In this study, we report that the steroid extract 5a, 8a-epidioxycholest-6-ene-3b-ol (MME) from Meretrix meretrix has the ability to inhibit growth of hepatoma cells and to induce G1-phase cell cycle arrest in two human hepatoma cell lines, HepG2 and Hep3B. HepG2 cells were more sensitive than Hep3B to MME. The extract markedly up-regulated the expression of p53 and p21WAF1/CIP1 in HepG2, suggesting that MME-induced G1 phase cell cycle arrest in HepG2 might be p53-dependent. Therefore, the up-regulation of p27KIP1and p16INK4A in both cell lines indicates that a p53-independent pathway might be involved in the mechanism of MME inducing cell cycle arrest. In conclusion, MME induces G1 phase cell cycle arrest via both p53-dependent and p53-independent pathways.

Understanding the molecular composition and the formation mechanism of shell matrix framework is of great interest for biomineralization in mollusk shell. The cDNAs encoding a novel matrix protein family (KRMP) were cloned from the mantle of pearl oyster, Pinctada fucata. Analysis of the deduced amino acid sequences revealed that KRMP have a high proportion of lysine, glycine, and tyrosine, and their predict isoelectric points are higher than any other identified shell matrix protein to our knowledge. The deduced amino acid sequences of KRMP can be divided into three regions, including an N-terminal signal peptide, a lysine-rich basic region interacting with acidic proteins or CO3 2, and a Gly/Tyr-rich region involved in the protein cross-link via quinone-tanning process. RT-PCR and in situ hybridization demonstrated that KRMP mRNA was specifically expressed in the mantle edge, involved in the prismatic layer formation. Taken together, it seems that KRMP is a matrix protein family participating in the framework formation of prismatic layer.

Tyrosinase (monophenol, L-DOPA: oxygen oxidoreductase, EC 1.14.18.1), a kind of copper-containing phenoloxidase, arouses great interests of scientists for its important role in periostracum formation. A cDNA clone encoding a putative tyrosinase, termed OT47 because of its estimated molecular mass of 47 kDa, was isolated from the pearl oyster, Pinctada fucata. This novel tyrosinase shares similarity with the cephalopod tyrosinases and other type 3 copper proteins within two conserved copper-binding sites. RT-PCR analysis showed that OT47 mRNA was expressed only in the mantle edge. Further in situ hybridization analysis and tyrosinase activity staining revealed that OT47 was expressed at the outer epithelial cells of the middle fold, different from early histological results in Mercenaria mercenaria, suggesting a different model of periostracum secretion in P. fucata. Taken together, these results suggest that OT47 is most likely involved in periostracum formation. The identification and characterization of oyster tyrosinase also help to further understand the structural and functional properties of molluscan tyrosinase.

Alkaline phosphatases (ALP, EC 3.1.3.1) are ubiquitous enzymes found in most species. ALP from a pearl oyster, Pinctada fucata (PALP), is presumably involved in nacreous biomineralization processes. Here, chemical modification was used to investigate the involvement of basic residues in the catalytic activity of PALP. The Tsou's plot analysis indicated that the inactivation of PALP by 2,4,6-trinitrobenzenesulfonic acid (TNBS) and phenylglyoxal (PG) is dependent upon modification of one essential lysine and one essential arginine residue, respectively. Substrate reaction course analysis showed that the TNBS and PG inactivation of PALP followed pseudo-first-order kinetics and the second-order inactivation constants for the enzyme with or without substrate binding were determined. It was found that binding substrate slowed the PG inactivation whereas had little effect on TNBS inactivation. Protection experiments showed that substrates and competitive inhibitors provided significant protection against PG inactivation, and the modified enzyme lost its ability to bind the specific affinity column. However, the TNBS-induced inactivation could not be prevented in presence of substrates or competitive inhibitors, and the modified enzyme retained the ability to bind the affinity column. In a conclusion, an arginine residue involved in substrate binding and a lysine residue involved in catalysis were present at the active site of PALP. This study will facilitate to illustrate the role ALP plays in pearl formation and the mechanism involved.

A cDNA clone encoding a novel G protein h subunit of h1 subclass, pfGh1 was isolated from the pearl oyster (Pinctada fucata). The deduced amino acid sequence of pfGh1 (341 amino acids) shares high homology to northern European squid (Loligo pealei) and great pond snail (Lymnaea stagnalis) Gh1, while it has diverged from bovine (Bos taurus) and human. The well-conserved amino acid domains in G protein h subunit, seven WD repeats, were founded in the deduced amino acid sequence. Alignment analysis showed that the beginning amino acid residues in variable fragment of the seventh WD motif are different from any other Gh. The prediction of 3D structure of pfGh showed that pfGh belongs to h-propeller family proteins whose members contain 4–8 antiparallel h-sheets resembling the blades of a propeller. In situ hybridization and Northern blotting analysis revealed that the pfGh mRNA hybridization signals were widely expressed in various tissues except muscle, with abundantly in epithelia of gill, gonad and outer fold of mantle. We also investigated the interactions between Ghg and calmodulin (CaM), and specific amino acid residues that may be critical for the binding of Ghg to CaM were also identified. Furthermore, the functional studies of the interaction showed that the binding of CaM and Ghg increases the alkaline phosphatase (ALP) activity, an indicator for mineralization in MC3T3-E1 cells. The ALP activity of the mutants of pfGhg that impaired the interactions of Ghg with CaM is higher than the Control group; however, it is lower than the WTC group. Together, these results suggest that the Ghg might interact with CaM and point to the important physiological function in modulating cellular functions.

Nacre formation is an ideal model to study biomineralization processes. Although much has been done about biomineralization mechanism of nacre, little is known as to how cellular signaling regulates this process. We are interested in whether G protein signaling plays a role in mineralization. Degenerate primers against conserved amino acid regions of G proteins were employed to amplify cDNA from the pearl oyster Pinctada fucata. As a result, the cDNA encoding a novel Gsa (pfGsa) from the pearl oyster was isolated. The Gsa cDNA encodes a polypeptide of 377 amino acid residues, which shares high similarity to the octopus (Octopus vulgaris) Gsa. The well-conserved A, C, G (switch I), switch II functional domains and the carboxyl terminus that is a critical site for interaction with receptors are completely identical to those from other mollusks. However, pfGsa has a unique amino acid sequence, which encodes switch III and interaction sites of adenylyl cyclase respectively. In situ hybridization and Northern blotting analysis revealed that the oyster Gsa mRNA is widely expressed in a variety of tissues, with highest levels in the outer fold of mantle and epithelia of gill, the regions essential for biomineralization. We also show that overexpression of the pfGsa in mammalian MC3T3-E1 cells resulted in increased cAMP levels. Mutant pfGsa that has impaired CTX substrate diminished its ability to induce cAMP production. Furthermore, the alkaline phosphatase (ALP) activity, an indicator for mineralization, is induced by the Gsa in MC3T3-E1 cells. These results indicated that Gsa may be involved in regulation of physiological function, particularly in biological biomineralization.

The shells of bivalves are mainly composed of calcium carbonate, a product of calcium metabolism. In the process of shell formation, the uptake, transport and recruitment of calcium ion are highly regulated and involved in many factors. Among these regulatory factors, calmodulin (CaM), a pivotal multifunction regulator of calcium metabolism in nearly all organisms, is thought to play an important role in the calcium metabolism involved in shell formation. In this study, a full-length CaM cDNA was isolated from the pearl oyster (Pinctada fucata). The oyster calmodulin encodes a 16.8 kDa protein which shares high similarity with vertebrate calmodulin. The oyster CaM mRNA shows the highest level of expression in the gill, a key organ involved in calcium uptake in oyster calcium metabolism. In situ hybridization results revealed that oyster CaM mRNA is expressed at the folds and the outer epithelial cells of the dorsal region of the mantle, suggesting that CaM is involved in regulation of calcium transport and secretion. Oyster CaM also showed a typical Ca2+ dependent electrophoretic shift characterization and calcium binding activity. Taken together, we have identified and characterized a pivotal calcium metabolism regulator of the oyster that may play an important role in regulation of calcium uptake, transport and secretion in the process of shell formation.

Understanding the molecular composition is of great interest for both nacre formation mechanism and biomineralization in mollusk shell. A cDNA clone encoding an MSI31 relative, termed MSI7 because of its estimated molecular mass of 7.3 kDa, was isolated from the pearl oyster, Pinctada fucata. This novel protein shares similarity with MSI31, a prismatic framework protein of P. fucata. It is peculiar that MSI7 is much shorter in size, harboring only the Gly-rich sequence that has been proposed to be critical for Ca2q binding. In situ hybridization result showed that MSI7 mRNA was expressed specifically at the folds and outer epithelia of the mantle, indicating that MSI7 participates in the framework formation of both the nacreous layer and prismatic layer. In vitro experiment on the function of MSI7 suggested that it accelerates the nucleation and precipitation of CaCO3. Taken together, we have identified a novel matrix protein of the pearl oyster, which may play an important role in determining the texture of nacre.