To clarify the adsorption mechanism of proteins onto calcium hydroxyapatite (Hap), the present study measured adsorption (DeltaHads) and desorption (DeltaHdes) enthalpies of bovine serum albumin (BSA; isoelectric point (iep) 4.7, molecular mass (Ms) 67,200 Da, acidic protein), myoglobin (MGB; iep=7.0, Ms=17,800 Da, neutral protein), and lysozyme (LSZ; iep=11.1, Ms=14,600 Da, basic protein) onto Hap by a flow microcalorimeter (FMC). Five kinds of large platelike particles of CaHPO4.2H2O (DCPD) after hydrolyzing at room temperature with different concentrations of NaOH aqueous solution ([NaOH]) for 1 h were used. DCPD converted completely to Hap after treatment at [NaOH]>or=2%, and the crystallinity of Hap was increased with an increase in [NaOH] up to 10%. The amounts of protein adsorbed (Deltanads) and desorbed (Deltandes) were measured simultaneously by monitoring the protein concentration downstream from the FMC with a UV detector. The Deltanads values were also measured statically by a batch method in each system. The Deltanads values measured by the FMC and static measurements fairly agreed with each other. Results revealed that DeltaHBSAads was decreased with an increase in [NaOH]; in other words, DeltaHBSAads was decreased with the improvement of Hap's crystallinity, suggesting that the BSA adsorption readily proceeded onto Hap. This fact indicated a high affinity of Hap to protein. This affinity was further recognized by DeltaHBSAdes because its positive value was increased by increasing [NaOH]. These opposite tendencies in DeltaHBSAads and DeltaHBSAdes revealed that Hap possessed a high adsorption affinity to BSA (i.e., enthalpy facilitated protein adsorption but hindered its desorption). The fraction of BSA desorption was also decreased with an increase in [NaOH], confirming the high affinity of Hap to protein. Similar results were observed on the LSZ system, though the enthalpy values were smaller than those of BSA. In the case of neutral MGB, DeltaHBSAads also exhibited results similar to those of the BSA and LSZ systems. However, due to its weak adsorption by the van der Waals force, DeltaHBSAdes was small and almost zero at [NaOH]>or=2%. Hence, the fraction of MGB desorption was less dependent on [NaOH].

Abstract. celecoxib plays antitumor roles via multiple mechanisms in a variety of human cancers. the aim of this study was to clarify the mechanism of action of celecoxib in canine mammary tumors. We examined the antitumor effects of celecoxib in aZacB canine mammary tumor cells expressing low levels of cyclooxygenase-2 (coX-2) to minimize the effect of coX-2 on its activity. our data revealed that celecoxib inhibited cell proliferation mainly via COX-2-independent mechanisms. Specifically, celecoxib decreased the proportion of cells in S phase and increased g2/M arrest, which was associated with increased expression of the cyclin-dependent kinase inhibitors (cDKis) p21 and p27. in addition, treatment with celecoxib downregulated coX-2 expression, and induced apoptosis via both the intrinsic and extrinsic pathways. These findings suggest that celecoxib might be a useful agent for the treatment of canine mammary tumors, regardless of coX-2 expression. in the future, it might be possible to use a combination of celecoxib and other antitumor agents to treat canine mammary tumors.

Thermodynamic parameters have obtained in the present study by measuring the adsorption isotherms of bovine serum albumin (BSA: isoelectric point (iep) = 4.7), myoglobin (MGB: iep = 7.0) and lysozyme (LSZ: iep = 11.1) onto calcium hydroxyapatite (Hap) at different temperatures (15°C and 35°C) in an attempt to clarify the adsorption mechanism of such proteins. The adsorption affinity of proteins strongly depended on the Ca/P molar ratio of the Hap particles. In the case of BSA, the initial slopes of the adsorption isotherms increased remarkably with an increase in the Ca/P ratio. In contrast, the initial slopes of the adsorption isotherms for LSZ changed gradually with increasing Ca/P ratio. Finally, no Ca/P ratio dependence was observed in the MGB system. These results were explained in terms of the electrostatic interaction between the proteins and Hap. The negative values of ∆G ads for the BSA system increased from -34.0 kJ/mol to -36.1 kJ/mol with increasing Ca/P ratio whereas those for the LSZ system decreased from -37.9 kJ/mol to -30.8 kJ/mol. A diminished dependence of ∆G ads on the Ca/P ratio was observed in the MGB system. The adsorptions of LSZ and MGB onto Hap were exothermic which again supports an electrostatic attraction mechanism. In contrast, the adsorption of BSA onto Hap exhibited a large positive value for ∆H ads , indicating that the process was endothermic. The ∆S ads value of the LSZ system was negative while those for the BSA and MGB systems were positive. Positive values for ∆S ads , especially for the BSA system, indicate that the adsorption processes for BSA and MGB (both with a lower structural stability) were driven by an entropy gain, i.e. ∆S ads > 0. Adsorption of these proteins onto the Hap surface was accompanied by a structural rearrangement.

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