Amorphous calcium carbonate biomineralization in the earthworm's calciferous gland: pathways to the formation of crystalline phases

In this study, we investigated the microstructural transformations that take place during carbonate formation in the earthworm’s calciferous gland by analysing the evolution from the precursor fluid of the solid phases (spherulites) to the final carbonate concretions released by the gland. Results from HREM and electron diffraction showed that the spherulithic deposits merely consisted of ACC partially transformed to vaterite. Furthermore, comparisons of the diffraction spectra and microstructural analyses allowed the identification of the transition sequences to more stable carbonates. And thus, transformations of ACC to calcite were observed on the surfaces of these amorphous globular aggregates as their smooth characteristic surface became rougher with time. This transition path was not unique, and the presence of aragonite, as an intermediate phase, has also been found. In this particular case, the transition process followed a completely different pathway with the crystallization starting in the centre of the sphere and progressively extending to the periphery, leading to the formation of radial aggregates. In situ experiments performed on the freshly extracted precursor fluid and analysed by FT-IR spectroscopy showed that ACC is the main constituent and is probably stabilised by macromolecules such as proteins and sugars. Furthermore, the Debye–Scherrer diffraction experiments showed that the carbonate phase present in this fluid remains stable as ACC for more than a week. All these features are indicative of this entire process being biologically controlled by the earthworms. The analysis of the amorphous structure factor of this ACC indicates that these transformations are preceded by short-range order modifications of the amorphous precursor phase.     

XRD studies of beta-chitin from squid pen with calcium solvent

The crystalline structure of β-chitin from squid pen was investigated by X-ray diffraction (XRD). The purified β-chitin was prepared from bigfin reefsquid pen. β-Chitin was treated with saturated calcium chloride dihydrate/alchohol (CaCl₂·2H₂O/MeOH) solvent system at different conditions for XRD studies. The change of crystallinity of β-chitin from squid pen was studied by using the fiber photographs on imaging plates. The results showed that the diffraction peak (0 1 0) was shifted. It means that the lattice plane (0 1 0) interplanarilly spreaded to 3.4 Å, when the squid pen was washed with water after treatment of Ca solvent. Furthermore, when the squid pen was dried after treatment of Ca solvent and washing with water, interplanar spacing of (0 1 0) inversely shrank to 1.1 Å. These results suggested that Ca solvent especially influences the plane (0 1 0) of β-chitin structure.     

Characterization of carbon metabolism in Opuntia ficus-indica Mill. exhibiting the idling mode of Crassulacean acid metabolism

Upon transfer from well-watered conditions to total drought, long-day-grown cladodes of Opuntia ficus-indica Mill. shift from full Crassulacean acid metabolism (CAM) to CAM-idling. Experiments using ¹⁴C-tracers were conducted in order to characterize the carbon-flow pattern in cladodes under both physiological situations. Tracer was applied by ¹⁴CO₂ fumigations and NaH¹⁴CO₃ injections during the day-night cycle. The results showed that behind the closed stomata, mesophyll cells of CAM-idling plants retained their full capacity to metabolize CO₂ in light and in darkness. Upon the induction of CAM-idling the level of the capacity of phosphoenolpyruvate carboxylase (EC 4.1.1.31) was maintained. By contrast, malate pools decreased, displaying finally only a small or no day-night oscillation. The capacity of NADP-malic enzyme (EC 1.1.1.40) decreased in parallel with the reduction in malate pools. Differences in the labelling patterns, as influenced by the mode of tracer application, are discussed.Abbreviations CAM Crassulacean acid metabolism - PEP-Case phosphoenolpyruvate carboxylase     

Variation in crystalline type with amylose content in maize starch granules: an X-ray powder diffraction study

Comparative studies of native maize starches with different amylose contents were carried out using X-ray powder diffraction. The results show a transition of crystalline type from A through C to B, accompanying a decrease in degree of crystallinity from 41.8% to 17.2% across a range of apparent amylose content from 0% to 84%. Hydration induces an increase in degree of granule crystallinity, but does not change the transition of crystal type. Progressively from A-type to C-type, crystallinity decreases rapidly with an increase in amylose content. From C-type to B-type, overall crystallinity decreases more slowly. The crystal type is strongly dependent on amylose content and on average chain length of the respective amylopectin. Waxy A-types have an average chain length of about 20, while in high amylose B-types this rises to ≈35. The proportion of short chains (10–13 glucose units) appears to affect crystal type significantly. Some V-type material was detected at high amylose levels. The proportion of this increased after prolonged exposure of the granules to iodine vapour. Implications for the arrangement of starch components in the granule are discussed.     

Role of intestinal mucus in crystal biogenesis: an electron-microscopical,diffraction and X-ray microanalytical study

Summary In the posterior intestine of the sea-water eel, mucus plays an important role in biocrystallization of calcium ions. By means of transmission and scanning electron microscopy associated with X-ray microanalysis and X-ray diffraction it has been possible to determine the role of mucous fibers as nucleation sites. Biocrystallization occurs in 2 steps: (1) Calcification of mucus. As soon as mucus is excreted in the intestinal lumen, it is loaded with calcium, as shown by lanthanum affinity and X-ray microanalysis on freeze-dried tissues. (2) Genesis of crystals. Needleshaped crystallites build up in coalescent spherites in the intestinal lumen near the microvilli. Genesis occurs as follows: (a) crystallite mineralization by nucleation in an organic matrix composed of glycoproteinaceous mucous fibers, followed by the appearance of spherites; (b) coalescence in spherites and association of spherites in rhombohedra; (c) extrusion of organic material during the final step of crystallization.     

Layered structures of hydrophobically modified chitosan derivatives

Two series of chitosan derivatives, N-aliphatic acyl chitosans (C_n-CS) and N-aliphatic-O-dicinnamoyl-chitosans (C_n-CinCS) with n=2, 4, 8, 12 and 18, were prepared through regioselective reactions. The solid state structures were studied by FTIR and X-ray diffraction techniques. Two different types of layered structures were found to exist in the powder samples of C_n-CS and C_n-CinCS. The C_n-CS series crystallized into a sheet-type structure, in which all the flexible side chains lied down in the sheet in a direction normal to the backbones and partially interdigitated with each other. For the C_n-CinCS series, the polar backbones were stacked into sheets, and the flexible side chains occupied the space between sheets in a direction inclined to the backbones. The relationship between the structures and the solubilities are discussed.     

Effects of drought on water relations and nonstructural carbohydrates in cladodes of Opuntia ficus-indica

Nocturnal acid accumulation, water content, osmotic pressure (π), and nonstructural carbohydrates were determined in the chlorenchyma and the water-storage parenchyma of Opuntia ficus-indica (L.) Miller for well-watered plants and those subjected to drought for 15 weeks. During the 15-week drought, total cladode water content decreased by 57%, the water-storage parenchyma losing a greater fraction of water than the chlorenchyma, which most likely helped maintain nocturnal acid accumulation in the latter tissue. Despite the preferential water loss from the water-storage parenchyma, it had a lower π than the chlorenchyma over the 15 weeks of drought, suggesting a substantial decrease in osmotically active solutes in the water-storage parenchyma. Also, the measured π increases of both tissues were much less than those predicted based on the loss of water during drought and the initial content of osmotically active solutes under well-watered conditions. A decrease in the amount of soluble sugars (glucose. fructose and sucrose) occurred in plants subjected to drought. accounting for 46% and 81% of the difference between the measured and the predicted increases in π of the chlorenchyma and the water-storage parenchyma. respectively. The decrease in soluble sugars was associated with an equivalenl increase in polysaccharides, presumably starch, in the water-storage parenchyma. but not in the chlorenchyma.     

Rapid propagation of tuna (Opuntia ficus-indica) and plant establishment in soil

Explants from young joints of mature plants of tuna (Opuntia ficus-indica Mill.) were cultured on Murashige and Skoog (MS) medium containing 8.8 M benzyladenine (BA) and 0.5 M naphthaleneacetic acid (NAA). Shoots produced were utilized as secondary explants. Each shoot was cut longitudinally from apex to base into two explants, and some of these explants were cut transversely into proximal and distal explants. The size and number of shoots produced was affected by size and position of the explant within its source. The shoots were rooted in vitro or ex vitro and plants were successfully established in soil from both rooting methods.Abbreviations AC activated charcoal - BA benzyladenine - IBA indolebutyric acid - MS Murashige & Skoog (1962) medium - NAA naphthaleneacetic acid     

Application of soft X-ray microradiography to observation of cystoliths in the leaves of various higher plants

Soft X-ray microradiography was applied to observation of the cystoliths, calcified bodies of higher plants, in the leaves ofMorus bombycis, Humulus scandens, Ficus elastica, F. retusa (Moraceae),Boehmeria platanifolia, Pilea viridissima (Urticaceae) andMomordica charantia (Cucurbitaceae). It was proved that this technique is useful for examination of the shape, size, distribution and number of cystoliths in fresh leaves. The microradiographs revealed large cigar-shaped cystoliths in the leaf ofP. viridissima, and neighbor-cystoliths in somewhat restricted areas of the leaves ofM. bombycis andH. scandens, and two to seven radially arranged cystoliths in the leaf ofM. charantia. The number of cystoliths per unit area of leaf (nos./cm²) was estimated to be from 1,090 to 3,900 by means of the microradiographs, varying from species to species. The CaCO₃ content of the leaf calculated from the volume and number of cystoliths was approximately 0.4 mg/cm² in all species exceptF. retusa. InF. retusa, it was about 1.06 mg/cm², the highest value among all species tested. Hand-sections of the leaves showed that the lithocysts were localized in the upper and/or lower epidermis, and they were associated with many photosynthetic cells in all species, suggesting some relationship between CaCO₃ deposition in cystoliths and photosynthesis. This paper is dedicated to Professor Kurazo Furuya, Department of Biology, Tokyo Gakugei University, on the occasion of his retirement (1986).     

New organoantimony complexes with the isomers of chlorophenylacetic acid: Syntheses, characterizations and crystal structures of 1D polymeric chain, 2D network structure and 3D framework

A series of novel organoantimony(V) complexes have been synthesised by the reactions of the isomers of chlorophenylacetic acids with triphenylantimony(V) dichloride or tetraphenylantimony(V) bromide in 1:2 or 1:1 stoichiometries. All the complexes have been characterized by elemental analysis, IR and NMR (¹H, ¹³C) spectra analyses; furthermore, complexes 1, 2, 3 and 4 have been determined by X-ray single crystal diffraction. The structure of complexes show that the five-coordinated and six-coordinated antimony(V) atoms adopt distorted trigonal bipyramidal geometry and octahedral geometry. And the structural analyses show that complexes 1 and 3 have 2D network structures; complex 2 possesses a 1D polymeric chain structure and complex 4 has a 3D supramolecular framework.     

Thermotropic ‘two-stage’ liquid crystalline crystalline lipid phase separation in microsomal membranes

The effect of temperature on native microsomal membrane vesicles isolated from Tetrahymena is investigated by wide angle X-ray diffraction. A 4.2 Å reflection, typical for lipids in the crystalline state, can be recorded in the temperature range between 0°C and 35°C. Quantitative evaluation of this reflection reveals a broad thermotropic ‘two-stage’ liquid crystallinecrystalline lipid phase separation with a ‘breakpoint’ at approx. 18°C. This ‘breakpoint’ coincides with the emergence of lipid-protein segregations in endomembranes of intact Tetrahymena cells as previously visualized by freeze-etch electron microscopy.     

Effect of local sequence inversions on the crystalline antiparallel β-sheet lamellar structures of periodic polypeptides: implications for chain-folding

The crystal structure and texture of the monodisperse periodic polypeptide [(AG)₃EG(GA)₃EG]₁₀ (poly(±AG)₃EG; A=alanine, G=glycine, E=glutamic acid) were analyzed by X-ray diffraction, Fourier transform infrared spectroscopy, and electron microscopy. Structure determination was aided by comparison with the recently described structure for the related periodic polypeptide [(AG)₃EG]₃₆ by Krejchi et al. (Macromolecules 1997;30:5012). Texture-oriented samples of poly(±AG)₃EG were obtained by crystallization of the polymer from aqueous formic acid solution. The evidence supports an antiparallel (ap) β-sheet protein structure and the X-ray diffraction signals index on an orthorhombic unit cell with parameters: a=0.950 nm (hydrogen-bond direction), b=1.052 nm (apβ-sheet stacking direction), c=6.95 nm (chain direction). The absence of the (010) diffraction signal, a prominent signal in the poly(AG)₃EG diffraction pattern, implies that the apβ-sheets are ‘apolar', i.e. both surfaces are equally populated with alanyl methyl groups. Selective line broadening of wide-angle diffraction signals with ℓ≠0 gives an estimated crystal size of 4 nm in the chain direction. This observation, coupled with the appearance of low-angle particle interference peaks, indicates a crystal thickness considerably less than the chain length and suggests an adjacent-re-entry chain-folded lamellar structure incorporating the apβ-sheet architecture. The polypeptide folds through γ-turns, in-phase with the pseudo-octapeptide repeat; the glutamic acid residues occur on the lamellar surfaces. These results and those from the crystalline lamellae of poly(AG)₃EG suggest that β-turns are not compatible with these repetitively stacked apβ-sheet structures. This implies that intersheet interactions of alanyl methyl groups and glycyl -protons are not sufficiently strong to dictate the folding geometry in these structures.     

The determination of the separate Ca2+ pump protein and phospholipid profile structures within reconstituted sarcoplasmic reticulum membranes via X-ray and neutron diffraction

We have previously compared the electron density profiles for several highly-functional reconstituted sarcoplasmic reticulum membranes with that for the isolated sarcoplasmic reticulum membrane (Herbette, L., Scarpa, A., Blasie, J.K., Wang, C.T., Saito, A. and Fleischer, S. (1981) Biophys. J. 36, 47–72). In this paper, we compare the separate calcium pump protein profile within these reconstituted sarcoplasmic reticulum membranes, as derived by X-ray and neutron diffraction methods, with that within isolated sarcoplasmic reticulum membranes. In addition, the time-average perturbation of the lipid bilayer by the incorporated calcium pump protein within these reconstituted sarcoplasmic reticulum membranes has been determined in some detail.     

Influence of phorbol esters on ionophore-mediated calcium exchange-diffusion in liposomes

The interference of phorbol esters upon the process of A23187-mediated calcium exchange diffusion was examined in multilamellar liposomes formed of different types of lipids and incubated at variable temperatures. Phorbol esters facilitated the process of calcium ionophoresis in liposomes formed of dipalmitoylphosphatidylcholine (DPPC) or dimyristoylphosphatidyl-choline (DMPC) and incubated below transition temperature. The magnitude of this facilitating action was negatively correlated with the tumor-promoting capacity of the phorbol esters. The phorbol esters also facilitated calcium ionophoresis in liposomes formed of a mixture of DPPC and cholesterol, provided that the temperature exceeded 34 degrees C. The magnitude of the latter facilitating action was positively correlated with both the temperature and the tumor-promoting potency of the phorbol esters. Thus, the existence of a parallelism between the biological potency of phorbol esters and their biophysical effect in this artificial system tightly depended on such factors as the lipid composition of the liposomal matrix and the ambient temperature.     

Fine structure of crystalline inclusions in B-cells of the islets of Langerhans in the alligator

Summary The ultrastructure of crystalline beta granules of the islets of Langerhans in the alligator has been investigated. From optical diffraction analysis and serial sectioning, the existence of four distinct types of crystalline inclusions was established in ultrathin sections. The first type is the most frequent and is interpreted as a rhombohedroni with a base, the ortho-hexagonal unit-cell edges being a=18.9 nm, c=23.0 nm. The second type of crystal (not observed in serial sections) is found compatible with a rhomb-dodecahedron which indexes on a cubic cell with a=9.6 nm. The third type of crystal was assigned to dipyramids. Dipyramids are extremely rare, and only two diffraction patterns were obtained; their crystal system could not be determined. Prisms, which are second in abundance, represent the fourth type of crystal. Spacings as well as the symmetry differ from those of the above three crystal types and indicate a tetragonal cell with a=4.2 nm, c=14.2 nm. The data for the prismatic crystals are strikingly similar to those of proinsulin and may represent the first case of agreement between crystals (i) formed in vitro and studied by X-ray diffraction and (ii) those investigated in situ by electron microscopy.     

Calcium sulfate hemihydrate (bassanite) statoliths in the cubozoan Carybdea sp.

The chemistry and physical structure of statoliths of young cubozoan medusae (Carybdea sp.) were examined by X-ray spectroscopy (EDX) and X-ray powder diffractometry (XRD). These concretions, associated with sensory receptors, were found to consist of bassanite (calcium sulfate hemihydrate), a dense but hygroscopic biomineral. Bassanite occurs in a cluster of radially oriented crystals in a druse, which contains perfect hexagonal crystals. This discovery provides evidence that the Rhopaliophora (Scyphozoa and Cubozoa) originated from an ancestor having statoliths of bassanite.