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Ribosome inactivating proteins (RIPs) are plant proteins with enzymatic activity identified as rRNA N-glycosidase (EC 3.2.2.22), which cleaves the N-glycosidic bond of a specific adenine on the ricin/sarcin region of rRNA, thus causing inhibition of protein synthesis. They also depurinate extensively DNA and other polynucleotides. The three-dimensional structure of dianthin 30, a type 1 (single-chain) RIP of Dianthus caryophyllus (leaves), is now described at 1.4 angstroms, a resolution never achieved before for any RIP. The fold typical of RIPs is conserved, despite some differences in the loop regions. The general structure comparison by superimposed alpha-carbon (249 atoms) and the sequence alignment by structure for dianthin 30 and saporin-S6 give a root mean square deviation of 0.625 angstroms. Despite the differences reported for the biological activities of the two RIPs, their structures fit quite well and both show a protein segment containing strands beta7, beta8, and beta9 shorter than other RIPs. However, the surface electrostatic potential in the active site region neatly distinguishes dianthin 30 from saporin-S6. The possible relationship between the charge distribution and the behavior of the proteins toward different substrates is discussed.

Chloroplast glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of higher plants uses both NADP(H) and NAD(H) as coenzyme and consists of one (GapA) or two types of subunits (GapA, GapB). AB-GAPDH is regulated in vivo through the action of thioredoxin and metabolites, showing higher kinetic preference for NADPH in the light than in darkness due to a specific effect on kcat(NADPH). Previous crystallographic studies on spinach chloroplast A4-GAPDH complexed with NADP or NAD showed that residues Thr33 and Ser188 are involved in NADP over NAD selectivity by interacting with the 2′-phosphate group of NADP. This suggested a possible involvement of these residues in the regulatory mechanism. Mutants of recombinant spinach GapA (A4-GAPDH) with Thr33 or Ser188 replaced by Ala (T33A, S188A and double mutant T33A/S188A) were produced, expressed in Escherichia coli, and compared to wild-type recombinant A4-GAPDH, in terms of crystal structures and kinetic properties. Affinity for NADPH was decreased significantly in all mutants, and kcat (NADPH) was lowered in mutants carrying the substitution of Ser188. NADH-dependent activity was unaffected. The decrease of kcat/Km of the NADPH-dependent reaction in Ser188 mutants resembles the behaviour of AB-GAPDH inhibited by oxidized thioredoxin, as confirmed by steady-state kinetic analysis of native enzyme. A significant expansion of size of the A4-tetramer was observed in the S188A mutant compared to wild-type A4. We conclude that in the absence of interactions between Ser188 and the 2′-phosphate group of NADP, the enzyme structure relaxes to a less compact conformation, which negatively affects the complex catalytic cycle of GADPH. A model based on this concept might be developed to explain the in vivo light-regulation of the GAPDH.

Photosynthetic glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of Spinacia oleracea belongs to a wide group of GAPDHs found in most organisms displaying oxygenic photosynthesis, including cyanobacteria, green and red algae, and higher plants. As a major catalytic difference with respect to glycolytic GAPDH, photosynthetic GAPDH exhibits dual cofactor specificity toward pyridine nucleotides with a preference for NADP(H). Here we report the crystal structure of NAD-complexed recombinant A(4)-GAPDH (NAD-A(4)-GAPDH) from Spinacia oleracea, expressed in Escherichia coli. Its superimposition onto native A(4)-GAPDH complexed with NADP (NADP-A(4)-GAPDH) pinpoints specific conformational changes resulting from cofactor replacement. In photosynthetic NAD-A(4)-GAPDH, the side chain of Asp32 is oriented toward the coenzyme to interact with the adenine ribose diol, similar to glycolytic GAPDHs (NAD-specific). On the contrary, in NADP-A(4)-GAPDH Asp32 moves away to accommodate the additional 2'-phosphate group of the coenzyme and to minimize electrostatic repulsion. Asp32 rotation is allowed by the presence of the small residue Ala40, conserved in most photosynthetic GAPDHs, replacing bulky amino acid side chains in glycolytic GAPDHs. While in NADP-A(4)-GAPDH two amino acids, Thr33 and Ser188, are involved in hydrogen bonds with the 2'-phosphate group of NADP, in the NAD-complexed enzyme these interactions are lacking. The crystallographic structure of NAD-A(4)-GAPDH highlights that four residues, Thr33, Ala40, Ser188, and Ala187 (Leu, Leu, Pro, and Leu respectively, in glycolytic Bacillus stearothermophilus GAPDH sequence) are of primary importance for the dual cofactor specificity of photosynthetic GAPDH. These modifications seem to trace the minimum evolutionary route for a primitive NAD-specific GAPDH to be converted into the NADP-preferring enzyme of oxygenic photosynthetic organisms.

Chemically modified mica sheets have been tested as heterogeneous nucleant surfaces for lysozyme, concanavalin A and thaumatin. Smooth mica surfaces with reduced hydrophilic properties and different density of ionisable groups have been prepared by a silanisation reaction using mixtures of n-propyltriethoxysilane and 3-aminopropyltriethoxysilane in different percentages starting from 0 to 100% of aminosilane. The crystallisation experiments were carried out with the hanging drop vapour diffusion technique. The results suggest that these mica surfaces act as heterogeneous nucleant agents, whose effectiveness is due to non-specific attractive and local interactions between charged residues of the protein and the ionisable groups on the mica surfaces.

Composites of beta-chitin with calcium carbonate polymorphs were prepared by precipitation of the mineral into a chitin scaffold by means of a double diffusion system. The beta-chitin was obtained from the pen of the Loligo sp. squid. The three main polymorphs of calcium carbonate: aragonite, calcite and vaterite, were observed. Their location within the matrix is a function of the polymorph. The supersaturation inside the compartmentalized space in the chitin governs the location and polymorphism of the crystals.

Here, we report the first crystal structure of a photosynthetic glyceraldehyde-3-phosphate dehydrogenase (GAPDH) complexed with NADP. The enzyme, purified from spinach chloroplasts, is constituted of a single type of subunit (A) arranged in homotetramers. It shows non-regulated NADP-dependent and NAD-dependent activities, with a preference for NADP. The structure has been solved to 3.0 A resolution by molecular replacement. The crystals belong to space group C 222 with three monomers in the asymmetric unit. One of the three monomers generates a tetramer using the space group 222 point symmetry and a very similar tetramer is generated by the other two monomers, related by a non-crystallographic symmetry, using a crystallographic 2-fold axis. The protein reveals a large structural homology with known GAPDHs both in the cofactor-binding domain and in regions of the catalytic domain. Like all other GAPDHs investigated so far, the A 4 -GAPDH belongs to the Rossmann fold family of dehydrogenases. However, unlike most dehydrogenases of this family, the adenosine 2′-phosphate group of NADP does not form a salt-bridge with any positively charged residue in its surroundings, being instead set in place by hydrogen bonds with a threonine residue belonging to the Rossmann fold and a serine residue located in the S-loop of a symmetry-related monomer. While increasing our knowledge of an important photosynthetic enzyme, these results contribute to a general understanding of NADP versus NAD recognition in pyridine nucleotide-dependent enzymes. Although the overall structure of A 4 -GAPDH is similar to that of the cytosolic GAPDH from bacteria and eukaryotes, the chloroplast tetramer is peculiar, in that it can actually be considered a dimer of dimers, since monomers are bound in pairs by a disulphide bridge formed across Cys200 residues. This bridge is not found in other cytosolic or chloroplast GAPDHs from animals, bacteria, or plants other than spinach.

A banded spherulite of bacterial poly(3-hydroxybutyrate) (band spacing 120 μm) was linearly scanned along the radial direction in 3 μm steps by means of microfocus X-rat diffraction using a synchroton source with a beam diameter of 3 μm. A large number of X-ray patterns was collected inside each band; Those taken at the center fo the bands represented the two limiting diffraction patterns from which the exact orientation of the unit cell was inferred. The intensity of the reflections (020) and (002) , taken as indicators of parellelism to the X-ray beam of the unit cell's c- or b-axis, respectively, changed periodically along the spherulite radius, alternating maximal with zero-intensity zones. When the reflection (020) showed a maximum, the reflection (002) was practically nil and vice versa. This behavior clearly shows that the unit cell's mean position smoothly rotates around the a-axis with increasing distance from the spherulite center. The identity of the cell rotation period with the band spacing derived from optical microscopy observations provides structural evidence that the extinction bands originate from a regular twisting of the lamellar crystals during their growth.

Polymeric films containing ionizable groups, such as sulfonated polystyrene, cross-linked gelatin films with adsorbed poly- l -lysine or entrapped poly- l -aspartate and silk fibroin with entrapped poly- l -lysine or poly- l -aspartate, have been tested as heterogeneous nucleant surfaces for proteins. Concanavalin A from jack bean and chicken egg-white lysozyme were used as models. It was found that the crystallization of concanavalin A by the vapor diffusion technique, is strongly influenced by the presence of ionizable groups on the film surface. Both the induction time and protein concentration necessary for the crystal nucleation decrease whereas the nucleation density increases on going from the reference siliconized cover slip to the uncharged polymeric surfaces and even more to the charged ones. Non-specific attractive and local interactions between the protein and the film surface might promote molecular collisions and the clustering with the due symmetry for the formation of the crystal nuclei. The results suggest that the studied polymeric film surfaces could be particularly useful for the crystallization of proteins from solutions at low starting concentration, thus using small quantities of protein, and for proteins with very long crystallization time.

The control of the polymorphism and architectural crystal assembly of calcium carbonate minerals in gels formed by means of collagenous matrices with entrapped polypeptides is reported. It has been observed that the calcium carbonate polymorphic selectivity is related to the local supersaturation within the microenvironment where nucleation and growth occur. This crucial parameter is controlled in terms of the entrapped additive concentration and of the tailoring of the biopolymeric scaffold by mechanical deformation. Specific orientation effects and crystal aggregation of the mineral phases can be controlled either by the charged polypeptide with a beta structure or by the structural organization of the triple helical stretches in the collagenous matrix. This results in the growth and assembly of crystals into desired shapes and sizes by molecular recognition at a definite crystal face or by the control of the organic macromolecular microenvironment fit in the emerging area of biologically inspired approach to structured inorganic materials with appropriate physical and chemical properties.

Xerogels, obtained by evaporating a water solution of gelatin, were used as templates for the growth of crystal assemblies of calcium phosphate. Octacalcium phosphate (OCP) crystals were grown inside the gelatin xerogel by diffusion of solutions of calcium and phosphate ions into the gelatin films from opposite ends. The structure, morphology and orientation of the crystals grown on the surfaces and inside the gelatin films were studied by optical and scanning electron microscopy and X-ray diffraction. OCP was the unique detectable crystalline phase. The crystals preferentially grew as thin plates elongated along their c axes with large {100} faces parallel to the sheets of gelatin films. When the gelatin films were uniaxially deformed the OCP crystals grew with their c axes almost parallel to the direction of deformation. According to a mechanism of templated nucleation, a structural model for the ordered growth of OCP on the portions of triple helical collagen molecules has been proposed.

X-ray diffraction and polarized optical microscopy investigations were carried out on thin sections of rabbit tibia in order to study the morphological organization of the structural components of this tissue, which often is utilized to test bone response to implants. In the optical microscope, the lateral face as well as the lateral portion of the caudal face exhibit a lamellar structure with an alternation of dark and bright lamellae running parallel to the long axis of the tibia. In contrast, both in the medial face and in the medial portion of the caudal face there are numerous osteonic structures. In spite of the complexity of this morphological organization, the results of small- and high-angle X-ray diffraction analyses indicate that the structural relationship between collagen fibrils and inorganic crystals is quite similar to that observed in single osteons and allows evaluation of the orientation of the two main structural components. Both collagen fibrils and apatitic crystallites are preferentially oriented parallel to the long axis of the tibia. The degree of orientation is greater in the thickness than in the plane of the lamellae, suggesting that collagen fibrils and inorganic crystallites lie preferentially in the plane of the lamellae, where they follow an oblique course. The degree of orientation of the apatitic crystallites is higher in the lateral face than in the medial and caudal faces, in agreement with the optical microscopic images. The results provide information that must be taken into account when evaluating the structural modifications of bone due to the insertion of a prosthetic device.

The results of a study on the fine structural distortion due to the two previously observed types of degradation in cyclically loaded single osteons (i.e., stiffness degradation and pinching effect) are presented. Fully calcified longitudinal and alternate osteons were isolated from 350-microns-thick longitudinal sections of human femoral cortical bone. The samples were prepared from 500-microns-long central cylindrical portions of an osteon, whose two ends were penetrating into rectangular lugs for fixation to an electromechanical device that cyclically loaded the samples. This device was connected to a microwave micrometer and a recorder. The structural distortions induced by cyclic loading were investigated by high- and low-angle X-ray diffraction on conventional and synchrotron radiation sources. Cyclic loading results in a reduction in the degree of orientation of apatite crystallites, especially in longitudinal osteons, in which the most abundant longitudinal lamellae are not protected against buckling by transverse lamellae as they are in alternate osteons. In contrast, the degree of orientation of collagen fibrils does not seem to be affected by cycling loading in the two osteon types, possibly because the disorientation of collagen fibrils is, within limits, a reversible process. Finally, the contrast between the disorientation of inorganic crystallites and the apparently unaltered distribution of collagen fibrils suggests that the degradation of cyclically loaded osteons may be due to a separation of the crystallites from the fibrils.

X-ray diffraction, infrared spectroscopy and chemical investigations have been carried out on the inorganic phases from rat cortical and trabecular bone. Although both inorganic phases consist of poorly crystalline B carbonated apatite, several significant differences have been observed. In particular, trabecular bone apatite displays reduced crystallite sizes, Ca/P molar ratio, and carbonate content, and exhibits a greater extent of thermal conversion into β-tricalcium phosphate than cortical bone apatite. These differences can be related to the different extents of collagen posttranslational modifications exhibited by the two types of bone, in agreement with their different biological functions.

The results of X-ray diffraction and morphological investigations carried out on calcium carbonate crystals grown from solutions containing different Mg 2+ /Ca 2+ molar ratios and polyelectrolytes are reported. Polyelectrolytes containing charged groups important in biomineralization processes are used. Polymers containing sulphate (carrageenans K and J) and carboxylate (polyacrylate, poly-L-glutamate and poly-L-aspartate) charged groups do not influence the magnesium substitution for calcium in calcite. When the Mg 2+ /Ca 2+ molar ratio is high, the control of polymorph type seems to be dictated almost exclusively by magnesium ions independent of the presence of poly-L-glutamate, polyacrylate or carrageenans. In these conditions only aragonite crystallizes. The behaviour of poly-L-aspartate, which assumes a β-sheet structure for long stretches of chain in the presence of calcium ions, is remarkably different. Since, in the presence of this polymer, calcite crystallizes together with aragonite also when Mg 2+ /Ca 2+ molar ratio is high, it can be assumed that the β-sheet structure of poly-L-aspartate plays an important role in determining the calcite crystallization in the presence of magnesium ions.

X-ray diffraction, infrared absorption, and chemical investigations have been carried out on hydroxyapatite synthesized in the presence of different magnesium concentrations in solution. Magnesium inhibits the crystallization of hydroxyapatite through a reduction of Ca/P molar ratio and crystal sizes of apatite. The reduction of the crystal sizes is also very great for very low magnesium content and increases on increasing magnesium concentration in solution up to 35 Mg atom percent with respect to the total metal ions. The samples are completely amorphous between 35 and 50 Mg atom percent. For higher magnesium concentration different crystalline phases are formed. The results of the x-ray powder pattern fitting indicate that the HA crystal structure at most hosts magnesium amounts of about seven percent. Magnesium substitutes only the calcium atoms which form the channels containing the hydroxy ions. Since magnesium content is much smaller than that found in the solid phase, the greatest amount of magnesium must not be lattice bound. The extent of hydroxyapatite conversion into magnesium substituted β-tricalcium phosphate on heat treatment appears strongly related to magnesium content of the apatitic solid phase. On the basis of these results, the key role of magnesium on the crystallization, crystal growth, and thermal stability of hydroxyapatite has been used to explain the relevant properties of biological apatites.

Continuous series of solid solutions of lead and calcium hydroxyapatites have been prepared by solid-state reaction as well as from aqueous solutions and characterized by X-ray diffraction and IR spectroscopic analyses. While the lattice constant a of the solid solutions varies linearly with composition, the variation of the lattice constant c does not follow Vegard's law but shows a discontinuity at about 50% lead atom content. A similar discontinuity has been observed in the variation of the IR frequencies with composition. Although lead incorporation seems to destabilize the apatite structure, the solid solutions prepared from aqueous media at a lead content around 50% exhibit a high thermal stability. The variation of c, thermal stability, as well as the frequencies and intensities of the IR bands of lead–calcium hydroxyapatite are interpreted on the basis of the inhomogeneous lead distribution in the two non-equivalent cation sites of the hydroxyapatite structure.

X-ray diffraction, infrared absorption spectroscopy, and chemical investigation have been carried out on deproteinated samples of turkey leg tendon at different degrees of calcification. The inorganic phase consists of poorly crystalline B carbonated apatite. On increasing calcification, the apatite crystal size, as well as its thermal stability, increase while the relative magnesium content is reduced. On the other hand, synchrotron X-ray diffraction data clearly indicate that apatite lattice parameters do not change as the crystals get larger. At the last stage of calcification the crystal size, chemical composition, and thermal conversion of the apatite crystallites approximate those of bone samples, which have been examined for comparison. The results provide a quantitative relationship between relative magnesium content and extent of apatite conversion into B-tricalcium phosphate by heat treatment. Furthermore, they suggest that the smaller crystallites laid down inside the gap region of the collagen fibrils are richer in magnesium than the longer ones that fill the space between collagen fibrils.

The three-dimensional structures of two type 1 RIPs, bouganin and lychnin, has been solved. Their adenine polynucleotide glycosylase activity was also determined together with other known RIPs: dianthin 30, PAP-R, momordin I, ricin A chain and saporin-S6. Saporin-S6 releases the highest number of adenine molecules from rat ribosomes, and poly(A), while its efficiency is similar to dianthin 30, bouganin and PAP-R on herring sperm DNA. Measures of the protein synthesis inhibitory activity confirmed that saporin-S6 is the most active. The overall structure of bouganin and lychnin is similar to the other considered RIPs and the typical RIP fold is conserved. The superimpositioning of their Cα atoms highlights some differences in the N-terminal and C-terminal domains. A detailed structural analysis indicates that the efficiency of saporin-S6 on various polynucleotides can be ascribed to a negative electrostatic surface potential at the active site and several exposed positively charged residues in the region around that site. These two conditions, not present at the same time in other examined RIPs, could guarantee an efficient interaction with the substrate and an efficient catalysis. © 2009 Elsevier Inc. All rights reserved.

Calcified leg flexor tendons in which the inorganic phase content had been lowered by progressive demineralization were studied by small angle X-ray diffraction and thermogravimetry. The X-ray diffraction results agree very well with the data previously obtained on calcified turkey tendon indicating that the method used to decalcify tendons provides good correspondence with the process of calcification. Up to five thermal processes can be detected in the thermogravimetric scans: (1) water release; (2) collagen decomposition; (3 and 4) combustion of the residual organic components; (5) carbonate removal from the apatitic phase. The temperature of collagen decomposition decreases at lower inorganic phase content in agreement with the higher thermal stability of calcified collagen fibrils compared with uncalcified ones. The decrease of collagen thermal stability upon decalcification is paralleled by a decrease of the structural order of the collagen fibrils as indicated by small angle X-ray diffraction data. Decalcification down to about 40% wt of inorganic phase does not significantly alter the inorganic blocks that are regularly arranged inside the gap zone of the collagen. Further removal of inorganic phase down to about 15% wt provokes a variation of the intensity distribution of the small angle meridional reflections that can be ascribed to a reduction of the mean height of the inorganic blocks. At inorganic phase contents below 15% wt the gap region is more free to contract upon air drying as a result of the reduction of the mean length of the inorganic blocks.

Collagen structure and collagen-apatite structural relationship has been investigated in human anterior ligament, where the mineral deposition occurs on collagen fibrils morphologically different from those of bone and tendons. Ultrastructural observations made on replicas of cryoprotected and freeze fractured uncalcified samples display a "helicoidal" morphology of the collagen fibrils. X-ray diffraction analysis carried out using conventional and synchrotron radiation sources revealed that the D-axial spacing is 65.0 nm and the electron density distribution inside the repeating period is very similar to those of tendon collagen in the same conditions of hydration. The short D-period can be interpreted as due to a greater angle of molecular crimping and/or molecular tilt compared to that of tendon. Air drying does not cause any appreciable variation in the D-axial period and induces an increase of the gap/overlap ratio that can be ascribed to telopeptide disorder. In spite of the different morphology of the collagen fibrils, the structural relationship between collagen and the mineral phase in calcified ligament is very close to that observed in bone and tendons. The apatitic phase is laid down in blocks along the collagen fibrils with the same axial periodicity, D = 65.0 nm, as that of uncalcified collagen fibrils. The mean height of the mineral blocks, which are 0.45D long, is even higher than in bone and masks any further fluctuation of the electron density due to the organic matrix.

Chloroplast glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a light-regulated, NAD(P)H-dependent enzyme involved in plant photosynthetic carbon reduction. Unlike lower photosynthetic organisms, which only contain A 4 –GAPDH, the major GAPDH isoform of land plants is made up of A and B subunits, the latter containing a C-terminal extension (CTE) with fundamental regulatory functions. Light-activation of AB–GAPDH depends on the redox state of a pair of cysteines of the CTE, which can form a disulfide bond under control of thioredoxin f , leading to specific inhibition of the NADPH-dependent activity. The tridimensional structure of A 2 B 2 –GAPDH from spinach chloroplasts, crystallized in the oxidized state, shows that each disulfide-containing CTE is docked into a deep cleft between a pair of A and B subunits. The structure of the CTE was derived from crystallographic data and computational modeling and confirmed by site-specific mutagenesis. Structural analysis of oxidized A 2 B 2 –GAPDH and chimeric mutant [A+CTE] 4 –GAPDH revealed that Arg-77, which is essential for coenzyme specificity and high NADPH-dependent activity, fails to interact with NADP in these kinetically inhibited GAPDH tetramers and is attracted instead by negative residues of oxidized CTE. Other subtle changes in catalytic domains and overall conformation of the tetramers were noticed in oxidized A 2 B 2 –GAPDH and [A+CTE] 4 –GAPDH, compared with fully active A 4 –GAPDH. The CTE is envisioned as a redox-sensitive regulatory domain that can force AB–GAPDH into a kinetically inhibited conformation under oxidizing conditions, which also occur during dark inactivation of the enzyme in vivo .

Composites of β-chitin with octacalcium phosphate (OCP) or hydroxylapatite (HAP) were prepared by precipitation of the mineral into a chitin scaffold by means of a double diffusion system. The β-chitin was obtained from the pen of the Loligo sp. squid. Only oriented precipitation of OCP was observed. The OCP crystals with the usual form of (001) blades grow inside chitin layers preferentially oriented with the {100} faces parallel to the surface of the squid pen and were more stable to the hydrolysis to HAP with respect to that precipitated in solution. Reasons are given why mechanical factors are thought to be the predominant cause for the orientation of the OCP crystals with the a -axis almost normal to the chitin fibers. We conclude that in these in vitro experiments the compartmentalized space in the chitin governs the orientation of the crystals, even if epitaxial factors may play a role in the nucleation processes.

The thermal conversion of octacalcium phosphate into hydroxyapatite has been investigated by a crystallographic, thermogravimetric, and calorimetric study. The conversion of octacalcium phosphate takes place through the remotion of three of its five water molecules and yields a poor crystalline apatitic phase. The three water molecules are lost in two steps. The first one, which is reversible, corresponds to the remotion of one water molecule and induces a slight contraction of the unit cell of OCP. The successive remotion of two water molecules, which provokes the structural conversion of OCP into apatite, is in irreversible process. The mechanism of the water loss of OCP is explained in terms of its crystal structure.

Magnesium calcite with unusual magnesium contents up to 14 mol% crystallizes from water–alcohol mixtures. Morphological changes result from interactions of solvent and Mg2+ with specific planes of the growing crystals.

Continuous series of solid solutions of lead and calcium hydroxyapatites have been prepared by solid-state reaction as well as from aqueous solutions and characterized by X-ray diffraction and IR spectroscopic analyses. While the lattice constant a of the solid solutions varies linearly with composition, the variation of the lattice constant c does not follow Vegard's law but shows a discontinuity at about 50% lead atom content. A similar discontinuity has been observed in the variation of the IR frequencies with composition. Although lead incorporation seems to destabilize the apatite structure, the solid solutions prepared from aqueous media at a lead content around 50% exhibit a high thermal stability. The variation of c, thermal stability, as well as the frequencies and intensities of the IR bands of lead–calcium hydroxyapatite are interpreted on the basis of the inhomogeneous lead distribution in the two non-equivalent cation sites of the hydroxyapatite structure.

The distribution and orientation of collagen fibres in unstretched and biaxially stretched pig aortic media have been determined by an X-ray diffraction technique in order to analyse the reorientation of collagen fibres in response to the presence of a notch in biaxially stretched samples. The results show that collagen fibres align themselves following the force trajectories. This results in preferential fibre orientation perpendicular to the advancing crack tip, in agreement with the theoretical predictions of stress concentration effect due to the presence of an elliptical notch in an elastic plate.

When isolated osteon samples are submitted to wide-angle X-ray diffraction, it is not possible to detect any preferential orientation of the hydroxyapatite crystallites of the lamellae with transversally arranged fiber bundles. So a complete and exhaustive X-ray diffraction analysis of an osteon needs adequately prepared osteonic subunits. For the present investigation, 2 types of samples were prepared from longitudinal and alternate osteons: osteonic radial sections and isolated straightened transversal lamellae. An X-ray diffraction microcamera has been used with a rotating anode X-ray generator. In accordance with the data provided by the polarizing microscope, the orientation of crystallites runs parallel to the osteon axis in longitudinally structured osteons, whereas in alternate osteons the orientation changes by about 90 degrees in successive lamellae. Neither crystallites associated with the collagen fibrils that run alongside the osteocyte canaliculi nor those associated with the fibrils that run transversally in longitudinally structured osteons are revealed by X-ray diffraction, because there are so few of them.

Structure d'hydroxyapatites obtenus a 1173 K par reaction a l'etat solide pour differentes concentrations en Pb substituant Ca (20, 45, 80% d'atomes Pb). Les atomes Pb preferent les sites (2) de la structure apatite

Octacalcium phosphate (OCP) and brushite (DCPD) have been proposed as precursor phases in the precipitation of hydroxyapatite (HAP) from supersaturated solutions. We have investigated the interaction of Mg 2+ , Pb 2+ , Sr 2+ , CO 3 2− , and F − with OCP, DCPD, and HAP under conditions of cyclic pH fluctuation. OCP converts completely into HAP when submitted to fluctuation either in the range of pH 4–7 or 4–9, while only in this last range DCPD converts partially into HAP. Pb 2+ and Sr 2+ do not greatly affect the conversion of OCP and DCPD into HAP. On the other hand, these conversions are promoted by the presence of F − in solution and inhibited by Mg 2+ and C0 3 2− , which favor the DCPD phase.

The structural organization of the collagen fibres in pig descending aortic media layer was studied by X-ray diffraction at various distending presusres. The degree of orientation of the collagen fibres along the circumference was evaluated. Furthermore, the variation of the mechanical behaviour of the aortic media layer was investigated as a function of the amount and distribution of collagen fibres in different portions along the artery. The results indicate that according to the model of Wolinsky and Glagov collagen is the effective structural component which bears most of the stressing forces while the elastic matrix distributes them uniformly.

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

Many natural collagen containing materials are highly extensible composites and their mechanical behaviour will depend on the amount of collagen fibres present, the mechanical properties of the fibres and their distribution and orientation. A characteristic feature of these materials is that the fibrous collagen network can change orientation during stretching and hence the mechanical response of the tissues is a non-linear function of stress. In order to study the effect of fibre orientation, samples of carotid artery have been prestrained by given amounts in the hydrated state and then allowed to dry. The mean orientation of the fibres has been derived using pole figures obtained from X-ray diffraction measurements and Young's modulus has been calculated in the direction of fibre reorientation and compared with the experimental measurements. The results obtained indicate that X-ray diffraction techniques can be used for the study of the mechanical properties of extensible fibrous materials.

An investigation of the small-angle X-ray scattering properties of aqueous solutions of an amylose derivative has been carried out. Experiments have been conducted in stable and fairly concentrated polymer solutions (up to 3.2%) by using a slightly substituted carboxymethylamylose having a degree of substitution of 0.08. Scattering intensities display a maximum in the low angle range which prevents extrapolation of the angular dependence to zero angle. Data obtained in the range of scattering vector 0.01 −1 yield 8 A as the radius of gyration of the chain cross-section and 140 dalton A −1 as the mass per unit length. These results are analysed in terms of the current model of amylose solution conformation and compared with the theoretical calculations of the Debye scattering function of the isolated chain.

Samples of turkey tendon at different degree of calcification have been investigated by high and small angle X-ray diffraction techniques using conventional and synchrotron radiation sources. The results indicate that the small angle diffraction patterns of samples at a degree of calcification ranging from 20 to 60% wt are due to the apatite phase which fits in the main band of collagen fibrils and exhibits the same axial D-periodicity, 67.0 nm, as native collagen fibrils in either wet or air-dried conditions. Analysis of these patterns reveals that the mineral phase is deposited along the collagen fibrils according to a step function. The length of the step is 0.46 D independent of the degree of calcification, while the height of the step increases with increasing calcification. The shift of the high angle equatorial diffraction spacing from 1.42 to 1.24 nm when the inorganic phase content passes from 40 to 60% wt suggests that needle-shaped crystals which compress the molecules are preferentially deposited during the advanced stages of calcification, in agreement with previuos electron microscopy observations. The structural relationship between inorganic deposits and collagen matrix is similar to that found for single osteons. This similarity increases as calcification proceeds.

Small-angle X-ray diffraction patterns of bovine, human and porcine media aortic wall show meridional reflections corresponding to a periodicity which suggests a molecular packing typical of tendon collagen. However the meridional intensity distribution of stretched air dried aortic samples appears different from that of air-dried tendon, probably because of the presence of a large amount of type III collagen or interactions of collagen with the environment, which are specific for aortic tissue. The stretched wet aortic samples show a marked decrease in intensity, revealing an extensive disorder in the axially-projected structure of the fibrils. When a loading system simulating the effect of blood pressure is applied to a ring of aorta, no evidence of orientation of collagen is seen by X-ray diffracton, as would be expected if collagen fibrils had an isotropic distribution inside the aorta media. Scanning electroni microscopy supports the existence of a network of collagen fibrils surrounding elastic lame...

X-ray diffraction techniques have been used to investigate the orientation under stress of collagen fibres in pig's descending aortic wall. The variation of the limit value of the incremental modulus as a function of the collagen content seems to suggest that the extra amount of collagen going from the aortic arch downwards is deposited in a preferred orientation corresponding to the circumferential direction. The results have been used to describe the uniaxial mechanical behaviour of this tissue taking into account its composite structure.

Human tendons affected by diffuse idiopathic skeletal hyperostosis — a disease characterized by calcification of tendons and ligaments — have been investigated by low and high angle X-ray diffraction using both conventional and synchroton radiation sources. The results reveal a close relationship between the preferential orientation of the c -axis of the hydroxyapatite crystallites and that of collagen fibres. The orientation of collagen fibres along the tendon axis decreases as the degree of calcification increases. The low angle diffraction analyses show that the mineral component is deposited preferentially along the collagen fibres according to a step function. Calcified collagen exhibits the same axial periodicity ( D =67.0 nm) as native collagen fibres. This periodicity remains unchanged in the dry calcified tissue. The length of the mineral block (0.45 D ) does not change with the degree of calcification. The apatite-collagen relationship closely resembles that found in bone tissue.

Fluoride and carbonate ions, which are present in the inorganic phase of bone, enamel, and dentine, are known to play an important and opposite role in the process of recrystallization. We have investigated the incorporation of fluoride and carbonate ions into hydroxyapatite structure under conditions of cyclic pH fluctuation and the effect of these incorporations on the conversion of hydroxyapatite into β-tricalcium phosphate after heat treatment. Fluoro-hydroxyapatite has been obtained as unique crystalline phase up to 20 fluoride at. %. The degree of substitution of fluoride for hydroxyl does not depend on the extent of carbonate incorporated into the apatite structure. On the other hand, the carbonate incorporation into the apatite structure seems to be hindered by the contemporary presence of fluoride. Both fluoride and carbonate exhibit a stabilizing effect on the apatite structure, as far as its conversion into β-tricalcium phosphate is concerned. The order of efficiency in stabilizing the apatite structure is F − > F − + CO 3 2− > C0 3 2− .

The structure and distribution of collagen fibres in Metridium senile mesoglea has been investigated using high and small angle X-ray diffraction techniques on conventional and synchrotron sources. The mesoglea collagen axial spacing appears very close to that of rat tail tendon, which is at variance with the values previously obtained from electron microscopic observations. The different intensity distribution of the small angle X-ray diffraction maxima recorded for mesoglea and rat tail tendon indicates a different distribution of electron density inside the repeating period. Furthermore the absence of the first order, the weak second order and the strong third and sixth orders in the patterns of wet and dry mesogleal collagen could explain that only a periodicity of 20–22 nm corresponding to one-third of the true axial period observed in the electron micrographs. The analysis of the reflections at 0.29 and 1.1–1.4 nm characteristics of the collagen molecular structure have been used to determine the distribution and orientation of the collagen fibres in unstretched and stretched samples

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

A continuous series of solid solutions of cadmium and calcium hydroxyapatite has been prepared from aqueous solution and characterized by X-ray diffraction and i.r. spectroscopic analyses. The lattice dimensions and the i.r. frequencies of the solid solutions vary linearly with the atom % of the cadmium. Upon heat treatment these solid solutions are converted into the β and α forms of cadmium-substituted tricalcium phosphate. Cadmium promotes these thermal conversions, having a destabilizing effect on the apatite structure. This effect cannot be explained by the small differences in the ionic radius of cadmium and calcium ions, but instead to differences in the nature of the cation–oxygen interactions.

Chemical and crystallographic characterization of the inorganic deposits present in the human aortic wall is reported. No evidence of calcification has been found in the adventitia layer of the aortic wall, while two different phases have been identified in the intima and media layers. The inorganic deposits localized in the atherosclerotic plaques of the intima layer have been identified as hydroxyapatite. The inorganic phase uniformly deposited in the human media aortic wall highly crystalline β-TCMP with a magnetism content which depends on the calcification degree.

The wide angle X-ray diffraction pattern of air-dried lens capsule collagen under tension is the same as the tendon collagen diffraction pattern with regard to the main reflections, and indicates that lens capsule collagen has the characteristic three-stranded helical structure with an axial repeat of 0.29 nm as tendon collagen. The low angle X-ray diffraction pattern shows several weak diffraction maxima corresponding to the meridional reflections of capsule collagen which show orders of 63.0 nm periodicity. This is an evidence of quarter staggered molecular assembly typical of tendon collagen even if less ordered. The results are consistent with the existence in lens capsule collagen of clearly defined molecular units, which can be oriented by stress and are packed in a poor-ordered fibrillar assembly.

The coordination polymer of zinc(II) with the diastereoisomeric mixture of bis-(1-methyl-butyl)-phosphinic acid, (Zn{[(CH3)(n-C3H7)CH]2PO2}2)n, has been prepared and its chain structure has been investigated by X-ray diffraction. The identity period along the fibre axis determined from the X-ray diffraction photographs of drawn fibres is 9.16 A. The reflections can be indexed on the basis of a hexagonal packing of chains. Comparison between observed intensities and the calculated intensity distribution on the layer lines of the X-ray pattern shows that zinc(II) bis-(1-methyl-butyl)-phosphinate, has a backbone structure with double phosphinate bridges between tetrahedral metal atoms instead of a structure with alternating single and triple phosphinate bridges as found when the organic side chains are phenyl or linear alkyl groups. This is accounted for by restrictions on the formation of the triple bridged cages due to the steric interactions between the branched alkyl groups. Es wurde das Koordinationspolymere von Zink(II) mit dem diastereoisomeren Gemisch der Bis-(1-methyl-butyl)-phosphinsaure (Zn{[(CH3)(n-C3H7)CH]2PO2}2)n dargestellt und seine Kettenstruktur mit Hilfe von Rontgenbeugungsaufnahmen untersucht. Aus den Streudiagrammen von verstreckten Fasern wurde eine Identitatsperiode langs der Faserachse von 9,16 A ermittelt. Die Reflexe konnen unter der Annahme einer hexagonalen Packung der Ketten indiziert werden. Der Vergleich der gemessenen Reflexintensitaten mit der fur die Schichtlinien berechneten Intensitatsverteilung zeigt, das das Zink(II)-bis-(1-methyl-butyl)-phosphinat eine Hauptvalenzkette mit doppelten Phosphinatbrucken zwischen tetraedrischen Metallatomen besitzt, wahrend eine Struktur mit alternierenden einfachen und dreifachen Phosphinatbrucken gefunden wurde, wenn die organischen Seitenketten aus Phenyl- oder linearen Alkylgruppen bestehen. Dies wird durch Behinderung bei der Bildung von raumlichen Dreifach-Brucken durch sterische Wechselwirkungen zwischen den Alkylgruppen erklart.

The results of the interpretation of the X-ray fibre photograph of the copolymer isomorphous with the homopolymer are reported. The unit cell has a monoclinic symmetry with . The molecular chains are characterized by a backbone strucure with tetrahedral metal atoms bonded by alternate single and triple bridging phosphinate groups. The best agreement between observed and calculated intensities was obtained assuming the space group P21/m. Pairs of parallel chains are packed so that the triple bridged cages of one chain fall midway those of the other chain in a way similar to that found in the β-form of the polymer Uber die Ergebnisse aus den Rontgen-Faser-Diagrammen von dem Copolymeren isomorph mit den Homopolymeren wird berichtet. Die Gitterzelle ist monoklin mit Die Kettenmolekule bilden eine Hauptkettenstruktur mit tetraedrisch angeordneten Metallatomen, abwechselnd von Phosphinat-Gruppen mit Einfach- und Dreifachbrucken ancinander gekoppelt. Die Raumgruppe P21/m gab die beste Ubereinstimmung mit den beobachteten Intensitaten. Die Kettenmolekule sind alternierend um b/2 gegeneinander verschoben, so das die kafigformigen Phosphinatgruppen mit Dreifachbrucken einer Kette in dem Zwischenraum zwischen denjenigen der Nachbarkette liegen, ahnlich wie bei der β-Form von .

Scanning electron microscopy and high angle X-ray diffraction were used to define the relationship between collagen and elastin of bovine aortic wall. The diffraction pattern shows on one hand that the broad rings at 4.5 A and 9 A, due to elastin, do not orient on stretching and on the other hand, that the collagen rings at 11 A and 2.9 A start to orient at low elongations. These data together with scanning electron microscopy suggest a tight structural relationship between collagen and elastin that should influence the mechanics of deformation at all degrees of elongation.