Magnetoferritin: in vitro synthesis of a novel magnetic protein.

The iron storage protein ferritin consists of a spherical polypeptide shell (apoferritin) surrounding a 6-nanometer inorganic core of the hydrated iron oxide ferrihydrite (5Fe2O3.9H2O). Previous studies have shown that the in vitro reconstitution of apoferritin yields mineral cores essentially identical to those of the native proteins. A magnetic mineral was synthesized within the nanodimensional cavity of horse spleen ferritin by the use of controlled reconstitution conditions. Transmission electron microscopy and electron diffraction analysis indicate that the entrapped mineral particles are discrete 6-nanometer spherical single crystals of the ferrimagnetic iron oxide magnetite (Fe3O4). The resulting magnetic protein, "magnetoferritin," could have uses in biomedical imaging, cell labeling, and separation procedures.

Crystallization at Inorganic-organic Interfaces: Biominerals and Biomimetic Synthesis.

Crystallization is an important process in a wide range of scientific disciplines including chemistry, physics, biology, geology, and materials science. Recent investigations of biomineralization indicate that specific molecular interactions at inorganic-organic interfaces can result in the controlled nucleation and growth of inorganic crystals. Synthetic systems have highlighted the importance of electrostatic binding or association, geometric matching (epitaxis), and stereochemical correspondence in these recognition processes. Similarly, organic molecules in solution can influence the morphology of inorganic crystals if there is molecular complementarity at the crystal-additive interface. A biomimetic approach based on these principles could lead to the development of new strategies in the controlled synthesis of inorganic nanophases, the crystal engineering of bulk solids, and the assembly of organized composite and ceramic materials.

The association of different urinary proteins with calcium oxalate hydromorphs. Evidence for non-specific interactions.

It has been proposed that various urinary proteins interact specifically with different calcium oxalate hydromorphs and these interactions have important implications regarding the understanding of the onset and progress of kidney stone disease. Calcium oxalate monohydrate and dihydrate crystals were grown and characterised thoroughly to establish sample purity. These crystals were then incubated in artificial urine samples containing isolated urinary macromolecules. Crystal growth was prevented by saturating the incubation mix with calcium oxalate, and this was confirmed through electron microscopy and calcium measurements of the incubation mix. The surface interactions between the different calcium oxalate hydrates and urinary proteins were investigated by the use of Western blots and immunoassays. The same proteins, notably albumin, Tamm-Horsfall protein, osteopontin and prothrombin fragment 1, associated with both hydrates. There was a trend for more protein to associate with calcium oxalate dihydrate, and greater quantities of different proteins associated with both hydrates when Tamm-Horsfall protein was removed from the incubation mix. There is no evidence from this study to indicate that particular proteins interact with specific calcium oxalate hydrates, which in turn suggests that these protein-mineral interactions are likely to be mediated through non-specific charge interactions.

Biomineralization: new directions in crystal science.

Effective protocols for controlling crystal structure, size, and morphology attract considerable interest given the requirement for particles of modal size and shape in many areas of materials fabrication and the importance of crystallochemical selectivity in determining the exploitable properties of inorganic solids. For this reason biomineralization merits particular attention since many biominerals are deposited in a highly controlled manner to produce crystals which are uniformly sized and crystallographically unique. Studies of biominerals have revealed that while a complex array of strategies have evolved for regulating their formation, one feature is common to the biological paradigm; interactions between organized biopolymeric assemblies and the nascent inorganic solids play a pivotal role in controlling the crystallization process. In order to gain a better understanding of the molecular interactions which take place at organic-inorganic interface and address the fundamental chemical problems of biomineralization, a crystal chemical approach has been adopted. Organized organic assemblies (phospholipid vesicles, Langmuir monolayers, polypetide templates) of precise molecular design (head group identity, packing conformation, peptide sequence, etc.) were assayed for their effectiveness in controlling the nucleation and growth of inorganic solids. This work has established that through systematic changes in the nature of the organic matrix the size, crystallographic orientation, and growth of the mineral phase can be controlled. Critical to this process was the translation of specific molecular information at the organic-inorganic interface: epitaxial alignment, stereochemical complementarity, and electrostatic interactions were an essential feature of this recognition event.

The ultrastructure of the rat incisor odontoblast in organ culture.

Extracted incisors were dissected free of adherent soft tissue, split by a longitudinal incision lingually and the pulp sac carefully removed. The predentine-dentine pieces with attached odontoblasts were incubated for up to 24 h in culture medium in 5 per cent CO2, humid air at 37 degrees C. The cultures were recovered and processed for light microscopy and transmission electron microscopy. Examination of the explants showed a homogeneous layer of odontoblasts attached to the dentinal surface. The odontoblasts had a normal structure and nuclear polarity similar to that seen in vivo. There were no apparent degenerative changes.

An ultrastructural study of calcium phosphate formation in multilamellar liposome suspensions.

Calcium phosphate precipitation can be induced within liposomes containing buffered inorganic phosphate by the ionophore-mediated loading of calcium ions. Negative staining, positive staining for thin sectioning, and freeze-fracture electron microscopy were used to characterize these synthetic vesicles and to evaluate the liposome-mineral interactions resulting from apatite formation. Suspensions of phosphate (0-50 mM KH2PO4)-encapsulated liposomes were prepared from mixtures of phosphatidylcholine, dicetyl phosphate, and cholesterol in the molar ratios of 7:2:1. Precipitation reactions were initiated by first suspending the liposomes in a buffered solution containing calcium (1.3-2.2 mM Ca(NO3)2) and then adding the cationic ionophore X-537A. All experiments were carried out at 22 degrees C, pH 7.4, and 240 mosm. Transmission electron microscopical analysis showed that the liposome preparation consisted of multilamellar, multicompartmental vesicular structures. The liposomes were typically heterogeneous with respect to both the size and number of phospholipid bilayers surrounding the aqueous cores. In Ca-loaded liposomes, discrete clusters of apatite mineral were present within the lumen, and in close proximity to the inner lipid membranes. These nascent crystallites eventually penetrated the lipid envelope to provide a focus for external precipitation events. Crystalline apatite phases were not observed when the incubation conditions prevented intraliposomal precipitation. The de novo calcification of these liposomes had many features in common with the sequence of mineral deposition occurring in matrix vesicle-mediated calcification. These results reinforce the conclusions of earlier chemical and kinetic studies and further support the use of this system as an experimental model for examining the membrane-mineral interactions associated with tissue mineralization.

An ultrastructural study of the effects of acidic phospholipid substitutions on calcium phosphate precipitation in anionic liposomes.

A model membrane system was used to investigate the ability of specific membrane constituents to modulate the precipitation of calcium phosphate. Intraliposomal precipitation was induced in phosphate-encapsulated liposomes composed of 7:2:1 molar mixtures of phosphatidylcholine (PC), dicetyl phosphate (DCP), and cholesterol (Chol) by ionophore-supported (X-537A) Ca2+ uptake. Extraliposomal precipitation occurred when these reactions were initiated in metastable external solutions. In this case, the endogenously formed crystals penetrated through the enclosing lipid bilayers and seeded the external solution phase. Transmission electron microscopy (TEM) was used to monitor the effect of acidic phospholipids [phosphatidic acid (PA), phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidylglycerol (PG)] on the precipitation reactions when these molecular species were incorporated into the liposome membranes. Compared with the precipitation reactions in 7PC:2DCP:1Chol liposomes containing no acidic phospholipids, calcium phosphate formation in the presence of monoester phosphate (PA) and amino- (PS) phospholipids was inhibited. Analyses of the lipid-mineral interactions in PA-containing (10 mol%) liposomes revealed close physical contact between the small crystals of apatite and the inner lipid bilayers; there was only minimal extraliposomal precipitation. A few small crystals adhered to the external surfaces of the liposomes. In PS-containing liposomes, lipid-mineral interactions were dependent upon the DCP content of the lipid membrane. Discrete clusters of crystals formed within the interior aqueous compartment when intraliposomal precipitation was initiated in 7PC:2DCP:1Chol liposomes doped with up to 10 mol% PS. There was no evidence for specific associations between these crystals and the enclosing lipid bilayers.(ABSTRACT TRUNCATED AT 250 WORDS)

Modulation of calcium phosphate formation by phosphatidate-containing anionic liposomes.

Liposome prepared from 7:2:1 molar mixtures of phosphatidylcholine, dicetyl phosphate, and cholesterol to which 1-20 mole % dioleoylphosphatidic acid (DOPA) was added were used to examine the effect of membrane-bound monoester phosphatidate anions on calcium phosphate formation in aqueous solutions at 22 degrees C, pH 7.4 and 240 mOsm. Results showed that up to 20 mole % DOPA in the liposomal envelope did not initiate mineralization in solutions made metastable with 2.25 mM CaCl2 and 1.50 mM KH2PO4. Results alos revealed that precipitation induced in metastable solutions by the seeding action of intraliposomally formed mineral was measurably reduced with as little as 1 mole % DOPA and completely suppressed with 5 mole % DOPA. In contrast, 10 mole % concentrations of diester phosphate lipids either had no effect on extraliposomal precipitation (e.g., phosphatidylglycerol and phosphatidylinositol) or, as reported previously for phosphatidylserine) only partially reduced the amount of precipitate formed. Transmission electron microscopical analysis suggests that DOPA-containing lipid bilayers adhering to the seed crystals inhibited extraliposomal mineralization by encapsulating the crystals within the liposomes and/or by blocking potential growth sites on the crystal faces. The polar head group of DOPA, being more negatively charged and sterically less encumbered than diester phosphate ligands, most probably was responsible for this adherence of the lipid bilayers to the crystal surfaces.

Extraction of calcium containing crystals from synovial fluids and articular cartilage.

A protocol for extracting minerals from biological tissues and fluids has been developed. Synovial fluid (SF) and articular cartilage samples were treated with papain and sodium hypochlorite to extract and concentrate calcium containing crystals. The method was validated by testing the ability to reextract synthetic calcium pyrophosphate dihydrate and calcium hydroxyapatite crystals added to SF in an unchanged form. The extracted crystals were analyzed by infrared spectroscopy and X-ray powder diffraction. The concentration of both calcium pyrophosphate dihydrate and hydroxyapatite crystals in SF was found to be higher than published figures.

The hydroxyapatite ion activity product in acid solutions equilibrated with human enamel at 37 degrees C.

Five samples of powered enamel were each sequentially equilibrated 5 times at 37 degrees C with either 4 or 17 mmol/l H3PO4, in contact with air, and the ionic activity product for hydroxyapatite (IHA) estimated. There was evidence for preferential release of Mg and Na, especially in the first equilibrations. In two experiments, raised values of IHA were observed in the first equilibration but otherwise IHA was reasonable constant within experimental error and was much closer to the solubility product of hydroxyapatite than many previous estimates, mainly at 25 degrees C, suggest. The mean value of IHA for the majority fraction, averaged over all samples, was 1.7 (+/- 0.7) x 10(-58). Non-apatitic solids formed in all systems but solubility appeared nevertheless to be controlled by an apatitic phase: either the enamel mineral itself or apatite reprecipitated during the course of equilibration. High values of IHA reported previously may be due to use of conditions favouring dissolution only of more soluble factions or to metastability artifacts associated with control of solubility by non-apatitic phases.

The density and protein content of calcium oxalate crystals precipitated from human urine: a tool to investigate ultrastructure and the fractional volume occupied by organic matrix.

One of the key debates in biomineralisation studies is the extent to which components of the organic matrix become occluded into the crystal lattice during growth. Here, the relationship between protein content and density of calcium oxalate crystals grown in human urine has been investigated in order to determine which fraction of crystal volume is non-mineral. The density of crystals varied from 1.84 to 2.08 g/cm3 while the protein content ranged from 0.1 to 2.1% (w/w). There was an inverse relationship between measured density and protein content which was qualitatively and quantitatively consistent with predictions based on reasonable densities for the mineral and non-mineral components. The coefficients of the fitted equation suggest that, at 2% protein (w/w), the volume of non-mineral would be 5.0% (v/v). The density values we observed are incompatible with fractional volumes of 20%. The results confirm that the occlusion of a small but possibly significant amount of protein into a crystal lattice is possible, but cast doubt on the hypothesis that protein acts as a major intracrystalline ultrastructural element. Moreover, the methodology developed for this study offers a simple and robust method for interrogating organic/inorganic associations in a range of biological and medical systems.

Formation of brushite, monetite and whitlockite during equilibration of human enamel with acid solutions at 37 degrees C.

The residues of 5 samples of powdered human enamel, each subjected to 5 sequential equilibrations at 37 degrees C with either 17 or 4 mmol/l phosphoric acid, were examined microscopically. With 17 mmol/l acid, both brushite and monetite were found after 1 equilibration but, after further equilibrations, brushite was no longer present and the abundance of monetite crystals increased. Formation of monetite probably contributed to the lower metastability of this system compared to similar low-pH systems at 25 degrees C, where monetite does not form. Neither brushite nor monetite were present after equilibration with 4 mmol/l acid. Whitlockite was identified by transmission electron microscopy and electron diffraction in all residues. In the 4 mmol/l systems, the ionic activity product (IMWH) for magnesium whitlockite, Ca9Mg(HPO4)(PO4)6, became constant after 1-3 equilibrations, at a mean value of 3.6 (+/-0.51 SE).10(-105), which may reflect saturation with respect to this solid. For the 17 mmol/l systems, higher values of IMWH, and supersaturation with respect to monetite, were interpreted as evidence for persistent metastability due to slow crystal growth of whitlockite and monetite. It is concluded that neither brushite nor monetite are likely to form within carious lesions, but the results are consistent with the known association of whitlockite with caries.

Ultrastructure, morphology and crystal growth of biogenic and synthetic apatites.

The morphology, structure and crystal growth of apatite crystals isolated from calcified turkey tendon and synthetic carbonated apatites have been examined using high resolution transmission electron microscopy. The biogenic apatite consisted of small (35 x 20 x 5 nm) platelike crystals. Despite their irregular shape and ill-defined edges, individual particles were single domain crystals. Lattice images recorded from isolated turkey tendon crystals indicated that the crystallographic c-axis (0001) of apatite lies in the plane of the plate and parallel to the length of the crystallites. Lattice images suggested that the top face corresponds to the (1100) face of carbonated apatite. Lattice fringes observed in platelike crystallites viewed from the side corresponded to the projection of the apatite structure viewed along the [1120] direction. Thus, it can be argued that crystal growth is constrained along the [1100] direction, extends laterally along the [1120] direction, and is maximal along the [0001] direction. This latter direction is aligned with the collagen fiber axis. A mean length to width ratio (1.7) was determined by systemically measuring the maximum distances parallel and perpendicular to the c-axis identified from lattice images of the crystals. Similar information was obtained from lattice images of crystals located in collagen fibres. This confirmed that the morphological and structural features of isolated turkey tendon apatite crystals correlate directly with the in vivo crystallochemical characteristics of apatite. Crystals of synthetic carbonated apatite prepared at 37 degrees C were also platelike and, although generally much larger, had length to width ratios comparable with the turkey tendon apatite. The synthetic carbonated apatites were noticeably more sensitive to radiolytic damage than the turkey tendon crystals. The crystallographic c-axis of the inorganic particles was aligned parallel with the long, physical axis of the plate and the top face was identified as (1100). Similar data were also obtained from noncarbonated synthetic apatite samples. The results of the present study offer critical information about the crystal growth of individual carbonated apatite crystals in calcified turkey tendon and its relationship to the morphology of the crystallites. As similar growth characteristics are expressed in synthetic analogues, the data bring into question the putative regulatory role of the collagen-based matrix upon the nucleation and growth of biogenic apatite.(ABSTRACT TRUNCATED AT 400 WORDS)

Two bovine models of osteogenesis imperfecta exhibit decreased apatite crystal size.

In recent years advances have been made in detailing the changes in both collagen and noncollagenous proteins caused by a variety of mutations leading to osteogenesis imperfecta. Much less, however, is known about the mineral phase in the affected bone. In this report, we measured the crystallinity of the apatite in bovine OI bone. Line broadening of the 002 reflection (which estimates changes in the long or c axis of the crystals) and of the 310 reflection (which estimates changes in the thickness of the crystals) both show large decreases (30 and 35% respectively). Transmission electron micrograph measurements indicate that these changes were most probably a result of smaller crystals. No decrease in the ash weight of the bone was observed.

Organ-specific crystalline structures of ferritin cores in beta-thalassemia/hemoglobin E.

The cores of ferritins isolated from different organs of human subjects with beta-thalassemia/hemoglobin E (beta-thal/HbE) disease have different size distributions and crystallinities depending on the source organ. These patients have not been treated by hypertransfusion regimen or iron chelation therapy. beta-Thal/HbE spleens and livers yield ferritin cores which are less crystalline than those isolated from normal spleens and livers, reflecting the more rapid deposition of iron in the diseased state. Ferritins isolated from the hearts and pancreases of beta-thal/HbE subjects were found to have larger, more crystalline cores than those from the beta-thal/HbE livers and spleens, possibly as a consequence of the role of the heart and pancreas as long-term iron deposition sites in this iron overload pathology.

Controlled Biomineralization of Magnetite (Fe(inf3)O(inf4)) and Greigite (Fe(inf3)S(inf4)) in a Magnetotactic Bacterium.

A slowly moving, rod-shaped magnetotactic bacterium was found in relatively large numbers at and below the oxic-anoxic transition zone of a semianaerobic estuarine basin. Unlike all magnetotactic bacteria described to date, cells of this organism produce single-magnetic-domain particles of an iron oxide, magnetite (Fe(inf3)O(inf4)), and an iron sulfide, greigite (Fe(inf3)S(inf4)), within their magnetosomes. The crystals had different morphologies, being arrowhead or tooth shaped for the magnetite particles and roughly rectangular for the greigite particles, and were coorganized within the same chain(s) in the same cell with their long axes along the chain direction. Because the two crystal types have different crystallochemical characteristics, the findings presented here suggest that the formation of the crystal types is controlled by separate biomineralization processes and that the assembly of the magnetosome chain is controlled by a third ultrastructural process. In addition, our results show that in some magnetotactic bacteria, external environmental conditions such as redox and/or oxygen or hydrogen sulfide concentrations may affect the composition of the nonmetal part of the magnetosome mineral phase.