Action Mechanism of Fibroblast Growth Factor-2 (FGF-2) in the Promotion of Periodontal Regeneration in Beagle Dogs

Fibroblast growth factor-2 (FGF-2) enhances the formation of new alveolar bone, cementum, and periodontal ligament (PDL) in periodontal defect models. However, the mechanism through which FGF-2 acts in periodontal regeneration in vivo has not been fully clarified yet. To reveal the action mechanism, the formation of regenerated tissue and gene expression at the early phase were analyzed in a beagle dog 3-wall periodontal defect model. FGF-2 (0.3%) or the vehicle (hydroxypropyl cellulose) only were topically applied to the defect in FGF-2 and control groups, respectively. Then, the amount of regenerated tissues and the number of proliferating cells at 3, 7, 14, and 28 days and the number of blood vessels at 7 days were quantitated histologically. Additionally, the expression of osteogenic genes in the regenerated tissue was evaluated by real-time PCR at 7 and 14 days. Compared with the control, cell proliferation around the existing bone and PDL, connective tissue formation on the root surface, and new bone formation in the defect at 7 days were significantly promoted by FGF-2. Additionally, the number of blood vessels at 7 days was increased by FGF-2 treatment. At 28 days, new cementum and PDL were extended by FGF-2. Moreover, FGF-2 increased the expression of bone morphogenetic protein 2 (BMP-2) and osteoblast differentiation markers (osterix, alkaline phosphatase, and osteocalcin) in the regenerated tissue. We revealed the facilitatory mechanisms of FGF-2 in periodontal regeneration in vivo. First, the proliferation of fibroblastic cells derived from bone marrow and PDL was accelerated and enhanced by FGF-2. Second, angiogenesis was enhanced by FGF-2 treatment. Finally, osteoblastic differentiation and bone formation, at least in part due to BMP-2 production, were rapidly induced by FGF-2. Therefore, these multifaceted effects of FGF-2 promote new tissue formation at the early regeneration phase, leading to enhanced formation of new bone, cementum, and PDL.     

Exploring a DNA Sequence for the Three-Dimensional Structure Determination of a Silver(I)-Mediated C-C Base Pair in a DNA Duplex By 1H NMR Spectroscopy

Recently, we discovered novel silver(I)-mediated cytosine–cytosine base pair (C–Ag^I–C) in DNA duplexes. To understand the properties of these base pairs, we searched for a DNA sequence that can be used in NMR structure determination. After extensive sequence optimizations, a non-symmetric 15-base-paired DNA duplex with a single C–Ag^I–C base pair flanked by 14 A–T base pairs was selected. In spite of its challenging length for NMR measurements (30 independent residues) with small sequence variation, we could assign most non-exchangeable protons (254 out of 270) and imino protons for structure determination.     

Correlation between R/S enantiomer ratio of lansoprazole and CYP2C19 activity after single oral and enteral administration

The purpose of this study was to investigate whether CYP2C19 activity can be estimated from plasma concentrations of lansoprazole enantiomers 4 h (C_4h) after single administration by oral and enteral routes. Sixty‐nine subjects, 22 homozygous extensive metabolizers (homEMs), 32 heterozygous EMs (hetEMs), and 15 poor metabolizers (PMs), participated in the study. After a single oral or enteral dose of racemic lansoprazole (30 mg), plasma concentrations of lansoprazole enantiomers were measured 4 h postdose. The R/S ratio of lansoprazole at 4 h differed significantly among the three groups (P < 0.0001) regardless of the administration route. The R/S ratio of lansoprazole in CYP2C19 PMs ranged from 3.0 to 13.7, whereas in homEMs and hetEMs the ratio ranged from 8.6 to 90 and 2.1 to 122, respectively. The relationship between (S)‐lansoprazole concentration and R/S ratio of lansoprazole at C_4h is given by the following formula: log₁₀ [R/S ratio] = 2.2 – 0.64 × log₁₀ [C_4h of (S)‐lansoprazole] (r = 0.867, P < 0.0001). Thus, phenotyping CYP2C19 using the R/S enantiomer ratio of lansoprazole seems unlikely. However, to obtain a pharmacological effect similar to that in CYP2C19 PMs, we can presume that lansoprazole has a sufficient effect in the patient with an R/S enantiomer ratio at 4 h ≤ 13.70 and (S)‐lansoprazole concentration at 4 h ≥ 50 ng/ml. Chirality 2010. © 2009 Wiley‐Liss, Inc.     

Expression,localization, and biological function of the R3 subtype of receptor-type protein tyrosine phosphatases in mammals

The R3 subtype of receptor-type protein tyrosine phosphatases (RPTPs) includes VE-PTP, DEP-1, PTPRO, and SAP-1. All of these enzymes share a similar structure, with a single catalytic domain and putative tyrosine phosphorylation sites in the cytoplasmic region and fibronectin type III–like domains in the extracellular region. The expression of each R3 RPTP is largely restricted to a single or limited number of cell types, with VE-PTP and DEP-1 being expressed in endothelial or hematopoietic cells, PTPRO in neurons and in podocytes of the renal glomerulus, and SAP-1 in gastrointestinal epithelial cells. In addition, these RPTPs are localized specifically at the apical surface of polarized cells. The structure, expression, and localization of the R3 RPTPs suggest that they perform tissue-specific functions and that they might act through a common mechanism that includes activation of Src family kinases. In this review, we describe recent insights into R3-subtype RPTPs, particularly those of mammals.     

Replacement of C305 in Heart/Muscle-Type Isozyme of Human Carnitine Palmitoyltransferase I with Aspartic Acid and Other Amino Acids

Liver- and heart/muscle-type isozymes of human carnitine palmitoyltransferase I (L- and M-CPTI, respectively) show a certain similarity in their amino acid sequences, and mutation studies on the conserved amino acids between these two isozymes often show essentially the same effects on their enzymatic properties. Earlier mutation studies on C305 in human M-CPTI and its counterpart residue, C304, in human L-CPTI showed distinct effects of the mutations, especially in the aspect of enzyme stability; however, simple comparison of these effects on the conserved Cys residue between L- and M-CPTI was difficult, because these studies were carried out using different expression systems and distinct amino acids as replacements. In the present study, we carried out mutation studies on the C305 in human M-CPTI using COS cells for the expression system. Our results showed that C305 was replaceable with aspartic acid but that substitution with other amino acids caused both loss of function and reduced expression.     

Sugar-inducible expression of the nucleolin-1 gene of Arabidopsis thaliana and its role in ribosome synthesis, growth and development

Animal and yeast nucleolin function as global regulators of ribosome synthesis, and their expression is tightly linked to cell proliferation. Although Arabidopsis contains two genes for nucleolin, AtNuc-L1 is the predominant if not only form of the protein found in most tissues, and GFP-AtNuc-L1 fusion proteins were targeted to the nucleolus. Expression of AtNuc-L1 was strongly induced by sucrose or glucose but not by non-metabolizable mannitol or 2-deoxyglucose. Sucrose also caused enhanced expression of genes for subunits of C/D and H/ACA small nucleolar ribonucleoproteins, as well as a large number of genes for ribosomal proteins (RPs), suggesting that carbohydrate availability regulates de novo ribosome synthesis. In sugar-starved cells, induction of AtNuc-L1 occurred with 10 mM glucose, which seemed to be a prerequisite for resumption of growth. Disruption of AtNuc-L1 caused an increased steady-state level of pre-rRNA relative to mature 25S rRNA, and resulted in various phenotypes that overlap those reported for several RP gene mutants, including a reduced growth rate, prolonged lifetime, bushy growth, pointed leaf, and defective vascular patterns and pod development. These results suggest that the rate of ribosome synthesis in the meristem has a strong impact not only on the growth but also the structure of plants. The AtNuc-L1 disruptant exhibited significantly reduced sugar-induced expression of RP genes, suggesting that AtNuc-L1 is involved in the sugar-inducible expression of RP genes.     

Immunostimulatory activities of monoglycosylated α-d-pyranosylceramides

We compared the immunostimulatory effects of chemically synthesized α-galactosylceramides (α-GalCers), α-glucosylceramides (α-GluCers), 6″-monoglycosylated α-GalCer and 6″- or 4″-monoglycosylated α-GluCer and made the following observations: (1) the length of the fatty acid side chain in the ceramide portions greatly affects the immunostimulatory effects of α-GalCers and α-GluCers; (2) the configuration of the 4″-hydroxyl group of the inner pyranose moiety plays an important role in the immunostimulatory effects of monoglycosylated α- -pyranosylceramides; (3) the free 4″-hydroxyl group of the inner pyranose of monoglycosylated α- -pyranosylceramides plays a more important role in their immunostimulatory effects than the free 6″-hydroxyl group.     

Affinity-based fluorogenic labeling of ATP-binding proteins with sequential photoactivatable cross-linkers

A specific illumination approach has been developed for identification of adenosine triphosphate (ATP)-binding proteins. This strategy utilizes a tandem photoactivatable unit that consists of a diazirine group as a carbene precursor and an o-hydroxycinnamate moiety as a coumarin precursor. The photolysis of diazirine induces a specific cross-link on target proteins and is followed by photoactivation of coumarin generation with a concomitant release of the pre-installed affinity ligand. The ATP, installed with this cross-linker at the γ-position, successfully transferred a coumarin onto ATP-binding proteins using only UV-irradiation.