Identification of the noncollagenous proteins of bovine bone by two-dimensional gel electrophoresis

Summary We have characterized the noncollagenous proteins of bovine bone using two high-resolution gel electrophoretic techniques. Proteins were extracted from bone tissue by extended dialysis against 0.5 M EDTA. In some cases, a preextraction was done in guanidine HCl. Bovine plasma was also examined to identify the proteins in bone that might also be present in blood. We have scored 160 major, reproducible spots on our standard preparation bone map. These comprise about 40 individual protein groups. There are many more minor spots present which puts the total number present over 200. Of these groups, 15 are not present on plasma maps. Bone proteins identified in this way include actin, bone Gla-protein, and osteonectin. The remainder are unknown. Bone Gla-protein is present in bovine bone in four isoelectric forms, pI=3.95−4.50. Electroblotting analysis of EDTA and guanidine HCl extracted material failed to reveal any higher molecular weight immunologically reactive species. The plasma proteins found in bone include, but are not limited to albumin, apo A-I lipoprotein, IgG, IgM, transferrin, α-2-HS-glycoprotein, and hemoglobin. Extraction with guanidine HCl plus EDTA significantly enriches the yield for the nonplasma proteins but does not appear to extract any additional bone proteins.     

Blood:Bone disequilibrium. VI. Studies of the solubility characteristics of brushite: Apatite mixtures and their stabilization by noncollagenous proteins of bone

Summary Recent evidence of the occurrence of brushite in newly formed bone mineral prompted a study of the solubility properties of brushite:apatite mixtures under physiological conditions and the influence on them of pH, lactate, pyruvate, and, particularly, noncollagenous bone proteins (NCBPs). Brushite alone was surprisingly stable in solution at pH 7.4, 37°C. In the presence of increasing amounts of apatite, hydrolysis of brushite to an insoluble phase occurred. A decrease of 0.1 pH unit or the addition of 1.5 mM pyruvate or 10 mM lactate increased the ion activity product (Ca²⁺×HPO ₄ ²⁻ ) 3 or more times. However, within the loose envelope of bone such conditions so different from those in the circulation might be only local or temporary. NCBPs, on the other hand, stabilized brushite in solution alone as well as in the presence of apatite for days. They probably act by adsorbing strongly to the crystal surface and preventing nucleation by apatite. This brushite-apatite-bone protein system exhibits solubility characteristics that can resolve the old problems presented by the participation of the skeleton in extracellular calcium homeostasis on the one hand, and by the apparent insolubility of the apatite mineral of bone on the other. Deceased.     

The composition and antigenicity of sheep cortical bone matrix proteins

Sheep cortical bone powder was extracted with EDTA under mildly alkaline conditions. The extract was fractionated by CM-cellulose chromatography and caesium chloride gradient ultracentrifugation. Three chemically distinct fractions were obtained, including a sialoprotein fraction similar to that found in bovine bone. The serum proteins, albumin and immunoglobulin-G, present in trace quantities, were the most antigenic components of bone extract. Antibodies were produced in rabbits against sheep bone glycoproteins and detected by passive haemagglutination; these antibodies were weaker than those produced against articular cartilage proteins. Adsorption experiments indicated that major antigenic glycoproteins of bone matrix were not present in homologous articular cartilage tissue.     

卵巢切除大鼠皮质骨氨基多糖和非胶原蛋白变化与矿盐丢失

本实验目的是观察卵巢切除（ＯＶＸ）大鼠皮质骨矿盐变化特点，研究其变化机理。选用ＳＤ．两月龄大鼠，摘除双侧卵巢，实验期６０天。观察了ＯＶＸ大鼠股骨干近、中和远１／３段矿盐含量的变化及股骨干近１／３段骨质中氨基多糖（ＧＡＧ）、白蛋白和骨钙素（ＢＧＰ）含量的变化。研究结果显示：ＯＶＸ６０天大鼠股骨干仅近１／３段矿盐含量显著降低，中和远１／３段矿盐含量无明显改变。股骨干近１／３段骨质中ＧＡＧ和ＢＧＰ含量显著增加，白蛋白含显显著降低。结果表明ＯＶＸ６０天大鼠股骨干近１／３段矿盐丢失与局部骨质内ＧＡＧ含量增加，白蛋白含量和ＢＧＰ质量降低有关。     

In vivo decomposition of phosphoserine and serine in noncollagenous protein from human dentin

Summary HCl-soluble proteins in human dentin ranging in age from 3 to 45 years exhibit amino acid compositional changes consistent with β-elimination and hydrolysis of phosphoserine as well as dehydration and aldol cleavage of serine. This is the first evidence of nonenzymatic mechanisms forin vivo degradation of hydroxy and substituted hydroxy amino acids in dentin. Decomposition of phosphoseryl residues reduces the calcium-binding capacity of phosphoproteins. Elimination and dehydration reactions can produce variability in molecular weight. The rates of decomposition may be rapid enough to cause the heterogeneity or “maturational” degradation seen in dentin phosphoproteins during mineralization.     

引导性骨再生中成骨细胞来源的实验观察

目的 研究 长管骨引导性骨再生成骨细胞来源,进一步完善引导性骨再生理论。方法 将４２只成年雄性新西兰兔在双侧桡骨中段制作标准骨缺损不愈合模型,用硅胶膜呈管状包囊一侧骨缺损,另一侧作为对照侧。１只兔术后１￣１２周分别于每周进行Ｘ线检查;３０只兔,随机分为６组,分别于术后３天、１、２、３、４、５周外死取材,分别进行常规ＨＥ染色,ＳＰ方法ＢＭＰ、ＢＧ抗体的免疫组化染以。结果 隔膜在骨缺损局部生成隔离密闭     

Purification,composition, and31P NMR spectroscopic properties of a noncollagenous phosphoprotein isolated from chicken bone matrix

Summary Fractionation of the EDTA-soluble, noncollagenous proteins of the organic matrix of chicken bone by Sephadex G-100 molecular sieving has revealed that the majority of the organic phosphorus is present in two fractions, from one of which a homogeneous phosphoprotein has been isolated. The purified phosphoprotein has an apparent molecular weight of 12,000 and contains bothO-phosphoserine andO-phosphothreonine.³¹P-NMR spectroscopy demonstrates that all of the organic phosphorus exists in the form of phosphomonoesters which have an average pK₂ of 6.8. The phosphoprotein is highly acidic due to its high content of dicarboxylic acids in addition to the presence of organic phosphorus. The characteristic amino acid composition of the phosphoprotein establishes its noncollagenous nature and highlights the differences among bone, dentin, and enamel phosphoproteins. The absence ofγ-carboxyglutamic acid distinguishes it from osteocalcin, the noncollagenousγ-carboxyglutamic acid-containing peptide of bone matrix.     

Impaired bone activity in aged rats: Alterations at the cellular and molecular levels

We have used a model of rapid bone induction and resorption in rats initiated by the removal of bone marrow to define age-associated deficits. Here we report the sequential expression of various genes implicated in the formation and removal of bone following marrow ablation. Significant increases in alkaline phosphatase and procollagen ₁(I) mRNA were observed by day 5, and of osteocalcin and osteopontin by day 6. At their peak, these mRNA levels were elevated three- to eight-fold and correlated with histological evidence of bone formation. No change in collagen II mRNA was observed, indicating that there was no cartilage phase. Collagenase activity increased 10-fold at day 9 and coincided with the beginning of bone resorption. Actin mRNA, a reference gene marker, remained at constant levels. Comparison of the response between adult (6 mo.) and old (24 mo.) rats showed the same temporal pattern, but a lower expression of bonerelated genes in older rats. Histological examination also showed that the bone volume and osteoblast number at day 6 were significantly lower in old rats. Furthermore, the percentage of mineralized bone was greatly reduced in the aged rat. This model system is currently being used to evaluate the effectiveness of interventions to up-regulate the bone activity in senescent rats.     

Immunocytochemical localization of γ-carboxyglutamic acid-containing proteins (osteocalcin) in rat bone and dentin

Summary Gla-protein or osteocalcin is one of the most abundant noncollagenous matrix proteins found in bone and dentin. The present study describes, with high resolution, the intracellular and extracellular distribution of Gla-protein in alveolar bone and incisor dentin. Sections of tissues embedded in Lowicryl K4M were incubated with rabbit antibodies to rat dentin Gla-protein. The site of the specific antigen-antibody reaction was revealed by the protein A-gold complex. Labeling was detected over bone and dentin while fewer gold particles were present over prebone and predentin. Gold particles were also seen over the protein synthetic organelles (rough endoplasmic reticulum, Golgi apparatus) of osteoblasts and odontoblasts. These findings confirm, with improved resolution, previous light immunohistochemical studies, and offer the possibility to examine the secretory pathway of the protein.     

Bone gla: Protein in blood derived directly from human bone tissue

Summary In order to find out whether the concentration of bone gla-protein (BGP) is elevated in blood derived directly from bone tissue compared to peripherically sampled blood a peroperative model was used. Blood was collected simultaneously from an armvein and from the osteotomized surface of the bone during total hip replacement in 18 patients Peripheral blood was also collected before the anaesthetic procedure. The anaesthetic and/or the operative procedure slightly decreased the level of BGP in the peripheral blood during this short time interval, but serum BGP was not different in the bone-marrow derived blood and in the peripheral blood taken simultaneously. These data suggest that BGP released from bone into the circulation is rapidly cleared from the marrow and that peripheral serum BGP is a valid index of the bone-derived levels of BGP.     

Postoophorectomy bone loss is associated with reduced bone Gla protein serum levels: A possible effect of osteoblastic insufficiency

Summary To further investigate the relationship between oophorectomy (OF) and mineral bone loss, 15 women who underwent total hysterectomy and bilateral oophorectomy were studied for 12 months after surgery. Mineralometric and metabolic data were obtained before and after 3, 6, and 12 months. The women lost bone mineral content (measured by single photon absorptiometry) at the same rate they lost cortical and trabecular bone, suggesting that bone loss after OF is a generalized phenomenon. Our data also show that in increase in bone resorption takes place only in the first period after OF; the persistency of a negative bone balance up to 12 months, accompanied by a reduction of osteocalcin serum levels, may be dependent on a reduced bone formation, probably due to osteoblastic insufficiency.     

Normal bone particles are preferentially resorbed in the presence of osteocalcin-deficient bone particlesIn vivo

Summary In anin vivo model of osteoclastic bone resorption, we previously showed that osteocalcin-deficient bone particles (BPs), derived from warfarin-treated rats, were resorbed 50% as well as normal BPs and that they recruited fewer osteoclastic cells with decreased tartrate-resistant acid phosphatase (TRAP) activity. In order to determine the specificity of the resorption response, we evaluated the fate of implanted mixtures of normal and osteocalcin-deficient BPs. Normal and warfarin-treated donor rats were prelabeledin vivo with oxytetracycline to permit identification of BPs from either source. Normal, osteocalcin-deficient, and 50∶50 mixtures of BPs (either labeled or unlabeled) were implanted into normal rats and recovered 12 days later for enzymatic (TRAP) and nondecalcified histomorphometric analyses. The incorporated oxytetracycline had no signficant effect on resorption of bone particles. The recovered osteocalcin-deficient BPs were surrounded by fewer osteoclastic cells, were resorbed less, and contained less extractable TRAP activity than normal BPs. In mixed BP implants with normal and osteocalcin-deficient BPs, each type of bone particle elicited the same tissue response as when implanted separately. Remarkably, the different particles evoked dissimilar osteoclastic responses and were resorbed to different extents, even when adjacent within the same implant. These data suggest that osteocalcin may act as a substrate signal for resorption and that osteocalcin in the normal BPs does not influence the cellular response to adjacent osteocalcin-deficient BPs.     

Osteocalcin and bone morphometric parameters in adults without bone disease

Summary Serum bone Gla-protein (s-BGP or osteocalcin) and other serum biochemical parameters were measured in 19 subjects (8 women and 11 men, aged 20–82 years) without any bone disease. Each subject simultaneously underwent an iliac crest biopsy; tetracycline double-labeling was performed in 11 subjects, allowing correlations between s-BGP and bone histomorphometric parameters. s-BGP was significantly correlated with static bone parameters: trabecular bone volume (r=0.74;P<0.001), osteoid surfaces (r=0.69;P<0.001), osteoblastic surfaces (r=0.68;P<0.002); dynamic bone formation parameters: total labeled surfaces (r=0.72;P<0.01); and the bone formation rate (r=0.69;P<0.01). We conclude that s-BGP is a valuable marker for evaluating bone formation in healthy adult subjects.     

Characterization of mitogenic activities extracted from bovine bone matrix

The mitogenic activity in the unfractionated mixture of proteins released from adult bovine bone matrix during demineralization with ethylenediaminetetraacetate (EDTA) has been examined. Bovine bone extract (BBE) from 1 to 25 μg protein per ml stimulated proliferation of chick embryo calvaria bone cells, newborn mouse bone cells, and osteoblastlike cell lines MMB-1 and ROS 17/2.8. BBE also stimulated DNA synthesis in cells from chick embryo cartilage, skin and skeletal muscle tissues and fibroblastlike BALB/c 3T3 and NRK cells. BBE contained β transforming growth factor (TGF) activity (NRK cell colony formation in soft agar in the presence of epidermal growth factor EGF). The cell specificity results suggest that (1) BBE contains more than one growth factor, including a βTGF and a factor that is not specific for bone cells, and (2) all of the bone derived growth factor activities that have been described previously, including SGF, are apparently present in BBE.

Maximal stimulation of chick embryo calvarial cell DNA synthesis by BBE was equal to or exceeded maximal stimulation by nonosseous growth factors that have been reported to stimulate DNA synthesis in bone organ cultures (EGF, fibroblast growth factor, platelet-derived growth factor, insulinlike growth factor I, and multiplication stimulating activity). Combinations of BBE with maximally stimulatory concentrations of each growth factor stimulated DNA synthesis to a greater magnitude than did each growth factor alone. These results suggest that combinations of bone derived and systemic factors can coordinately stimulate bone cell proliferation.     

Serum bone Gla protein as a marker of bone turnover in acromegaly

Summary Serum bone Gla protein, a sensitive and specific marker of bone turnover, was measured in 35 acromegalic patients (14 untreated, 8 clinically active, and 13 cured) and 21 controls. We also examined 10 acromegalic patients before and after transsphenoidal surgery. Untreated and clinically active acromegalic patients had significantly higher serum bone Gla protein concentrations than the control subjects. Other nonspecific biochemical markers of bone metabolism, such as urinary hydroxyproline and urinary calcium, were also present in significantly greater amounts in active acromegalic patients. After treatment, a significant decrease in levels was observed, with return to control levels. In acromegalic patients, positive correlations were found among serum bone Gla protein and serum growth hormone and serum insulin-like growth factor I levels, as well as among levels of insulin-like growth factor I and serum phosphorus, serum alkaline phosphatase, and urinary hydroxyproline. These results suggest that serum bone Gla protein is a sensitive marker of the action of growth hormone in bone metabolism in acromegaly, a role that is probably mediated by insulin-like growth factor I.     

多发性骨髓瘤骨代谢指标的改变

本文测定了１０例多发性骨髓瘤患者的血钙、磷、ＡＫＰ、ＢＧＰ、２４小时尿ＨＯＰ及腰椎２～４正位ＢＭＤ，并且经化疗完全缓解后再次复查。结果（１）多发性骨髓瘤患者血Ｃａ、Ｐ、ＡＫＰ与正常对照无显著差异，化疗前后也无显著差异。（２）血ＢＧＰ低于正常对照，２４小时尿ＨＯＰ高于正常对照，化疗后ＢＧＰ增高，ＨＯＰ下降。（３）ＢＭＤ改变：１０例患者中有３例骨量减低，６例骨质疏松，化疗后ＢＭＤ有所增加。因此，血ＢＧＰ、尿ＨＯＰ和ＢＭＤ对观察多发性骨髓瘤治疗效果有一定的临床指导意义。还初步探讨了破骨细胞因子对骨质破坏的机理。     

Indicators of bone formation in weight lifters

Physical activity has been suggested to be one of the determinants of bone turnover and to prevent age-related bone loss. To examine this we measured the serum levels of osteocalcin (bone Gla-protein, BGP), C-terminal procollagen peptide (PICP), serum alkaline phosphatase, bone-specific alkaline phosphatase, and S-calcium as indices of bone formation in 19 actively performing and 15 ex-lifters. All were nationally or internationally ranked male athletes. Their values were compared with those from 38 age- and gendermatched controls. Actively performing weight lifters had 35% higher (P<0.05) serum concentration of osteocalcin than the controls. The ex-lifters did not differ from the agematched controls. Also serum calcium was elevated in active lifters (6%) (P<0.01) but not in ex-lifters. No difference was found for serum-ALP, B-ALP, or PICP in either of the groups. Our study indicates that in addition to an already documented and well-known higher bone mineral density in heavily exercising athletes, they have an indication of higher bone formation as measured by biochemical markers. In athletes who have retired from competitional training, however, the bone formation does not differ from that of more sedentary controls.     

糖尿病患者血清BGP与骨密度变化的研究

本文应用放射免疫分析方法对60例正常人及74例糖尿病患者的血清BGP进行了测定，并就其与BMD、血Ca、P及AKP等进行比较分析。结果表明：糖尿病患者血清BGP明显降低，而血Ca、P及AKP仍在正常范围．血清BGP与BMD呈负相关。说明血清BGP测定与BMD一样可以作为骨质疏松、糖尿病等代谢性骨病诊断与研究的一种敏感指标。     

Evaluation of bone turnover in type I osteoporosis using biochemical markers specific for both bone formation and bone resorption

The aims of the study were to evaluate the use of bone-specific biochemical markers of turnover in type I osteoporosis, to test for evidence of heterogeneity of bone turnover in this condition, and to attempt to devise an uncoupling index by using the relationship between bone-specific biochemical markers of bone formation and bone resorption. In women with type I osteoporosis (mean age 64 years, SD 5;n=63) the mean level of serum osteocalcin, a specific biochemical marker of bone formation, was 9.9 ng/ml (SD 2.0), which was higher than the level in normal postmenopausal women (mean age 65 years, SD 6;n=8.9 ng/ml (SD 2.0;p<0.01). The variance of serum osteocalcin levels in the two groups was similar. Compared with this 11% increase in the biochemical marker for bone formation, the markers of bone resorption, total urinary deoxypyridinoline (bone-specific), pyridinoline and hydroxyproline were increased by 40% (p<0.0001), 61% (p<0.0001) and 25% (p<0.01), respectively. Furthermore, these biochemical markers of bone resorption had greater variance in women in type I osteoporosis than in the normal postmenopausal women (p<0.001). The urinary excretion of the free crosslinks deoxypyridinoline, pyridinoline and glycosylated pyridinoline were increased by 26% (p<0.001), 17% (p<0.01) and 13% (NS) respectively. An uncoupling index was calculated for the difference between urinary deoxypyridinoline and serum osteocalcin using the results from the normal women and expressed asz-scores. We conclude that the pyridinium crosslinks of collagen enable better discrimination between normal and osteoporotic women than does hydroxyproline. In osteoporosis there appears to be heterogeneity of bone resorption. Finally, an uncoupling index indicated that in osteoporosis bone resorption was increased to a greater extent than bone formation as compared with normal postmenopausal women.     

Effect of short-term hypomagnesemia on the chemical and mechanical properties of rat bone.

Magnesium is known to have an essential role in determining the properties of bone, but the way in which Mg exerts its actions remains unclear. Although long-term Mg deficiency is known to produce osteopenia, the effects of short-term Mg deficiency have not been established. To test the hypothesis that Mg deficiency results in an altered pattern of initial mineralization and concomitant altered bone properties, the radiographic, histologic, chemical, and mechanical properties of the bones of rats given a Mg-deficient diet were compared to those of rats pair-fed the same diet supplemented with Mg. Short-term Mg-deficiency in the diet of growing rats produced a significant decrease in both the trabecular bone volume and the mineral content of the newly formed metaphysis, a significant increase in the Ca:P ratio, and a slight, but significant increase in hydroxyapatite crystallite size and/or perfection in the metaphysis. Comparable, but not significant, trends were found in the diaphyses. Metaphyseal bone osteocalcin levels were reduced in the Mg-deficient rats and lipid was more easily extracted from their bones. No detectable alterations in radiographic microstructure were noted. Mechanically, a significant decrease in the maximum three-point bend strength of the femurs of Mg-deficient rats was observed. These data support the hypothesis that short-term Mg deficiency affects the pattern of bone mineral formation.