In situ analysis of mineral content and crystallinity in bone using infrared micro-spectroscopy of the nu(4) PO(4)(3-) vibration.

Measurements of bone mineral content and composition in situ provide insight into the chemistry of bone mineral deposition. Infrared (IR) micro-spectroscopy is well suited for this purpose. To date, IR microscopic (including imaging) analyses of bone apatite have centered on the nu(1),nu(3) PO(4)(3-) contour. The nu(4) PO(4)(3-) contour (500-650 cm(-1)), which has been extensively used to monitor the crystallinity of hydroxyapatite in homogenized bone samples, falls in a frequency region below the cutoff of the mercury-cadmium-telluride detectors used in commercial IR microscopes, thereby rendering this vibration inaccessible for imaging studies. The current study reports the first IR micro-spectroscopy spectra of human iliac crest cross sections in the nu(4) PO(4)(3-) spectral regions, obtained with a synchrotron radiation source and a Cu-doped Ge detector coupled to an IR microscope. The acid phosphate (HPO(4)(2-)) content and mineral crystallite perfection (crystallinity) of a human osteon were mapped. To develop spectra-structure correlations, a combination of X-ray powder diffraction data and conventional Fourier transform IR spectra have been obtained from a series of synthetic hydroxyapatite crystals and natural bone powders of various species and ages. X-ray powder diffraction data demonstrate that there is an increase in average crystal size as bone matures, which correlates with an increase in the nu(4) PO(4)(3-) FTIR absorption peak ratio of two peaks (603/563 cm(-1)) within the nu(4) PO(4)(3-) contour. Additionally, the IR results reveal that a band near 540 cm(-1) may be assigned to acid phosphate. This band is present at high concentrations in new bone, and decreases as bone matures. Correlation of the nu(4) PO(4)(3-) contour with the nu(2) CO (3)(2-) contour also reveals that when acid phosphate content is high, type A carbonate content (i.e., carbonate occupying OH(-) sites in the hydroxyapatite lattice) is high. As crystallinity increases and acid phosphate content decreases, carbonate substitution shifts toward occupation of PO(4)(3-) sites in the hydroxyapatite lattice. Thus, IR microscopic analysis of the nu(4) PO(4)(3-) contour provides a straightforward index of both relative mineral crystallinity and acid phosphate concentration that can be applied to in situ IR micro-spectroscopic analysis of bone samples, which are of interest for understanding the chemical mechanisms of bone deposition in normal and pathological states.     

IR and Raman study on the interactions of the 5′-GMP and 5′-CMP phosphate groups with Mg(II), Ca(II), Sr(II), Ba(II), Cr(III), Co(II), Cu(II), Zn(II), Cd(II), Al(III) and Ga(III)

The chief motive behind this research is the interest provoked by the presence of metal ions as necessary stabilizers of the negative charges of phosphate groups in nucleic acids. The effect that the presence of different metal ions produces on the band principally assigned to the nu(s) PO(3)(2-) mode has been studied using FT-IR and FT-Raman spectroscopy. The results obtained reveal the diagnostic capacity of these techniques in determining the type of metal ion interaction with respect to the mononucleotides that form DNA and RNA, providing a tool for improving the knowledge of the stabilizing or destabilizing effects of these ions on such macromolecules. The metal complexes of the ribonucleotides 5'-CMP and 5'-GMP with Mg(II), Ca(II), Sr(II), Ba(II), Cr(III), Co(II), Cu(II), Zn(II), Cd(II), Al(III) and Ga(III) were obtained in this study. After studying and analyzing the IR and Raman spectra of all these complexes and comparing them with the spectra of the corresponding disodium salts, it was verified that, independently of the type of nucleotide involved, the presence of the metal in the vicinity of the phosphate group produces an alteration in the aforementioned nu(s) PO(3)(2-) band. This effect is related to the type of interaction that the phosphate group has with the metal. Three components are observed: (1) one near 983-975 cm(-1) (detectable in IR and Raman), associated with phosphate groups in an electrostatic type of interaction with the metal ion, separated by two or more water molecules; (2) another near 989-985 cm(-1) (only in IR), associated with phosphate groups in indirect interaction through the water molecules of the coordination sphere of the metal ions; and (3) the IR and Raman bands near 1014-1001 cm(-1), which represent phosphate groups directly bonded to the metal ion. These results are supported by the behavior of 5'-CMP in aqueous solution in the presence of Mg(II) ions.     

Fabrication of gelatin/calcium phosphate composite nanofibrous membranes by biomimetic mineralization

Based on the principles of biomimetic mineralization, biocomposite nanofibrous membranes were fabricated by the growth of CaP crystals on electrospun gelatin nanofibers to mimic both the physical architecture and chemical composition of natural bone ECM. Plenty more CaP crystals formed on the nanofibrous membrane containing Ca(2+) ion precursors, in which these crystals were also observed on the inner side of membrane. The release rate of Ca(2+) ion precursors from the nanofibrous membrane was slower than that of PO(4)(3-) ion precursors, suggesting the existence of more strong intermolecular interaction between gelatin and Ca(2+) ions. ATR-FTIR and XRD results clearly revealed the formation of CaP crystals mixed with apatite and CaCO(3), or apatite and TCP on the membranes. The Ca/P molar ratio of crystals obtained from the XPS data was 2.03 and 1.60, which depended on the mineralization conditions. Higher amount of CaP crystals significantly accelerated the deposit rate of bone-like apatite on the surface of composite membrane, meaning to the improved in vivo bone bioactivity.     

Superoxide dismutase activity of human plasma; the effect of Cu2+-complex compounds

The superoxide-dismutase (EC 1.15.1.1) activity was revealed in blood plasma after its successive treatment with alcohol, chloroform and one- or two-substituted phosphate. Its value is the highest when treating plasma with KH2PO4. The boiling of the supernatant liquid obtained after addition of K2HPO4 leads to a complete loss of the activity. The treatment of plasma with KH2PO4 provides higher stability of the enzyme to thermal actions. The analysis of EPR-spectra has shown the presence of different complex compounds of copper ions in the supernatant liquid. Certain experiments with the use of aqueous solutions of CuCl2 and histidine have shown that Cu2+ inhibits the superoxide-dismutase activity in samples treated with KH2PO4 and increases it in samples treated with K2HPO4.     

Requirement of calcium and phosphate ions in expression of sodium-dependent vitamin C transporter 2 and osteopontin in MC3T3-E1 osteoblastic cells

Osteoclasts dissolve mineralized bone matrix at bone resorption sites and release large amounts of calcium (Ca(2+)) and phosphate (PO(4)(3-)) ions into the extracellular fluid. However, the exact nature of Ca(2+) and PO(4)(3-) on osteoblasts remains unclear. We proposed that Ca(2+) and PO(4)(3-) ions are required for the expression of sodium-dependent vitamin C transporter (SVCT) 2 and a differentiation marker, osteopontin (OPN), in osteoblasts as a response to the osteoclastic degradation. Results from Northern blotting indicated that a deficiency of Ca(2+) or PO(4)(3-) inhibited both SVCT2 and OPN expression in a time-dependent manner, whereas elevated Ca(2+) (1 to 4 mM) or PO(4)(3-) (1 to 4 mM) dose-dependently induced SVCT2, OPN expression and OPN promoter activity. In addition, the L-type calcium channel blocker, nifedipine (5 to 20 micro M) and the phosphate transporter inhibitor, foscarnet (0.15 to 0.6 mM), dose-dependently abolished Ca(2+)- and PO(4)(3-)-induced SVCT2, OPN expression and OPN promoter activity. Furthermore, the results from L-ascorbic acid uptake assay and Western blotting indicated that the stimulatory effect of Ca(2+) and PO(4)(3-) on functional SVCT2 protein expression. These findings suggested that Ca(2+) and PO(4)(3-) regulate osteoblastic phenotype by entering into cells to stimulate SVCT2 and OPN expression.     

Nucleoside phosphorylation by phosphate minerals

In the presence of formamide, crystal phosphate minerals may act as phosphate donors to nucleosides, yielding both 5'- and, to a lesser extent, 3'-phosphorylated forms. With the mineral Libethenite the formation of 5'-AMP can be as high as 6% of the adenosine input and last for at least 10(3) h. At high concentrations, soluble non-mineral phosphate donors (KH(2)PO(4) or 5'-CMP) afford 2'- and 2':3'-cyclic AMP in addition to 5'-and 3'-AMP. The phosphate minerals analyzed were Herderite Ca[BePO(4)F], Hureaulite Mn(2+)(5)(PO(3)(OH)(2)(PO(4))(2)(H(2)O)(4), Libethenite Cu(2+)(2)(PO(4))(OH), Pyromorphite Pb(5)(PO(4))(3)Cl, Turquoise Cu(2+)Al(6)(PO(4))(4)(OH)(8)(H(2)O)(4), Fluorapatite Ca(5)(PO(4))(3)F, Hydroxylapatite Ca(5)(PO(4))(3)OH, Vivianite Fe(2+)(3)(PO(4))(2)(H(2)O)(8), Cornetite Cu(2+)(3)(PO(4))(OH)(3), Pseudomalachite Cu(2+)(5)(PO(4))(2)(OH)(4), Reichenbachite Cu(2+)(5)(PO(4))(2)(OH)(4), and Ludjibaite Cu(2+)(5)(PO(4))(2)(OH)(4)). Based on their behavior in the formamide-driven nucleoside phosphorylation reaction, these minerals can be characterized as: 1) inactive, 2) low level phosphorylating agents, or 3) active phosphorylating agents. Instances were detected (Libethenite and Hydroxylapatite) in which phosphorylation occurs on the mineral surface, followed by release of the phosphorylated compounds. Libethenite and Cornetite markedly protect the beta-glycosidic bond. Thus, activated nucleic monomers can form in a liquid non-aqueous environment in conditions compatible with the thermodynamics of polymerization, providing a solution to the standard-state Gibbs free energy change (DeltaG degrees ') problem, the major obstacle for polymerizations in the liquid phase in plausible prebiotic scenarios.     

Biomimetic apatite coatings--carbonate substitution and preferred growth orientation

Biomimetic apatite coatings were obtained by soaking chemically treated titanium in SBF with different HCO(3)(-) concentration. XRD, FTIR and Raman analyses were used to characterize phase composition and degree of carbonate substitution. The microstructure, elemental composition and preferred alignment of biomimetically precipitated crystallites were characterized by cross-sectional TEM analyses. According to XRD, the phase composition of precipitated coatings on chemically pre-treated titanium after exposure to SBF was identified as hydroxy carbonated apatite (HCA). A preferred c-axis orientation of the deposited crystals can be supposed due to the high relative peak intensities of the (002) diffraction line at 2theta=26 degrees compared to the 100% intensity peak of the (211) plane at 2theta=32 degrees . The crystallite size in direction of the c-axis of HCA decreased from 26 nm in SBF5 with a HCO(3)(-) concentration of 5 mmol/l to 19 nm in SBF27 with a HCO(3)(-) concentration of 27 mmol/l. Cross-sectional TEM analyses revealed that all distances correspond exactly to the hexagonal structure of hydroxyapatite. The HCO(3)(-) content in SBF also influences the composition of precipitated calcium phosphates. Biomimetic apatites were shown to have a general formula of Ca(10-x-y)Mg(y)(HPO(4))(x-z)(CO(3))(z)(PO(4))(6-x)(OH)(2-x-w)(CO(3))(w/2). According to FTIR and Raman analyses, it can be supposed that as long as the HCO(3)(-) concentration in the testing solutions is below 20 mmol/l, only B-type HCA (0相似文献   

Effect of phosphate ions on the fluorescence of tryptophan derivatives. Implications in fluorescence investigation of protein-nucleic acid complexes

In order to test the ability of phosphate groups to quench the fluorescence of tryptophan in protein-nucleic acid complexes we have studied the effect of various phosphate ions on the fluorescence of tryptophan derivatives. Unsubstituted and monoalkyl monoanions (H2PO4- and CH3OPO3H-) quench the fluorescence of all investigated indole derivatives while the dimethyl anion (CH3O)2 PO2- does not. This suggests that quenching of tryptophan fluorescence by phosphate monoanions requires the presence of an acidic OH group and could be due to a proton transfer from the phosphate ion to the indole chromophore. Trianions (PO4 3-4) which are strong proton acceptors quench the fluorescence of all tryptophan derivatives except N(1)methyl tryptophan. This result strongly supports our proposal that quenching of tryptophan fluorescence by phosphate trianions occurs through deprotonation of the NH indole group. Bianions (HPO '4(7), and CH3O PO3 2-3) quench the fluorescence of several indole derivatives including N-acetyl tryptophanamide but have no effect on tryptophan or N(1)-methyl tryptophan. From our results we conclude that phosphate groups of nucleic acids are not able to quench the fluorescence of tryptophyl residues in protein-nucleic acid complexes except if an accessible residue is located near a phosphorylated polynucleotide chain end.     

Carbonic anhydrase inhibitors. Interaction of isozymes I, II, IV, V, and IX with phosphates, carbamoyl phosphate, and the phosphonate antiviral drug foscarnet

A detailed inhibition study of five carbonic anhydrase (CA, EC 4.2.1.1) isozymes with inorganic phosphates, carbamoyl phosphate, the antiviral phosphonate foscarnet as well as formate is reported. The cytosolic isozyme hCA I was weakly inhibited by neutral phosphate, strongly inhibited by carbamoyl phosphate (K(I) of 9.4 microM), and activated by hydrogen- and dihydrogenphosphate, foscarnet and formate (best activator foscarnet, K(A)=12 microM). The cytosolic isozyme hCA II was weakly inhibited by all the investigated anions, with carbamoyl phosphate showing a K(I) of 0.31 mM. The membrane-associated isozyme hCA IV was the most sensitive to inhibition by phosphates/phosphonates, showing a K(I) of 84 nM for PO(4)(3-), of 9.8 microM for HPO(4)(2-), and of 9.9 microM for carbamoyl phosphate. Foscarnet was the best inhibitor of this isozyme (K(I) of 0.82 mM) highly abundant in the kidneys, which may explain some of the renal side effects of the drug. The mitochondrial isozyme hCA V was weakly inhibited by all phosphates/phosphonates, except carbamoyl phosphate, which showed a K(I) of 8.5 microM. Thus, CA V cannot be the isozyme involved in the carbamoyl phosphate synthetase I biosynthetic reaction, as hypothesized earlier. Furthermore, the relative resistance of CA V to inhibition by inorganic phosphates suggests an evolutionary adaptation of this mitochondrial isozyme to the presence of high concentrations of such anions in these energy-converting organelles, where high amounts of ATP are produced by ATP synthetase, from ADP and inorganic phosphates. The transmembrane, tumor-associated isozyme hCA IX was on the other hand slightly inhibited by all these anions.     

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.     

In situ examination of the time-course for secondary mineralization of Haversian bone using synchrotron Fourier transform infrared microspectroscopy.

At the tissue level it is well established that the rate of remodeling is related to the degree of mineralization. However, it is unknown how long it takes for an individual bone structural unit (BSU) to become fully mineralized during secondary mineralization. Using synchrotron Fourier transform infrared microspectroscopy (FTIRM) we examined the time required for newly formed bone matrix to reach a physiological mineralization limit. Twenty-six, four-month old female New Zealand white rabbits were administered up to four different fluorochrome labels at specific time points to evaluate the chemical composition of labeled osteons from the tibial diaphysis that had mineralized for 1, 8, 18, 35, 70, 105, 140, 175, 210, 245, 280, 315, 350, and 385 days. Interstitial bone from 505 day old rabbits was used as a reference value for the physiological limit to which bone mineralizes. Using synchrotron FTIRM, area integrations were carried out on protein (Amide I: 1688-1623 cm(-1)), carbonate (v(2)CO(3)(2-): 905-825 cm(-1)), and phosphate (v(4)PO(4)(3-): 650-500 cm(-1)) IR bands. IR spectral data are presented as ratios of phosphate/protein (overall matrix mineralization) and carbonate/protein. The rate of mineralization of osteonal bone proceeded rapidly between day 1 and 18, reaching 67% of interstitial bone levels. This was followed by a slower, more progressive accumulation of mineral up to day 350. By 350 days the rate of increase plateaued. The ratio of carbonate/protein also increased rapidly during the first 18 days, reaching 73% of interstitial bone levels. The ratio of carbonate/protein plateaued by day 315, reaching levels not significantly different to interstitial bone levels. In conclusion, our data demonstrate that bone accumulates mineral rapidly during the first 18 days (primary mineralization), followed by a more gradual increase in the accumulation of mineral (secondary mineralization) which we found to be completed in 350 days.     

Effect of hemodialysis on the distribution of phosphates in blood

Phosphate retention is commonly associated with chronic renal failure. Previously we presented a technique to calculate the distribution of phosphate constituents in body fluids. We studied it in pre- and postdialysis serum of 62 patients. After hemodialysis, there was some decrease in the concentration of each constituent, with significant changes in the molar fraction of H2PO4- and CaHPO4. There is a significant negative correlation between the molecular mass of the constituent (X) and the effect of dialysis (Y): urea and H2PO4- 50%; NaHPO4-, HPO4(2-), and creatinine 41-44%; MgHPO4 39%, and CaHPO4 28%. Y = 69.7 - 0.26x, r = -0.833, p less than 0.001. This limited removal of CaHPO4 may contribute to the vascular calcification and peripheral ischemic necrosis of chronic uremic patients.     

A comparative study of calf thymus DNA binding to Cr(III) and Cr(VI) ions. Evidence for the guanine N-7-chromium-phosphate chelate formation

Chromium(VI) salts are well known to be mutagens and carcinogens and to easily cross the cell membranes. Because they are powerful oxidizing agents, Cr(VI) reacts with intracellular materials to reduce to trivalent form, which binds DNA. This study was designed to investigate the interaction of calf thymus DNA with Cr(VI) and Cr(III) in aqueous solution at pH 6.5-7.5, using Cr(VI)/DNA(P) molar ratios (r) of 1:20 to 2:1 and Cr(III)/DNA(P) molar ratios (r) of 1:80 to 1:2. UV-visible and Fourier transform infrared (FTIR) difference spectroscopic methods were used to determine the metal ion-binding sites, binding constants, and the effect of cation complexation on DNA secondary structure. Spectroscopic results showed no interaction of Cr(VI) with DNA at low anion concentrations (r = 1:20 to 1:1), whereas some perturbations of DNA bases and backbone phosphate were observed at very high Cr(VI) contents (r > 1) with overall binding constant of K = 508 M(-1). Cr(III) chelates DNA via guanine N-7 and the nearest PO(2) group with overall binding constant of K = 3.15 x 10(3) M(-1). Evidence for cation chelate formation comes from major shiftings and intensity variations of the guanine band at 1717 and the phosphate asymmetric stretching vibration at 1222 cm(-1). At low Cr(III) concentration (r = 1:40), the number of Cr(III) ions bound to DNA were 6-7 cations/500 base pairs, and this increased to 30-35 cations/500 base pairs at high metal ion content (r = 1:4). DNA condensation occurred at high cation concentration (r = 1:10). No major alteration of DNA conformation was observed, and the biopolymer remained in the B family structure upon chromium complexation.     

The pH dependence of red cell membrane transport of titratable anions studied by NMR spectroscopy

The effects of varying extracellular pH on the rates of uptake of titratable anions by human erythrocytes under conditions of constant intracellular pH have been determined for a series of highly related anions, the phosphate "analogs." These compounds are simply substituted phosphorus oxyacids, differing in the number and acidity of titratable protons: phosphate (HPO4(2-), pKa 6.8); phosphite (HPO3(2-), pKa 6.4); hypophosphite (H2PO2-); methylphosphonate ((CH3)PO3(2-), pKa 7.4); dimethylphosphinate ((CH3)2PO2-); fluorophosphate [PO3F2-, pKa 4.7); and thiophosphate (HSPO3(2-), pKa 5.5). Suspensions of intact, Cl(-)-loaded erythrocytes (intracellular pH, 7.2) were incubated at 37 degrees C in isotonic buffers (pH 4-8) containing 60 mM phosphate analog for specified time intervals, whereupon influx was halted by the addition of 1 mM 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS), an inhibitor of anion exchange. The intracellular anion concentrations were determined from 31P or 19F nuclear magnetic resonance spectra from the erythrocyte suspensions. The influx rates for the titratable phosphate analogs exhibited bimodal pH dependence, reaching maximal levels at pH values that increased with increasing anion pK. This pH-dependent behavior is consistent with a transport channel that contains a titratable regulatory site which interacts with the translocated anion. Based upon the Henderson-Hasselbalch equation, the probability that a titratable anion will have an electric charge of equal magnitude to that of the titratable carrier is highest at a pH value exactly midway between the pK of the regulatory site and that of the anion. The pH maxima observed for the phosphate analogs indicate a pK for this site of 5.5 at 37 degrees C. Intracellular pH changes associated with influx indicated that transport of the "fast" anion phosphite is largely in monoionized form. Intracellular pH changes associated with transport of slow anions were predominantly determined by partial ionic equilibrium effects and did not indicate the ionization state of the transported anion.     

Control of COD/N ratio for nutrient removal in a modified membrane bioreactor (MBR) treating high strength wastewater

A modified membrane bioreactor (MBR) system has been developed to evaluate the efficiency of nutrient removal in treating synthetic high strength water. This study examined the effect of influent COD/N ratio on this system. Results showed that above 95.0% removal efficiencies of organic matter were achieved; indicating COD removal was irrespective of COD/N ratio. The average removal efficiencies of total nitrogen (TN) and phosphate (PO(4)(3-)-P) with a COD/N ratio of 9.3 were the highest at 90.6% and 90.5%, respectively. Furthermore, TN removal was primarily based on simultaneous nitrification and denitrification (SND) process occurred in the aerobic zone. Decreased COD/N ratios to 7.0 and 5.3, TN removal efficiencies in steady-states were 69.3% and 71.2%, respectively. Both aerobic SND and conventional biological nitrification/denitrification contributed to nitrogen removal and the latter played dominant effect. PO(4)(3-)-P-release and uptake process ceased in steady-states of COD/N 7.0 and 5.3, which decreased its removal efficiency significantly.     

The effects of ruthenium tetraammine compounds on vascular smooth muscle.

The time course of the relaxation effect induced by a single dose (3 x 10(-6) mol/L) of trans-[Ru(NH3)4L(NO)]3+ (L=nic, 4-pic, py, imN, P(OEt)3, SO(3)(2-), NH3, and pz) species and sodium nitroprusside (4 x 10(-9) mol/L) was studied in aortic rings without endothelium and pre-contracted with noradrenaline (1 x 10(-6) mol/L). All the compounds induced a relaxing effect in the aortic rings, but the intensity and time of relaxation were different. Only the species where L=py, 4-pic, and P(OEt)3 were able to induce 100% (99-100%) of the relaxing effect during the assay. trans-[Ru(NH3)4(L)(NO)]3+ (L=SO(3)(2-) and NH3) showed the lowest relaxing effect (36 and 37%, respectively) when compared with the other compounds. Relationship was observed between the time corresponding to half of the maximum relaxation intensity observed and, respectively, k-NO, E0'[Ru(NO)]3+/[Ru(NO)]2+ in trans-[Ru(NH3)4(L)(NO)]3+ species and E0'Ru(III)/Ru(II) in trans-[Ru(NH3)4(L)(H2O)]3+ ions. These relationships strongly suggested that the NO liberation from the reduced nitrosyl complexes was responsible for the observed relaxation.     

Mechanism of arsenate resistance in the ericoid mycorrhizal fungus Hymenoscyphus ericae

Arsenate resistance is exhibited by the ericoid mycorrhizal fungus Hymenoscyphus ericae collected from As-contaminated mine soils. To investigate the mechanism of arsenate resistance, uptake kinetics for arsenate (H(2)AsO(4)(-)), arsenite (H(3)AsO(3)), and phosphate (H(2)PO(4)(-)) were determined in both arsenate-resistant and -non-resistant H. ericae. The uptake kinetics of H(2)AsO(4)(-), H(3)AsO(3), and H(2)PO(4)(-) in both resistant and non-resistant isolates were similar. The presence of 5.0 microM H(2)PO(4)(-) repressed uptake of H(2)AsO(4)(-) and exposure to 0.75 mM H(2)AsO(4)(-) repressed H(2)PO(4)(-) uptake in both H. ericae. Mine site H. ericae demonstrated an enhanced As efflux mechanism in comparison with non-resistant H. ericae and lost approximately 90% of preloaded cellular As (1-h uptake of 0.22 micromol g(-1) dry weight h(-1) H(2)AsO(4)(-)) over a 5-h period in comparison with non-resistant H. ericae, which lost 40% of their total absorbed H(2)AsO(4)(-). As lost from the fungal tissue was in the form of H(3)AsO(3). The results of the present study demonstrate an enhanced H(3)AsO(3) efflux system operating in mine site H. ericae as a mechanism for H(2)AsO(4)(-) resistance. The ecological significance of this mechanism of arsenate resistance is discussed.     

Two-dimensional vibrational correlation spectroscopy of in vitro hydroxyapatite maturation

Two-dimensional (2-D) Raman and 2-D IR correlation spectroscopy are applied to analyze changes in the nu(4) region of the IR spectrum and in the nu(1) region of the Raman spectrum during the maturation of hydroxyapatite (HA) following the solution-mediated conversion of amorphous calcium phosphate (ACP) to HA. The nu(1) region of the Raman spectrum exhibits a frequency shift and sharpening during the maturation. Comparison of the experimental and simulated 2-D plots for this process suggests that the shift of a single peak, rather than a change in the relative intensity of two overlapped bands, is responsible for the observed spectral changes. The nu(4) mode of the PO(3-)(4) ion (T(2) symmetry in the free species) splits into a triplet with components near 563, 575, and 603 cm(-1) in HA. In addition, broad features appear at 540 and 617 cm(-1). During the latest stages of the maturation, an OH(-) librational mode develops at approximately 632 cm(-1). Changes in the relative intensities of three components of the nu(4) mode are not all correlated with each other. The synchronous 2-D plots reveal that the 563 and 603 cm(-1) pair are positively correlated while the feature at 575 cm(-1) is absent. A 587 cm(-1) mode arising from ACP is negatively correlated with the 563 and 603 cm(-1) pair and is both synchronously (positively) and asynchronously correlated with the 540 cm(-1) feature during the early stages of the maturation but is absent from 2-D plots of the later stages of the maturation. Cross correlations between the nu(4) mode and the nu(1),nu(3) contour generally confirm and extend previous assignments for the latter spectral region. Finally, the suitability of the 2-D approach for analysis of IR spectral images is examined through studies of HA crystallinity in a human iliac crest biopsy sample. Trabecular bone contains a fraction of HA that is more crystalline and mature than could be achieved in vitro during the room temperature ACP --> HA interconversion.     

Influence of protic ionic liquids on the structure and stability of succinylated Con A

We report the synthesis of a series of ionic liquids (ILs) from various ions having different kosmotropicity including dihydrogen phosphate (H(2)PO(4)(-)), hydrogen sulfate (HSO(4)(-)) and acetate (CH(3)COO(-)) as anions and chaotropic cation such as trialkylammonium cation. To characterize the biomolecular interactions of ILs with protein, we have explored the stability of succinylated Con A (S Con A) in the presence of these aqueous ILs, which are varied combinations of kosmotropic anion with chaotropic cation such as triethylammonium dihydrogen phosphate [(CH(3)CH(2))(3)NH][H(2)PO(4)] (TEAP), trimethylammonium acetate [(CH(3))(3)NH][CH(3)COO] (TMAA), trimethylammonium dihydrogen phosphate [(CH(3))(3)NH][H(2)PO(4)] (TMAP) and trimethylammonium hydrogen sulfate [(CH(3))(3)NH][HSO(4)] (TMAS). Circular dichroism (CD) and fluorescence experiments have been used to characterize the stability of S Con A by ILs. Our data distinctly demonstrate that the long alkyl chain IL TEAP is a strong stabilizer for S Con A. Further, our experimental results reveal that TEAP is an effective refolding enhancer for S Con A from a thermally denatured protein structure.