyme complex. Calculations of molecular electrostatic potential show that this conformation stabilizes binding of negatively charged ligands at the Mg²⁺ ion more strongly than the open conformation observed in the native enolase. This closed conformation is complementary to the transition state, which also has a negatively charged ion, hydroxide, at Mg²⁺. The synergism of inhibition by F^? and P_i most probably is due to the requirement of P_i, for the closed conformation. It is possible that other Mg²⁺-dependent enzymes that have OH^? ions bound to the metalion in the transition state also will be inhibited by fluoride ions. © Wiley-Liss, Inc.     

Micromechanical evaluation of mineralized multilayers

The biomechanical stability of osseointegrated implants is of particular importance, especially the stability which is achieved from structural manipulation at the interface between the implant surface and the bone tissues. Nanoscale β-tricalcium phosphate-immobilized titanium was prepared by discharge into a physiological buffered saline solution. Compared with hydroxyapatite, it has been shown to be effective in generating a bone-like chemical structure on the surface by cooperative interaction between osteoblastic cells and the β-tricalcium phosphate. The present study, after cell cultivation, investigates the nanostructures and biomechanical property differences of a mineralized layer formed on two samples of nano-calcium phosphate-immobilized titanium. A scanning probe microscope study revealed that the mineralized tissue formed on the β-tricalcium phosphate samples after 1 week of cell culture showed significantly higher roughness, compared with hydroxyapatite samples. Nanoindentation micromechanical evaluation of the in vitro generated multilayered structures exhibited thicker bone-like mineralized layers on the β-tricalcium phosphate samples. A successful modification of titanium implants through the cooperative interaction between osteoblastic cells and nano β-tricalcium phosphate is anticipated.     

Factors affecting the transport of β-amino acids in rat renal brush-border membrane vesicles. The role of external chloride

The effect of a variety of ions and other solutes on the accumulation of the β-amino acid, taurine, was examined in rat renal brush-border membrane vesicles. Initial taurine uptake (15 and 30 s) is sodium-dependent with a typical overshoot. This Na⁺ effect was confirmed by exchange diffusion and gramicidin inhibition of taurine uptake. External K⁺ or Li⁺ do not increase taurine accumulation more than Na⁺-free mannitol, except that the combination of external K⁺ and Na¹ in the presence of nigericin enhances uptake. Of all anions tested, including more permeant (SCN⁻ and NO₃⁻) or less permeant (SO₄²⁻), chloride supported taurine accumulation to a significantly greater degree. Preloading vesicles with choline chloride reduced taurine uptake, suggesting that external Cl⁻ stimulates uptake. Since this choline effect could be related to volume change, due to the slow diffusion of choline into vesicles, brush-border membrane vesicles were pre-incubated with LiCl, LiNO₃ and LiSO₄. Internal LiCl, regardless of the final Na⁺ anion mixture, reduced initial rate (15 and 60 s) and peak (360 s) taurine uptake. Internal LiNO₃ or LiSO₄ with external NaCl resulted in similar or higher values of uptake at 15, 60 and 360 s, indicating a role for external Cl⁻ in taurine uptake in addition to Na⁺ effect. Although uptake by vesicles is greatest at pH 8.0 and inhibited at acidic pH values (pH less than 7.0), an externally directed H⁺ gradient does not influence uptake. Similarly, amiloride, an inhibitor of the Na⁺/H⁺ antiporter, had no influence on taurine accumulation over a wide variety of concentrations or at low Na⁺ concentrations. Taurine uptake is blocked only by other β-amino acids and in a competitive fashion. d-glucose and p-aminohippurate at high concentrations (> 10⁻³ M) reduce taurine uptake, possibly by competing for sodium ions, although gramicidin added in the presence of d-glucose inhibits taurine uptake even further. These studies more clearly define the nature of the renal β-amino acid transport system in brush-border vesicles and indicate a role for external Cl in this uptake system.     

Biomimetic Synthesis of Collagen/Nano-Hydroxyapitate Scaffold for Tissue Engineering

This paper reports a precipitation method for the fabrication of compositionally graded biomimetic collagen/nano-hydroxyapatite (HA) composite scaffold. The method is centrifugation based and produce the precipitation of nano-HA crystallites in situ (calcium ions (Ca²⁺) react phosphate ions (PO₄³⁻) and precipitate a non-stoichiometric hydroxyapatite). It was observed that prism needle-like nano-HA crystallites (about 2.5 nm × 3 nm× 25 nm) precipitated on collagen fibrils in the interior of collagen matrix. Chemical and microstructure analysis revealed a gradient of the Ca to P ratio across the width of the scaffold, lead to the formation of a HA-rich side and a HA-deplete side of scaffold. The HA-rich side featured low porosity and agglomerates of the nano-HA crystallites; while HA-depleted side featured higher porosity and nano-HA crystallites integrated with collagen fibrils to form a porous network structure.     

A novel highly specific colorimetric fluorescent probe for the detection of nitrite in aqueous solution

Developing an effective method for the detection of nitrite (NO₂⁻) ions in the natural environment especially in environmental waters and soils is very necessary, since they will cause serious damage to human health once excess NO₂⁻ ions enters the human body. Therefore, a new colorimetric fluorescent probe NB-NO₂⁻ for determining NO₂⁻ ions was designed, which possesses good water-solubility and satisfactory selectivity over other common ions for NO₂⁻ ions. The addition of NO₂⁻ ions changed the color of solution from blue to colorless seen by the naked-eye. Furthermore, through test and calculation, the detection limit of the probe NB-NO₂⁻ is 129 nM. Based on the earlier excellent characteristics, the probe NB-NO₂⁻ was successfully used for monitoring NO₂⁻ ions in environmental waters and soils.     

The effect of chaotropic agents on photosynthetic reactions

The influence of a series of anions on photosynthetic reaction rates in spinach chloroplasts is descibed. For the most part, the stimulatory and inhibitory effects of these ions can be related to their chaotropic properties, although F⁻, a nonchaotropic anion, inhibits photosystem II reactions and SO ₄ ²⁻ and F⁻ inhibit photophosphorylation. Other exceptions include less severe effects of nitrate than expected and unusual sensitivity to iodide by photosystem I. Since free iodine inhibits photosystem I the iodine effect may be related to photooxidation of I⁻ to I⁰ by photosystem I. Cyclic and noncyclic photophosphorylation usually show greater sensitivity to each chaotrope than photosystems I and II activity, which suggests that phosphorylation factors, such as CF₁, are easily detached or dissociated. Bromide is unusual in that it appears to affect photophosphorylation and electron transport at similar low concentrations. The type of cation appears to influence the response to the chaotropic anion, especially as increased inhibition by chloride in the presence of magnesium in photophosphorylation reactions.     

Thermal behavior of bone and synthetic hydroxyapatites submitted to magnesium interaction in aqueous medium

The thermal behavior of the products obtained from magnesium interaction with powdered femoral bone and carbonate containing synthetic hydroxyapatite under conditions of pH fluctuation in aqueous medium has been investigated. The products, heat treated at different temperatures from 100 to 1300 degrees C, have been characterized by infrared spectroscopy and X-ray diffraction technique. The results show that the interaction with magnesium ion destabilizes the apatitic structure and favours its thermal conversion into beta-tricalcium phosphate (beta-TCP). The replacement of magnesium with calcium in the beta-TCP crystal lattice hinders its subsequent thermal conversion into the alpha form. The influence of magnesium on the thermal stability is much more evident for carbonate-containing synthetic hydroxyapatite than for bone apatite.     

Photochemistry of Co(EDTA)−-I− System in Aqueous Solutions

The Co(EDTA)⁻ complex in aqueous solution gives rise to a specific interaction with I⁻ ions as evidenced by a new, relatively intense band formed at 290–300 nm. This specific interaction is attributed to the formation of an ion-pair between Co(EDTA)⁻ and I⁻, even though they are like charged ions. Irradiation of this ion-pair in air- equilibrated solutions with 313 nm light, causes the reduction of the Co(EDTA)⁻ to Co(EDTA)²⁻ and the oxidation of the I⁻ ion to I₃⁻. The results obtained are interpreted on the basis of a mechanism in which Co(EDTA)²⁻ and I^· are the primary photoproducts. The I^· radical is then scavenged by I⁻ to yield I₂⁻, which subsequently disproportionates to I₃⁻ and I⁻ and reoxidizes Co(EDTA)²⁻ to Co(EDTA)⁻. At the beginning of the photoreaction, the I₂⁻ decay is equally distributed on the two reaction pathways. It was possible to determine a value of 0.2 ± 0.05 for the photoreaction quantum yield and an efficiency of the primary photochemical step almost unitary. A schematic representation of the energetics of the overall reaction is reported.     

Thermodynamics and coordination characteristics of the hydronium-uranium(VI)-dicyclohexano-24-crown-8 extraction complex

The extraction equilibrium of the hydronium-uranium(VI)-dicyclohexano-24-crown-8 complex was carried out in the crown ether1,2-dichloroethaneHCl aqueous solution system at different temperatures. The extraction complex has the overall composition (L)₂·(H₃O⁺·χH₂O)₂·UO₂Cl₄²⁻ (L = dicyclohexano-24-crown-8). The values of the extraction equilibrium constants (K_ex) increase steadily with a decrease in temperature: 13.5 (298 K), 7.96 (301 K), 4.20 (303 K) and 2.07 (305 K). A plot of log K_ex against 1/T shows a straight line. The value of the enthalpy change, ΔH°, was calculated from the slope and equals −212 kJ mol⁻¹. The value of the entropy change, ΔS°, was calculated from ΔH° and K_ex and equals −690 J K⁻¹ mol⁻¹, whereas ΔG° = −6.45 kJ mol⁻¹. Comparing these thermodynamic parameters with those of the dicyclohexano-18-crown-6 isomer A [1] (ΔS° = −314 J K⁻¹ mol⁻¹, ΔH° = −101 kJ mol⁻¹ and ΔG° = −8.37 kJ mol⁻¹), it can be seen that ΔH° and ΔS° are more negative for the former than for the latter, and both are enthalpy-stabilized complexes. The molecular structure of the complex has the feature that there are two H₅O₂⁺ ions in it, in contrast to the H₃O⁺ ions in the dicyclohexano-18-crown-6 isomer A complex [1]. Each of the H₅O₂⁺ ions is held in the crown ether cavity by four hydrogen bonds. The H₅O₂⁺ ion has a central bond. The uranium atom forms UO₂Cl₄²⁻ as a counterion away from the crown ether. The formation of this complex is in good agreement with more negative entropy change and less negative free energy change, as mentioned above.     

Crystal structure of a new form of copper(II)—inosine 5′-monophosphate—2,2′-dipyridylamine ternary complex

Two crystalline forms of the [Cu(II) (IMP) (DPA) (H₂O)]₂·nH₂O (IMP=inosine 5′-monophosphate, DPA=2,2′-dipyridylamine) complex were obtained from aqueous solution at pH=6.2. The crystals of the two forms belong to the monoclinic system, space group P2₁. The cell parameters are: a=9.445(2), b=33.902(4), c=7.802(2) Å, β=90.48(2)°, Z= 2, D_c=1.69g cm⁻³ and μ(Mo Kα) = 10.49cm⁻¹ (form α, n=4), and a=7.828(2), b=18.552(3), c=17.378(3) Å, β=91.16(2)°, Z=2, D_c=1.66 g cm⁻³, μ(Mo Kα) = 10.40 cm⁻¹ (form β, n=3.62). Bau and coworkers reported the preparation of form α by vapor diffusion of CH₃CN into aqueous solution containing Cu(NO₃)₂, Na₂IMP and DPA in a 1:1:1 molar ratio and the analysis of the compound by single crystal X-ray diffraction [1].Intensities for 3412 reflections were collected from a crystal of form β in the present work. Graphite-monochromatized Mo Kα radiation was employed. The structure was refined to final R and R_w values of 0.1000 and 0.1115 respectively. The dimeric units contain two copper ions in square-pyramidal coordination polyhedra. Each polyhedron consists of two nitrogen atoms of DPA, two oxygen atoms from two phosphate groups and a water molecule in the axial position. A statistical disorder was found in a nucleotide moiety of the dimer. Two sets of atomic positions corresponding to the purine system were refined with site occupation factors of 0.62(1) and 0.38(1) respectively. Also the ribose ring shows a disorder with two possible conformations. The puckering mode of the prevailing conformation is C(3′)-endo. In the other nucleotide molecule of the dimer the furanose puckering mode is C(3′)-endo. The rotation around the glycosyl linkages can be described as ‘anti’ in the structure of form β. The C(4)N(9)C(1′)O(4′) torsion angle values are −97(2) and −94(3)° for the disordered nucleotide molecule and +91(2)^o for the other nucleotide moiety. Strong intermolecular DPADPA and purine-purine stacking interactions stabilize the crystal lattice. The differences on the nucleotide conformation between the structure of form α and form β can probably be ascribed to differences in the hydrogen bonds and stacking interactions.     

Complexes of magnesium(II) and other divalent metal ions with adenosine 5′-triphosphate and 2,2′-dipyridylamine in aqueous solution

Binary and ternary systems involving adenosine 5′-triphosphate (ATP), 2,2′-dipyridylamine (DPA) and magnesium, calcium, strontium, manganese, cobalt, copper, and zinc(II) metal ions have been investigated in aqueous media by potentiometric titrations. The analysis of the titration curves shows the existence of M(ATP)²⁻, M(ATP)(H)⁻, and M(ATP)₂(H)₂⁴⁻ species for alkaline-earth metal ions, while no ternary complex can be detected. For transition metal ions both binary and ternary species are found. Binary M(ATP)₂(H)₂⁴⁻ complexes are present in solutions containing manganese and cobalt(II) metal ions but these species cannot be revealed in the case of copper and zinc(II). Ternary complexes as M(ATP)(DPA)²⁻ and M(ATP)(DPA)(H)⁻ are common to all transition metals. Binuclear and hydroxo complexes as M₂(ATP)(OH)⁻ and M(ATP)(OH)³⁻ are found only for copper and zinc(II). A hypothesis on the possible role of the species M-ATP in 1:2 ratio in the dephosphorylation mechanism is advanced on the basis of a comparison between the equilibrium data in the solution phase and the solid state structures of the magnesium, calcium, and manganese(II)- ATP-DPA systems.     

Control of Vertebrate Skeletal Mineralization by Polyphosphates

Background

Skeletons are formed in a wide variety of shapes, sizes, and compositions of organic and mineral components. Many invertebrate skeletons are constructed from carbonate or silicate minerals, whereas vertebrate skeletons are instead composed of a calcium phosphate mineral known as apatite. No one yet knows why the dynamic vertebrate skeleton, which is continually rebuilt, repaired, and resorbed during growth and normal remodeling, is composed of apatite. Nor is the control of bone and calcifying cartilage mineralization well understood, though it is thought to be associated with phosphate-cleaving proteins. Researchers have assumed that skeletal mineralization is also associated with non-crystalline, calcium- and phosphate-containing electron-dense granules that have been detected in vertebrate skeletal tissue prepared under non-aqueous conditions. Again, however, the role of these granules remains poorly understood. Here, we review bone and growth plate mineralization before showing that polymers of phosphate ions (polyphosphates: (PO₃ ⁻)_n) are co-located with mineralizing cartilage and resorbing bone. We propose that the electron-dense granules contain polyphosphates, and explain how these polyphosphates may play an important role in apatite biomineralization.

Principal Findings/Methodology

The enzymatic formation (condensation) and destruction (hydrolytic degradation) of polyphosphates offers a simple mechanism for enzymatic control of phosphate accumulation and the relative saturation of apatite. Under circumstances in which apatite mineral formation is undesirable, such as within cartilage tissue or during bone resorption, the production of polyphosphates reduces the free orthophosphate (PO₄ ³⁻) concentration while permitting the accumulation of a high total PO₄ ³⁻ concentration. Sequestering calcium into amorphous calcium polyphosphate complexes can reduce the concentration of free calcium. The resulting reduction of both free PO₄ ³⁻ and free calcium lowers the relative apatite saturation, preventing formation of apatite crystals. Identified in situ within resorbing bone and mineralizing cartilage by the fluorescent reporter DAPI (4′,6-diamidino-2-phenylindole), polyphosphate formation prevents apatite crystal precipitation while accumulating high local concentrations of total calcium and phosphate. When mineralization is required, tissue non-specific alkaline phosphatase, an enzyme associated with skeletal and cartilage mineralization, cleaves orthophosphates from polyphosphates. The hydrolytic degradation of polyphosphates in the calcium-polyphosphate complex increases orthophosphate and calcium concentrations and thereby favors apatite mineral formation. The correlation of alkaline phosphatase with this process may be explained by the destruction of polyphosphates in calcifying cartilage and areas of bone formation.

Conclusions/Significance

We hypothesize that polyphosphate formation and hydrolytic degradation constitute a simple mechanism for phosphate accumulation and enzymatic control of biological apatite saturation. This enzymatic control of calcified tissue mineralization may have permitted the development of a phosphate-based, mineralized endoskeleton that can be continually remodeled.     

Kinetics of the formation and hydrolysis reactions of some thiomolybdate(VI) anions in aqueous solution

The kinetics of the formation of the thiomolybdate ions MoOS₃²⁻ and MoS₄²⁻ were determined spectroscopically from the addition of excess sulphide to MoO₂S₂²⁻ in pH buffered media (6–8) at 30 °C. The reverse (hydrolysis) reactions of MoO₂S₂²⁻ and MoOS₃²⁻ were measured under the same conditions. The reaction rates measured are shown below:

Values of the rate-constants (s⁻¹) obtained at pH 7.0 were k₁₀ 2.4 × 10⁻³, k₂₁ 1.5 × 10⁻⁵, k₃₀ 2.1 × 10⁻⁵, k₂₃ 6.0 × 10⁻⁴, and k₃₄ 1.9 × 10⁻⁵; where the results are comparable they are in good agreement with those obtained by earlier workers, although different conditions were used. However, in this work it was found that certain reactions had to be mathematically treated as two consecutively occurring reactions. There is also a difference in interpretation of the mechanism of the hydrolysis reactions of the tri- and tetrathio ions. In general the lability towards further S replacement of O atoms, and the reverse reaction, decreased with increased S substitution. All reaction rates increased with increasing H⁺ ion concentration, mostly this was a linear relationship over the limited pH range examined. The effect of the H⁺ ion is interpreted in terms of protonation of the oxythiomolybdate ions at an O atom leading to increased lability.     

Bis(benzoyl) phosphate inactivators of beta-lactamase C from Mtb

The class A β-lactamase BlaC is a cell surface expressed serine hydrolase of Mycobacterium tuberculosis (Mtb), one of the causative agents for Tuberculosis in humans. Mtb has demonstrated increased susceptibility to β-lactam antibiotics upon inactivation of BlaC; thus, making BlaC a rational enzyme target for therapeutic agents. Herein, we present the synthesis and structure-activity-relationship data for the 1st-generation library of bis(benzoyl) phosphates (1–10). Substituent effects ranged from σ_p = −0.27 to 0.78 for electronic and π = −0.41 to 1.98 for hydrophobic parameters. Compounds 1, 4 and 5 demonstrated the greatest inhibitory potency against BlaC in a time-dependent manner (k_obs = 0.212, 0.324, and 0.450 mn⁻¹ respectively). Combined crystal structure data and mass spectrometric analysis of a tryptic digest for BlaC inactivated with 4 provided evidence that the mechanism of inactivation by this bis(benzoyl) phosphate scaffold occurs via phosphorylation of the active-site Ser-70, ultimately leading to an aged form of the enzyme.