R-spondin1 synergizes with Wnt3A in inducing osteoblast differentiation and osteoprotegerin expression

R-spondins are a new group of Wnt/beta-catenin signaling agonists, however, the role of these proteins in bone remains unclear. We reported herein that R-sponin1 (Rspo1) acted synergistically with Wnt3A to activate Wnt/beta-catenin signaling in the uncommitted mesenchymal C2C12 cells. Furthermore, we found that Rspo1 at concentrations as low as 10 ng/ml synergized strongly with Wnt3A to induce C2C12 osteoblastic differentiation and osteoprotegerin expression. These events were blocked by Wnt/beta-catenin signaling antagonist Dickkopf-1. Finally, we demonstrated that Rspo1 synergized with Wnt3A to induce primary mouse osteoblast differentiation. Together, these findings suggest that Rpos1 may play an important role in bone remodeling.     

Determination of trace alkaline phosphatase by solid-substrate room-temperature phosphorimetry based on Triticum vulgare lectin labeled with fullerenol

Fullerenol (F) shows a strong and stable room-temperature phosphorescence (RTP) signal on the surface of nitrocellulose membrane (NCM) at lambda ex max/ lambda em max =542.0/709.4 nm. When modified by dodecylbenzenesulfonic acid sodium salt (DBS), fullerenol emits a stronger signal. It was also found that quantitative specific affinity-adsorption reaction can be carried out between Triticum vulgare lectin (WGA) labeled with DBS-F and alkaline phosphatase (ALP) on the surface of NCM, and the product obtained (WGA-ALP-WGA-F-DBS) emits a strong and stable RTP signal. Furthermore, the content of ALP was proportional to the DeltaI(p) value. Based on the facts above, a new method for the determination of trace amounts of ALP by affinity-adsorption solid-substrate room-temperature phosphorimetry (AA-SS-RTP) was established, using fullerenol modified with DBS to label WGA. The detection limit was 0.011 fg spot(-1) (corresponding concentration: 2.8x10(-14) g ml(-1), namely 2.8x10(-16) mol l(-1)). This method with high sensitivity, accuracy, and precision has been successfully applied to the determination of the content of ALP in human serum survey and forecast human disease, and the results are tallied with those using alkaline phosphatase kits. The mechanism for the determination of ALP using AA-SS-RTP was also discussed.     

Alkaline phosphatase interference in immuno-enzymatic assays

BackgroundAlkaline phosphatase (ALP) enzymes are widely used as signal amplifiers in immunoenzymatic methods. Conditions that cause ALP elevations, such as bone or liver diseases, can cause interference in immunoenzymatic methods. We aimed to examine ALP''s effect on immunoenzymatic assay by adding isolated pure ALP to the prepared serum pool.MethodsWe prepared a serum pool and divided it into 4 groups. By adding isolated pure ALP at different concentrations to each group, we obtained sample groups containing ALP enzyme at concentrations of 85 U/L, 340 U/L, 870 U/L, and 1570 U/L. 20-repetition of bhCG, ferritin, FT4, TSH, troponin I, and Vit B12 tests were performed in each group. The coefficient of variation, bias, and total error was calculated. All groups were compared by using the Friedman test for paired samples.ResultsAfter ALP addition, the calculated total error values of FT4, bhCG and troponin I tests were above the acceptable error limits. There were statistically significant differences in bhCG, FT4, troponin I, and Vit B12 tests compared to the baseline ALP level (P<0.0125).ConclusionsIsolated ALP elevations can be a source of interference for immunoenzymatic methods.     

Synthesis and biological evaluation of heterocyclic analogues of pregnenolone as novel anti-osteoporotic agents

The structural modifications of pregnenolone have been described via the introduction of heterocyclic moieties at C-17 position by limiting the acyl group. Novel heterocyclic analogues of pregnenolone have been synthesized by using Friedlander and Claisen-Schmidt reactions, and the synthesized compounds were evaluated for their osteogenic activity. Among the synthesized derivatives, four compounds showed significantly increased ALP activity. Among all four active compounds, the novel compound 3a has shown significant bone matrix mineralization and mRNA expressions of osteogenic marker genes, BMP2, RUNX-2 and OCN at 1 pM concentration.     

Synthesis of novel 5,6-dehydrokawain analogs as osteogenic inducers and their action mechanisms

An imbalance between bone resorption by osteoclasts and bone formation by osteoblasts can cause bone loss and bone-related disease. In a previous search for natural products that increase osteogenic activity, we found that 5,6-dehydrokawain (1) from Alpinia zerumbet promotes osteoblastogenesis. In this study, we synthesized and evaluated series of 5,6-dehydrokawain analogs. Our structure-activity relationships revealed that alkylation of para or meta position of aromatic ring of 1 promote osteogenic activity. Among the potential analogs we synthesized, (E)-6-(4-Ethylstyryl)-4-methoxy-2H-pyran-2-one (14) and (E)-6-(4-Butylstyryl)-4-methoxy-2H-pyran-2-one (21) both significantly up-regulated Runx2 and Osterix mRNA expression at 10 µM. These osteogenic activities could be mediated by bone morphogenetic protein (BMP) and activation of p38 MAPK signaling pathways. Compounds 14 and 21 also inhibited RANKL-induced osteoclast differentiation of RAW264 cells. These results indicated that novel 5,6-dehydrokawain analogs not only increase osteogenic activity but also inhibit osteoclast differentiation, and could be potential lead compounds for the development of anti-osteoporosis agents.     

A comprehensive review of signal peptides: Structure,roles, and applications

Signal peptides (SP) are short peptides located in the N-terminal of proteins, carrying information for protein secretion. They are ubiquitous to all prokaryotes and eukaryotes. SPs have been of special interest in several scientific and industrial fields, including recombinant protein production, disease diagnosis, immunization, and laboratory techniques. Recently, the role of SPs in recombinant protein production has gained too much attention. Herein, several studies have been reviewed to elucidate the precise structure and function of SPs, particularly the optimized ones for recombinant protein production. However, some features of SPs still have remained obscure. In this review, some approaches concerning elucidation and optimization of SPs are discussed, and pragmatic conclusions and suggestions for future studies are also proposed. Moreover, a summary of secretory pathways, evolutionary changes, functions, applications, and different types of SPs is mentioned. At last, current limitations and prospects are discussed.     

Developmental changes of plasma alkaline phosphatase,calcium, and inorganic phosphorus in relation to the growth of bones in the Japanese macaque (Macaca fuscata)

The plasma levels of alkaline phosphatase (ALP), calcium, and inorganic phosphorus in the Japanese macaque were measured during its growing period. The plasma ALP level within two days after birth was 818±186 IU/1, and the level showed a temporary peak at about 2 months of age (2747±763 IU/1). The ALP level formed a plateau from 1 to 3 years of age, then decreased again and reached finally an adult level (211±71 IU/1). Sex differences were observed twice during the animal's lifetime, from 6 to 9 months of age and from 3 to 7 years of age. On integration of this complicated curve, a correlation was observed with the development of the length of the bones. The plasma levels of calcium and inorganic phosphorus which were high within two days after birth, reached adult levels at 3 months and 5 years of age, respectively.