Evidence of low molecular weight components in the organic matrix of the reef building coral, Stylophora pistillata

Biominerals contain both inorganic and organic components. Organic components are collectively termed the organic matrix, and this matrix has been reported to play a crucial role in mineralization. Several matrix proteins have been characterized in vertebrates, but only a few in invertebrates, primarily in Molluscs and Echinoderms. Methods classically used to extract organic matrix proteins eliminate potential low molecular weight matrix components, since cut-offs ranging from 3.5 to 10 kDa are used to desalt matrix extracts. Consequently, the presence of such components remains unknown and these are never subjected to further analyses. In the present study, we have used microcolonies from the Scleractinian coral Stylophora pistillata to study newly synthesized matrix components by labelling them with 14C-labelled amino acids. Radioactive matrix components were investigated by a method in which both total organic matrix and fractions of matrix below and above 5 kDa were analyzed. Using this method and SDS-PAGE analyses, we were able to detect the presence of low molecular mass matrix components (<3.5 kDa), but no free amino acids in the skeletal organic matrix. Since more than 98% of the 14C-labelled amino acids were incorporated into low molecular weight molecules, these probably form the bulk of newly synthesized organic matrix components. Our results suggest that these low molecular weight components may be peptides, which can be involved in the regulation of coral skeleton mineralization.     

Ultrastructural localization of spicule matrix proteins in normal and metalloproteinase inhibitor-treated sea urchin primary mesenchyme cells

Studies of the sea urchin larval skeleton have contributed greatly to our understanding of the process of biomineralization. In this study we have undertaken an investigation of the morphology of skeleton formation and the localization of proteins involved in the process of spicule formation at the electron microscope level. Sea urchin primary mesenchyme cells undergo a number of morphological changes as they synthesize the larval skeleton. They form a large spicule compartment that surrounds the growing spicule and, as spicule formation comes to an end, the density of the cytoplasm decreases. Inhibition of spicule formation by specific matrix metalloproteinase inhibitors or serum deprivation has some subtle effects on the morphology of cells and causes the accumulation of specific classes of vesicles. We have localized proteins of the organic matrix of the spicule and found that one protein, SM30, is localized to the Golgi apparatus and transport vesicles in the cytoplasm as well as throughout the occluded protein matrix of the spicule itself. This localization suggests that SM30 is an important structural protein in the spicule. Another spicule matrix protein, SM50, has a similar cytoplasmic localization, but in the spicule much of it is localized at the periphery of the spicule compartment, and consequently it may play a role in the assembly of new material onto the growing spicule or in the maintenance of the integrity of the matrix surrounding the spicule.     

Ultrastructural localization of proteins involved in sea urchin biomineralization.

Three skeletal tissues of the adult echinoid Paracentrotus lividus (the pedicellaria primordium, the test, and the tooth) were immunolabeled with three sera raised against the total mineralization organic matrix and two specific matrix proteins (SM30 and SM50) from the embryo of the echinoid Strongylocentrotus purpuratus. Two conventional chemical fixation protocols and two high-pressure freezing/freeze-substitution protocols were tested. One conventional protocol is recommended for its good preservation of the ultrastructure, and one high-pressure freezing/freeze-substitution protocol is recommended for its good retention of antigenicity. Immunolabeling was obtained in the three adult tissues. It was confined to the active skeleton-forming cells and to the structured organic matrix. The results indicate that the matrix proteins follow the classical routes of secretory protein assembly and export and suggest that SM30 and SM50 are a part of the tridimensional network formed by the organic matrix before the onset of mineralization. They show that the genetic program of part of skeletogenesis is conserved among different calcification models and developmental stages.     

Differential distribution of spicule matrix proteins in the sea urchin embryo skeleton

Spicule matrix proteins are the products of primary mesenchyme cells, and are present in calcite spicules of the sea urchin embryo. To study their possible roles in skeletal morphogenesis, monoclonal antibodies against SM50, SM30 and another spicule matrix protein (29 kDa) were obtained. The distribution of these proteins in the embryo skeleton was observed by immunofluorescent staining. In addition, their distribution inside the spicules was examined by a 'spicule blot' procedure, direct immunoblotting of proteins embedded in crystallized spicules. Our observations showed that SM50 and 29 kDa proteins were enriched both outside and inside the triradiate spicules of the gastrulae, and also existed in the corresponding portions of growing spicules in later embryos and micromere cultures. The straight extensions of the triradiate spicules and thickened portions of body rods in pluteus spicules were also rich in these proteins. The SM30 protein was only faintly detected along the surface of spicules. By examination using the spicule blot procedure, however, SM30 was clearly detectable inside the body rods and postoral rods. These results indicate that SM50 and 29 kDa proteins are concentrated in radially growing portions of the spicules (normal to the c-axis of calcite), while SM30 protein is in the longitudinally growing portions (parallel to the c-axis). Such differential distribution suggests the involvement of these proteins in calcite growth during the formation of three-dimensionally branched spicules.     

Expression of spicule matrix proteins in the sea urchin embryo during normal and experimentally altered spiculogenesis

During its embryonic development, the sea urchin embryo forms an endoskeletal calcitic spicule. This instance of biomineralization is experimentally accessible and also offers the advantage of occurring within a developmental context. Here we investigate the time course of appearance and localization of two proteins among the four dozen that constitute the protein matrix of the skeletal spicule. SM50 and SM30 have been studied in some detail, and polyclonal antisera have been prepared against them (C. E. Killian and F. H. Wilt, 1996, J. Biol. Chem. 271, 9150-9159). Using these antibodies we describe here the localization and time course of accumulation of these two proteins in Strongylocentrotus purpuratus, both in the intact embryo and in micromere cultures. We also investigate the disposition of the matrix proteins, SM50, SM30, and PM27, in the three-dimensional spicule by studying changes in protein localization during experimental manipulation of isolated skeletal spicules. We conclude that SM50, PM27, and SM30 probably play different roles in biomineralization, based on their localization and patterns of expression. It is unlikely that these proteins are solely structural elements within the mineral. SM50 and PM27 may play a role in defining the extracellular space in which spicule deposition occurs, while SM30 may play a role in secretion of spicule components. Finally, we report on the effects of serum on expression of some primary mesenchyme-specific proteins in micromere cultures; withholding serum severely depresses accumulation of SM30 but has only modest effects on the accumulation of other proteins.     

Optimization of the enzyme-linked lectin assay for enhanced glycoprotein and glycoconjugate analysis

Lectins are proteins capable of recognizing and binding to specific oligosaccharide structures found on glycoproteins and other biomolecules. As such, they have utility for glycoanalytical applications. One common difficulty encountered in the application of these proteins, particularly in multiwell plate assay formats known as enzyme-linked lectin assays (ELLAs), is finding appropriate blocking solutions to prevent nonspecific binding with plate surfaces. Many commonly used blocking agents contain carbohydrates and generate significant background signals in ELLAs, limiting the utility of the assays. In this study, we examined the suitability of a range of blocking reagents, including protein-based, synthetic, and commercially available carbohydrate-free blocking reagents, for ELLA applications. Each blocking reagent was assessed against a panel of 19 commercially available biotinylated lectins exhibiting diverse structures and carbohydrate specificities. We identified the synthetic polymer polyvinyl alcohol (PVA) as the best global blocking agent for performing ELLAs. We ultimately present an ELLA methodology facilitating broad spectrum lectin analysis of glycoconjugates and extending the utility of ELLAs.     

The skeletal organic matrix from Mediterranean coral Balanophyllia europaea influences calcium carbonate precipitation

Scleractinian coral skeletons are made mainly of calcium carbonate in the form of aragonite. The mineral deposition occurs in a biological confined environment, but it is still a theme of discussion to what extent the calcification occurs under biological or environmental control. Hence, the shape, size and organization of skeletal crystals from the cellular level through the colony architecture, were attributed to factors as diverse as mineral supersaturation levels and organic mediation of crystal growth. The skeleton contains an intra-skeletal organic matrix (OM) of which only the water soluble component was chemically and physically characterized. In this work that OM from the skeleton of the Balanophyllia europaea, a solitary scleractinian coral endemic to the Mediterranean Sea, is studied in vitro with the aim of understanding its role in the mineralization of calcium carbonate. Mineralization of calcium carbonate was conducted by overgrowth experiments on coral skeleton and in calcium chloride solutions containing different ratios of water soluble and/or insoluble OM and of magnesium ions. The precipitates were characterized by diffractometric, spectroscopic and microscopic techniques. The results showed that both soluble and insoluble OM components influence calcium carbonate precipitation and that the effect is enhanced by their co-presence. The role of magnesium ions is also affected by the presence of the OM components. Thus, in vitro, OM influences calcium carbonate crystal morphology, aggregation and polymorphism as a function of its composition and of the content of magnesium ions in the precipitation media. This research, although does not resolve the controversy between environmental or biological control on the deposition of calcium carbonate in corals, sheds a light on the role of OM, which appears mediated by the presence of magnesium ions.     

Soluble organic matrix of two Scleractinian corals: partial and comparative analysis

This study is a biochemical and molecular analysis of the soluble organic matrix (SOM) of two Scleractinian corals differing in their morphological characteristics: Stylophora pistillata, a branched robust coral and Pavona cactus, a leafy complex coral. Soluble organic matrix of both coral species were shown to contain high amounts of potentially acidic amino acids and glycine. However, proportions of glycosaminoglycans and SDS-PAGE analyses of soluble organic matrix proteins were very different. Three proteins of S. pistillata and at least five proteins of P. cactus were detected by silver staining, some of them being able to bind calcium. Internal peptide sequences of two matrix proteins (one from each species) were obtained. One sequence of S. pistillata is unusual because it contains a long poly-aspartate domain, as described in proteins belonging to the calsequestrin family and in proteins from molluscan species. This domain suggests an essential role for this protein in the control of mineralization.     

Sex-dependent correlations between the personality dimension of harm avoidance and the resting-state functional connectivity of amygdala subregions

Harm avoidance (HA) is a personality dimension involving the tendency to respond intensely to signals of aversive stimuli. Many previous neuroimaging studies have associated HA scores with the structural and functional organization of the amygdala, but none of these studies have evaluated the correlation between HA score and amygdala resting-state functional connectivity (rsFC). Moreover, the amygdala is not a homogeneous structure, and it has been divided into several structurally and functionally distinct subregions. Investigating the associations between HA score and properties of subregions of the amygdala could greatly improve our understanding of HA. In the present study, using a large sample of 291 healthy young adults, we aimed to uncover correlations between HA scores and the rsFCs of each amygdala subregion and to uncover possible sex-based differences in these correlations. We found that subregions of the amygdala showed different rsFC patterns, which contributed differently to individual HA scores. More specifically, HA scores were correlated with rsFCs between the laterobasal amygdala subregion and temporal and occipital cortices related to emotional information input, between the centromedial subregion and the frontal cortices associated with emotional output control, and between the superficial subregion and the frontal and temporal areas involved in both functions. Moreover, significant gender-based differences were uncovered in these correlations. Our findings provide a more detailed model of association between HA scores and amygdala rsFC, extend our understanding of the connectivity of subregions of the amygdala, and confirm sex-based differences in HA associations.     

Spiculogenesis in the sea urchin embryo: Studies on the SM30 spicule matrix protein

When proteins isolated from spicules of Strongylocentrotus purpuratus embryos were examined by western blot analysis, a major protein of approximately 43 kDa was observed to react with the monoclonal antibody, mAb 1223. Previous studies have established that this antibody recognizes an asparagine-linked, anionic carbohydrate epitope on the cell surface glycoprotein, msp130. This protein has been shown to be specifically associated with the primary mesenchyme cells involved in assembly of the spicule. Moreover, several lines of evidence have implicated the carbohydrate epitope in Ca²⁺ deposition into the growing spicule. The 43 kDa, spicule matrix protein detected with mAb 1223 also reacted with a polyclonal antibody to a known spicule matrix protein, SM30. Further characterization experiments, including deglycosylation using PNGaseF, two-dimensional electrophoresis, and immunoprecipitation, verified that the 43 kDa spicule matrix protein had a pl of approximately 4.0, contained the carbohydrate epitope recognized by monoclonal antibody mAb 1223 and reacted with anti-SM30. Electron microscopy confirmed the presence of proteins within the demineralized spicule that reacted with mAb 1223 and anti-SM30. We conclude that the spicule matrix protein, SM30, is a glycoprotein containing carbohydrate chains similar or identical to those on the primary mesenchyme cell membrane glycoprotein, msp130.     

Posterior Midgut Epithelial Cells Differ in Their Organization of the Membrane Skeleton from Other Drosophila Epithelia

In epithelial cells, the various components of the membrane skeleton are segregated within specialized subregions of the plasma membrane, thus contributing to the development and stabilization of cell surface polarity. It has previously been shown that, in various Drosophila epithelia, the membrane skeleton components ankyrin and alphabeta-spectrin reside at the lateral surface, whereas alphabeta(H)-spectrin is restricted to the apical domain. By use of confocal immunofluorescence microscopy, the present study characterizes the membrane skeleton of epithelial cells in the posterior midgut, leading to a number of unexpected results. First, ankyrin and alphabeta-spectrin are not detected on the entire lateral surface but appear to be restricted to the apicolateral area, codistributing with fasciclin III at smooth septate junctions. The presumptive ankyrin-binding proteins neuroglian and Na(+),K(+)-ATPase, however, do not colocalize with ankyrin. Second, alphabeta(H)-spectrin is enriched at the apical domain but is also present in lower amounts on the entire lateral surface, colocalizing apicolaterally with ankyrin/alphabeta-spectrin. Finally, despite the absence of zonulae adherentes, F-actin, beta(H)-spectrin, and nonmuscle myosin-II are enriched in the midlateral region. Thus, the model established for the organization of the membrane skeleton in Drosophila epithelia does not hold for the posterior midgut, and there is quite some variability between the different epithelia with respect to the organization of the membrane skeleton.     

How corals made rocks through the ages

Hard, or stony, corals make rocks that can, on geological time scales, lead to the formation of massive reefs in shallow tropical and subtropical seas. In both historical and contemporary oceans, reef‐building corals retain information about the marine environment in their skeletons, which is an organic–inorganic composite material. The elemental and isotopic composition of their skeletons is frequently used to reconstruct the environmental history of Earth's oceans over time, including temperature, pH, and salinity. Interpretation of this information requires knowledge of how the organisms formed their skeletons. The basic mechanism of formation of calcium carbonate skeleton in stony corals has been studied for decades. While some researchers consider coral skeletons as mainly passive recorders of ocean conditions, it has become increasingly clear that biological processes play key roles in the biomineralization mechanism. Understanding the role of the animal in living stony coral biomineralization and how it evolved has profound implications for interpreting environmental signatures in fossil corals to understand past ocean conditions. Here we review historical hypotheses and discuss the present understanding of how corals evolved and how their skeletons changed over geological time. We specifically explain how biological processes, particularly those occurring at the subcellular level, critically control the formation of calcium carbonate structures. We examine the different models that address the current debate including the tissue–skeleton interface, skeletal organic matrix, and biomineralization pathways. Finally, we consider how understanding the biological control of coral biomineralization is critical to informing future models of coral vulnerability to inevitable global change, particularly increasing ocean acidification.     

Interaction of MAR-sequences with nuclear matrix proteins.

The recent discovery of DNA sequences responsible for the specific attachment of chromosomal DNA to the nuclear skeleton (MARs/SARs) was an important step towards our understanding of the functional and structural organization of eukaryotic chromatin [Mirkovitch et al.: Cell 44:273-282, 1984; Cockerill and Garrard: Cell 44:273-282, 1986]. A most important question, however, remains the nature of the matrix proteins involved in the specific binding of the MARs. It has been shown that topoisomerase II and histone H1 were capable of a specific interaction with SARs by the formation of precipitable complexes [Adachi et al.: EMBO J8:3997-4006, 1989; Izaurralde et al.: J Mol Biol 210:573-585, 1989]. Here, applying a different approach, we were able to "visualize" some of the skeletal proteins recognizing and specifically binding MAR-sequences. It is shown that the major matrix proteins are practically the same in both salt- and LIS-extracted matrices. However, the relative MAR-binding activity of the individual protein components may be different, depending on the method of matrix preparation. The immunological approach applied here allowed us to identify some of the individual MAR-binding matrix proteins. Histone H1 and nuclear actin are shown to be not only important components of the matrix, but to be involved in a highly efficient interaction with MAR-sequences as well. Evidence is presented that proteins recognized by the anti-HMG antibodies also participate in MAR-interactions.     

Identification of organic phosphorus covalently bound to collagen and non-collagenous proteins of chicken-bone matrix. The presence of O-phosphoserine and O-phosphothreonine in non-collagenous proteins, and their absence from phosphorylated collagen

Non-collagenous phosphoproteins, almost all of which can be extracted in EDTA at neutral pH in the presence of proteinase inhibitors, are identified in the matrix of chicken bone, and are therefore not covalently bound to collagen. Similarly, all the peptides containing gamma-carboxyglutamic acid are present in the EDTA extract and none in the insoluble residue, confirming that none is covalently linked to chicken bone collagen. However, organic phosphorus is also found to be present in chicken bone collagen, principally in the alpha2-chains. Of the total protein-bound organic phosphorus present in chicken bone matrix, approx. 80% is associated with the non-collagenous proteins and 20% with collagen. The soluble non-collagenous proteins contain both O-phosphoserine and O-phosphothreonine and these account for essentially of their organic phosphorus content. In contrast, collagen contains neither O-phosphoserine nor O-phosphothreonine. Indeed, no phosphorylated hydroxy amino acid, phosphoamidated amino acid or phosphorylated sugar could be identified in purified components of collagen, which contain approximately four to five atoms of organic phosphorus per molecule of collagen. Peptides containing organic phosphorus were isolated from partial acid hydrolysates and enzymic digests of purified collagen components, which contain an as-yet-unidentified cationic amino acid. These data, the very high concentrations of glutamic acid in the phosphorylated peptides, and the pH-stability of the organic phosphorus moiety in intact collagen chains strongly suggest that at least part of the organic phosphorus in collagen is present as phosphorylated glutamic acid. This would indicate that the two major chemically different protein fractions in chicken bone matrix that contain organic phosphorus may represent two distinct metabolic pools of organic phosphorus under separate biological control.     

我国及邻近地区斑腿蝗科区系及其起源研究

根据斑腿蝗科(Catantopidae)在我国各动物地理区的种类组成和区系特点,发现华中区和华北区的区系特点不明显,前者为南北成份的混交区,后者特有种可跨区分布,这与蝗虫有明显的区域和地带分布的特点不相符。为此,作者同意马世骏先生把江汉平原、秦阳山山地、辽黄淮平原划为古北区,成为东方区系。根据我国北方种类与前苏联西伯利亚区系关系密切,而极少数种类与前苏联欧洲部分甚至欧洲部分所共有这种事实,认为世界动物地理区划中应把西伯利亚单独作为一个亚区较合适。     

Desmocytes in the calicoblastic epithelium of the stony coral Mycetophyllia reesi and their attachment to the skeleton

Desmocytes scattered over the surface of the corallum of the scleractinian Mycetophyllia reesi attach the calicoblastic tissue to the skeleton. The structure of the desmocyte is generally consistent with that of other scleractinians except for their more rectangular profiles and greater size. However, the extent of attachment is distinctive, and the mode of attachment to mineral is described for the first time. The skeleton contains dual rows of interconnected pits between the septa, within and among which desmocytes form virtually uninterrupted sheets. Desmocytes terminate with hemidesmosomes that attach the epithelium to a fibrillar basal lamina. Fibrils extend from the basal lamina into the skeletal matrix anchoring tissue firmly to the skeleton. In addition, the basal lamina itself appears to be incorporated within the organic matrix during growth, partitioning the skeleton into compartments. Because the skeletal organic matrix is physicochemically labile during demineralization, these intraskeletal details cannot be observed unless polycationic dyes such as Ruthenium red or other glycan precipitating agents are employed in the fixative sequence.     

Biologically controlled mineralization in the hypercalcified sponge Petrobiona massiliana (Calcarea, Calcaronea)

Hypercalcified sponges, endowed with a calcium carbonate basal skeleton in addition to their spicules, form one of the most basal metazoan group engaged in extensive biomineralization. The Mediterranean species Petrobiona massiliana was used to investigate biological controls exerted on the biomineralization of its basal skeleton. Scanning and transmission electron microscopy (SEM, TEM) confirmed that basopinacocytes form a discontinuous layer of flattened cells covering the skeleton and display ultrastructural features attesting intense secretory activity. The production of a highly structured fibrillar organic matrix framework by basopinacocytes toward the growing skeleton was highlighted both by potassium pyroantimonate and ruthenium red protocols, the latter further suggesting the presence of sulfated glycosaminoglycans in the matrix. Furthermore organic material incorporated into the basal skeleton was shown by SEM and TEM at different structural levels while its response to alcian blue and acridine orange staining might suggest a similar acidic and sulfated chemical composition in light microscopy. Potassium pyroantimonate revealed in TEM and energy electron loss spectroscopy (EELS) analysis, heavy linear precipitates 100-300 nm wide containing Ca(2+) and Mg(2+) ions, either along the basal cell membrane of basopinacocytes located toward the decalcified basal skeleton or around decalcified spicules in the mesohyl. Based on the results of the previous mineralogical characterization and the present work, an hypothetical model of biomineralization is proposed for P. massiliana: basopinacocytes would produce an extracellular organic framework that might guide the assemblage of submicronic amorphous Ca- and Mg-bearing grains into higher structural units.     

Mitochondrial diversity of Musca domestica housefly populations in the Asian and western Pacific biogeographical regions

Houseflies (Musca domestica L., Diptera: Muscidae) are cosmopolitan, colonizing, and eusynanthropic. Their distribution in the Malaysian archipelago provides an opportunity to study successive waves of colonization and extinction during the Pleistocene and Recent epochs. We scored single-strand conformation polymorphisms (SSCPs) at 16S2 and COII mitochondrial loci in 47 housefly samples from the Australian, Austro-Malayan, Indo-Malayan, Manchurian and Indo-Chinese subregions of Wallace's zoogeographical classification. We discuss the results in light of the Pleistocene vs. post-Pleistocene dispersal and faunal exchange in the Asia-Pacific area. Fourteen haplotypes were detected, of which 10 were confined to a single subregion. No haplotype was ubiquitous and only one was found in four subregions. Population diversity, HS, was greatest in the Indo-Malayan (0.36) and heterogeneous among subregions. The mean subregional diversity was 0.21 +/- 0.03, representing the probability that two randomly chosen flies, from any subregion, had different haplotypes. The hierarchical partition of diversity indicated restricted maternal gene flow among subregions (GRT = 0.60, Nm approximately 0.32). These results suggest long-standing genetic isolation of houseflies in the Malaysian archipelago and support the hypothesis that they dispersed widely during the Pleistocene. Haplotypes common among mainland populations but shared with island groups in low frequencies (<1%) indicate surprisingly little recent gene flow.