N4-aminocytidine, a nucleoside analog that has an exceptionally high mutagenic activity.

The reaction of cytidine with hydrazine to give N4-aminocytidine was greatly promoted by addition of a less-than-stoichiometric amount of bisulfite, and the product was isolated in a good yield. N4-Aminocytidine was strongly mutagenic to bacteria (Salmonella typhimurium TA100 and TA1535, and E. coli WP2 uvrA) and to phage (phi X174 am3). The activity did not require the presence of mammalian microsomal fraction in the system. The mutagenic potency of N4-aminocytidine in these systems was two orders of magnitude greater than that of N4-amino-2'-deoxycytidine, and more than two orders of magnitude greater than that of N4-hydroxycytidine. The greater activity of the riboside than the deoxyriboside was ascribed to the lack of deoxycytidine kinase in these cells. This compound may be useful as a powerful mutagen to induce a transition mutation in microorganisms.     

Fibroblast growth factor‐2 stimulates directed migration of periodontal ligament cells via PI3K/AKT signaling and CD44/hyaluronan interaction

Fibroblast growth factor‐2 (FGF‐2) regulates a variety of functions of the periodontal ligament (PDL) cell, which is a key player during tissue regeneration following periodontal tissue breakdown by periodontal disease. In this study, we investigated the effects of FGF‐2 on the cell migration and related signaling pathways of MPDL22, a mouse PDL cell clone. FGF‐2 activated the migration of MPDL22 cells and phosphorylation of phosphatidylinositol 3‐kinase (PI3K) and akt. The P13K inhibitors, Wortmannin and LY294002, suppressed both cell migration and akt activation in MPDL22, suggesting that the PI3K/akt pathway is involved in FGF‐2‐stimulated migration of MPDL22 cells. Moreover, in response to FGF‐2, MPDL22 showed increased CD44 expression, avidity to hyaluronan (HA) partly via CD44, HA production and mRNA expression of HA synthase (Has)‐1, 2, and 3. However, the distribution of HA molecular mass produced by MPDL22 was not altered by FGF‐2 stimulation. Treatment of transwell membrane with HA facilitated the migration of MPDL22 cells and an anti‐CD44 neutralizing antibody inhibited it. Interestingly, the expression of CD44 was colocalized with HA on the migrating cells when stimulated with FGF‐2. Furthermore, an anti‐CD44 antibody and small interfering RNA for CD44 significantly decreased the FGF‐2‐induced migration of MPDL22 cells. Taken together, PI3K/akt and CD44/HA signaling pathways are responsible for FGF‐2‐mediated cell motility of PDL cells, suggesting that FGF‐2 accelerates periodontal regeneration by regulating the cellular functions including migration, proliferation and modulation of extracellular matrix production. J. Cell. Physiol. 226: 809–821, 2011. © 2010 Wiley‐Liss, Inc.     

Fibroblast growth factor-2 regulates expression of osteopontin in periodontal ligament cells

Osteopontin is a protein found in the bone-related matrix and plays multiple regulatory roles in mineralizing and non-mineralizing tissue. In osteogenic cell-lines, the expression of osteopontin increases with the progression of differentiation, but both the expression and function of osteopontin vary with the cell type and its activation state. In this study, we examined the expression of osteopontin by clones established from mouse periodontal ligament, in response to inorganic phosphate and fibroblast growth factor (FGF)-2, which can induce periodontal tissue regeneration. The involvement of inorganic phosphate in the expression of osteopontin during the course of cell differentiation of a clone MPDL22 was confirmed by addition of foscarnet, an inorganic phosphate transport inhibitor. Although FGF-2 decreased the mRNA expression of almost every bone-related protein in MPDL22, FGF-2 upregulated the expression of osteopontin in MPDL22 at both mRNA and protein levels. Interestingly, FGF-2 enhanced the concentration of osteopontin in the culture supernatant of MPDL22, whereas inorganic phosphate did not. The FGF-2-induced osteopontin in the culture supernatant seems to be involved in cell survival activity. An immunohistochemical study showed that the FGF-2-induced osteopontin was mainly present in perinuclear matrices while the inorganic phosphate-induced osteopontin was associated with extracellular matrices in addition to perinuclear matrices. The present results indicated that FGF-2 induces unique expression of osteopontin, which may play a role different from the other bone-related proteins during the process of periodontal tissue regeneration by FGF-2.     

CD73-generated adenosine promotes osteoblast differentiation

CD731 is a GPI-anchored cell surface protein with ecto-5'-nucleotidase enzyme activity that plays a crucial role in adenosine production. While the roles of adenosine receptors (AR) on osteoblasts and osteoclasts have been unveiled to some extent, the roles of CD73 and CD73-generated adenosine in bone tissue are largely unknown. To address this issue, we first analyzed the bone phenotype of CD73-deficient (cd73(-/-)) mice. The mutant male mice showed osteopenia, with significant decreases of osteoblastic markers. Levels of osteoclastic markers were, however, comparable to those of wild-type mice. A series of in vitro studies revealed that CD73 deficiency resulted in impairment in osteoblast differentiation but not in the number of osteoblast progenitors. In addition, over expression of CD73 on MC3T3-E1 cells resulted in enhanced osteoblastic differentiation. Moreover, MC3T3-E1 cells expressed adenosine A(2A) receptors (A(2A)AR) and A(2B) receptors (A(2B)AR) and expression of these receptors increased with osteoblastic differentiation. Enhanced expression of osteocalcin (OC) and bone sialoprotein (BSP) observed in MC3T3-E1 cells over expressing CD73 were suppressed by treatment with an A(2B)AR antagonist but not with an A(2A) AR antagonist. Collectively, our results indicate that CD73 generated adenosine positively regulates osteoblast differentiation via A(2B)AR signaling.     

Preliminary Analysis of Length and GC Content Variation in the Ribosomal First Internal Transcribed Spacer (ITS1) of Marine Animals

Length and guanine–cytosine (GC) content of the ribosomal first internal transcribed spacer (ITS1) were compared across a wide variety of marine animal species, and its phylogenetic utility was investigated. From a total of 773 individuals representing 599 species, we only failed to amplify the ITS1 sequence from 87 individuals by polymerase chain reaction with universal ITS1 primers. No species was found to have an ITS1 region shorter than 100 bp. In general, the ITS1 sequences of vertebrates were longer (318 to 2,318 bp) and richer in GC content (56.8% to 78%) than those of invertebrates (117 to 1,613 bp and 35.8% to 71.3%, respectively). Specifically, gelatinous animals (Cnidaria and Ctenophora) were observed to have short ITS1 sequences (118 to 422 bp) with lower GC content (35.8% to 61.7%) than the other animal taxa. Mollusca and Crustacea were diverse groups with respect to ITS1 length, ranging from 108 to 1,118 and 182 to 1,613 bp, respectively. No universal relationship between length and GC content was observed. Our data indicated that ITS1 has a limited utility for phylogenetic analysis as obtaining confident sequence alignment was often impossible between different genera of the same family and even between congeneric species.     

Design, synthesis, and enzymatic property of a sulfur-substituted analogue of trigalacturonic acid

A sulfur-substituted analogue of trigalacturonic acid (3) was synthesized. The synthesis features the application of 3-cyano-3-(tert-butyldimethylsilyl)oxypropylthioether (CSP) as a novel protective group for thiols. This analogue was designed with the expectation that it would be a stable analogous substrate for endo-polygalacturonase isolated from Stereum purpureum based on computer modeling experiments. Surface plasmon resonance experiments revealed that 3 forms a stable complex with the target enzyme.     

Neuronal Glucoprivation Enhances Hypothalamic Histamine Turnover in Rats

Abstract: Histamine (HA) turnover in the rat hypothalamus following insufficient energy supply due to glucoprivation was examined after administration of insulin or 2-deoxy- d -glucose (2-DG). HA turnover was assessed by accumulation of tele -methylhistamine ( t -MH), a major metabolite of brain HA, following administration of pargyline. Intraperitoneal injection of 1, 2, and 4 U/kg of insulin, which had no influence on steady-state levels of HA and t -MH, increased pargyline-induced accumulation of t -MH. Accumulation of t -MH due to pargyline was inversely related to the concomitant plasma glucose concentration after different doses of insulin. The level of t -MH accumulated by pargyline did not change compared with that of controls, when a euglycemic condition was maintained or insulin at a dose of 6 mU per rat was infused into the third cerebroventricle. Intracerebroventricular infusion of 24 µmol per rat of 2-DG, which had no influence on steady-state levels of HA and t -MH, increased the level of t -MH enhanced by pargyline. The results indicate that an increase in hypothalamic HA turnover in response to glucoprivation may be involved in homeostatic regulation of energy metabolism in the brain.     

Pyrrolinone derivatives as a new class of P2X3 receptor antagonists. Part 1: Initial structure-activity relationship studies of a hit from a high throughput screening

The P2X3 receptor is primarily expressed in the peripheral sensory nerves, and therefore, antagonists of this receptor may be useful for the treatment of chronic pain. Pyrrolinone derivatives have been identified as a novel class of P2X3 receptor antagonists. A lead structure with moderate activity was discovered through a high-throughput screening assay. A structure-activity study led to the discovery of several P2X3 receptor antagonists. Compound 34 showed potent and specific antagonistic activity and analgesic efficacy.     

Retinoic acid given at late embryonic stage depresses sonic hedgehog and Hoxd-4 expression in the pharyngeal area and induces skeletal malformation in flounder (Paralichthys olivaceus) embryos

During the development of pharyngeal cartilages, signal molecules, including sonic hedgehog (shh) and various growth factors, as well as Hox genes are expressed in the pharyngeal area. To elucidate whether shh and Hoxd-4 function in pharyngeal cartilage formation in teleost jaw and gill primordia, spatial and temporal patterns of shh expression in flounder (Paralichthys olivaceus) embryonic pharynx were examined. The effects of retinoic acid (RA) on shh and Hoxd-4 expression and the patterning of pharyngeal cartilages were analyzed. At the prim-5 stage, when cartilage precursor cells aggregate in the pharyngeal primordia, pharyngeal endoderm expressed shh in two domains, in portions of the mandibular and hyoid primordia and in the gill primordia. After a further 40 h, shh domains expanded at the posterior edge of the endoderm of each mandibular, hyoid and gill primordium, concurrent with the growth of the primordia. A new shh expression domain appeared at the endodermal border of the mouth. Retinoic acid treatment depressed shh and Hoxd-4 expression, and also reduced the amount of expansion of the shh expression domains. Pharyngeal cartilages that formed in these embryos were malformed; their growth direction was shifted posteriorly and size was reduced. This provides the possibility that shh and Hoxd-4 regulate the growth and direction of pharyngeal cartilage precursor cells and that RA disturbs their expression, causing skeletal malformation.