Phosphoproteins of the chicken eggshell calcified layer

The chicken eggshell matrix is a complex mixture of proteins and proteoglycans. It also contains phosphoproteins that are thought to affect mineralization of the matrix. Several of the matrix phosphoproteins, such as the major component osteopontin, have already been identified as phosphoproteins in other tissues, but the phosphorylation status of the eggshell matrix forms was unknown. The phosphopeptides, obtained after cleavage of the matrix proteins with several different cleavage methods, were enriched by anion-exchange chromatography and reversible binding to titanium oxide and identified by LC-MS(n) or pseudo-MS(n) analysis following neutral loss scanning. Altogether we identified 39 phosphorylated matrix proteins, 22 of which were not known to be phosphorylated before. Eight of the proteins were identified as eggshell matrix components for the first time. Together these proteins contained more than 150 different phosphorylation sites, 103 of which were determined with high confidence. Among the major phosphorylated proteins of the chicken eggshell matrix were osteopontin and the eggshell-specific proteins ovocleidin-17, ovocleidin-116, and ovocalyxin-32.     

Phosphoproteins of murine hepatitis viruses.

Four strains of the coronavirus murine hepatitis virus were examined for the presence of phosphorylated proteins. The nucleocapsid protein was determined to contain phosphate covalently linked to serine but not to threonine residues. The nucleocapsid protein was the only phosphorylated protein detected in these strains of murine hepatitis virus.     

Cartilage tissue engineering of nasal septal chondrocyte-macroaggregates in human demineralized bone matrix

Tissue Engineering is an important method for generating cartilage tissue with isolated autologous cells and the support of biomaterials. In contrast to various gel-like biomaterials, human demineralized bone matrix (DBM) guarantees some biomechanical stability for an application in biomechanically loaded regions. The present study combined for the first time the method of seeding chondrocyte-macroaggregates in DBM for the purpose of cartilage tissue engineering. After isolating human nasal chondrocytes and creating a three-dimensional macroaggregate arrangement, the DBM was cultivated in vitro with the macroaggregates. The interaction of the cells within the DBM was analyzed with respect to cell differentiation and the inhibitory effects of chondrocyte proliferation. In contrast to chondrocyte-macroaggregates in the cell-DBM constructs, morphologically modified cells expressing type I collagen dominated. The redifferentiation of chondrocytes, characterized by the expression of type II collagen, was only found in low amounts in the cell-DBM constructs. Furthermore, caspase 3, a marker for apoptosis, was detected in the chondrocyte-DBM constructs. In another experimental setting, the vitality of chondrocytes as related to culture time and the amount of DBM was analyzed with the BrdU assay. Higher amounts of DBM tended to result in significantly higher proliferation rates of the cells within the first 48 h. After 96 h, the vitality decreased in a dose-dependent fashion. In conclusion, this study provides the proof of concept of chondrocyte-macroaggregates with DBM as an interesting method for the tissue engineering of cartilage. The as-yet insufficient redifferentiation of the chondrocytes and the sporadic initiation of apoptosis will require further investigations.     

Phosphoproteins: structural components of oncornaviruses.

Oncornaviruses, which contain a virion-associated protein kinase, were found to possess phosphoproteins as virion structural components. One major phosphoprotein common to strains of laboratory and wild mouse oncornaviruses and a strain of feline leukemia virus was shown to be a polypeptide of about 12, 000 mol wt. In addition to this, the Kirsten strain of murine sarcoma virus contained a second major phosphoprotein of about 10, 000 mol wt, and mouse erythroblastosis virus contained a second major phosphoprotein that was either identical to or comigrated with the virion glycoprotein of about 74, 000 mol wt. The major phosphoprotein of RD-114 virus was found to be of about 16, 000 mol wt. The major phosphoamino acid of the 12, 000-mol wt polypeptide of the mouse erythroblastosis virus was identified as phosphoserine, and that of the 16, 000-mol wt polypeptide of the RD-114 virus was identified as phosphothreonine.     

Recombinant TGF-beta1 stimulates bone marrow osteoprogenitor cell activity and bone matrix synthesis in osteopenic, old male mice.

We have previously hypothesized that the osteopenic changes seen in the skeletons of old male BALB/c mice are due to reductions in the availability and/or synthesis of bone TGF-beta which results in fewer, less osteogenic marrow osteoprogenitor cells (CFU-f; OPCs) and lower levels of bone formation. Among other things, this hypothesis would predict that introducing exogenous TGF-beta into old mice (growth factor replacement) should stimulate marrow CFU-f and increase bone formation. In the present study, we have tested this prediction and, indirectly the hypothesis, by injecting human recombinant TGF-beta1, i.p., into both young adult (4 month) and old mice (24 month). The effects of the growth factor on the skeleton were then assessed by measurements of trabecular bone volume, bone formation, fracture healing, and the number, proliferative, apoptotic, and alkaline phosphatase activity of marrow CFU-f/OPCs. Our data show that the introduction of 0.5 or 5.0 ug/day of TGF-beta1 into old mice for 20 days 1) increases trabecular bone volume, bone formation and the mineral apposition rate, 2) augments fracture healing, 3) increases the number and size of CFU-f colonies, and 4) increases proliferation and diminishes apoptosis of CFU-f in primary bone marrow cultures. Importantly, these stimulatory effects of injected growth factor are apparently age-specific, i.e., they are either not seen in young animals or, if seen, are found at much lower levels. While these observations do not exclude other possible mechanisms for the osteopenia of old mice, they provide further support for the hypothesis that, with age, diminished TGF-beta synthesis or availability results in a reduction in the marrow osteoprogenitor pool and bone formation. The findings also demonstrate that the latter changes can be reversed, at least transiently, by introducing exogenous TGF-beta1.     

Involvement of bone tissue in chicken infected with the myeloblastic MAV 2-0 virus. Generalized cortical hyperostosis

An affection of osseous tissue appears in chicken have been injected at the embryonic state with retrovirus myeloblastic MAV 2-0 in solution. The structural lesions are different from these examined in human or murine osteopetrosis, considered to be an general cortical hyperostosis.     

Vitreous cryopreservation of tissue engineered bone composed of bone marrow mesenchymal stem cells and partially demineralized bone matrix

Cryopreservation of tissue engineered products by maintaining their structure and function is a prerequisite for large-scale clinical applications. In this study, we examined the feasibility of cryopreservation of tissue engineered bone (TEB) composed of osteo-induced canine bone marrow mesenchymal stem cells (cBMSCs) and partially demineralized bone matrix (pDBM) scaffold by vitrification. A novel vitreous solution named as VS442 containing 40% dimethyl-sulfoxide (DMSO), 40% EuroCollins (EC) solution and 20% basic culture medium (BCM) was developed. After being cultured in vitro for 8 days, cell/scaffold complex in VS442 was subjected to vitreous preservation for 7 days and 3 months, respectively. Cell viability, proliferation and osteogenic differentiation of cBMSCs in TEB after vitreous cryopreservation were examined with parallel comparisons being made with those cryopreserved in VS55 vitreous solution. Compared with that cryopreserved in VS55, cell viability and subsequent proliferative ability of TEB in VS442 after being rewarmed were significantly higher as detected by live/dead staining and DNA assay. The level of alkaline phosphatase (ALP) expression and osteocalcin (OCN) deposition in VS442 preserved TEB was also higher than those in the VS55 group since 3 days post-rewarm. Both cell viability and osteogenic capability of the VS55 group were found to be declined to a negligible level within 15 days post-rewarm. Furthermore, it was observed that extending the preservation of TEB in VS442 to 3 months did not render any significant effect on its survival and osteogenic potential. Thus, the newly developed VS442 vitreous solution was demonstrated to be more efficient in maintaining cellular viability and osteogenic function for vitreous cryopreservation of TEB over VS55.     

The influence of mesenchymal stem cells on bone tissue regeneration upon implantation of demineralized bone matrix

Mesenchymal stem cells (MSC) are resident pluripotent cells of bone marrow stroma. MSC are able to differentiate into chondroblasts, adipocytes, neurons, glia, cardiomyocytes, or osteoblasts. The problem of MSC usage in cell therapy of bone defects is widely discussed at present. The experiments were carried out using rats of inbred line Wistar-Kyoto. MSC were isolated from bone marrow and cultivated in vitro. Demineralized bone matrices (DBM) were obtained from parietal bones of rats and hens. Part of DBM was loaded with MSC. Bone defects were made in cranium parietal regions. DBM with or without MSC or metal plates were transplanted in these regions. It was shown that the application of MSC increased angiogenesis and osteogenesis in the damaged bone. The implantation of rat's DBM with MSC led to the formation of a full value bone. MSC suppressed inflammation, when transplantation of hen's DBM was carried out. The application of MSC always improved bone tissue regeneration.     

Protein synthesis initiation factors from chicken erythroblasts.

Two factors (IF-I and IF-II) necessary for the initiation of protein synthesis have been partially purified from a 0.5 M KC1 wash of chicken erythroblast polysomes. IF-I mediates the binding of the initiator tRNA and GTP to a 40 S ribosomal subunit, resulting in the formation of a 44 S initiation intermediate. In the presence of IF-II and a suitable RNA template, the 44 S initiation intermediate combines with a 60 S ribosomal subunit to form a functional 80 S initiation complex. The methionyl moiety of the initiator tRNA in the 80 S initiation complex is able to react with puromycin to form methionylpuromycin.     

Appearance of adipose cells in connective tissue at the implantation site of bone matrix gelatin and bupivacaine injection.

Two types of adipose cells were found in the connective tissue on day 7 after bone matrix gelatin (BMG) implantation and an injection of bupivacaine: mature adipose cells with a large lipid droplet (2-140 microns) and immature adipose cells with many small lipid droplets (0.1-2 microns). On day 10 after BMG implantation, typical adipose tissue was observed near the implant. The immature adipose cells had small, spherical mitochondria, glycogen granules and cytoplasmic microvesicles, and they might differentiate from undifferentiated mesenchymal cells in the connective tissue or the peripheral cells around the vessels as a white adipose tissue. These findings suggest that the differentiation of adipose cells in the connective tissue near heterotopic bone formation might be induced not only by mechanical and/or bupivacaine injury, but also by some factor or factors of the BMG.     

Increased collagen synthesis in myeloblastosis-associated virus-infected chicken embryo fibroblasts.

Chicken embryo fibroblasts infected with a non-transforming derivative of avian myeloblastosis virus [MAV-2(0)] showed a threefold increase in the biosynthesis of collagen compared to values in normal counterparts. In contrast, non-collagen protein synthesis was unchanged.     

Isolation and chemical characterization of the phosphoproteins of chicken bone matrix: heterogeneity in molecular weight and composition

Ethylenediaminetetraacetic acid and HCl extracts of calcified chicken bone were fractionated by a variety of techniques, including molecular sieving in guanidinium chloride, ion-exchange chromatography on DEAE-cellulose, high-performance liquid chromatography (HPLC), reverse-phase HPLC, and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Using several different experimental schemas, we isolated 14 apparently homogeneous components varying in molecular weight from approximately 150K to approximately 4K-5K. The compositions of all of the phosphoproteins were characterized by high concentrations of Asp, Glu, Ser, Gly, and Ala. Seven of the components which were analyzed contained concentrations of carbohydrate varying from approximately 4% to approximately 17%. Three of the components containing O-phosphoserine which behaved as single bands on SDS-PAGE with molecular weights of approximately 150K, approximately 90K, and approximately 70K contained Hyp and Hyl or Hyl alone and may represent covalently bonded or strongly associated collagen-phosphoprotein complexes or hydroxylated Pro and/or Lys residues of the phosphoproteins. The findings that the amino acid compositions of several of the components were very similar and that N-terminal partial amino acid sequences of the approximately 90- and approximately 60-kilodalton (kDa) and of the approximately 150- and approximately 32-kDa components, respectively, were identical make it clear that some of the lower molecular weight components are derived by proteolysis from higher molecular weight species. In addition to proteolysis, we speculate that it is possible, from the N-terminal amino acid sequence data and preliminary cross-reaction studies of antibodies to four of the phosphoproteins, that the heterogeneity observed in the phosphoprotein components may also be due in part to there being more than one independent gene product for chicken bone phosphoproteins.     

Identification and characterization of the major chicken bone phosphoprotein. Analysis of its synthesis by cultured embryonic chick osteoblasts

The major phosphoprotein synthesized by cultured chicken embryo osteoblasts had a molecular mass of approximately 66 kDa. The 32P label on the protein was cleaved by acid phosphatase treatment and O-[32P]phosphoserine and O-[32P]phosphothreonine could be identified after partial acid hydrolysis. The phosphoprotein contributed approximately 2.0% of the total protein synthesized by osteoblasts and was shown to be secreted, as shown by its presence in the culture media. Glycosylation was demonstrated by the fact that it could be labelled with [3H]galactosamine. The major approximately 66-kDa phosphoprotein was resolved by isoelectric focusing into three major variants with pI values ranging over 3.7 - 3.9; all three forms appear to be the result of variation in the extent of protein phosphorylation. An identical approximately 66-kDa phosphoprotein could be extracted from chicken bones which had both the same range of pI values and an identical elution position following DEAE-Sephacel chromatography. Analysis of the protein isolated from bone demonstrated the presence of sialic acid and, while amino-terminal sequence analysis and internal tryptic fragment sequence analysis of about 25% of the protein revealed little similarity to the rat phosphoprotein osteopontin, a conserved nine-residue sequence spanning the Arg-Gly-Asp cell-binding site of the rat protein osteopontin, was identified in the approximately 66-kDa chicken protein. Peptide mapping with Staphylococcus aureus V8 protease of the in vivo protein compared to the in vitro synthesized protein demonstrated identical peptide fingerprints. The two proteins also had comparable amino acid compositions. Several smaller-molecular-mass phosphoproteins ranging in size over about 55 - 29 kDa were also observed in the HCl extracts of bone. Peptide mapping of these species demonstrated that the approximately 66-kDa, approximately 55-kDa, and approximately 45-kDa species had a common core of peptide fragments. Pulse/chase experiments in culture revealed no evidence for a defined pathway of intracellular proteolysis associated with the approximately 66-kDa species since this phosphoprotein remained the prevalent species after a 24-h chase. Because of the predominant association of all the smaller-molecular-mass forms with the cell layer and an absence of a quantitative conversion to any of the smaller forms over a 24-h chase, these results suggested that the lower-molecular-mass species were not the result of proteolytic processing during synthesis or secretion, but rather represent proteolysis of the approximately 66-kDa component in the extracellular matrix.(ABSTRACT TRUNCATED AT 400 WORDS)     

Post-translational processing of chicken bone phosphoproteins. Identification of the bone phosphoproteins of embryonic tibia.

After periodate oxidation and incubation with a dihydrazide, cross-linking of the two heavy chains of immunoglobulins G from several species proceeds specifically through their oligosaccharides. We have used malonic acid dihydrazide, adipic acid dihydrazide and dithiodipropionic acid dihydrazide. The last compound is introduced in this work as a cleavable-carbohydrate-specific cross-linker. It was found that in rabbit and human immunoglobulins the degree of cross-linking was strongly dependent on the oxidation conditions but only very weakly dependent on the concentration and size of the dihydrazides. Papain cleavage of the cross-linked rabbit IgG indicated that the cross-linking occurred predominantly, if not exclusively, in the Fc region, probably through the two glycans linked to Asn-297 in the CH2 domain of each of the two heavy chains. The immunoglobulins from sheep, pig, goat and guinea pig show a comparable cross-linking pattern, indicating that the sugar chains from these immunoglobulins have a spatial structure closely related to that of rabbit and human IgG. When dithiodipropionic acid dihydrazide was used as the cross-linker, the cross-link could be cleaved by mercaptoethanol.     

Post-translational processing of chicken bone phosphoproteins. Identification of bone (phospho)protein kinase.

We have detected a protein kinase which phosphorylates bone phosphoproteins (BPPs) in the detergent extract of the membranous fractions in the periosteal bone strips of 12-day-embryonic-chick tibia. This enzyme, tentatively named BPP kinase, has a catalytic subunit of Mr approximately 39,000, utilizes GTP as well as ATP as a phospho-group donor, is inhibited by 2,3-bisphosphoglycerate and heparin, and is therefore similar to casein kinase II. The enzyme can phosphorylate dephosphorylated proteins such as casein, phosvitin and chicken BPPs, but the last-named are preferred substrates. The in vitro-phosphorylation-assay products of this enzyme in the extract were indistinguishable on an SDS/polyacrylamide gel from the major [32P]phosphoproteins metabolically labelled in the embryonic-chick bone tissue. The regulatory mechanisms of the phosphorylation process of BPPs by BPP kinase as well as the potential role of this enzyme in mineralization are discussed.