Comparison of aragonitic molluscan shell proteins

Acidic macromolecules, as a nucleation factor for mollusc shell formation, are a major focus of research. It remains unclear, however, whether acidic macromolecules are present only in calcified shell organic matrices, and which acidic macromolecules are crucial for the nucleation process by binding to chitin as structural components. To clarify these questions, we applied 2D gel electrophoresis and amino acid analysis to soluble shell organic matrices from nacre shell, non-nacre aragonitic shell and non-calcified squid shells. The 2D gel electrophoresis results showed that the acidity of soluble proteins differs even between nacre shells, and some nacre (Haliotis gigantea) showed a basic protein migration pattern. Non-calcified shells also contained some moderately acidic proteins. The results did not support the correlation between the acidity of soluble shell proteins and shell structure.     

Organic biopolymers of venus clams: Collagen-related matrix in the bivalve shells with crossed-lamellar ultrastructure

BackgroundBiochemical studies and spectroscopic techniques have shown that chitin-silk fibroins are common in nacroprismatic bivalve shells. However, the nature of organic biopolymers in the less well studied shell architectures, such as crossed lamellar shells, remain unknown. Here, two venus shells, Callista disrupta and Callista kingii, with crossed lamellar ultrastructure have been studied.MethodsWe employed thermal gravimetric analysis, optical-, confocal- and scanning electron-microscopes, gel-sodium dodecyl sulfate (gel-SDS), FTIR, ultra-performance liquid chromatography and high-performance anion-exchange chromatography system with pulsed amperometric detection to analyse organic macromolecules in the shells.ResultsThermal analysis showed a low concentration of organic macromolecules in C. disrupta (1.38 wt%) and in C. kingii (1.71 wt%). A combination of biochemical protocols, including Calcofluor White staining and FTIR spectroscopic assessment, indicate that amino-polysaccharide chitin together with proteins, are present in the organic scaffolding of the shells. Scanning electron microscope of insoluble acid biopolymer extracts as well as FTIR technique show that the hierarchical structural organizations of organic biopolymers consist collagen-related matrix. Our histochemical fixing and staining techniques reveal many discrete proteins and glycoproteins from soluble organic macromolecules on the gel-SDS. We show here ‘singlet’ and ‘doublet’ glycosaminoglycan bands that are far above 260 kDa.General significance/conclusionsThe presence of collagen matrix in Callista shells shows promise for the new source of biomaterials. Most importantly, the structural organization of the proteinaceous motif is predominantly helical structures and not silk-fibroin unlike in nacreous bivalve shells.     

Phylogenetic implications and diagenetic stability of macromolecules from pleistocene and recent shells of Mercenaria mercenaria (mollusca,bivalvia)

The phylogeny and diagenesis of Pleistocene and Recent bivalves were studied immunologically by use of a conventional antiserum elicited against an EDTA‐soluble macromolecular extract from shells of the modern bivalve mollusc Mercenaria mercenaria. ELISA tests of the antiserum with shell fragments of a wide range of modern bivalves gave taxonomically significant results. The antiserum reacted with palaeoheterodonts and heterodonts but not with representatives of other bivalve subclasses. This phylogenetic reactivity was also apparent in tests with fossil shells, although the specificity and overall strength of the reaction were both reduced. Absorption of the antiserum with etched shell powders of various (palaeo)heterodonts yielded more specific antibody preparations.

Investigations of shell matrix diagenesis, using the anti‐Mercenaria serum, demonstrated that small amounts of original determinants could be detected even in fossils over one million years old. The reactivity of the serum with extracts of fossil Mercenaria decreased with sample age. The relationship between serum reactivity and the degree of amino acid racemization was almost linear. Clearly, the various determinants to which antibodies were elicited were being destroyed at different rates.     

Organic matrixlike macromolecules associated with the mineral phase of sea urchin skeletal plates and teeth

The skeletal plates and teeth of the echinoid Paracentrotus lividus contain a heterogeneous assemblage of macromolecules that are not part of the connective tissue, but are presumably intimately associated with the mineral phase. Upon dissolution of the Mg-calcite mineral phase, some of these molecules are insoluble. The insoluble fractions of the teeth and skeletal plates are quite different, the former being predominantly protein and the latter, primarily some unknown nonproteinaceous material. The soluble constituents are similar in both tissues. These hydrophilic macromolecules have been partially separated and characterized. In both hard parts, two distinct classes of macromolecules are present, as indicated by the amino acid compositions of their protein constituents. These two classes of macromolecules are also present in the shells of a foraminifer and in various mollusks, both of which are formed by the "organic matrix-mediated" biomineralization process. The locations of these macromolecules in the teeth and skeletal plates are not known, nor whether they form coherent structures. It is therefore premature to conclude that these macromolecules do function as an organic matrix, although the results presented are in agreement with such an interpretation.     

Sero‐taxonomy of skeletal macromolecules in living Terebratulid brachiopods

Antibodies prepared against macromolecules isolated from the shells of three living brachiopod genera have proved to be of considerable taxonomic significance, in that the pattern of cross‐reactivity of all three antisera consistently points to a new interpretation for the evolution of the largest extant brachiopod order, the Terebratulida. This new molecular evidence actually complements rather than contradicts the existing morphology‐based taxonomy, since detailed systematic investigation of the taxa in question has already demonstrated subtle but significant morphological differences in the major taxonomic characters which appear to reflect this new interpretation.

As fragments of skeletal macromolecules, including antigenic determinants, are known to survive for many millions of years within the protected micro‐environments provided by enclosing biominerals, these results suggest that such molecular fossils could well provide important insights on at least the high‐level taxonomic relationships of fossil organisms.     

Biomacromolecules of Algae and Plants and their Fossil Analogues

A review of our current understanding of resistant biomacromolecules derived from present and past algae and higher plants is presented. Insight in the nature of recent and fossil macromolecules is strongly hampered by the difficulties in obtaining the material in pure and unaltered form. For the extant material, avoiding artificial condensation and structural alteration as a result of chemical isolation and purification of biomacromolecules requires constant attention. To date, several types of sporopollenin seem to occur. One type is characterised by oxygenated aromatic building blocks, in particular p-coumaric acid and ferrulic acid. The other type is thought to consist predominantly of an aliphatic biopolymer. In this review it is concluded that extant sporopollenin consists of the aromatic type, whereas the aliphatic component of fossil sporopollenin is due to early-diagenetic oxidative polymerization of unsaturated lipids. The cuticles of most higher plants contain the aliphatic biopolyester cutin. Additionally, cuticles of drought-adapted, mostly CAM plants, seem to contain the non-hydrolysable aliphatic biopolymer cutan. Only a very few algae are able to biosynthesize resistant, (fossilisable) cell walls: some Chlorophyta, Eustigmatophyta and Prasinophyta produce the aliphatic biopolymer algaenan. Some Dinophyta are also capable of producing algaenan cell walls. Additionally, some taxa produce highly resistant cyst-walls with a high proportion of aromatic moieties. For the morphologically well-preserved fossil material, contamination by organic particles other than the target taxon is hard to eliminate and can contribute to either the aliphatic or aromatic signal. Furthermore, post-mortem migration of aliphatic moieties into, and their condensation onto the macromolecule might occur, e.g. by oxidative polymerization. These phenomena hamper the evaluation of the aliphatic signature of fossil plant material and may for example explain the preservation of initially cutin-based cuticles or non-algaenan containing algae. The extent to which migration and in situ formation of aromatic moieties plays a role in modifying resistant algal macromolecules, notably under elevated temperature and/or pressure conditions, still remains an open question.     

Dopamine β-Hydroxylase and Other Glycoproteins from the Soluble Content and the Membranes of Adrenal Chromaffin Granules: Isolation and Carbohydrate Analysis

Abstract: Chromogranin A and two other proteins (A₁ and A₂) of the soluble proteins of bovine chromaffin granules were isolated by extraction from polyacrylamide gels after electrophoresis. The carbohydrate content of these proteins was 5%, with galactose, N -acetylgalactosamine, and sialic acid as the main sugars. Membranes of chromaffin granules were solubilized with sodium dodecyl sulphate (SDS) and three glycoproteins were isolated by sequential affinity chromatography on Concanavalin A (Con A) and wheat germ lectin (WGL) Sepharose columns. Two glycoproteins, designated GP II and III, were found to have a high carbohydrate content of about 30%. Mannose, galactose, N -acetylgalactosamine, and sialic acid were the main sugars. In addition membrane-bound dopamine β-hydroxylase was isolated by this procedure. No significant differences between the carbohydrate composition of the membrane-bound and the soluble enzyme were revealed. It was shown that all four subunits of dopamine β-hydroxylase possess carbohydrate chains with an affinity for Con A. The isolation methods established in this study will be useful for immunological studies on these glycoproteins.     

Carbon-Isotope Composition of Biochemical Fractions and the Regulation of Carbon Balance in Leaves of the C3-Crassulacean Acid Metabolism Intermediate Clusia minor L. Growing in Trinidad

Carbon-isotope ratios ([delta]13Cs) were measured for various bio-chemical fractions quantitatively extracted from naturally exposed and shaded leaves of the C3-Crassulacean acid metabolism (CAM) intermediate Clusia minor, sampled at dawn and dusk on days during the wet and dry seasons in Trinidad. As the activity of CAM increased in response to decreased availability of water and higher photon flux density, organic acids and soluble sugars were enriched in 13C by approximately 3.5 to 4%[per mille (thousand) sign] compared to plants sampled during the wet season. The induction of CAM was accompanied by a doubling in size of the reserve carbohydrate pools. Moreover, stoichiometric measurements indicated that degradation of both chloroplastic reserves and soluble sugars were necessary to supply phosphoenolpyruvate for the synthesis of organic acids at night. Results also suggest that two pools of soluble sugars exist in leaves of C. minor that perform CAM, one a vacuolar pool enriched in 13C and the second a transport pool depleted in 13C. Estimates of carbon-isotope discrimination expressed during CAM, derived from the trafficking among inorganic carbon, organic acids, and carbohydrate pools overnight, ranged from 0.9 to 3.1%[per mille (thousand) sign]. The [delta]13C of structural material did not change significantly between wet and dry seasons, indicating that most of the carbon used in growth was derived from C3 carboxylation.     

Fructan Hydrolysis Drives Petal Expansion in the Ephemeral Daylily Flower

Dry weight, water content, soluble carbohydrate content, and carbohydrate composition of daylily (Hemerocallis hybrid cv Cradle Song) flower petals were monitored in the 3 d leading up to full opening and in the first day of senescence. Timing of events was related to the time (hour 0) when flower expansion was 60% complete. Petal dry weight increased linearly from hour -62 (tight bud) to hour 10 (fully developed flower), then fell rapidly to hour 34 as senescence advanced. Increase in water content was proportional to dry weight increase from hour -62 to hour -14, but was more rapid as the bud cracked and the flower opened, giving an increase in fresh weight/dry weight ratio. Soluble carbohydrate was 50% of petal dry weight up to hour 10, then decreased during senescence to reach 4% by hour 34. Up until hour -14, fructan accounted for 80% of the soluble carbohydrate in the petals, whereas hexose accounted for only 2%. Fructan hydrolysis started just prior to bud crack at hour -14, reaching completion by hour 10 when no detectable fructan remained, and fructose plus glucose accounted for more than 80% of the total soluble carbohydrate. The proportion of sucrose remained constant throughout development. Osmolality of petal cell sap increased significantly during fructan hydrolysis, from 0.300 to 0.340 osmolal. Cycloheximide applied to excised buds between hour -38 and hour -14 halted both fructan hydrolysis and flower expansion. The findings suggest that onset of fructan hydrolysis, with the concomitant large increase in osmoticum, is an important event driving flower expansion in daylily.     

Variant specific glycoproteins of Trypanosoma equiperdum: cross reacting determinants and chemical studies

Two variant specific surface antigens (VSSA) (BoTat-1 and BoTat-28) were purified from clones of $\text{Trypanosoma equiperdum}$ and tested for immunological cross reactivity. Competitive radioimmunoassay revealed the presence of cross reacting determinants. Limited tryptic cleavage of BoTat-28 resulted in the purification of two fragments F₁ and F₂ with respectively 38,000 and 28,000 apparent molecular weight. F₁ represents a large N-terminal fragment containing 30 % of the total sugar content. F₂ is characterized by an N-terminal amino acid sequence different from that of the native glycoprotein and contains all the carbohydrate. Inhibition of the heterologous precipitation reaction was only achieved with F₂ and reaches 100 % as with the native glycoproteins. These results demonstrate the presence of cross reacting determinants located in the C-terminal part of the molecule.     

Studies of Biominerals Relevant to the Search for Life on Mars

The evidence of the water erosion on Mars is particularly interesting since present climatic conditions are such that liquid water cannot exist at the surface. But, if water was present on the planet in the past, there may have been life, too. Since the discovery of carbonates on Mars also may have very important implications on the possibility that life developed there, we are studying minerals that can have biotic or abiotic origin: calcite (CaCO₃) and aragonite, a metastable state of calcite. We have analysed biomineral aragonite, in the form of recent sea shells, as well as crystals of mineral aragonite. Infrared spectroscopy in the 2–25 μm wavelength range reveals that, after thermal processing, the biotic samples have a different spectral behaviour from the abiotic ones. As a result, it is possible to distinguish abiotic mineral aragonite from aragonite of recent biological origin. Obviously, if life existed in the past on the Red Planet, we could expect to find “ancient” biotic carbonates, which should therefore be investigated, in order to search for a way of discriminating them from abiotic minerals. For this reason, at the beginning we have considered samples of crushed fossil shells of aragonite composition. Afterwards, in order to take into account that fossilization processes almost always produce a transformation of metastable form (aragonite) into more stable form (calcite), we also studied samples of mineral calcite and different types of fossils completely transformed into calcite. All these biotic fossil samples show the same spectral behaviour as the fresh biotic material after thermal annealing at 485°C. Instead, the calcite behaves like abiotic aragonite. Furthermore, it is known that seashells and other biominerals are formed through an intimate association of inorganic materials with organic macromolecules. The macromolecules control the nucleation, structure, morphology, crystal orientation and spatial confinement of the inorganic phase: this differentiates biominerals from minerals. Analysing the aragonite or calcite fossils with a Scanning Electron Microscope, we found that the fossilization process did not modify the structure of the biominerals which maintain their microscopic characteristics. Looking at the morphology of fossil biominerals, it is evident that the crystals are arranged in complex architectures compared with the compact structure of the mineral crystals. In conclusion, the properties and structure of the biominerals are different from those of the minerals. The rapid increase of the crystalline structure developed under biotic conditions makes these minerals less resistant to thermal treatments, compared with samples of abiotic origin. This result holds both for recent shells as well as all fossil samples. The spectroscopic behaviour of all analysed calcium carbonates of biotic origin is different from that of the abiotic one. Therefore, the infrared spectroscopy is a valid technique to discern the origin of the samples and a powerful tool for analysing in-situ and “sample-return” Mars missions specimens. Also Optical and Scanning Electron Microscopy can be useful to support this type of studies. ^*Presented at: National Workshop on Astrobiology: Search for Life in the Solar System, Capri, Italy, 26 to 28 October, 2005     

Biochemical and calorific content of deep-sea aspidochirotid holothurians from the northeast atlantic ocean

• 1.1. The organic composition of the body tissues of eight species of deep-sea aspidochirotid holothurian, collected between 500 and 4100m depth in the NE Atlantic Ocean, was obtained by the biochemical analysis of protein, lipid, carbohydrate and % ash.
• 2.2. The major organic class was protein with soluble lipid the major soluble fraction in the ovary. Carbohydrate values were consistently low.
• 3.3. The calorific value was significantly higher in the ovary than in the other tissues.
• 4.4. The total body calorific content for two selected species, Benthothuria funebris and Mesothuria lactea, was 25.62 and 26.24J/mg ash-free dry weight (AFDW).

     

Immunological responses from brachiopod skeletal macromolecules; a new technique for assessing taxonomic relationships using shells

The phylogenetic relationships of living, calcareous-shelled brachiopods have been assessed on the basis of immunological responses from intracrystalline macromolecules. Calculations of immunological distances between representatives of the order Terebratulida have revealed a primary threefold division which correlates precisely with a proposed subdivision of the order into three superfamilies but refutes attempts to establish a fourth superfamily. This conclusion was confirmed by carrying out immunological investigations of small shell fragments from other brachiopod genera which were so rare that no antisera could be prepared. The immunological results also indicate a fundamental subdivision of the long-looped brachiopods, with one group perhaps being derived from short-looped terebratuloids rather than long-looped terebratelloids. Sero-taxonomy of skeletal macromolecules provides an ideal method of acquiring molecular phylogenetic data in many groups because a large number of taxa can be surveyed in a short period of time, and microscopic pieces of shell contain sufficient antigenic determinants for many reactions. The technique can also be applied to specimens which have been stored without special treatment in museum collections, making the technique particularly applicable to rare taxa for which no other form of molecular data is available. □ Immunology, Brachiopoda, taxonomy, intracrystalline molecules.     

Reducing effect of feeding powdered scallop shell on the body fat mass of rats

The lipolytic effect of powdered scallop shells was estimated in vitro and in vivo. The scallop shells consisted of 98% calcium carbonate and 2% organic compounds, the extracted organic components promoted lipolysis in 3T3-L1 adipocyte cells. Male Wistar rats were fed on an experimental diet containing either the scallop shell powder or calcium carbonate (control) for 28 d. Feeding the scallop shell powder resulted in a decrease in body weight and in the weight of white adipose tissue. While the organ weights of the liver, kidney, testis, pancreas, and spleen, and of the brown adipose tissue relative to the body weight were no different between the scallop shell powder diet and control diet, the white adipose tissue weight relative to the body weight significantly decreased in the rats fed on the scallop shell powder. The glycerol concentration in the serum increased in the rats fed on the scallop shell powder, suggesting that this promoted lipolysis in the adipose tissue. These results show that the organic components in the scallop shells induced the decrease in weight of the adipose tissue due to the promotion of lipolysis.     

A new hydroid from the Upper Cretaceous of Mississippi

A new fossil hydroid is reported as an organic impression on a calcareous gastropod steinkern from the Prairie Bluff Chalk (Maastrichtian), Oktibbeha County, Mississippi. This is the first such hydroid reported from the Upper Cretaceous of the Atlantic Coastal Plain. The fossil organism consists of anastomosing hydrorhizae forming a holdfast, a fascicled hydrocaulus, and elongated, crenulated and ribbed hydrothecae. The fossil is unlike other Mesozoic hydroids that have been reported from Europe and North America; it is described as Mesodendrium oktibbehaensis gen. et sp. nov. and tentiatively referred to an extant family, the Campanulariidae (Calyptoblastina). The complete preservation of the holdfast, hydrocaulus and hydrothecae suggests that this hydroid lived inside gastropod shells. In analogy with Recent symbiotic hydroids inhabiting mollusc shells, the new specimen described here possibly represents the oldest known example of a symbiotic relationship between hydroids and hermit crabs.     

Blood-group-ABH antigens of human erythrocytes. Quantitative studies on the distribution of H antigenic sites among different classes of membrane components

The contribution of blood-group-active glycolipids and glycoproteins to the blood-group-ABH character of human erythrocytes was investigated. For that purpose the blood-group-H sites of human O cells were converted in vitro into group-A sites by transfer of alpha-N-acetyl-D-[14C]galactosamine residues with the aid of the blood-group-A gene-dependent alpha-N-acetylgalactosaminyl transferase prepared from human A1 plasma. Upon partition of the red cell membranes between water and organic solvent, about 5% of the label was found in the organic phase and about 20% in the water phase, thus reflecting the distribution of blood-group antigenic sites between glycosphingolipids with short carbohydrate chains and polyglycosylceramides, respectively. The fact that about 70% of the radioactivity remained tightly bound to the membranes and could only be released by treatment with pronase provided good evidence that the bulk of blood-group-H determinants is bound to glycoprotein material. Following these results it can thus be assumed that blood-group-ABH activity of human erythrocytes is determined preferentially by group-specific glycoproteins rather than glycolipids.     

Discrete molecular weight components of the organic matrices of mollusc shells

Discrete molecular weight components form part of the soluble fraction of the organic matrices of mollusc shells. The number of these components, their relative migration distances and concentrations vary greatly in the specimens analysed. The discrete molecular weight constituents from the organic matrices of species of the same genus are most similar. The presence of these components shows that organic matrices are heterogeneous and presumably capable of performing diverse functions.     

Studies on the Dissociation of the Soluble Ovomucin by Sonication

The soluble ovomucin obtained from the liquid part of thick white by gel filtration on a Sepharose 4B was an aggregated and polymerized molecule (intrinsic viscosity was 365 ml/g and molecular weight was 8.3 × 10⁶) and it was unable to dissociate the soluble ovomucin into two components without modifications.

Molecular weight and reduced viscosity of the soluble ovomucin decreased markedly with time of sonication. By the sonication for 10 min, it was successful to fractionate it into carbohydrate rich and poor component by density gradient electrophoresis, cellulose acetate electrophoresis and DEAE-cellulose column chromatography.

Concerning carbohydrate and amino acid compositions of two components obtained from the sonicated soluble ovomucin, it was found that the carbohydrate poor component corresponded to the reduced S-component or the reduced α-ovomucin, and the carbohydrate rich component to the reduced F-component or the reduced β-ovomucin.

It was considered that the sonicated soluble ovomucin was an intermediate of the aggregated, polymerized ovomucin (the soluble ovomucin) and the monomeric ovomucin (the sonicated and reduced soluble ovomucin).     

Radiation inactivation of human intestinal mucin: determination of the size of the functional antigenic unit

Previously we have shown that the major antigenic determinant of human intestinal mucin is associated with its glycopeptide monomers and not the 118 kDa 'link' component. In the present study, the size and nature of the functional unit containing the antigenic determinant has been assessed by radiation inactivation and immunological assays. Increasing doses of radiation led to a monoexponential decay in antigenic reactivity due to a progressive loss of antigenic determinants. From three independent mucin preparations, a value of 78500 +/- 7000 was determined for the Mr of the functional antigenic unit. Prolonged pronase digestion of native mucin released large degraded glycopeptide monomers containing all the mucin carbohydrate, and low molecular weight peptides. The antigenicity of the glycopeptides decreased with digestion but could not be recovered in the peptide fractions, suggesting that determinants were released and destroyed by the enzyme. Treatment of native mucin with trifluoromethanesulphonic acid caused a major loss of carbohydrate (approx. 70%), but the protein component was unchanged in amino acid profile and remained antigenic. Subsequent thiol reduction, however, abolished the antigenicity of the deglycosylated mucin. We conclude that antigenicity is associated with a non-glycosylated segment of the peptide backbone of the glycopeptides and that a large functional unit of Mr 78500 which is stabilized by disulphide bonds is important for full antigenic activity.     

L'intensité lumineuse,son influence sur la teneur en phosphore des carapaces d'Ostracodes

Actual and fossil shells of ostracodes have beenanalyzed with an electronic microprobe: all of them contain phosphorus. X—pictures teach us that the repartition of this chemical element is homogeneous in all the points of the shell. There is no relationship between phosphorus contents in the shells of ostracodes and salinity. In return, there is a relationship between phosphorus contents and temperature and, chiefly, organic phosphorus contents in the environment. However, it is interesting to observe that the analyzed shells of fresh-water ostracodes contain less phosphorus that the generality of the shells of marine ostracodes. The contents of organic phosphorus, in the biotope, depends on the growth of phytoplancton in the upper layers of water which needs a minimum light intensity.