Background: Thermodynamically unstable magnesium calcite is deposited in the shell of pearl oysters at ambient pressure. Results: The novel acidic matrix protein PfN44 interacts with magnesium to inhibit the deposition of aragonite. Conclusion: PfN44 participates in shell formation by inhibiting aragonite formation. Significance: Results of this study suggest a connection between the matrix protein and magnesium.

The initial growth of the nacreous layer is crucial for comprehending the formation of nacreous aragonite. A flat pearl method in the presence of the inner-shell film was conducted to evaluate the role of matrix proteins in the initial stages of nacre biomineralization in vivo. We examined the crystals deposited on a substrate and the expression patterns of the matrix proteins in the mantle facing the substrate. In this study, the aragonite crystals nucleated on the surface at 5 days in the inner-shell film system. In the film-free system, the calcite crystals nucleated at 5 days, a new organic film covered the calcite, and the aragonite nucleated at 10 days. This meant that the nacre lamellae appeared in the inner-shell film system 5 days earlier than that in the film-free system, timing that was consistent with the maximum level of matrix proteins during the first 20 days. In addition, matrix proteins (Nacrein, MSI60, N19, N16 and Pif80) had similar expression patterns in controlling the sequential morphologies of the nacre growth in the inner-film system, while these proteins in the film-free system also had similar patterns of expression. These results suggest that matrix proteins regulate aragonite nucleation and growth with the inner-shell film in vivo.

MCP-1 production by macrophages is stimulated by neutrophil elastase and oxygen radicals generated by hypoxia, probably due to microthrombus formation after ischemia/reperfusion of the rat liver.

No abstract

The growth of molluscan shell crystals is generally thought to be initiated from the extrapallial fluid by matrix proteins, however, the cellular mechanisms of shell formation pathway remain unknown. Here, we first report amorphous calcium carbonate (ACC) precipitation by cellular biomineralization in primary mantle cell cultures of Pinctada fucata. Through real-time PCR and western blot analyses, we demonstrate that mantle cells retain the ability to synthesize and secrete ACCBP, Pif80 and nacrein in vitro. In addition, the cells also maintained high levels of alkaline phosphatase and carbonic anhydrase activity, enzymes responsible for shell formation. On the basis of polarized light microscopy and scanning electron microscopy, we observed intracellular crystals production by mantle cells in vitro. Fourier transform infrared spectroscopy and X-ray diffraction analyses revealed the crystals to be ACC, and de novo biomineralization was confirmed by following the incorporation of Sr into calcium carbonate. Our results demonstrate the ability of mantle cells to perform fundamental biomineralization processes via amorphous calcium carbonate, and these cells may be directly involved in pearl oyster shell formation.

Neutrophils mediate microvascular and parenchymal cell dysfunction in a variety of organs after ischemia-reperfusion. 1,2 Reperfusion of ischemic tissue results in the formation of proinflammatory mediators that promote the adherence and emigration of neutrophils in postcapillary venules. 3 The activated neutrophils subsequently injure tissue by releasing oxidants and proteases. 4,5 This hypothesis is based largely on studies demonstrating that tissue injury induced by ischemia-reperfusion is attenuated by rendering the animals neutropenic with antineutrophil serum. 6,7 Additional support for this view is derived from several studies showing that monoclonal antibodies against either the CD11a or CD11b ␣ subunits of the CD11/CD18 on neutrophils 8,9 or intercellular adhesion molecule-1 (ICAM-1) on endothelial cells 10 afford significant protection against ischemia-reperfusion injury. Thus, ICAM-1 is important for the development of a neutrophil-dependent injury mechanism.Alterations in the endothelial cell in response to cytokines may be of critical importance in interactions between circulating leukocytes and the vessel wall, because changes in the expression of adhesion receptors may regulate the number and type of leukocytes emigrating from the blood into inflamed tissues. 11 Tumor necrosis factor ␣, interleukin-1, and interferon gamma have been reported to induce the production of ICAM-1 message and protein in isolated murine hepatocytes, 12 human hepatocytic cell lines, 13 and endothelial cells. 14,15 These observations suggest that ICAM-1 expression may be induced by other proinflammatory cytokines.Monocyte chemoattractant protein 1 (MCP-1), originally described as the JE gene, is a 148-amino acid chemotactic cytokine [16][17][18] that is expressed by a number of cells including monocytes, leukocytes, fibroblasts, endothelial cells, smooth muscle cells, chondrocytes, and pulmonary epithelial cells. [16][17][18][19][20] Jiang et al. 21 and Vaddi et al. 22 demonstrated that MCP-1 induced expression of CD11c (p150,95, ␣-subunit) and CD11b (Mac-1 ␣-subunit), suggesting that MCP-1 is not only a chemoattractant, but also a cytokine with the capacity to regulate monocyte function. Thus, MCP-1 may modulate leukocyte-endothelial cell interactions, specifically by affecting ICAM-1 expression. Kupffer cells generate a wide range of inflammatory mediators including cytokines, eicosanoids, reactive oxygen intermediates, and other factors. The production of several cytokines following Kupffer cell activation may be important in the pathophysiology of liver ischemiareperfusion injury. Therefore, the present study was undertaken to investigate the effects of MCP-1 produced by Kupffer cells on ICAM-1 expression after ischemia-reperfusion of the rat liver. MATERIALS AND METHODSAnimals. Male Wistar rats were obtained from a commercial supplier (Kyudo, Inc., Kumamoto, Japan). All animals were maintained under standard conditions and were fed water and rodent chow ad libitum. The animals weighed between 225 and 250 g.Preparatio...

Matrix proteins play important roles in shell formation. Our group firstly isolated three cDNAs encoding lysine-rich matrix protein from Pinctada fucata in 2006. However, the functions of KRMPs are not fully understood. In addition, KRMPs contain two functional domains, the basic domain and the Gly/Tyr domain respectively. Based on the modular organization, the roles of their two domains were poorly characterized. Furthermore, KRMPs were then reported in other two species, P. maxima and P. margaritifera, which indicated that KRMPs might be very important for shell formation. In this study, the characterization and function of KRMP-3 and its two functional domains were studied in vitro through purification of recombinant glutathione S-transferase tagged KRMP-3 and two KRMP-3 deletion mutants. Western blot and immunofluorescence revealed that native KRMP-3 existed in the EDTA-insoluble matrix of the prismatic layer and was located in the organic sheet and the prismatic sheath. Recombinant KRMP-3 (rKRMP-3) bound tightly to chitin and this binding capacity was duo to the Gly/Tyr-rich region. rKRMP-3 inhibited the precipitation of CaCO3, affected the crystal morphology of calcite and inhibited the growth of aragonite in vitro, which was almost entirely attributed to the lysine-rich region. The results present direct evidence of the roles of KRMP-3 in shell biomineralization. The functional rBR region was found to participate in the growth control of crystals and the rGYR region was responsible to bind to chitin.

The immunosuppressants AZA, CsA, FK506, and RPM reduce neutrophil accumulation and attenuate ischemia/reperfusion injury of the liver.