Vorkommen von chitin bei coelenteraten

The occurrence of chitin has been demonstrated in heavily calcified Milleporidae (3 species) and Hexacorallia (4 species), but not in Octocorallia. Microfibrils, probably containing chitin, are arranged in a dispersed texture in these species and are embedded in a protein-containing matrix.     

Identification and localization of lysozyme as a component of eggshell membranes and eggshell matrix.

The avian eggshell is a composite biomaterial composed of non-calcifying eggshell membranes and the overlying calcified shell matrix. The calcified shell forms in a uterine fluid where the concentration of different protein species varies between the initial, rapid calcification and terminal phases of eggshell deposition. The role of these avian eggshell matrix proteins during shell formation is poorly understood. The properties of the individual components must be determined in order to gain insight into their function during eggshell mineralization. In this study, we have identified lysozyme as a component of the uterine fluid by microsequencing, and used western blotting, immunofluorescence and colloidal-gold immunocytochemistry to document its localization in the eggshell membranes and the shell matrix. Furthermore, Northern blotting and RT-PCR indicates that there is a gradient to the expression of lysozyme message by different regions of the oviduct, with significant albeit low levels expressed in the isthmus and uterus. Lysozyme protein is abundant in the limiting membrane that circumscribes the egg white and forms the innermost layer of the shell membranes. It is also present in the shell membranes, and in the matrix of the calcified shell. Calcite crystals grown in the presence of purified hen lysozyme exhibited altered crystal morphology. Therefore, in addition to its well-known anti-microbial properties that could add to the protective function of the eggshell during embryonic development, shell matrix lysozyme may also be a structural protein which in soluble form influences calcium carbonate deposition during calcification.     

Ex-vivo observation of calcification process in chick tibia slice: Augmented calcification along collagen fiber orientation in specimens subjected to static stretch

Bone formation through matrix synthesis and calcification in response to mechanical loading is an essential process of the maturation in immature animals, although how mechanical loading applied to the tissue increases the calcification and improves mechanical properties, and which directions the calcification progresses within the tissue are largely unknown. To address these issues, we investigated the calcification of immature chick bone under static tensile stretch using a newly developed real-time observation bioreactor system. Bone slices perpendicular to the longitudinal axis obtained from the tibia in 2- to 4-day-old chick legs were cultured in the system mounted on a microscope, and their calcification was observed up to 24 h while they were stretched in the direction parallel to the slice. Increase in the calcified area, traveling distance and the direction of the calcification and collagen fiber orientation in the newly calcified region were analyzed. There was a significant increase in calcified area in the bone explant subjected to tensile strain over ∼3%, which corresponds to the threshold strain for collagen fibers showing alignment in the direction of stretch, indicating that the fiber alignment may enhance tissue calcification. The calcification progressed to a greater distance to the stretching direction in the presence of the loading. Moreover, collagen fiber orientation in the calcified area in the loaded samples was coincided with the progression angle of the calcification. These results clearly show that the application of static tensile strain enhanced tissue calcification, which progresses along collagen fibers aligned to the loading direction.     

The differentiation and calcification of chondrocytes in primary cell cultures.

The cartilage from a non-immobilized fracture undergoes a series of morphological and biochemical changes resembling the in vivo differentiation and calcification in the epiphyseal plate. The studies reported here demonstrate that a homogeneous population of chondrocytes isolated from fracture callus fibrocartilage undergoes the same changes in vitro. Chondrocyte primary cultures were grown for 28 days during which time the morphological, histological and histochemical properties of the cultures were studied. Demonstrated by various histological procedures, chondrocytes synthesized the characteristic cartilage matrix, and progressively calcified with increased culture age. This system can be used to elucidate the cellular and molecular mechanisms of calcification.     

Molecular analyses of protein components of the organic matrix in the exoskeleton of two scleractinian coral species

Biochemical and histological studies on the exoskeleton of scleractinian corals had demonstrated presence of the organic matrix containing proteins, lipids and chitin. Examination at the electron microscopic level had shown that the initial phase of calcification occurred in close association with organic substances secreted by calicoblastic cells. The possibility was thereby proposed that certain organic substances induce formation of calcium carbonate crystals, presumably functioning as templates for nucleation. In search for such a molecule, biochemical and molecular analyses were initiated on protein components of the organic matrix extracted from the calcified exoskeleton of the hermatypic coral, Galaxea fascicularis and the ahermatype, Tubastrea aurea. In SDS-PAGE analyses of the extracts, one major protein and a few other minor bands were detected in each of the two species. A cDNA encoding the major protein (named galaxin) in G. fascicularis was cloned and its primary structure was deduced. It consisted mostly of tandem repeats of a unit sequence of about 30 residues, and its sequence did not exhibit significant similarity to known proteins. Preliminary characterization of the T. aurea proteins showed that two proteins bound Ca²⁺, and suggested that the major protein of 46 kDa was not homologous to galaxin.     

Osteopontin is associated with bioprosthetic heart valve calcification in humans

Calcification of non-osseous tissues such as heart valves or vessels is a major concern in clinical practice. The exact mechanism is still unknown. Numerous studies have shown that mineral deposits of crystalline hydroxyapatite within these tissues were associated with increased non-collagenous protein content. More recently osteopontin was found to be associated with calcification in living tissues such as vessels and native human aortic valves. The aim of this study was to determine whether or not non-collagenous proteins can also be found in non-living tissues such as glutaraldehyde-pretreatedporcine valves after implantation in humans. Thirty-eight glutaraldéhyde pretreated porcine bioprostheses were studied: 16 not implanted and 22 after 11 years of implantation in the aortic and mitral valve position in humans. In areas of calcification vizualized by Von Kossa staining and microradiography, immunostaining using polyclonal antibodies against calcium-bindingproteins showed osteopontin positive staining and no staining for osteocalcin, bone sialoprotein or osteonectin. In uncalcified areas and in non-implanted valves, staining for osteopontin or other calcium-binding proteins was negative. Western blot analysis of macroscopically calcified and uncalcified areas showed that several proteins were adsorbed in implanted valves and confirmed the presence of osteopontin in the calcified areas, while no immunolabelling was found in non-calcified areas, in uncalcified valves and in non-implanted valves. Thus the presence of osteopontin in the calcified areas of bioprosthetic heart valves implanted in human indicates that this protein is associated with bioprosthetic valvular calcification. Since these valves are made of non-living connective tissue, and no cell immunostained for osteopontin was found around the calcified area, this study suggests that a non-cellular mediated mechanism involving protein adsorption may play a role in bioprosthetic valvular calcification.     

Osteocalcin and matrix Gla protein in zebrafish (Danio rerio) and Senegal sole (Solea senegalensis): comparative gene and protein expression during larval development through adulthood

Bone Gla protein (Bgp or osteocalcin) and matrix Gla protein (Mgp) are important in calcium metabolism and skeletal development, but their precise roles at the molecular level remain poorly understood. Here, we compare the tissue distribution and accumulation of Bgp and Mgp during larval development and in adult tissues of zebrafish (Danio rerio) and throughout metamorphosis in Senegal sole (Solea senegalensis), two fish species with contrasting environmental calcium levels and degrees of skeletal reorganization at metamorphosis. Mineral deposition was investigated in parallel using a modified Alizarin red/Alcian blue protocol allowing sensitive simultaneous detection of bone and cartilage. In zebrafish, bgp and mgp mRNAs were localized in all mineralized tissues during and after calcification including bone and calcified cartilage of branchial arches. Through immunohistochemistry we demonstrated that these proteins accumulate mainly in the matrix of skeletal structures already calcified or under calcification, confirming in situ hybridization results. Interestingly, some accumulation of Bgp was also observed in kidney, possibly due to the presence of a related protein, nephrocalcin. Chromosomal localization of bgp and mgp using a zebrafish radiation hybrid panel indicated that both genes are located on the same chromosome, in contrast to mammals where they map to different chromosomes, albeit in regions showing synteny with the zebrafish location. Results in Senegal sole further indicate that, during metamorphosis, there is an increase in expression of both bgp and mgp, paralleling calcification of axial skeleton structures. In contrast with results obtained for previously studied marine fishes, in zebrafish and Senegal sole Mgp accumulates in both calcified tissues and non-mieralized vessel walls of the vascular system. These results suggest different patterns of Mgp accumulation between fish and mammals.     

Matrix Gla protein is differentially expressed during the deposition of a calcified matrix by vascular pericytes

PCR-based subtractive hybridisation was used to identify genes up-regulated when pericytes undergo osteogenic differentiation and deposit a calcified matrix. cDNA pools were generated from confluent pericytes and from pericyte cultures containing calcified nodules. A pericyte cDNA library was screened with the product of the subtraction procedure (calcified minus confluent cDNA) and the majority of the positive clones were identified as matrix Gla protein (MGP). Northern analysis and immunohistochemistry demonstrated that MGP was only expressed by pericytes in calcified nodules. Antibodies to MGP inhibited the deposition of a calcified matrix by pericytes, suggesting that MGP regulates both cell differentiation and calcification.     

The calcified puparium of the face fly, Musca autumnalis (Diptera: Muscidae)

The general composition of the calcified puparium of Musca autumnalis was determined. Ash-weight analyses show that 62% of the puparium is inorganic material. The major components of the puparium are calcium, magnesium, phosphate and carbonate. Calcium and magnesium phosphate are the predominant salts in the puparium, with a lesser contribution by their respective carbonates. Scanning electron microscopy revealed that the exocuticle was the site of calcification in the puparium. Inorganic analyses of the insoluble salt isolated from face fly larvae showed it to be compositionally identical with the puparial salt. Major organic components of the puparium are lipid, chitin and protein. The presence of carbonic anhydrase and alkaline phosphatase activity in post-feeding larvae was confirmed.     

Vitamin-D-dependent calcium-binding protein (CaBP-9K) in rat growth cartilage

The presence of vitamin-D-dependent calcium-binding protein (CaBP-9K) in tibial growth-plate cartilage was immunohistochemically demonstrated using a specific antibody to rat duodenal CaBP-9K. The protein was found to be mainly localized in the cytoplasm of maturing chondrocytes. In hypertrophic chondrocytes, CaBP-9K concentrations decreased, and the protein was found in the cytoplasmic processes. No CaBP-specific immunoreactivity was seen in the hypertrophic chondrocytes of the lower calcified hypertrophic zone; in contrast, the protein was found in the extracellular lateral edges of longitudinal septa, i.e. where matrix vesicles are preferentially localized and where cartilage mineralization is initiated. These findings suggest that vitamin D has a direct function in this tissue. It also seems likely that CaBP-9K is an indicator of chondrocyte maturation, and that it is involved in the matrix vesicle-associated process of cartilage calcification.     

EVIDENCE FOR CHANGES IN PROTEIN POLYSACCHARIDE ASSOCIATED WITH THE ONSET OF CALCIFICATION IN CARTILAGE

Past work has suggested that protein polysaccharide may play a role in the calcification of cartilage. Recent electron microscopic studies on noncalcified cartilage have indicated that protein polysaccharide in cartilage matrix is represented by granules associated with collagen fibers. The present work has been designed for comparison of the matrix of noncalcified cartilage to that of calcified cartilage, with particular reference to these granules. Small blocks of tibia from 16-day embryos were fixed in cacodylate-buffered glutaraldehyde and postfixed in either phosphate- or Veronal-buffered osmium tetroxide. Special care was taken to maintain the pH above 7.0 at all times. For electron microscopy the tissues were dehydrated, embedded in Epon 812, sectioned, and stained with uranyl acetate or lead citrate. A marked decrease in the size of granules in the matrix of calcified cartilage compared to noncalcified cartilage was noted. Associated with the decrease in the size of granules was a condensation of matrix components and the presence of an amorphous electron-opaque material that was not seen in noncalcified areas. These results are interpreted to represent either a drop in concentration or a change in state of protein polysaccharide with the onset of calcification in cartilage.     

Vitamin-D-dependent calcium-binding protein (CaBP-9K) in rat growth cartilage

Summary The presence of vitamin-D-dependent calcium-binding protein (CaBP-9K) in tibial growth-plate cartilage was immunohistochemically demonstrated using a specific antibody to rat duodenal CaBP-9K. The protein was found to be mainly localized in the cytoplasm of maturing chondrocytes. In hypertrophic chondrocytes, CaBP-9K concentrations decreased, and the protein was found in the cytoplasmic processes. No CaBP-specific immunoreactivity was seen in the hypertrophic chondrocytes of the lower calcified hypertrophic zone; in contrast, the protein was found in the extracellular lateral edges of longitudinal septa, i.e. where matrix vesicles are preferentially localized and where cartilage mineralization is initiated. These findings suggest that vitamin D has a direct function in this tissue. It also seems likely that CaBP-9K is an indicator of chondrocyte maturation, and that it is involved in the matrix vesicle-associated process of cartilage calcification.     

Morphological and histochemical study of supragingival human calculus and dental plaque using ruthenium hexammine trichloride and acridine orange.

Ruthenium hexammine trichloride (RHT) and acridine orange were used to preserve and visualize anionic groups in human plaque and dental calculus. RHT-reacting material was present on the membrane of micro-organisms and in intermicrobial spaces of the calcifying areas, and seems to correspond to, and derive from, acidic glyco- and phospholipids of the plasma membrane of the micro-organisms. However, the presence of acidic salivary peptidoglycans cannot be ruled out. Two types of calcification were found: extramicrobial and intramicrobial. The former consisted of calcified deposits irregularly scattered in the intermicrobial matrix. They were in close relationship with RHT-reacting material, or were placed inside vesicular structures delimited by a membrane. Intramicrobial calcification consisted of small aggregates of needle-shaped crystals and/or of granular deposits; in both cases, they either masked the whole cytoplasm of the micro-organisms, or were located only over the plasma membrane. These results suggest that mineral deposition occurs in connection with acidic components of intermicrobial matrix, microbial plasma membranes, and cytoplasms. The addition of RHT and acridine orange to fixing and decalcifying solutions yields satisfactory preservation of dental calculus and plaque, and apparently reduces loss of their anionic organic components and increases their electron density. However, these substances are not sufficient to preserve all ultrastructural details in decalcified areas, probably because the inorganic substance prevents reaction of acridine orange and RHT with the organic components of the calcified matrix.     

Immunocytochemical localization of sulfhydryl oxidase in mammalian epidermis suggests that the enzyme cross‐links keratins in the granular and transitional corneous layers

The epidermis of representative mammalian species including humans has been examined for the presence of sulfhydryl oxidase, an enzyme likely involved in the oxidation of corneous proteins containing sulfhydryl groups in the epidermis. A database search indicates that the enzyme shares common sequences in numerous mammalian species so that an antibody against the human sulfhydryl oxidase 2 has been utilized on other species. The immunofluorescent study on the epidermis of the platypus (monotreme), red kangaroo (marsupials), hamster and human (placentals) reveals a prevalent labelling in the granular, transitional and lowermost part of the stratum corneous layer. The detailed ultrastructural immunogold study of the human epidermis reveals a diffuse and uneven labelling in the paler component of the composite keratohyalin granules or among keratin filaments of the transitional layer while the labelling disappears in the corneous layer. The study supports the hypothesis of the participation of the enzyme in the oxidative process that determines the formation of stable disulphide groups among keratins and other corneous proteins of the stratum corneum. This process gives rise to the resistant cell corneous envelope of keratinocytes in addition to the isopeptide bonds that derive from the catalytic action of epidermal transglutaminase on several corneous proteins.     

Immunohistochemical investigation on the presence of chondroitin sulfate in calcification nodules of epiphyseal cartilage.

Chondroitin sulfate localization in mouse epiphyseal cartilage was studied using CS-56 monoclonal antibody immunospecific for the glycosaminoglycan portion of the molecule. For light and fluorescence microscopy, decalcified specimens were embedded in paraffin, Lowicryl, or were frozen and cryostat-sectioned, and the antigen-antibody reaction was demonstrated by treating sections with IgM-peroxidase, IgM-alkaline phosphatase, or IgM-fluorescein conjugates. For electron microscopy, decalcified and undecalcified specimens were embedded in Lowicryl; ultrathin sections from undecalcified specimens were decalcified by flotation on EDTA; sections from both types of specimens were treated with IgM-immunogold conjugate for demonstration of CS-56 reaction. Before immunoreaction, part of all decalcified sections were digested with Streptomyces or testicular hyaluronidase. Control sections were treated with either mouse and goat non-immune serum, or mouse monoclonal antiserum to human dendritic reticulum cells. Both light and electron microscopy show CS-56 reaction with cytoplasmic components of maturing and hypertrophic chondrocytes. Under the light microscope, immunoreaction was not visible in calcified matrix, and was visible in uncalcified matrix only after hyaluronidase digestion. Under the electron microscope, it was evident both in uncalcified and calcified matrix, although the latter showed few immunogold particles, usually placed on areas which appeared incompletely calcified. Gold particles were chiefly distributed at the periphery of calcification nodules and fully calcified matrix. These results show that CS-56, besides reacting with cytoplasm of maturing and hypertrophic chondrocytes, binds to crystal ghosts and other components of cartilage matrix, immunoreactivity decreasing as calcification increases. This suggests that chondroitin sulfate molecules are either degraded during calcification, or segregated into macromolecular complexes, or both degraded and segregated. The second possibility is supported by the increase of immunosensitivity induced by hyaluronidase digestion.     

Influence of intermittent compressive force on proteoglycan content in calcifying growth plate cartilage in vitro

We investigated the effect of mechanical stimulation by an intermittent compressive force (ICF) on proteoglycan (PG) synthesis and PG structure in calcified and noncalcified cartilage of fetal mouse long bone rudiments. Uncalcified cartilaginous long bone rudiments were cultured for 5 days in the presence of [35S]sulfate and [3H]glucosamine under control conditions (atmospheric pressure) or under the influence of ICF. ICF was generated by intermittently compressing the gas phase above the culture medium (130 mbar, 0.3 Hz). During culture, the center of the rudiments started to calcify. ICF stimulated calcification such that, after 5 days, the diaphysis of calcified cartilage was about two times as long as in the control cultures. At the end of the experiment, the rudiments were divided in a central calcified diaphysis and two noncalcified epiphyses. Diaphysis and epiphyses were pooled separately. PGs were extracted with 4 M guanidinium chloride and isolated by cesium chloride density gradient centrifugation. PGs (predigested with proteinase K or chondroitinase ABC) were characterized for hydrodynamic size of aggregates, monomers, and chondroitin sulfate chains by gel permeation chromatography and for degree of sulfation by ion exchange chromatography on high pressure liquid chromatography columns. ICF increased the amount of incorporated sulfate per tissue volume unit in the noncalcified epiphyses, but decreased this parameter in the calcified diaphysis. However, in both calcified and noncalcified cartilage, ICF increased the degree of sulfation of the chondroitin sulfate chains. No effects were found on the hydrodynamic size of the PG aggregates or monomers, but in the epiphyses ICF increased the size of the chondroitin sulfate chains. No other changes of structural characteristics of the macromolecules were observed. This study demonstrates that ICF generally stimulated the incorporation of [35S]sulfate into chondroitin sulfate chains. We conclude from the lowered [35S]sulfate content in calcified cartilage that ICF reduced the number of chondroitin sulfate chains and probably PGs while accelerating matrix calcification. It seems likely that the two effects are linked, indicating that a reduction of the number of chondroitin sulfate chains is part of the complicated process of cartilage calcification.     

Studies on fish scale formation and resorption. I. Fine structure and calcification of the scales in Fundulus heteroclitus (Atheriniformes: Cyprinodontidae)

Fine structure of the scales of Fundulus heteroclitus was examined by scanning and transmission electron microscopy. The concentric ridges of the scale surface were characterized by the presence of minute, highly calcified, denticles or tooth-like processes. Needle-shaped crystals of hydrox-yapatite were precipitated not only in the osseous layer but in the intimate lamellae of the fibrillary plate except in portions just below the grooves. The calcification of the osseous layer was observed to proceed by filling the matrix with patches of crystals. The fibrillary plate appeared to calcify by invasion of crystals from the upper calcified zone into spaces between collagen fibers.     

An in vitro model quantifying the effect of calcification on the tissue–stent interaction in a stenosed aortic root

Transcatheter Aortic Valves rely on the tissue-stent interaction to ensure that the valve is secured within the aortic root. Aortic stenosis presents with heavily calcified leaflets and it has been proposed that this calcification also acts to secure the valve, but this has never been quantified. In this study, we developed an in vitro calcified aortic root model to quantify the role of calcification on the tissue-stent interaction. The in vitro model incorporated artificial calcifications affixed to the leaflets of porcine aortic heart valves. A self-expanding nitinol braided stent was deployed into non-calcified and artificially calcified porcine aortic roots and imaged by micro computed tomography. Mechanical tests were then conducted to dislodge the stent from the aortic root and it was found that, in the presence of calcification, there was a significant increase in pullout force (8.59 ± 3.68 N vs. 2.84 ± 1.55 N p = 0.045), stent eccentricity (0.05 ± 0.01 vs. 0.02 ± 0.01, p = 0.049), and coefficient of friction between the stent and aortic root (0.36 ± 0.12 vs. 0.09 ± 0.05, p = 0.018), when compared to non-calcified roots. This study quantifies for the first time the impact of calcification on the friction between the aortic tissue and transcatheter aortic valve stent, showing the role of calcification in anchoring the valve stent in the aortic root.     

Putative Chitin Synthases from Branchiostoma floridae Show Extracellular Matrix-related Domains and Mosaic Structures

The transition from unicellular to multicellular life forms requires the development of a specialized structural component,the extracellular matrix(ECM).In Metazoans,there are two main supportive systems,which are based on chitin and collagen/hyaluronan,respectively.Chitin is the major constituent of fungal cell walls and arthropod exoskeleton.However,presence of chitin/chitooligosaccharides has been reported in lower chordates and during specific stages of vertebrate development.In this study,the occurrence of chitin synthases(CHSs) was investigated with a bioinformatics approach in the cephalochordate Branchiostoma floridae,in which the presence of chitin was initially reported in the skeletal rods of the pharyngeal gill basket.Twelve genes coding for proteins containing conserved amino acid residues of processive glycosyltransferases from GT2 family were found and 10 of them display mosaic structures with novel domains never reported previously in a chitin synthase.In particular,the presence of a discoidin(DS) and a sterile alpha motif(SAM) domain was found in nine identified proteins.Sequence analyses and homology modelling suggest that these domains might interact with the extracellular matrix and mediate protein-protein interaction.The multi-domain putative chitin synthases from B.floridae constitute an emblematic example of the explosion of domain innovation and shuffling which predate Metazoans.     

ELECTRON MICROSCOPIC STUDIES OF INDUCED CARTILAGE DEVELOPMENT AND CALCIFICATION

Cultured, human, amniotic cells (FL strain) injected into the thigh muscles of cortisone-conditioned mice proliferated to form discrete colonies which, over a period of 5 days, became invested by numerous fibroblasts. Cartilage cells and matrix appeared within the fibroblastic zones during the succeeding 2–4 days. Cartilage matrix calcified within 12 days following FL-cell injection. Cartilage cells closely resembled fibroblasts from which they appeared to be derived, and were readily distinguished from FL cells by their prominent ergastoplasm and Golgi complexes. Cartilage matrix was composed of a distinctive feltwork of randomly arranged, collagen fibrils (~600 A axial period and ~250 A width) from which small electron-opaque, leaflike matrix particles extended. Matrix calcification occurred with the deposition of radially arranged needle-like structures resembling hydroxyapatite. Dense centers were often identified within these clusters. Examination of heavily calcified areas revealed confluent masses of apatite-like material. In general, the fine structure of induced cartilage formation and calcification resembled that of cartilage development and calcification as previously described in the normal epiphysis.