Ultrastructural Characteristics of the Nacre in Some Gastropods

The nacreous layer in Gibbula, Calliostoma, Trochus and Haliotis is described on the basis of scanning electron microscopic studies. The central part of each nacreous tablet contains a significant amount of calcified organic matrix which is insoluble in a chromium sulphate and a 25% glutaraldehyde solution. In most cases, the tablet is subdivided by radial vertical organic membranes into a varying number (2 to 50) of crystalline sectors. These sectors represent polysynthetically twinned crystal individuals which form cyclic or interpenetrant twins. The nacreous tablets in gastropods are compared with those in bivalves, and with the non-biogenic aragonite. The mechanical properties of the nacre, and the effects of the interlamellar conchiolin membranes upon the nucleation of the tablets, are discussed.     

Soluble proteins of the nacre of the giant oyster Pinctada maxima and of the abalone Haliotis tuberculata: extraction and partial analysis of nacre proteins

Several proteins from nacre of the oyster Pinctada maxima and the abalone Haliotis tuberculata were extracted and partly characterized. Proteins dispersed in aragonite were solubilized during demineralization with acetic acid whereas proteins adsorbed on conchiolin were extracted with sodium dodecyl sulfate and beta-mercaptoethanol. The matrix of Pinctada maxima nacre is composed of one main protein with an apparent molecular weight of 20 kDa (p20). This protein was found in the acetic acid soluble fraction of nacre, as well as in the Laemmli-solubilized extract of conchiolin. In addition, the p20 solubilized with acetic acid can form oligomers made of 6 monomers linked together by disulfide bridges. The first N-terminal 21 amino acids of p20 were determined and no homology with known proteins was found. In Haliotis tuberculata nacre, 5 main proteins were solubilized during demineralization and 3 glycoproteins were detected. Stains-all and Alcian blue staining revealed polyanionic proteins in the extracts isolated from Pinctada maxima and Haliotis tuberculata nacre.     

Proteomics Analysis of the Nacre Soluble and Insoluble Proteins from the Oyster Pinctada margaritifera

Shell nacre is laid upon an organic cell-free matrix, part of which, paradoxically, is water soluble and displays biological activities. Proteins in the native shell also constitute an insoluble network and offer a model for studying supramolecular organization as a means of self-ordering. Consequently, difficulties are encountered in extraction and purification strategies for protein characterization. In this work, water-soluble proteins and the insoluble conhiolin residue of the nacre of Pinctada margaritifera matrix were analyzed via a proteomics approach. Two sequences homologous to nacre matrix proteins of other Pinctada species were identified in the water-soluble extract. One of them is known as a fundamental component of the insoluble organic matrix of nacre. In the conchiolin, the insoluble residue, four homologs of Pinctada nacre matrix proteins were found. Two of them were the same as the molecules characterized in the water-soluble extract. Results established that soluble and insoluble proteins of the nacre organic matrix share constitutive material. Surprisingly, a peptide in the conchiolin residue was found homologous to a prismatic matrix protein of Pinctada fucata, suggesting that prismatic and nacre matrices may share common proteins. The insoluble properties of shell matrix proteins appear to arise from structural organization via multimerization. The oxidative activity, found in the water-soluble fraction of the nacre matrix, is proposed as a leading process in the transformation of transient soluble proteins into the insoluble network of conchiolin during nacre growth.     

Ultrastructure of the Interlamellar Membranes of the Nacre of the Bivalve Pteria hirundo,Determined by Immunolabelling

The current model for the ultrastructure of the interlamellar membranes of molluscan nacre imply that they consist of a core of aligned chitin fibers surrounded on both sides by acidic proteins. This model was based on observations taken on previously demineralized shells, where the original structure had disappeared. Despite other earlier claims, no direct observations exist in which the different components can be unequivocally discriminated. We have applied different labeling protocols on non-demineralized nacreous shells of the bivalve Pteria. With this method, we have revealed the disposition and nature of the different fibers of the interlamellar membranes that can be observed on the surface of the nacreous shell of the bivalve Pteria hirundo by high resolution scanning electron microscopy (SEM). The minor chitin component consists of very thin fibers with a high aspect ratio and which are seemingly disoriented. Each fiber has a protein coat, which probably forms a complex with the chitin. The chitin-protein-complex fibers are embedded in an additional proteinaceous matrix. This is the first time in which the sizes, positions and distribution of the chitin fibers have been observed in situ.     

Mineral bridges in nacre

We confirm with high-resolution techniques the existence of mineral bridges between superposed nacre tablets. In the towered nacre of both gastropods and the cephalopod Nautilus there are large bridges aligned along the tower axes, corresponding to gaps (150–200 nm) in the interlamellar membranes. Gaps are produced by the interaction of the nascent tablets with a surface membrane that covers the nacre compartment. In the terraced nacre of bivalves bridges associated with elongated gaps in the interlamellar membrane (>100 nm) have mainly been found at or close to the edges of superposed parental tablets. To explain this placement, we hypothesize that the interlamellar membrane breaks due to differences in osmotic pressure across it when the interlamellar space below becomes reduced at an advanced stage of calcification. In no cases are the minor connections between superimposed tablets (<60 nm), earlier reported to be mineral bridges, found to be such.     

Ultrastructural studies on the brachiopod pedicle

Studies of the ultrastructure of representative articulate and inarticulate pedicles show that there are similarities in secretory activity between pedicle epithelia of both classes and the outer mantle epithelium of the inarticulates. Rootlet epithelial cells of the articulate pedicle produce small vesicles which pass to the junction between rootlet and substrate and probably play a part in the breakdown of the substrate. Scanning electron microscopy shows that dissolution of a bivalve shell acting as a substrate often follows the boundary of a single nacre tablet, and transmission electron microscopy shows that the rootlet extends its infiltration along the conchiolin walls. The inarticulate pedicle ending is modified to collect adherent sand grains.     

Structure,crystallography, and morphogenesis of the cryptic shell of the terrestrial slug Limax maximus (Mollusca,gastropoda)

The structure and crystallography of the internal shell of the pulmonate gastropod slug Limax maximus were studied at the levels of light and scanning electron microscopy, revealing patterns of shell ontogeny and morphogenesis. The calcified portion of the slightly convex ovoid shell is composed of a single palisade layer of calcitic crystals. Numerous projections, 100 μm in width at the dorsal tip, are found on the dorsal surface of the shell and coincide with local nucleation sites of primordial calcium salt deposition onto the periostracum. With continued calcification these projections coalesce ventrally, forming the single crystalline shell layer. The organic portion of the shell includes the periostracum and an extensive PAS-staining conchiolin. In EDTA-etched preparations, conchiolin appears as a spongy network of fibers throughout the shell. Both horizontal and vertical components of the conchiolin are present, the former of variable thickness and occurring in an intercrystalline manner, the latter always occurring normal to the horizontal set. Macromorphogenic growth is characterized by three distinct temporal stages. Primary growth occurs radially from the umbonal region. Secondary growth is synonymous with shell thickening. Tertiary growth is characterized by both a lateral component, in which the shell extends beyond the primary growth boundaries, and a ventral component, in which the shell continues to grow in thickness. SEM of the ventral shell surface reveals a pattern of growth at the crystalmatrix interface. Proteinaceous fibers of the conchiolin occur unidirectionally in horizontal rows. Zones of incipient calcitic crystallization onto these hypostracal fiber bundles are contrasted by zones of increasing crystallization until the fibrous template (reduced hypostracum) is completely covered by crystals.     

Macromolecular structure of the organic framework of nacre in Haliotis rufescens: implications for growth and mechanical behavior

We have performed a macromolecular structural analysis of the interlamellar and intertabular parts of the organic framework of the nacreous part of the shell of Haliotis rufescens, including the identification of structural chitin. Using histochemical optical microscopy we have mapped the locations of carboxylates and sulfates of proteins and chitin on the surfaces and within the core of the interlamellar layers and the intertabular matrix that together form the external organic matrix of composite nacre. This extends the earlier work of Nudelmann et al. [Nudelman, F., Gotliv, B.A., Addadi, L. and Weiner, S. 2006. Mollusk shell formation: mapping the distribution of organic matrix components underlying a single aragonite tablet in nacre. J. Struct. Biol. 153, 176–187] and Crenshaw and Ristedt [Crenshaw, M.A., Ristedt, H. 1976. The histochemical localization of reactive groups in septal nacre from Nautilus pompilius. In: Omori, M., Watabe, N. (Eds.) The Mechanisms of Biomineralization in Animals and Plants. Tokai University Press, Toyko] on Nautilus pompilius. Our mapping identifies distinct regions, defined by the macromolecular groups, including what is proposed to be the sites of CaCO₃ nucleation and that play a key role in nacre growth. Using AFM scanning probe microscopy we have identified a fibrous core within the framework that we associate with chitin. The structural picture that is evolved is then used to develop a simple structural model for the organic framework which is shown to be consistent with mechanical property measurements. The role of the intracrystalline matrix within the nacre tablets in mediating nacre’s mechanical response is noted within the framework of our model.     

Topography of the organic components in mother-of pearl

1. The topography of the organic components (conchiolin) has been investigated on positive, postshadow-cast, formvar, and carbon replicas of mother-of-pearl from shells of a Cephalopod, of two Gastropods, and of six Pelecypods. All these shells are characterized by a true nacreous inner shell layer. 2. The material included normal shell surfaces, fragments of cleavage obtained by fracture, and surfaces polished tangentially and transversally to the inner surface of the shells. Replicas of these surfaces were prepared before and after etching of graded heaviness, induced by a chelating agent (sequestrene NA 2, titriplex III). Micrographs of the successive steps of the process of corrosion have been recorded. 3. Corrosion unmasked, on the nacreous surfaces, organic membranes or sheets, running as continuous formations in between adjacent mineral lamellae, and separating the individual crystals of aragonite which are aligned in rows and constitute each lamella. 4. The interlamellar sheets of material exhibit a reticulated structure, which is especially visible in preparations orientated tangentially to the lamellae and to the tabular surface of the aragonite crystals. The pattern of this lace-like structure, different in the various species studied, appeared in the same species as closely similar to that reported previously in leaflets of thoroughly decalcified mother-of-pearl, dissociated by ultrasonic waves. The present results support former conclusions with regard to the existence of taxonomic differences between Cephalopods, Gastropods, and Pelecypods in the morphological organization of the organic phase within mother-of-pearl.     

Soluble silk-like organic matrix in the nacreous layer of the bivalve Pinctada maxima.

Nacre organic matrix has been conventionally classified as both 'water-soluble' and 'water-insoluble', based on its solubility in aqueous solutions after decalcification with acid or EDTA. Some characteristics (aspartic acid-rich, silk-fibroin-like content) were specifically attributed to either one or the other. The comparative study on the technique of extraction (extraction with water alone vs. demineralization with EDTA) presented here, seems to reveal that this generally accepted classification may need to be reconsidered. Actually, the nondecalcified soluble organic matrix, extracted in ultra-pure water, displays many of the characteristics of what until now has been called 'insoluble matrix'. We present the results obtained on this extract and on a conventional EDTA-soluble matrix, with various characterization methods: fractionation by size-exclusion and anion-exchange HPLC, amino acid analysis, glycosaminoglycan and calcium quantification, SDS/PAGE and FTIR spectroscopy. We propose that the model for the interlamellar matrix sheets of nacre given by Nakahara [In: Biomineralization and Biological Metal Accumulation, Westbroek, P. & deJong, E.W., eds, (1983) pp. 225-230. Reidel, Dordrecht, Holland] and Weiner and Traub [Phil. Trans. R. Soc. Lond. B (1984) 304, 425-434] may no longer be valid. The most recent model, proposed by Levi-Kalisman et al. [J. Struct. Biol. (2001) 135, 8-17], seemed to be more in accordance with our findings.     

Histochemical studies on the localization and activity of acid phosphatase in calcifying tissues

Summary Acid phosphatase activity has been studied in cold microtome sections and using simultaneous azo coupling method in developing teeth and bone, and serial sections were made for the demonstrations of alkaline phosphatase.1. In developing teeth, strongest activity of acid phosphatase was found in the distal portion of high columnar ameloblasts associated with heavy calcification in the rodent incisor, and ameloblasts and odontoblasts in adjacent occlusal surface in molar teeth. However, the activity of immatured ameloblast and crevicular aspects of molar were weaker.2. In the epiphyseal bone trabeculae a striking acid phosphatase reaction was found.3. As regards to the effects of decalcifying solutions to the enzymatic activity, the use of EDTA decalcifying agent (10% and pH 7 to 4) showed the best results. That is, a decrease of decalcifying time and a greater preservation of acid phosphatase activity.With 11 Figures in the Text     

TOPOGRAPHY OF THE ORGANIC COMPONENTS IN MOTHER-OF-PEARL

1. The topography of the organic components (conchiolin) has been investigated on positive, postshadow-cast, formvar, and carbon replicas of mother-of-pearl from shells of a Cephalopod, of two Gastropods, and of six Pelecypods. All these shells are characterized by a true nacreous inner shell layer. 2. The material included normal shell surfaces, fragments of cleavage obtained by fracture, and surfaces polished tangentially and transversally to the inner surface of the shells. Replicas of these surfaces were prepared before and after etching of graded heaviness, induced by a chelating agent (sequestrene NA 2, titriplex III). Micrographs of the successive steps of the process of corrosion have been recorded. 3. Corrosion unmasked, on the nacreous surfaces, organic membranes or sheets, running as continuous formations in between adjacent mineral lamellae, and separating the individual crystals of aragonite which are aligned in rows and constitute each lamella. 4. The interlamellar sheets of material exhibit a reticulated structure, which is especially visible in preparations orientated tangentially to the lamellae and to the tabular surface of the aragonite crystals. The pattern of this lace-like structure, different in the various species studied, appeared in the same species as closely similar to that reported previously in leaflets of thoroughly decalcified mother-of-pearl, dissociated by ultrasonic waves. The present results support former conclusions with regard to the existence of taxonomic differences between Cephalopods, Gastropods, and Pelecypods in the morphological organization of the organic phase within mother-of-pearl.     

Characteristics of the interrelationships of gliocytes with neurocyte processes in rat celiac ganglia

While studying the ultrastructure of rat celiac nodes, it was stated that lemmocytes from the intercellular plexus develop around separate neuronal processes spiral membranes and multilayer membranes in the shape of concentric "musses". Similar membranes but of simpler structure develop perisomatic glyocytes. In old rats these types of glial membranes occur considerably rare. Both varieties of complicated types of glial membranes serve, evidently, for the isolation of preterminal sections of neuronal processes from probable unwanted contacts with other terminals.     

STRUCTURE OF THE CONCHIOLIN CASES OF THE PRISMS IN MYTILUS EDULIS LINNE

The prisms in the shell of Mytilus edulis Linné are calcite needles. Their small size and their thin conchiolin cases distinguish them from the prisms of many other species of mollusks. These Mytilus prisms have been studied with the electron microscope. The material consisted of positive replicas of surfaces of the prismatic layer, etched with chelating agents, and of preparations of tubular cases from decalcified prisms which were compared with the conchiolin from decalcified mother-of-pearl of the same species. In the replicas, the cases appear as thin pellicles in the intervals between the prism crystals. Both the prism cases and the nacreous conchiolin, disintegrated by exposure to ultrasonic waves and sedimented on supporting films, appear in the form of tightly meshed, reticulated sheets, described as "tight pelecypod pattern" in former studies on nacreous conchiolin of Mytilus. The results show that in the shell of this species the same conchiolin structure is associated with aragonite in mother-of-pearl and with calcite in the prismatic layer.     

Forming nacreous layer of the shells of the bivalves Atrina rigida and Pinctada margaritifera: an environmental- and cryo-scanning electron microscopy study

A key to understanding control over mineral formation in mollusk shells is the microenvironment inside the pre-formed 3-dimensional organic matrix framework where mineral forms. Much of what is known about nacre formation is from observations of the mature tissue. Although these studies have elucidated several important aspects of this process, the structure of the organic matrix and the microenvironment where the crystal nucleates and grows are very difficult to infer from observations of the mature nacre. Here, we use environmental- and cryo-scanning electron microscopy to investigate the organic matrix structure at the onset of mineralization in the nacre of two mollusk species: the bivalves Atrina rigida and Pinctada margaritifera. These two techniques allow the visualization of hydrated biological materials coupled with the preservation of the organic matrix close to physiological conditions. We identified a hydrated gel-like protein phase filling the space between two interlamellar sheets prior to mineral formation. The results are consistent with this phase being the silk-like proteins, and show that mineral formation does not occur in an aqueous solution, but in a hydrated gel-like medium. As the tablets grow, the silk-fibroin is pushed aside and becomes sandwiched between the mineral and the chitin layer.     

Ultrastructural Properties of the Extra Membranes of Escherichia coli O111a as Revealed by Freeze-Fracturing and Negative-Staining Techniques

Escherichia coli O111a is a thermosensitive strain which, when grown at 40 C, accumulates large quantities of intracellular membranes. The ultrastructure of these membranes in cells which have been chemically fixed, embedded, and examined as thin sections has been compared with that of membranes in cells negatively stained or freeze-fractured. Results indicate that the extra membranes are present in the three types of preparations examined and, therefore, clearly are not artifacts of chemical fixation. Negative staining has proved also to be a valuable tool as a rapid means of monitoring cells for the accumulation of large amounts of extra membranes. Also, examination of thin sections has shown that distinct continuities between the plasma membrane and the extra membranes exist. In general, membrane surfaces in freeze-fractured cells containing extra membranes appear smooth and lack the particles associated with the plasma membranes of many cells.     

Abalone nacre insoluble matrix induces growth of flat and oriented aragonite crystals

Mollusc shell formation takes place in a preformed extracellular matrix, composed of insoluble chitin, coated with proteins and dissolved macromolecules. The water-soluble matrix is known to have a strong influence on the growth of CaCO(3), whereas the role of the insoluble matrix on mineralization is unclear. Therefore, we mineralized the EDTA (ethylenediaminetetraacetic acid) insoluble organic matrix of abalone nacre with a modified double-diffusion set-up, where the diffusing solutions were constantly renewed. Control experiments were performed with cellulose and chitosan foils. The mineralized matrices/foils were analyzed with SEM. We show that the insoluble matrix of abalone nacre induces the growth of flat and roughly polygonal CaCO(3) crystals. In some of the experiments with the insoluble matrix, the growth of three-dimensional parallel sheets of densely packed platelets inside the insoluble matrix was observed. XRD on these samples revealed that they consist of oriented aragonite.     

Purification and immuno-electron microscopical characterization of crystalline inclusions from plant peroxisomes

Summary This paper describes the first purification method for crystalline inclusions (cores) from plant peroxisomes and an ultrastructural characterization of these isolated cores. 5-day-old sunflower (Helianthus annuus L.) cotyledon fractions which were highly enriched in cores showed negligible activity of the matrix enzyme glycolate oxidase but high catalase activity. As proven by electron microscopy, crystalline particles were surrounded neither by matrix material nor by membranes. Their geometrical outlines and ultrastructure were identical to those of cores in tissue sections, as was their reactivity with three different polyclonal catalase antibodies in the immunogold technique. Three-dimensional reconstruction, based on the geometrical outlines and ultrastructure of sectioned isolated cores from sunflower, suggested that they were quadrangular blocks. Ultrastructural analysis revealed an even periodic arrangement of repeating units which are probably cubes with 20 nm long edges. Isolated peroxisomal cores from potato (Solanum tuberosum L.) tubers had outlines which suggested that they were even rhomboidal prisms. They showed a granular ultrastructure without any repeating units and contained catalase, demonstrated by immunogold labelling and enzyme activity measurement. The results presented here suggested the hypothesis that the structural elements in plant peroxisomal cores are made of enzymatically active catalase, although the substructure may vary from species to species.Abbreviations ACOx acyl-CoA oxidase - BSA bovine serum albumin - EDTA ethylenediamine-tetraacetate - GDH glutamate dehydrogenase - GOx glycolate oxidase - KPB potassium phosphate buffer     

Ultrastructural studies of isolatedZea mays L. Chloroplasts

Summary The ultrastructure of isolated mesophyll chloroplasts ofZea mays L. was studied using the shadow casting technique. Grana and interconnecting fret membranes were observed in the same basic arrangement as they appear in thin sections. Quantasome units, as previously described by other authors for other species, were detected. Portions of the peripheral reticulum and envelope were also observed. Swollen membranes, comparable to those observed when isolated plastids are placed in water, were frequent in the preparations. It can be assumed, then, that in any preparation of this nature the hydrophillic spaces will absorb a large amount of water, swelling the intramembranous spaces and giving the membranes the appearance of large vesicles which will appear as flattened sacs when dried on the electron microscope grids. As there is no indication of any quantasome pattern in these membranes it is assumed that the particle arrangement of these membranes was altered during the procedure. It was not possible to determine whether the membranes observed in the swollen vesicles belonged to the grana or frets. A change in the morphology of the membranes during the processing may be observed with the light microscope. Recognition of this change is frequently difficult in electron micrographs, consequently, membranes with similar appearance under the electron microscope may actually have arisen from different portions of the plastid membrane systems.