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Hard tissues (e.g., bone, enamel, dentin) in vertebrates perform various and different functions, from sustaining the body to haematopoiesis. Such complex and hierarchal tissue is actually a material composite whose static and dynamic properties are controlled by the subtle physical and chemical interplay between its components, collagen (main organic part) and hydroxylapatite-like mineral. The knowledge needed to fully understand the properties of bony and dental tissues and to develop specific applicative biomaterials (e.g., fillers, prosthetics, scaffolds, implants, etc.) resides mostly at the atomic scale. Among the different methods to obtains such detailed information, atomistic computer simulations (in silico) have proven to be both corroborative and predictive tools in this subject. The authors have intensively worked on quantum mechanical simulations of bioapatite and the present work reports a detailed review addressed to the crystal-chemical, physical, spectroscopic, mechanical, and surface properties of the mineral phase of bone and dental tissues. The reviewed studies were conducted at different length and time scales, trying to understand the features of hydroxylapatite and biological apatite models alone and/or in interaction with simplified collagen-like models. The reported review shows the capability of the computational approach in dealing with complex biological physicochemical systems, providing accurate results that increase the overall knowledge of hard tissue science.

Adsorbed and structurally incorporated carbonate in bioapatite, the primary mineral phase of bone, is observed across vertebrates, typically at 2–8 wt%, and supports critical physiological and biochemical functions. Several turtle species contain elevated bone-associated carbonate, a property linked to pH buffering and overwintering survival. Prior studies of turtle bone utilized bulk analyses, which do not provide spatial resolution of carbonate. Using Raman spectroscopy, the goals of this study were to: (1) quantify and spatially resolve carbonate heterogeneity within the turtle shell; (2) determine if cortical and trabecular bone contain distinct carbonate concentrations; and (3) assess if simulated overwintering conditions result in decreased bioapatite carbonation. Here, we demonstrate the potential for Raman spectroscopic analysis to spatially resolve bioapatite carbonation, using the western painted turtle as a model species. Carbonate concentration was highly variable within cortical and trabecular bone, based on calibrated Raman spot analyses and mapping, suggesting heterogeneous carbonate distribution among crystallites. Mean carbonate concentration did not significantly differ between cortical and trabecular bone, which indicates random distribution of crystallites with elevated and depleted carbonate. Carbonate concentrations (range: 5–22 wt%) were not significantly different in overwintering and control animals, deviating from previous bulk analyses. In reconciling bulk and Raman analyses, two hypotheses explain how overwintering turtles potentially access carbonate: (1) mobilization of mineral-associated, surface components of bone crystallites; and (2) selective, dispersed crystallite dissolution. Elevated bioapatite carbonate in the western painted turtle, averaging 11.8 wt%, represents the highest carbonation observed in vertebrates, and is one physiological trait that facilitates overwintering survival.

Porous hydroxyapatite-biochar composites with layered microstructures (SC–HA/C) were prepared by carbonizing sugarcane stem nodes and then soaking them in lime water and (NH4)2HPO4 solutions in rotation. The surface area of SC–HA/C ranges from 8.52 to 28.44 m2/g, and its microstructure inherits various macro-, meso-, and micro-pores in the cell walls of sugarcane and in the pits of the vessel walls. The maximum removal capacities were 11.50, 14.65, and 19.81 mg/g for the Cu (II) immobilization at 25 °C, 35 °C, and 45 °C with the solution Cu (II) concentration of 10~320 mg/L, respectively, which were in accordance with the copper sorption capacities of synthesized nano-hydroxylapatites. The Cu (II)-removal kinetics and isotherm followed the pseudo-second-order equation and the Langmuir equation very well. The formation of the Cu-containing hydroxylapatite solid solutions ((CuxCa1−x)5(PO4)3(OH)) through adsorption, ion exchange (x = 0.01~0.04), and dissolution-coprecipitation (x = 0.13~0.35) was the dominant process for the Cu (II) removal by the SC–HA/C composite.

The current study evaluated a newer method, which includes a dehydration step, of immobilizing Saccharomyces cerevisiae L-77 and S. cerevisiae L-73 onto hydroxylapatite and chamotte ceramic supports. The efficiency of cell immobilization on chamotte was significantly higher than hydroxylapatite. Immobilized yeast preparations were investigated for their ethanol-producing capabilities. The glucose concentration in a fermentation medium was 100 mg/mL. Immobilized preparations produced the same amount of ethanol (48 ± 0.5 mg/mL) as free cells after 36 H of fermentation. During the early stages of fermentation, immobilized yeast cells produced ethanol at a higher rate than free cells. Yeast preparations immobilized on both supports (hydroxylapatite and chamotte) were successfully used in six sequential batch fermentations without any loss of activity. The chamotte support was more stable in the fermentation medium during these six cycles of ethanol production. In addition to the high level of ethanol produced by cells immobilized on chamotte, the stability of this support and its low cost make it a promising material for biotechnologies associated with ethanol production.

The diversity in the structural and chemical state of apatites allows implant manufacturers to fine-tune implant properties. This requires suitable manufacturing processes and characterization tools to adjust the amorphous phase and hydroxyl content from the source hydroxylapatite. Hydroxylapatite was processed by high-velocity oxy-fuel spraying, plasma spraying and flame spraying, and primarily analyzed by Raman spectroscopy. Investigation of rounded splats, the building blocks of thermal spray coatings, allowed correlation between the visual identity of the splat surface and the Raman spectra. Splats were heat-treated to crystallize any remaining amorphous phase. The ν1 PO4 stretching peak at 950–970 cm−1 displayed the crystalline order, but the hydroxyl peak at 3572 cm−1 followed the degree of dehydroxylation. Hydroxyl loss was greatest for flame-sprayed particles, which create the longest residence time for the melted particle. Higher-frequency hydroxyl peaks in flame- and plasma-sprayed splats indicated a lower structural order for the recrystallized hydroxylapatite within the splats. Crystallization at 700 °C has shown potential for revealing hydroxyl ions previously trapped in amorphous calcium phosphate. This work compares Fourier transform infrared and Raman spectroscopy to measure the hydroxyl content in rapidly solidified apatites and shows that Raman spectroscopy is more suitable.

We here report a novel biomimetic mineralization strategy for enamel remineralization by intergration of calcium phosphate loaded and thermally triggered liposomes and a self-assembly amelogenin-inspired peptide. Firstly, calcium and phosphate loaded temperature sensitive liposomes were synthesized by Interdigitation-fusion method with 1,2-bis(palmitoyl)-sn-glycero-3-phosphor- choline (DPPC) and 1,2-bis(myristoyl)-sn-glycero- 3-phosphocholine (DMPC) at mass ratio of 9:1 . The liposomes were stable at room temperature, but slowly released calcium and phosphate ions if heated to 37 °C. Secondly, a novel polyanion amelogenin-inspired oligropeptide (Gln-Pro-Ala)4-Thr-Lys-Arg-Glu-Glu-Val-Asp ) was synthesized by standard solid-phase. Lastly, the mixture of peptide and liposomes solution was exposed to enamel surface at 37 °C. The results showed oriented enamel-like hydroxylapatite evenly deposited on enamel surface.

The effect of a bovine whey protein concentrate partially digested with lipase to release free fatty acids (WPD) on growth and viability of Streptococcus mutans , and its adherence to hydroxylapatite (HA) in phosphate-buffered saline were examined. The effects of other whey mixtures and individual whey proteins, β-lactoglobulin (BLG), lactoferrin (LF), caseinoglycomacropeptide (CGMP) and α-lactalbumin were also investigated. Bacterial viability was assessed by incubation with whey protein and measuring colony-forming units. S. mutans binding was quantified using Syto-13 nucleic-acid binding dye and Alamar Blue, which measures cellular metabolic activity. WPD was bactericidal, and inhibited S. mutans growth and adhesion to HA. Other wheys inhibited S. mutans adhesion, particularly acid whey, as well as the parent compound for WPD, Carbelac 80, and a whey protein isolate. BLG, CGMP and LF inhibited S. mutans adhesion. A mixture containing 56% BLG and 20% CGMP was as effective an inhibitor of bacterial adhesion as WPD.

This article deals with infrared spectroscopic studies, in the range 400-4000 cm−1, of some calcium phosphates having an apatitic structure. The bands are assigned and small differences between some spectra are explained. Occurrence of free OH− groups in the apatitic cell is confirmed; acidic phosphate groups, linked together by hydrogen bonds, have been detected in the spectra of compounds having a Ca/P value lower than the hydroxylapatite one. Heat effect upon the constitution of these compounds has also been studied; these infrared data get information which could not be obtained by X rays spectrography; i.e. the occurrence of pyrophosphate groups in solid solution in the apatitic cell. The experimental data are satisfactorily explained by the assumption that H+ ions are able to «substitute» progressively Ca++ ions in the apatitic structure. An original formula is proposed for the α tricalcium phosphate.

In this paper, chemically synthesized Mg2SiO4 (MS) powder was plasma-sprayed onto a titanium alloy substrate to evaluate its application potentials in biomedicine. The phase composition and surface morphology of the MS coating were analyzed. Results showed that the MS coating was composed mainly of Mg2SiO4 phase, with a small amount of MgO and glass phases. Mechanical testing showed that the coating exhibited good adhesion strength to the substrate due to the close thermal expansion coefficient between the MS ceramic and the titanium alloy substrate. The measured bonding strength was as high as 41.5 ± 5.3 MPa, which is much higher than the traditional HA coating. In vitro cytocompatibility evaluation of the MS coating was performed using canine bone marrow stem cells (MSCs). The MSCs exhibited good adhesion, proliferation and differentiation behavior on the MS coating surface, which can be explained by the high protein adsorption capability of the MS coating, as well as the stimulatory effects of Mg and Si ions released from the coating. The proliferation rate of the MSCs on MS coating was very close to that on the hydroxylapatite (HA) coating. Alkaline phosphatase (ALP) activity analysis demonstrated that the ALP level of the MSCs on the MS coating remained high even after 21 days, implying that the surface characteristics of the coating are beneficial for the differentiation of MSCs. In summary, our results suggest that MS coating might be a new approach to prepare bone implants.

The aim of this study was to investigate the inhibitory effect of various dairy powders and milk constituents on the adhesion of a clinical isolate of Streptococcus mutans to hydroxylapatite (HA), an analogue of tooth enamel. Adhesion of a microorganism to a cell surface such as epithelial cells or tooth enamel is considered to be the first step in pathogenesis. Inhibiting this process may have therapeutic effects in vivo. The adherence assays were performed by incubating S. mutans with HA in the presence of each test material for 45 min, followed by centrifugal separation of the HA. Unbound bacteria were then quantified using a fluorescent dye. Sweet and Acid WPC80, buttermilk powder and cream powder were found to very effectively inhibit adherence of S. mutans to phosphate-buffered saline coated HA (PBS-HA). Sodium caseinate and the casein fractions a-,b- and j-casein were also found to show high levels of anti-adhesive activity. A selection of test materials were assessed using saliva-coated HA (S-HA), and similar trends were observed. The results suggest commercial dairy powders, and certain milk proteins, can inhibit adhesion of S. mutans to HA and may have potential to control dental caries.

Binding isotherms and heats of interaction have been determined at 15, 25, and 40 degrees C for a coaggregating and a non-coaggregating oral bacterial pair. Heats of interaction were measured upon three consecutive injections of streptococci into an actinomyces Suspension using isothermal titration calorimetry. After each injection, the number of streptococci injected remaining free in Suspension was quantified microscopically and the degree of binding between the two bacterial strains was established. The coaggregating pair shows positive cooperative binding. The highest cooperativity, at 25 degrees C. correlates with a strong, macroscopically visible coaggregation. The non-coaggregating pair shows low cooperativity and lacks macroscopically visible coaggregation. Interactions between the coaggregating partners seem to be mainly due to specific. enthalpically saturable and favorable binding sites. Even though the enthalpic part of the interaction is saturated, cooperativity increases with consecutive injections, implying that the coaggregation phenomenon is driven by entropy gain. The change in heat capacity (Delta C-p) is positive for the non-coaggregating pair from 15-40 degrees C as well as for the coaggregating pair beyond 25 degrees C. At lower temperatures the coaggregating pair causes a negative ACPI The decrease in heat capacity together with an increase in entropy is considered to be indicative of hydrophobic interactions playing an important role in the formation of large coaggregates as observed for the coaggregating pair at 25 degrees C. 2005 Elsevier Inc. All rights reserved.

The thermal reactions in the mixtures of hydroxylapatite or fluorapatite and (NH4)2SO4up to 500°C were studied with the purpose of elaborating the conditions of obtaining calcium–ammonium cyclophosphate that could be used as fertilizer. Thermal analysis with a simultaneous FTIR analysis of the evolved gases as well as the analyses of chemical and phase composition of solid products were performed. The thermal changes in the mixtures could be divided into three steps: (1) decomposition of (NH4)2SO4and reactions of apatite with these products at 250–420°C, (2) calcium ammonium polyphosphate formation at 290–450°C, and (3) reaction of CaSO4with CaNH4P3O9at 320–500°C. Higher concentrations of NH3in the gas phase promote the formation of CaNH4P3O9and increase its stability. Calcination at temperatures above 350°C causes decomposition of CaNH4P3O9with a decrease in the content of water-soluble phosphorus and evolvement of SO2.

Dentin matrix protein-1 (DMP1) is a mineralized tissue matrix protein synthesized by osteoblasts, hypertrophic chondrocytes, and ameloblasts as well as odontoblasts. DMP1 is believed to have multiple in vivo functions, acting both as a signaling molecule and a regulator of biomineralization. Using a cell-free system in vitro, we evaluated the action of DMP1 in the regulation of hydroxylapatite (HA) formation and crystal growth. The non-phosphorylated recombinant protein acted as an HA nucleator, increasing the amount of mineral formed in a gelatin gel HA growth system relative to protein-free controls. The recombinant protein phosphorylated in vitro had no detectable effect on HA formation and growth. In contrast, phosphorylated bovine DMP1 expressed in marrow stromal cells with an adenovirus vector containing 29.7 phosphates/mol was an effective inhibitor of HA formation and growth. The native full-length protein appeared to be absent or present in only small amounts in the extracellular matrix of bones and teeth. However, two highly phosphorylated fragments representing the N- and C-terminal portions of DMP1 have been identified, apparently arising from proteolytic cleavage of four X-Asp bonds. The highly phosphorylated C-terminal 57-kDa fragment (containing 42 phosphates/mol), like the non-phosphorylated DMP1, was an HA nucleator. These data suggest that, in its native form, DMP1 inhibits mineralization, but when cleaved or dephosphorylated, it initiates mineralization. These in vitro data are consistent with the findings in the DMP1 knockout mouse.

Adherence to the tooth surface by Streptococcus mutans is an important step in initiation of dental caries. Current in vitro methods used to study bacterial adherence are time-consuming and may involve the use of radiolabels. The aim of this study was to develop a more convenient, high-throughput, microtitre-plate assay of bacterial adherence to hydroxylapatite. S. mutans was labelled with the fluorescent indicator BCECF/AM and fluorescence measured using a spectrofluorometer. Fluorescence microscopy confirmed label uptake. Optimal labelling occurred at 120 min with 50 μM BCECF/AM in DMSO. Viability was similar in control untreated bacterial cells, bacteria treated with DMSO alone or with the label for up to 4 h. Preliminary adherence experiments were performed using four commercially available types of hydroxylapatite. Fluorescence from pre-labelled bacteria was measured for bound cells. The assay was then optimised with respect to time and bacterial concentration using Fluka crude hydroxylapatite. Time course studies demonstrated that adherence reached saturation by 30 min incubation when using 1×10 7 cfu/ml labelled bacteria to 1 mg hydroxylapatite, coated with PBS or saliva. The fluorescence-based adherence assay was highly reproducible in repeated analyses and was useful in demonstrating interference with adherence. In conclusion, this microtitre-plate assay offers a more convenient approach to examine streptococcal adherence and could be used to screen for potential anti-adhesive agents.

A series of fossil tooth enamel samples was investigated by Fourier transform infrared (FTIR) spectroscopy, 13 C and 19 F magic-angle spinning nuclear magnetic resonance (MAS NMR) and scanning electron microscopy (SEM). Tooth remains were collected in Mio-Pliocene deposits of the Tugen Hills in Kenya. Significant transformations were observed in fossil enamel as a function of increasing fluorine content (up to 2.8 wt.%). FTIR spectroscopy revealed a shift of the ν 1 PO 4 stretching band to higher frequency. The ν 2 CO 3 vibrational band showed a decrease in the intensity of the primary B-type carbonate signal, which was replaced by a specific band at 864 cm −1 . This last band was ascribed to a specific carbonate environment in which the carbonate group is closely associated to a fluoride ion. The occurrence of this carbonate defect was consistently attested by the observation of two different fluoride signals in the 19 F NMR spectra. One main signal, at ∼−100 ppm, is related to structural F ions in the apatite channel and the other, at −88 ppm, corresponds to the composite defect. These spectroscopic observations can be understood as resulting from the mixture of two phases: biogenic hydroxylapatite (bioapatite) and secondary fluorapatite. SEM observations of the most altered sample confirmed the extensive replacement of the bioapatite by fluorapatite, resulting from the dissolution of the primary bioapatite followed by the precipitation of carbonate-fluorapatite. The ν 2 CO 3 IR bands can be efficiently used to monitor the extent of this type of bioapatite transformation during fossilization.

Bisphosphonates are widely used in diagnosis and therapy of different bone diseases. They are useful agents in osteotic vectorization of antitumor and antiinflammatory drugs because of their potential to accumulate in the inorganic bone matrix hydroxylapatite. Hence, there is great interest in alkyl-bisphosphonates, containing functional groups advantageous for further synthetic modification, as structural units for coupling with the drug. We report on the synthesis of tetraethyl-4-hydroxyphenylmethylene-1,1-bisphosphonate 8 by hydroxy-de-diazoniation of the diazonium salt 7, prepared by diazotation of the corresponding mine tetraethyl-4-aminophenylmethylene-1,1-bisphosphonate 6.

Glutamate decarboxylase (GAD) was purified 300-fold from green cowpea ( Vigna unguiculata L.) pods using a combination of PEG precipitation, DEAE cellulose chromatography, hydroxylapatite chromatography, and Q-resin chromatography. The partially purified preparation demonstrated 2 primary bands in SDS-polyacrylamide gel electrophoresis with up to 3 additional minor bands. Cowpea GAD has a pH optimum at between pH 5.5–6.0, and an apparent K M for glutamate of 3.2 mM at pH 5.8. Both crude GAD preparations and preparations partially purified through the hydroxylapatite step can be stimulated by Ca 2+ and calmodulin when assayed at pH 5.8. However, the purified enzyme does not show activation by Ca 2+ and/or calmodulin at pH 5.8 or at pH 7.0. © 1997 Elsevier Science Ltd. All rights reserved

An extracellular lipase from Flavobacterium odoratum was purified 14-fold with 18% recovery by three-phase partitioning, anion-exchange chromatography, and hydroxylapatite chromatography. The purified enzyme had a monomeric molecular mass of approximately 32,500. The enzyme is fairly thermostable and has an optimum temperature between 50–65°C. An alkali optimum pH was observed between pH 9.0–10.5. A wide substrate specificity with a preference toward esters of unsaturated fatty acids was obtained. The effect of metal ions indicated that the enzyme was probably metal ion dependent.

Squalene synthase (SQS) activity was detected in the microsomal fractions of suspension-cultured cells of dandelion (Taraxacum officinale), which produce cycloartane (involved in phytosterol biosynthesis) and other classes (e.g. oleanane and ursane) of triterpenoids. High SQS activity was detected at the late logarithmic growth stage of the culture. The activity was solubilized from the microsomes by the use of detergents (glycodeoxycholate and octylthioglucopyranoside), and chromatographed on triple-joint-columns composed of cation-exchange, anion-exchange and hydroxylapatite resins. Active fractions were further subjected to chromatography on hydroxylapatite and anion-exchange reins, followed by fast protein liquid chromatography (FPLC) on a gel-filtration column. About a 50-fold purification compared with the microsomal activity was achieved at the hydroxylapatite stage, and, although the recovery was poor, the activity was retained in the final FPLC fractions.

Objective: To characterize the receptor for 1,25-dihydroxycholecalciferol [1,25-(OH)2D3], we purified it from nuclear fractions of human placentae. Methods: Human placental fractions were concentrated with ammonium sulfate, extracted from hydroxylapatite, and then chromatographed on Sephacryl S-200 and DEAE-cellulose. Results: The receptor for [1,25-(OH)2D3] was purified approximately 1,500-fold. The molecular weight of the receptor was estimated to be 55 K dalton by gel filtration. The receptor fractions showed a dissociation constant (Kd) of 3.0 × 10−10 mol/l, and adsorbed to the DNA cellulose column. D3 analogs, estradiol, and progesterone had almost no effect on 1,25-(OH)2D3 binding. Conclusion: These properties of the 1,25-(OH)2D3 receptor in human placenta are similar to those of the chicken intestinal 1,25-(OH)2D3 receptor.

Serum is an essential requirement for the growth and long-term survival of human endothelial cells, even in the presence of such defined elements such as polypeptide growth factors and hormones. A polypeptide from fetal bovine serum was isolated and characterized on the basis of long-term survival of human endothelial cells in serum-free culture. The endothelial cell viability maintaining factor has been purified to homogeneity by a combination of polyethylene glycol precipitation, hydroxylapatite, gel permeation and reverse-phase high performance liquid chromatography. The final purified endothelial cell viability maintaining factor has a molecular weight of 65 000 (reduced) and has been identified as bovine apolipoprotein H by amino-terminal amino acid sequence analysis and Western blot analysis. Endothelial cell viability maintaining factor improved a long-term viability of human endothelial cells at maximal concentrations of 2.5–5 μg protein/ml in serum-free medium.

X-Ray diffraction and spectrophotometric analysis have been used to investigate the role of zinc on hydroxylapatite (HA) crystallization. The presence of zinc in solution strongly inhibits the crystallization of hydroxylapatite, which can be synthesized as a unique crystalline phase only up to zinc concentration of about 25 atom %. This phase exhibits a reduction of Ca/P molar ratio and crystal sizes with increasing zinc concentration. Although the Ca/Zn ratio in the solid phase is almost equivalent to that in solution, the values of the cell parameters of the apatitic phase indicate that zinc cannot appreciably substitute for calcium in HA structure. Therefore, zinc must be assumed to be adsorbed on the surface of apatite crystallites and/or in the amorphous phase. The extent of thermal conversion of HA into s-tricalcium phosphate (s-TCP) increases with increasing zinc concentration in the solid phase, either when it is obtained by means of synthesis in solution or after cyclic pH fluctuation. The decrease of the lattice constants of s-tricalcium phosphate with increasing zinc concentration in the solid phase indicates that zinc partially replaces calcium in this structure. The inhibiting effect of zinc on HA crystallization and its preference for s-TCP structure closely resembles the behavior previously observed for magnesium.

Two species [Penicillium aurantiogriseum and Pseudomonas sp. ( PI18 89 )] having high abilities in solubilizing inorganic phosphates (hydroxylapatite and brushite) were used to examine solubilization mechanisms. No direct contact between microorganisms and calcium phosphates (Ca-Ps) were necessary for effective solubilization. The P-concentration in solution did not increase according to a sigmoid curve type. Therefore, sampling time is of particular importance for estimating P mobilization. The widespread theory that solubilization is always caused by the release of organic acids may prove wrong for our two organisms. In our opinion the most probable reason for solubilization without acid production is the release of protons accompanying respiration or NH4+ assimilation. It was shown that solubilization of Ca-Ps with two different solubility products may lead to a short-term increase of the amount of at least one CaP. Precipitation and subsequent resolubilization of different organic or inorganic phosphates result in hardly predictable P-concentrations in culture solution.

An improved assay for measuring ligand binding to extracted nuclear type II estrogen binding sites which involves preimmobilization on glass fiber filters is described. At least two classes of specific estrogen binding sites have been demonstrated in rat uterus as well as in a variety of other tissues and species and have been designated as type I and type II. Although the endogenous ligand to the type II binding site has recently been identified as methyl p-hydroxyphenyllactate (MeHPLA), tritiated estrogens are generally used for radiolabeling this site due to the susceptibility of MeHPLA to enzymatic hydrolysis in in vitro assays. After extracting the type II site from the nuclear matrix, ligand binding and protein stability appear to be significantly enhanced by first immobilizing the site on an artificial matrix, such as hydroxylapatite, before incubating with radiolabeled ligand. Immobilization of the extracted site on glass fiber filters results in higher specific binding and lower nonspecific binding when compared to hydroxylapatite and a number of other immobilization matrices. The glass fiber ligand exchange procedure for measuring type II binding can also be performed on smaller samples and requires less time than other methods. Type II sites are significantly stabilized when immobilized on glass and exhibit sigmoidal binding curves when incubated with increasing concentrations of [3H]estradiol and [3H]estrone and display inhibition data characteristic of that observed using more traditional assays. Since the type II binding site can be readily extracted from glass, glass immobilization provides a very rapid method for the desalting and concentration of type II sites prior to subsequent characterization and purification steps and has implications for the electroblotting and postlabeling of type II preparations purified using nondenaturing electrophoresis.

Sulfite dehydrogenase in the membrane fraction of Thiobacillus thiooxidans JCM7814 was solubilized with n-heptyl-β-D-thioglucoside, and then purified by DEAE-Sepharose and hydroxylapatite columns containing Triton X-100. The purified enzyme was electrophoretically homogeneous. The enzyme had an apparent molecular mass of 400 kDa, and was composed of three subunits whose molecular masses were 74, 70, and 62 kDa. The purified enzyme was much more labile than the membrane-bound enzyme (membrane fraction). The optimal temperature of the membrane fraction was 25°C, while that of the purified enzyme was 20°C. The optimal pHs of both the enzymes were 7.5. The Km’s for sulfite of the membrane fraction and the purified enzyme were 4.88 mM and 1.95 mM, respectively. The activities of both the enzymes were increased by adding Triton X-100 and inhibited by sulfhydryl-binding reagents.

The aim of our research was to study how the conditions of immobilization influence cell attachment to two different ceramic surfaces: hydroxylapatite and chamotte tablets. Three fermentative yeast strains, namely brewery TT, B4 (ale, lager) and distillery Bc15a strains belonging to Saccharomyces spp., and one strain of Debaryomyces occidentalis Y500/5 of weak fermentative nature, but with high amylolytic activity due to extracellular α-amylase and glucoamylase, were used in this study. Different media, including cell starvation, were applied for immobilization of yeast strains as well as different phases of cell growth. Immobilization of selected yeasts on a hydroxylapatite carrier was rather weak. However, when incubation of starved yeast cells was conducted in the minimal medium supplemented by calcium carbonate, the scale of immobilization after 24 h was higher, especially for the D. occidentalis strain. Adhesion to hydroxylapatite carriers in wort broth was of reversible character and better results of adhesion were observed in the case of another ceramic carrier-chamotte. The number of immobilized cells was about 10(6)-10(7) per tablet and cell adhesion was stable during the whole fermentation process. The comparison of the volatile products that were formed during fermentation did not show any significant qualitative and quantitative differences between the free and the immobilized cells. This is the first time when a cheap, porous chamotte surface has been applied to yeast adhesion and fermentation processes.

Nuclear membrane bound testosterone 5 alpha-reductase solubilized in active form from human prostatic tissue by 0.5% n-octyl beta-D-glucopyranoside was purified by a four-step chromatographic procedure including DEAE-Trisacryl ion exchange, hydroxylapatite adsorption, testosterone-Sepharose affinity and Sepharose 4B gel filtration. A purification of approximately 30-fold was achieved judging from the increase in the specific enzymatic activity. We have purified the acidic pH-optimum 5 alpha-reductase type 2 isoenzyme. The apparent molecular weight of the purified enzyme was estimated as 42,000 by SDS-PAGE. At the same time we isolated a 38 kDa protein characterized by a real affinity for testosterone and by a possible association to the 5 alpha-reductase enzyme.

Bovine aminopeptidase H was detected and purified from fresh bovine skeletal muscle by ammonium sulfate fractionation and successive column chromatographies of DEAE-cellulose, Ultrogel AcA34, a phenyl-Sepharose CL-4B, hydroxylapatite, and DEAE-cellulose again. The purified enzyme migrated as a single band on SDS-PAGE. The molecular weight of this enzyme was found to be 52 000 and 400 000 by SDS-PAGE and Ultrogel AcA34 column chromatographies, respectively. The optimum pH for hydrolysis of L-leucine-2-naphthylamide (Leu-NA) was 7.5. This activity was strongly inhibited by monoiodoacetic acid and leupeptin but was not affected by EDTA, pepstatin, and phenylmethanesulfonyl fluoride. Bovine aminopeptidase H was shown to hydrolyze alpha-N-benzoyl-L-arginine-2-naphthylamide (BANA) as well as L-leucine-2-naphthylamide (Leu-NA). The Km values for the hydrolysis of Leu-NA and BANA were 3.13 and 2.38 mM, respectively. This enzyme strongly acted on Met-, Leu-, Lys-, Ala-, Glu-, and Ser-NA and hardly acted on Pro-NA.

The title compound, designed as a bone-seeking estrogen prodrug, was synthesized, characterized and shown to bind to hydroxylapatite, the mineral constituent of bone tissue.

An unacceptable loss of tritiated 1,25-dihydroxyvitamin D 3 [1,25-(OH) 2 D 3 ] to the wall of the reaction tubes constituted an obstacle when examining C3H/10T1/2 Cl 8 cells for 1,25-(OH) 2 D 3 receptor. The loss of tracer in low protein cell extracts could be strongly reduced by incubating the cell extracts in polyethylene tubes and in the presence of inert peptides prepared by digestion of gluten proteins. When incubated in buffer the recovery of tracer increased from 3 to 45% by using polyethylene tubes instead of sodium-glass tubes. However, the presence of a sufficient amount of inert peptides in the buffer significantly increased the recovery of tracer to 89%. This procedure improved the saturation binding analysis of the 1,25-(OH) 2 D 3 receptor in the C3H/10T1/2 Cl 8 cells using the hydroxylapatite assay.

1. 1. A purified vitellin was isolated from the mature ovaries of the prawn ( Penaeus monodon ) after purification with gel filtration, hydroxylapatite, DEAE-Sephacel and polyacrylamide gel electrophoresis. 2. 2. Eight polypeptide subunits and a molecular weight of 492 kDa were suggested as being present in the purified vitellin. 3. 3. Polypeptide subunits are associated with each other through non-disulfide bonds, and the subunits of the purified vitellin were easily rearranged during the purification. 4. 4. It has been suggested that the purified vitellin is a lipo-glyco-carotenoprotein. The amino acid composition of the vitellin has also been analyzed.

A method for the partial purification of rat liver paraoxonase is presented. The method consists of the following steps: preparation of microsomes, solubilization with Triton X-100, adsorption on hydroxylapatite and chromatography on DEAE-52 cellulose. A partially purified preparation of rat liver paraoxonase has been obtained, showing a specific activity of 422 mU/mg with a yield of about 22% and a purification factor of 77-fold.

Summary: Positive cooperativity is a mechanism proposed to account for the adhesion of bacteria to surfaces. In this paper, two methods that both claim to assess experimentally cooperative phenomena, viz. Scatchard analysis of adhesion data (using adhesion to vials) and analysis of the spatial arrangement of adhering cells (using a flow chamber), were compared and critically evaluated. Three oral strains were used and the substrata involved were glass (hydrophilic) and silicone-coated glass (hydrophobic) employed with or without a salivary coating. Scatchard analysis and near-neighbour analysis of adhering cells yield similar conclusions with regard to the mechanism of adhesion of the cells, provided that adhering cells are sufficiently immobilized under the experimental conditions. In the case of incomplete immobilization, near-neighbour collection results from sliding of adhering cells rather than from cooperative phenomena. Also, the agreement between the conclusions from both methods seems to be better, the more reversibly the cells adhere. Positive cooperativity can be absent or present on saliva-coated substrata with a distinct effect of the substratum hydrophobicity, despite the presence of an adsorbed film. This suggests that a different pellicle develops on a hydrophobic substratum than on a hydrophilic substratum. This is confirmed by our observation that the amino acid composition of salivary films in different on both types of substratum.

A two-step method using hydroxylapatite chromatography and gel filtration is described for the purification of two murine monoclonal antibodies of the IgM class. Ascites fluid from each hybridoma was diluted in sodium phosphate buffer (0.01 M, pH 6.8), loaded onto a hydroxylapatite column and eluted with a stepwise sodium phosphate gradient. The immunoreactive protein peaks were concentrated and subjected to gel filtration using either Sephadex G-200 or Sephacryl S-200HR. The biological activity of the end-products was confirmed by complement lysis assay and by indirect immunofluorescence and flow cytometry. The purity of the end-products as assessed by SDS polyacrylamide gel electrophoresis and densitometry was at least 90%. The methods described produced immunoreactive material with a high level of purity. The procedure for each antibody was reproducible and provides a reliable method for purification of monoclonal antibodies of the IgM class.

In the CaO-P2O5-H2O system it may be separated crystalline precipitate which gives X-ray photographs corresponding to stoichiometric calcium hydroxylapatite (SCHA) but differs from it by composition. CaO/P2O5 mole ratio of those compounds varies in the range from 2.66 to 3.34 whereas that of SCHA is 3.34.

The influence of the physicochemical properties of biomaterials on microbial cell adhesion is well known, with the extent of adhesion depending on hydrophobicity, surface charge, specific functional groups and acid–base properties. Regarding yeasts, the effect of cell surfaces is often overlooked, despite the fact that generalisations may not be made between closely related strains. The current investigation compared adhesion of three industrially relevant strains of Saccharomyces cerevisiae (M-type, NCYC 1681 and ALY, strains used in production of Scotch whisky, ale and lager, respectively) to the biomaterial hydroxylapatite (HAP). Adhesion of the whisky yeast was greatest, followed by the ale strain, while adhesion of the lager strain was approximately 10-times less. According to microbial adhesion to solvents (MATS) analysis, the ale strain was hydrophobic while the whisky and lager strains were moderately hydrophilic. This contrasted with analyses of water contact angles where all strains were characterised as hydrophilic. All yeast strains were electron donating, with low electron accepting potential, as indicated by both surface energy and MATS analysis. Overall, there was a linear correlation between adhesion to HAP and the overall surface free energy of the yeasts. This is the first time that the relationship between yeast cell surface energy and adherence to a biomaterial has been described.

This is the first report on a unique vitronectin molecule, yolk vitronectin, which is similar to its blood homologue in cell spreading activity but different in molecular size, bound carbohydrate, and heparin and collagen binding activity. Yolk vitronectin was purified 2,500-fold from chick egg yolk by a combination of hydroxylapatite, DEAE-cellulose, and anti-vitronectin-Sepharose column chromatographies. In SDS-polyacrylamide gel electrophoresis under reducing conditions, yolk vitronectin was separated into 54- and 45-kDa bands, which are 16 and 25 kDa smaller, respectively, than the 70-kDa major band of chick blood vitronectin. The 54-kDa band shares the same NH2-terminal sequence as chick blood vitronectin. In contrast, the NH2-terminal sequence of the 45-kDa band is somewhat homologous with the internal sequences of mammalian vitronectins beginning at the 50th amino acid from the NH2 terminus. The bound carbohydrate of the 54- and 45-kDa species of yolk vitronectin is similar to, but distinct from, that of blood vitronectin. Unlike blood vitronectin, yolk vitronectin cannot bind to either heparin or collagen.

General procedures for the rapid, large-scale purification of recombinant Lactobacillus casei thymidylate synthase and its mutants have been established. An effective method employs sequential phosphocellulose and hydroxylapatite chromatography. Crude cell extracts are directly applied to phosphocellulose, and the enzyme is obtained in a nearly pure state by stepwise elution with KCl. The eluate is directly applied to hydroxylapatite, and the homogeneous enzyme is eluted with a gradient of potassium phosphate. The method has been successful for the purification of recombinant wild-type enzyme and all mutants thus far examined. The entire purification procedure has been automated using a commonly available FPLC system and can be performed in several hours with minimal operator time.

Versiconal cyclase catalyzes the dehydration of versiconal to versicolorin B or versicolorin C [versicolorin B(C)]. The enzyme was purified from mycelia of Aspergillus parasiticus by DEAE-cellulose, hydroxylapatite, and Mono Q column chromatography. The protein contains two identical subunits of molecular weight 72,000 per molecule of native protein. The p I of the enzyme is 3.95. The pH activity curve had a broad maximum with a peak at 5.5. The K m and V max for versiconal at 30 °C and pH 6.0 are 3.1μ m and 0.15 μmol min −1 mg −1 , respectively. Most of the formation of versicolorin B(C) in the cell is attributed to the action of versiconal cyclase.

We purified the high-molecular-weight perforin inhibitor protein from normal human serum using DEAE-cellulose, HPLC-gel filtration and hydroxylapatite chromatography. This protein was shown to be identical to the serum apolipoprotein B-100 in terms of amino acid composition and the sequence of the digested peptides. This inhibitor protein not only inhibits the membrane binding activity of perform but also the pore insertion activity of membrane-bound perform.

A new method for the purification of manganese superoxide dismutase from human liver is described. The procedure involves essentially three steps: DEAE-cellulose, hydroxylapatite, and butyl-Toyopearl chromatographies. The method has several advantages: (i) its simplicity and rapidity (it takes less than 3 days), (ii) its high yield (62%) with a high specific activity (5660 units/mg of purified SOD), and (iii) its suitability for both large- and small-scale purifications.

The enzymes which metabolize 2-oxoaldehyde compounds in a crude extract of chicken liver were examined using 3-deoxyglucosone (3-DG) and methylglyoxal (MG). NADH- and NADPH-dependent reductase activities were observed and the latter was more than the former.NADPH-dependent 2-oxoaldehyde reductase was purified from chicken liver by ammonium sulfate fractionation, and DEAE-cellulose, CM-cellulose, hydroxylapatite, and Sephadex G-100 column chromatographies. The molecular weight of the enzyme was estimated to be 38,000 and 32,000 by SDS-PAGE and gel filtration, respectively. The optimum pH was 6.5–7.0 and this enzyme was stable at pH 7.0–8.0. The Km for 3-DG and MG were 3.75 mm and 1.52 mm, respectively. This enzyme had high activities towards 2-oxoaldehyde compounds, such as 3-DG, MG, and phenylglyoxal. This enzyme converted 3-deoxyglucosone (3-DG) to 3-deoxyfructose, and methylglyoxal (MG) to acetol.

Monoclonal antibodies of both the IgG and the IgM type were purified by hydroxylapatite HPLC (HA-HPLC) under very mild conditions. The IgM type antibodies, which were isolated from ascites fluid and separated from other proteins also by means of size exclusion HPLC. It was shown that the most frequently observed disadvantage of HA-HPLC, that is the relative short life of the columns (P. Steffen (1989) GIT Fachz. Lab., Suppl. 3/89 (Chromatogr.), 50-90), is due to microbial contamination rather than lower mechanical stability. In order to monitor column performance, a test was developed based on the use of standard proteins under isocratic separation conditions. This allows a direct comparison between the respective performances of columns made from different materials, hydroxylapatite or fluoroapatite, from different sources and with different particle sizes. A problem which often occurs with HA-HPLC in the case of IgM antibody isolation, namely precipitation of the antibodies at low salt concentrations at the beginning of a chromatographic run, was avoided by adding sodium chloride to both separation buffers.

Protein kinase C from rat brain and human neutrophils was chromatographically separated on a hydroxylapatite column connected to a high-performance liquid chromatography (HPLC) system. Protein kinase C from rat brain was separated into three peaks (types I, II, and III). In contrast, only two types were obtained from human neutrophils matching type II and type III in brain. In cell membranes from unstimulated human neutrophils type III was predominant, but mainly type II was translocated to the membranes upon stimulation with phorbolmyristatacetate (PMA). Both types were equivalently activated by phosphatidylserine, diolein, and magnium and presented identical Km for ATP.

In the effort to improve the performance of hydroxylapatite (HA) in removing lead ions from aqueous solutions, millimeter-sized granules with 50 % porosity were synthesized. Such HA particles, after drying at 100°C, or heating at 800°C or 1100°C, exhibited the specific surface areas of 50 m2 g−1, 25 m2 g−1, and 5 m2 g−1, respectively. It was found that heavy metal sorption capacity of HAs can be related to their surface area. Non-calcined granules were difficult to handle and easy to crush. Hardened granules showed heavy metal absorption on their outer surfaces. Absorption capacity of sintered HA particles towards lead was lower but adsorbed lead ions were spread inside the porous structure of HA granules more evenly. Under flow conditions, lead ions were captured by HA at a rate of 0.5 mg g−1 min−1. Small lead phosphate aggregates were released from the HA sorbent together with calcium ions. Size of the aggregates depended on the lead concentration and ranged from 1–50 μm in diameter; the aggregates could be removed by ultrafiltration. Results show that porous hardened HA granules can be used as an efficient phosphate source for the immobilization of lead ions from aqueous media. Organic ligands tend to interfere with the water purification procedure.

Two 'trimeric' allophycocyanin complexes could be isolated from the hemidiscoidal phycobilisomes of Rhodella violacea. AP = (alpha *AP alpha 2AP beta 2AP beta *AP) and APB = (alpha *AP alpha AP alpha APB beta 2AP beta *AP). Lc13.5APB. The isolation was performed by combined methods of gradient centrifugation, hydroxylapatite chromatography and 'native' polyacrylamide gel electrophoresis. AP showed the well-known spectral characteristics of allophycocyanin without linker polypeptide. APB is characterized by its long wavelength absorbing shoulder (675 nm) and fluorescence emission (682 nm), respectively. The existence of two low molecular linker polypeptides Lc12.5 and Lc13.5APB in the phycobilisomes of Rhodella violacea, their stoichiometric calculations and the localization of Lc13.5APB in allophycocyanin B facilitated the construction of a model of the phycobilisome core.

Bovine C5 has been isolated from fresh bovine serum by a five-step procedure: polyethylene glycol precipitation, sequential ion-exchange chromatography on DEAE-Sephacel and CM-Sephadex, hydroxylapatite chromatography, and affinity chromatography. The purified C5 was a protein of apparent molecular weight 202,000 +/- 9,000 composed of two chains: an alpha-chain of molecular weight 127,000 +/- 5,000 and a beta-chain of molecular weight 74,000 +/- 2,000. The alpha-chain was cleaved by Sepharose-CVF.Bb (a cobra venom factor (CVF)-induced C3/C5 alternative pathway convertase) in the absence of any C3 or C3b. The monocarboxylic acid form of K-76, a sesquiterpene compound isolated from the culture filtrates of Stachybotris complementi, inhibited the alternative pathway of bovine serum, and the inhibited hemolytic activity was restored, in a dose dependent manner, by bovine C5. This provided the basis for a C5 functional assay throughout the purification procedure. The purified C5 showed species specificity and was functionally distinct from bovine C3.

Multicatalytic proteinase (MCP) was solubilized from human erythrocyte membrane with 0.1% Triton X-100 and purified to homogeneity using a combination of DEAE-cellulose, hydroxylapatite, and Ultrogel AcA34 chromatographies. This membranous MCP had similar properties to MCP purified in parallel from the cytosol. Both MCPs had a molecular mass of 570 kDa, were composed of apparently nine subunits of 22-36 kDa and had trypsin- and chymotrypsin-like activities. These activities were latent and required heating for the induction. However, slight differences were observed in the effects of reagents (DFP, monoiodoacetic acid, Mg2+, and Ca2+) between membranous and cytosolic MCP. The amount of MCP identified on membranes was estimated to be three-quarters or one-half of that found in the cytosol based on its trypsin- or chymotrypsin-like activity, respectively.

Human insulin-like growth factor II (IGF-II) has been purified to homogeneity from bone which contained 10-15 times more IGF-II than insulin-like growth factor-I (IGF-I). After extraction of IGF-II by demineralization of human bone powder with 10% EDTA containing 4M guanidine HCl at pH 7.4, IGF-II was separated from IGF binding proteins by hydroxylapatite chromatography in the presence of 4M guanidine HCl. The hydroxylapatite unbound fraction containing IGF-II was purified by affinity chromatography using Sm 1.2. monoclonal antibodies, which bind both IGF-I and IGF-II. The final purification of IGF-II was achieved by FPLC mono S ion-exchange chromatography in which IGF-II was separated from IGF-I. Human IGF-II thus purified was shown to be pure by (1) HPLC reverse-phase chromatography, (2) SDS-PAGE, and (3) N-terminal amino acid sequence. From 300 g of bone, 0.18 mg IGF-II was obtained with an overall recovery of 42%. These studies demonstrate the usefulness of (1) bone as a source of IGF-II purification and (2) antibodies that cross-react with both IGF-I and IGF-II for affinity purification of IGFs.