We employed a well-standardized murine rib fracture model to assess the distribution, in the cortical bone, of three important osteocyte-derived molecules—dentine matrix protein 1 (DMP1), sclerostin and fibroblast growth factor 23 (FGF 23). Two days after the fracture, the periosteum thickened, and up to the seventh day post-fracture, the cortical surfaces were promoting neoformation of two tissue types depending on the distance from the fracture site: chondrogenesis was taking place near the fracture, and osteogenesis distant from it. The cortical bones supporting chondrogenesis featured several empty lacunae, while in the ones underlying newly-formed woven bone, empty lacunae were hardly seen. DMP1-immunopositive osteocytic lacunae and canaliculi were seen both close and away from the fracture. In contrast, the region close to the fracture had only few sclerostin- and FGF23-immunoreactive osteocytes, whereas the distant region revealed many osteocytes immunopositive for these markers. Mature cortical bone encompassing the native cortical bone was observed at two-, three- and four-weeks post-fracture, and the distribution of DMP1, sclerostin and FGF23 appeared to have returned to normal. In summary, early stages of fracture healing seem to be important for triggering chondrogenesis and osteogenesis that may be regulated by osteocytes via their secretory molecules.
Tissue-nonspecific alkaline phosphatase (TNSALP) in serum comprises liver alkaline phosphatase (liver-ALP) and bone alkaline phosphatase (bone-ALP). Liver-ALP is a marker of liver disease; thus a specific method for its measurement would be useful. Measurement of ALP by electrophoresis is difficult, although all of the isozymes can be assessed simultaneously. Total ALP can also be measured by automated analyzer, but it is difficult to determine the cause of a high ALP value because bone-, intestine-, placenta-, and tumor-ALP are measured together. Thus, anti-TNSALP monoclonal antibodies that can resolve these problems are needed. Here we have generated an anti-TNSALP monoclonal antibody, 3-29-3R. This clone has specificity to liver-ALP rather than to bone-ALP. In electrophoresis, 3-29-3R reacted with TNSALP and shifted the bands. The use of 3-29-3R enabled easy interpretation of the results. Furthermore, we tested 3-29-3R by developing an immunocapture enzymatic assay (IEA). Preliminary results of the IEA show that this method is effective for measurement of liver-ALP. Thus, the monoclonal antibody that we have established may be a useful tool for clinical diagnosis.
Osteoclasts are multinucleated cells that resorb bone. Although osteoclasts originate from the monocyte/macrophage lineage, osteoclast precursors are not well characterized in vivo. The relationship between proliferation and differentiation of osteoclast precursors is examined in this study using murine macrophage cultures treated with macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-κB (RANK) ligand (RANKL). Cell cycle–arrested quiescent osteoclast precursors (QuOPs) were identified as the committed osteoclast precursors in vitro. In vivo experiments show that QuOPs survive for several weeks and differentiate into osteoclasts in response to M-CSF and RANKL. Administration of 5-fluorouracil to mice induces myelosuppression, but QuOPs survive and differentiate into osteoclasts in response to an active vitamin D3 analogue given to those mice. Mononuclear cells expressing c-Fms and RANK but not Ki67 are detected along bone surfaces in the vicinity of osteoblasts in RANKL-deficient mice. These results suggest that QuOPs preexist at the site of osteoclastogenesis and that osteoblasts are important for maintenance of QuOPs.
Therapy using recombinant human bone morphogenetic protein-2 (rhBMP-2) is expected to promote bone healing and regeneration. Previous studies using protein or virus vectors for direct clinical application had problems, including a lack of efficiency, safety, and simplicity of the delivery system, and required an expensive protein, carrier matrix, or antigenic viral vector. In vivo gene transfer by electroporation is a simple and inexpensive method that only requires a plasmid and an electroporation device. Here, we created a plasmid-based human BMP-2 construct (pCAGGS-BMP-2) and examined the induction of bone in the skeletal muscle of rats after transferring different doses of this plasmid (25 microg, 100 microg, and 400 microg) by transcutaneous electroporation (8 electrical pulses of 100 V and 50 msec, in 1 to 5 sessions). First, we verified the gene transfer by transcutaneous electroporation using pCAGGS-lacZ. Next, the BMP-2 gene transfer and the production and localization of BMP-2 were identified by reverse transcription-polymerase chain reaction (RT-PCR), Western blots, and immunohistochemistry. Ectopic bone formation was verified by radiography, histologic and immunohistochemical analyses, and quantitative examination. Ectopic bone formation, consisting of active osteoblasts and osteoclasts, was observed in all rats treated with electroporation. Thus, transcutaneous electroporation with pCAGGS-BMP-2 induced ectopic bone formation in the skeletal muscle of rats. This supports the possibility of applying human BMP-2 gene transfer using transcutaneous electroporation clinically.
Minor contaminants occasionally found in conventionally prepared rat serum albumin were easily and completely removed by concanavalin A-Sepharose chromatography. The unadsorbed fraction from a concanavalin A-Sepharose column contained albumin which was homogeneous on polyacrylamide gel electrophoresis. The recovery of albumin from rat serum was approximately 30%. Approximately 2% of the added protein obtained as an albumin peak in DEAE-cellulose chromatography was adsorbed on and eluted with α-methyl-D-glucoside from the concanavalin A-Sepharose column, and resolved into three components by gel electrophoresis. There was one major glycoprotein, possibly α1-antitrypsin and two minor proteins one of which was albumin.
α 1 ‐Protease inhibitor was purified from rat serum by two different methods, of which an immunoaffinity method should be preferentially used to obtain all of the multiple forms. The protein thus obtained showed a single protein band in dodecylsulphate/polyacrylamide gel electrophoresis corresponding to a molecular weight of 54000, and contained 13.2% carbohydrate by weight. By column isoelectric focusing in a pH 3.5–5.0 gradient the purified α 1 ‐protease inhibitor was separated into five forms with pl values from 4.3–4.7. The amino acid composition of each form was identical, while sialic acid content was significantly different from each other. The most acidic form contained 6.7 residues/molecule, the most basic form, 5.1 residues/molecule, and three forms between them showed proportionally intermediate values between the two. When α 1 ‐protease inhibitor was treated with neuraminidase, the five forms were converted finally into three major forms with pl values of 5.3–5.7. In addition, the major form (band 3) of the inhibitor was also converted into three forms after complete removal of sialic acid. These results suggest that α 1 ‐protease inhibitor originally exists as three forms with different charges, possibly due to modification of amino acids which might not be detectable by the amino acid composition analysis in the present study. A possible explanation was presented for involvement of sialic acid in appearance of multiple forms originating from three parental forms.
Using an immunoblotting technique, we have studied the processing of plasma proteins in subcellular fractions of rat liver including rough and smooth micro-somes and the Golgi subtractions. Each subcellular fraction was directly subjected to SDS-polyacrylamide gel electrophoresis and analyzed by immunoblotting with antibodies against α1-protease inhibitor, haptoglobin, and the third component of complement (C3) in combination with 125I-protein A or 125I-rabbit anti-(goat IgG)-IgG. The results demonstrated that proteolytic processing of precursors of complement C3 and haptoglobin occurs in different compartments along the secretory pathway; conversion of prohaptoglobin takes place in the endoplasmic reticulum, while that of pro-C3 occurs in the Golgi complex. The processing in oligosaccharide chains of glycoproteins was also analyzed. The Golgi fraction was characterized by the presence of the mature 56 kDa α1-protease inhibitor, which was indistinguishable from the serum α1-protease inhibitor in SDS-polyacrylamide gel electrophoresis. In contrast, the immature 51 kDa form was the only form of α1-protease inhibitor found in the microsomal fraction. Similar results were obtained for the β subunit of haptoglobin; the immature 33 kDa form was detected in the microsomal fraction, while the mature 36 kDa form was found in the Golgi fraction. Taken together, these results identified the intracellular sites where these plasma proteins are modified by selective proteolysis and/or glycosylation.
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