Hongzhong Wang

Daidzein enhances osteoblast growth that may be mediated by increased bone morphogenetic protein (BMP) production.

Daidzein, a natural isoflavonoid found in Leguminosae, has received increasing attention because of its possible role in the prevention of osteoporosis. In the present investigation, primary osteoblastic cells isolated from newborn Wistar rats were used to investigate the effect of this isoflavonoid on osteoblasts. Daidzein (2-50 microM) increased the viability (P<0.05) of osteoblasts by about 1.4-fold. In addition, daidzein (2-100 microM) increased the alkaline phosphatase activity and osteocalcin synthesis (P<0.05) of osteoblasts by about 1.4- and 2.0-fold, respectively. Alkaline phosphatase and osteocalcin are phenotypic markers for early-stage differentiated osteoblasts and terminally differentiated osteoblasts, respectively. Our results indicated that daidzein stimulated osteoblast differentiation at various stages (from osteoprogenitors to terminally differentiated osteoblasts). We also investigated the effect of daidzein on bone morphogenetic protein (BMP) production in osteoblasts that display the mature osteoblast phenotype. The results indicated that BMP2 synthesis was elevated significantly in response to daidzein (the mRNA increased 5.0-fold, and the protein increased 7.0-fold), suggesting that some of the effects of daidzein on the cell may be mediated by the increased production of BMPs by the osteoblasts. In conclusion, daidzein has a direct stimulatory effect on bone formation in cultured osteoblastic cells in vitro, which may be mediated by increased production of BMPs in osteoblasts.

A novel ferritin subunit involved in shell formation from the pearl oyster (Pinctada fucata)

Iron is one of the most important minor elements in the shell of bivalves. This study was designed to investigate the involvement of ferritin, the principal protein for iron storage, in shell formation. A novel ferritin cDNA from the pearl oyster (Pinctada fucata) was isolated and characterized. The ferritin cDNA encodes a 206 amino acid polypeptide, which shares high similarity with snail soma ferritin and the H-chains of mammalian ferritins. Oyster ferritin mRNA shows the highest level of expression in the mantle, the organ for shell formation. In situ hybridization analysis revealed that oyster ferritin mRNA is expressed at the highest level at the mantle fold, a region essential for metal accumulation and contributes to metal incorporation into the shell. Taken together, these results suggest that ferritin is involved in shell formation by iron storage. The identification and characterization of oyster ferritin also helps to further understand the structural and functional properties of molluscan ferritins.

Cloning and Characterization of Prisilkin-39, a Novel Matrix Protein Serving a Dual Role in the Prismatic Layer Formation from the Oyster Pinctada fucata

Molluscs form their shells out of CaCO3 and a matrix of biomacromolecules. Understanding the role of matrices may shed some light on the mechanism of biomineralization. Here, a 1401-bp full-length cDNA sequence encoding a novel matrix protein was cloned from the mantle of the bivalve oyster, Pinctada fucata. The deduced protein (Prisilkin-39), which has a molecular mass of 39.3 kDa and an isoelectric point of 8.83, was fully characterized, and its role in biomineralization was demonstrated using both in vivo and in vitro crystal growth assays. Prisilkin-39 is a highly repetitive protein with an unusual composition of Gly, Tyr, and Ser residues. Expression of Prisilkin-39 was localized to columnar epithelial cells of the mantle edge, corresponding to the calcitic prismatic layer formation. Immunostaining in situ and immunodetection in vitro revealed the presence of a characteristic pattern of Prisilkin-39 in the organic sheet and in sheaths around the prisms. Prisilkin-39 binds tightly with chitin, an insoluble polysaccharide that forms the highly structured framework of the shell. Antibody injection in vivo resulted in dramatic morphological deformities in the inner shell surface structure, where large amounts of CaCO3 were deposited in an uncontrolled manner. Moreover, Prisilkin-39 strictly prohibited the precipitation of aragonite in vitro. Taken together, Prisilkin-39 is the first protein shown to have dual function, involved both in the chitinous framework building and in crystal growth regulation during the prismatic layer mineralization. These observations may extend our view on the rare group of basic matrices and their functions during elaboration of the molluscan shell.

EFFECTS OF GENISTEIN AND DAIDZEIN ON THE CELL GROWTH, CELL CYCLE, AND DIFFERENTIATION OF HUMAN AND MURINE MELANOMA CELLS

Genistein and daidzein are two major isoflavonoids in dietary soybean that have inhibition effect on the cell growth of different tumor cell lines. We previously reported the anti-tumor activities of genistein and daidzein in human co1on tumor (HCT) cells and their different ability to enhance the activation of murine lymphocytes. In the present study, the effect of genistein and daidzein on the cell growth, cell cycle progression, and differentiation of murine K1735M2 and human WM451 cel1s was investigated. It was found that genistein could inhibit the cell growth of two metastatic melanoma cell lines, murine Kl735M2 and human WM45l in a dose-dependent manner. Flow cytometry showed that genistein could cause arrest of both Kl735M2 and WM45l at G(2)/M phase, while daidzein increased the cell numbers at S phase, decreased the cell numbers at G(1) phase. Detection of melanin and morphological observation showed that genistein can induce Kl735M2 and WM45l to produce dendrite-like structure and produce more melanin by 80%. In contrast, daidzein only retarded the growth of K1735M2 and did not induce differentiation in either K1735M2 or WM451. These results suggest that genistein and daidzein in soybean can inhibit certain malignant phenotype of melanoma via different mechanisms and be potential medical candidates for melanoma cancer therapy.

Controlled crystal growth of calcium phosphate on titanium surface by NaOH-treatment

A simple supersaturated calcification solution (SCS) was used to deposit calcium phosphate (Ca-P) on the surface of NaOH-treated titanium (NaOH-Ti), in order to study the corresponding deposition mechanism of calcium phosphate (Ca-P). No Ca-P coating from SCS was found on titanium without NaOH-treatment, while NaOH-Ti possessed the capability to induce a Ca-P coating on the titanium surface. Octacalcium phosphate (OCP) crystals were first grown on an NaOH-Ti surface, followed by hydroxyapatite (HA) with a [0 0 1] preferential orientation on OCP. It is found that two factors controlled the growth of Ca-P crystals on NaOH-Ti from SCS. First, the surface morphology of NaOH-Ti characterized with crevices seems to be beneficial for inducing a Ca-P coating from SCS; second, the basic hydroxyl, Ti-OH, radical has increased in NaOH-Ti with the increase of treating time and concentration, which facilitate the nucleation of Ca-P crystals.

The role of matrix proteins in the control of nacreous layer deposition during pearl formation

To study the function of pearl oyster matrix proteins in nacreous layer biomineralization in vivo, we examined the deposition on pearl nuclei and the expression of matrix protein genes in the pearl sac during the early stage of pearl formation. We found that the process of pearl formation involves two consecutive stages: (i) irregular calcium carbonate (CaCO3) deposition on the bare nucleus and (ii) CaCO3 deposition that becomes more and more regular until the mature nacreous layer has formed on the nucleus. The low-expression level of matrix proteins in the pearl sac during periods of irregular CaCO3 deposition suggests that deposition may not be controlled by the organic matrix during this stage of the process. However, significant expression of matrix proteins in the pearl sac was detected by day 30–35 after implantation. On day 30, a thin layer of CaCO3, which we believe was amorphous CaCO3, covered large aragonites. By day 35, the nacreous layer had formed. The whole process is similar to that observed in shells, and the temporal expression of matrix protein genes indicated that their bioactivities were crucial for pearl development. Matrix proteins controlled the crystal phase, shape, size, nucleation and aggregation of CaCO3 crystals.

Influence of solution conditions on deposition of calcium phosphate on titanium by NaOH-treatment

The present paper demonstrated a biomimetic method to coat calcium phosphate (Ca-P) on the surface of titanium induced by NaOH-treatment from a simple supersaturated hydroxyapatite solution (SHS). The influence of pH value and calcium ions concentration on the precipitation process was investigated. It is necessary for the solution to be supersaturated than the critical concentration of octacalcium phosphate (OCP) to get Ca-P coatings on titanium surface. In the precipitating process, it seems that amorphous calcium phosphate (ACP) precipitated first, then OCP, and finally hydroxyapatite (HA). The system was in continuous evolution and the phase transitions occurred in sequence.

Novel Basic Protein, PfN23, Functions as Key Macromolecule during Nacre Formation

Background: The microstructure of nacre is controlled by the proteins in them. Results: When PfN23 was knocked down, the shell formation in adults and larvae was suppressed. Conclusion: The basic protein PfN23 is important for the control of crystal growth in nacre. Significance: This might provide a valuable complementary to the classic view that acidic proteins control nacre formation. The fine microstructure of nacre (mother of pearl) illustrates the beauty of nature. Proteins found in nacre were believed to be “natural hands” that control nacre formation. In the classical view of nacre formation, nucleation of the main minerals, calcium carbonate, is induced on and by the acidic proteins in nacre. However, the basic proteins were not expected to be components of nacre. Here, we reported that a novel basic protein, PfN23, was a key accelerator in the control over crystal growth in nacre. The expression profile, in situ immunostaining, and in vitro immunodetection assays showed that PfN23 was localized within calcium carbonate crystals in the nacre. Knocking down the expression of PfN23 in adults via double-stranded RNA injection led to a disordered nacre surface in adults. Blocking the translation of PfN23 in embryos using morpholino oligomers led to the arrest of larval development. The in vitro crystallization assay showed that PfN23 increases the rate of calcium carbonate deposition and induced the formation of aragonite crystals with characteristics close to nacre. In addition, we constructed the peptides and truncations of different regions of this protein and found that the positively charged C-terminal region was a key region for the function of PfN23 Taken together, the basic protein PfN23 may be a key accelerator in the control of crystal growth in nacre. This provides a valuable balance to the classic view that acidic proteins control calcium carbonate deposition in nacre.

Improvement in hard magnetic properties of FePt films by N addition

The structure and magnetic properties of (Fe56Pt44)100−xNx alloy films with x=0–15 at. % prepared by rf magnetron sputtering were investigated. Compared to an Fe56Pt44 binary film, (Fe56Pt44)100−xNx alloy films showed great enhancement in hard magnetic properties. A coercivity Hc of 1027 kA/m, a remanence Mr of 1.24 T, and a maximum energy product (BH)max of 260 kJ/m3 were obtained for an (Fe56Pt44)93N7 film annealed at 600 °C for 10 min, whereas the Fe56Pt44 film annealed at the same conditions gave Hc=682 kA/m, Mr=0.87 T, and (BH)max=132 kJ/m3. Structural analysis revealed that N addition influenced the phase structure and microstructure of FePt films. N atoms incorporated in the disordered FePt phase in as-deposited films, they released rapidly out of the FePt phase by vacuum annealing. It is suggested that the rapid release of N atoms from the FePt phase promotes the transformation to the ordered phase, results in a higher coercivity of the film. The high remanence of the (Fe56Pt44)93N7 film is attrib...

Enhancement in ordering of FePt films by magnetic field annealing

Effect of magnetic field annealing on chemical ordering of FePt films has been investigated. It is found that the ordering rate in FePt films is enhanced by applying a magnetic field during postdeposition annealing. Measurements of the structure and magnetic properties of FePt films reveal that the disorder–order transformation starts at or below 450°C when the film is annealed in a magnetic field of 40kOe. The possible reason for the enhancement in the ordering of FePt films by magnetic field annealing is discussed.