Chimeric mice with hepatocyte-humanized liver as an appropriate model to study human peroxisome proliferator-activated receptor-α.

Peroxisome proliferator (PP)-activated receptor-α (PPARα) agonists exhibit species-specific effects on livers of the rodent and human (h), which has been considered to reside in the difference of PPARα gene structures. However, the contribution of h-hepatocytes (heps) to the species-specificity remains to be clarified. In this study, the effects of fenofibrate were investigated using a hepatocyte-humanized chimeric mouse (m) model whose livers were replaced with h-heps at >70%. Fenofibrate induced hepatocellular hypertrophy, cell proliferation, and peroxisome proliferation in livers of severe combined immunodeficiency (SCID) mice, but not in the h-hep of chimeric mouse livers. Fenofibrate increased the expression of the enzymes of ß- and ω-hydroxylation and deoxygenation of lipids at both gene and protein levels in SCID mouse livers, but not in the h-heps of chimeric mouse livers, supporting the studies with h-PPARα-transgenic mice, a hitherto reliable model for studying the regulation of h-PPARα in the h-liver in most respects, except the induction of the peroxisome proliferation. This study indicates the importance of not only h-PPARα gene but also h-heps themselves to correctly predict effects of fibrates on h-livers, and, therefore, suggests that the chimeric mouse is a currently available, consistent, and reliable model to obtain pharmaceutical data concerning the effects of fibrates on h-livers.

Spatial immobilization of bone morphogenetic protein-4 in a collagen-PLGA hybrid scaffold for enhanced osteoinductivity.

The introduction of bioactive molecules into three-dimensional porous scaffolds to mimic the in vivo microenvironment is a promising strategy for tissue engineering and stem cell research. In this study, bone morphogenetic protein-4 (BMP4) was spatially immobilized in a collagen-PLGA hybrid scaffold with a fusion BMP4 composed of an additional collagen-binding domain derived from fibronectin (CBD-BMP4). CBD-BMP4 bound to the collagen-PLGA hybrid scaffold and the BMP4-immobilized hybrid scaffold supported cell adhesion and proliferation. The osteogenic induction effect of the immobilized CBD-BMP4 was investigated with three-dimensional culture of human bone marrow-derived mesenchymal stem cells in the BMP4-immobilized collagen-PLGA hybrid scaffold. The in vivo implantation experiment demonstrated that the immobilized CBD-BMP4 maintained its osteoinductive activity, being capable of up-regulating osteogenic gene expression and biomineralization. The strong osteoinductivity of the BMP4-immobilized scaffold suggests it should be useful for bone tissue engineering, stem cell function manipulation and bone substitutes.

Comprehensive analysis of dipeptides in alcoholic beverages by tag-based separation and determination using liquid chromatography/electrospray ionization tandem mass spectrometry and quadrupole-time-of-flight mass spectrometry.

Fermented foods and beverages contain several different types of dipeptides, which are believed to be important components for taste. To date, however, a method for the comprehensive analysis of dipeptides in these products has not yet been established. In this study, comprehensive analysis of dipeptides in alcoholic beverages was performed by a high-resolution separation method based on the structural characteristics of 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC)-derivatized dipeptides as well as dipeptide quantification and structural estimation using ultra-high-pressure liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) and UHPLC-quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOFMS), respectively. Dipeptide content was found to differ considerably among Japanese sake, beer, and wine; UHPLC-MS/MS analysis revealed that many types of dipeptides are present in sake. Dipeptide quantification analysis identified 32 types of dipeptides within the concentration range of 1.1-97.2 µM in sake. The analysis was validated by dipeptide recovery of 64.0-107.2% (2.5 µM of standard) with a relative standard deviation of ≤33.2% from an actual alcoholic sample. Furthermore, UHPLC-Q-TOFMS analysis suggested the existence of more than 35 types of dipeptides in sake. Thus, by the combined analysis methods, we discovered that more than 60 dipeptides are present in sake. This research is the first report of dipeptide profiling of fermented alcoholic beverages by comprehensive analysis.

Proteome analysis of human vitreous proteins.

PURPOSE: Various protein contents such as enzymes, growth factors, and structural components are responsible for biological activities in organs. We have created a map of vitreous proteins and developed a proteome analysis of human vitreous samples to understand the underlying molecular mechanism and to provide clues to new therapeutic approaches in eyes with proliferative diabetic retinopathy (PDR). METHODS: Vitreous and serum samples were obtained from subjects with idiopathic macular hole (MH, 26 cases) and PDR (33 cases). The expressed proteins in the samples were separated by two-dimensional (2-D) polyacrylamide gel electrophoresis. Protein spots were visualized by silver staining, and their expression patterns were analyzed. Some protein spots of concern were excised from the 2-D gels, digested in situ with trypsin, and analyzed by mass spectrometry. RESULTS: More than 400 spots were detected on 2-D gels of MH cases, of which 78 spots were successfully analyzed. The spots corresponded to peptide fragments of 18 proteins, including pigment epithelium-derived factor, prostaglandin-D2 synthase, and interphotoreceptor retinoid-binding protein. These were not identified in the corresponding serum samples. These proteins were also expressed in PDR samples, with no distinct tendency to increase or decrease compared with the MH samples. More than 600 spots were detected on 2-D gels of PDR cases, of which 141 spots were successfully analyzed. The spots corresponded to peptide fragments of 38 proteins. Enolase and catalase were identified among four detected spots. Neither was found in MH vitreous or in PDR serum samples. CONCLUSION: A map of protein expression was made in human vitreous from eyes with MH and PDR. In the PDR eyes, the increased protein expression observed was due to barrier dysfunction and/or production in the eye. Proteome analysis was useful in systematic screening of various protein expression in human vitreous samples.