Ameloblastin in Hertwig’s Epithelial Root Sheath Regulates Tooth Root Formation and Development

Tooth root formation begins after the completion of crown morphogenesis. At the end edge of the tooth crown, inner and outer enamel epithelia form Hertwig’s epithelial root sheath (HERS). HERS extends along with dental follicular tissue for root formation. Ameloblastin (AMBN) is an enamel matrix protein secreted by ameloblasts and HERS derived cells. A number of enamel proteins are eliminated in root formation, except for AMBN. AMBN may be related to tooth root formation; however, its role in this process remains unclear. In this study, we found AMBN in the basal portion of HERS of lower first molar in mice, but not at the tip. We designed and synthesized small interfering RNA (siRNA) targeting AMBN based on the mouse sequence. When AMBN siRNA was injected into a prospective mandibular first molar of postnatal day 10 mice, the root became shorter 10 days later. Furthermore, HERS in these mice revealed a multilayered appearance and 5-bromo-2′-deoxyuridine (BrdU) positive cells increased in the outer layers. In vitro experiments, when cells were compared with and without transiently expressing AMBN mRNA, expression of growth suppressor genes such as p21^Cip1 and p27^Kip1 was enhanced without AMBN and BrdU incorporation increased. Thus, AMBN may regulate differentiation state of HERS derived cells. Moreover, our results suggest that the expression of AMBN in HERS functions as a trigger for normal root formation.     

Problem-based learning is suitable for the curriculum of “Sleep disorders and disease” for students in dentistry

Sleep and Biological Rhythms -     

MELK-T1, a small-molecule inhibitor of protein kinase MELK,decreases DNA-damage tolerance in proliferating cancer cells

Maternal embryonic leucine zipper kinase (MELK), a serine/threonine protein kinase, has oncogenic properties and is overexpressed in many cancer cells. The oncogenic function of MELK is attributed to its capacity to disable critical cell-cycle checkpoints and reduce replication stress. Most functional studies have relied on the use of siRNA/shRNA-mediated gene silencing. In the present study, we have explored the biological function of MELK using MELK-T1, a novel and selective small-molecule inhibitor. Strikingly, MELK-T1 triggered a rapid and proteasome-dependent degradation of the MELK protein. Treatment of MCF-7 (Michigan Cancer Foundation-7) breast adenocarcinoma cells with MELK-T1 induced the accumulation of stalled replication forks and double-strand breaks that culminated in a replicative senescence phenotype. This phenotype correlated with a rapid and long-lasting ataxia telangiectasia-mutated (ATM) activation and phosphorylation of checkpoint kinase 2 (CHK2). Furthermore, MELK-T1 induced a strong phosphorylation of p53 (cellular tumour antigen p53), a prolonged up-regulation of p21 (cyclin-dependent kinase inhibitor 1) and a down-regulation of FOXM1 (Forkhead Box M1) target genes. Our data indicate that MELK is a key stimulator of proliferation by its ability to increase the threshold for DNA-damage tolerance (DDT). Thus, targeting MELK by the inhibition of both its catalytic activity and its protein stability might sensitize tumours to DNA-damaging agents or radiation therapy by lowering the DNA-damage threshold.     

A catalytically inactive beta 1,4-N-acetylglucosaminyltransferase III (GnT-III) behaves as a dominant negative GnT-III inhibitor.

beta 1,4-N-Acetylglucosaminyltransferase III (GnT-III) plays a regulatory role in the biosynthesis of N-glycans, and it has been suggested that its product, a bisecting GlcNAc, is involved in a variety of biological events as well as in regulating the biosynthesis of the oligosaccharides. In this study, it was found, on the basis of sequence homology, that GnT-III contains a small region that is significantly homologous to both snail beta 1,4GlcNAc transferase and beta1,4Gal transferase-1. Subsequent mutational analysis demonstrated an absolute requirement for two conserved Asp residues (Asp321 and Asp323), which are located in the most homologous region of rat GnT-III, for enzymatic activity. The overexpression of Asp323-substituted, catalytically inactive GnT-III in Huh6 cells led to the suppression of the activity of endogenous GnT-III, but no significant decrease in its expression, and led to a specific inhibition of the formation of bisected sugar chains, as shown by structural analysis of the total N-glycans from the cells. These findings indicate that the mutant serves a dominant negative effect on a specific step in N-glycan biosynthesis. This type of 'dominant negative glycosyltransferase', identified has potential value as a powerful tool for defining the precise biological roles of the bisecting GlcNAc structure.     

Basic fibroblast growth factor protects cardiac myocytes from iNOS-mediated apoptosis.

Basic fibroblast growth factor (bFGF) is an important angiogenic factor produced by hearts subjected to ischemia. However, the direct effects of bFGF on myocardial cells are unknown. Primary cultured cardiac myocytes from neonatal rats were stimulated with lipopolysaccharide (LPS), a potent inducer of inducible nitric oxide synthase (iNOS), in the presence or the absence of bFGF. LPS induced the expression of iNOS in cardiac myocytes, demonstrated at both mRNA and protein levels. We showed that LPS activated the apoptotic pathway, evidenced by TUNEL staining, DNA ladder formation, and morphologic features. LPS-induced apoptosis was blocked by the administration of L-NAME, an inhibitor of NOS. This indicates that LPS induces apoptosis via an iNOS-dependent pathway. Administration of bFGF completely inhibited myocardial cell apoptosis induced by hydrogen peroxide or acidic medium as well as LPS. To determine signaling pathways for this inhibitory effect, we utilized PD098059, an MEK-1-specific inhibitor. PD098059 blocked bFGF-induced activation of ERK (extracellularly responsive kinase)-1/2 and neutralized the apoptotic inhibitory effect of bFGF. These findings demonstrate that LPS induces myocardial cell apoptosis in an iNOS-dependent manner. The results also suggest that bFGF is a protective factor against myocardial cell apoptosis and that this protection requires the MEK-1-ERK pathway.     

Augmentation of Prolactin Release by alpha-Melanocyte Stimulating Hormone Is Possibly Mediated by Melanocortin 3-Receptors in the Mouse Anterior Pituitary Cells

Suckling- and estrogen-induced prolactin release from the anterior pituitary is mediated by alpha-melanocyte stimulating hormone (alpha-MSH) secreted by the intermediate lobe of the pituitary in the rat. Melanocortin 5-receptors are expressed in the anterior pituitary and probably mediate the alpha-MSH function. In contrast, the mouse anterior pituitary does not express the receptor. To examine whether or not alpha-MSH regulates prolactin release in mice, we performed cell immunoblot assay using anterior pituitary cells from adult female mice. We found that alpha-MSH acted on mammotrophs (prolactin-secreting cells) and stimulated prolactin release in a dose dependent manner. A series of RT-PCR using oligonucleotide primer pairs specific for each subtypes of melanocortin receptors revealed that the melanocortin 3-receptor is the sole receptor expressed in the mouse anterior pituitary. These results suggest that alpha-MSH-induced prolactin release is mediated by melanocortin 3-receptors in female mice.     

Gene expression profiles of endothelial progenitor cells by oligonucleotide microarray analysis

Among the many tissue stem or progenitor cells recently being unveiled, endothelial progenitor cells (EPCs) have attracted particular attention, not only because of their cardinal role in vascular biology and embryology but also because of their potential use in the therapeutic development of a variety of postnatal diseases, including cardiovascular and peripheral vascular disorders and cancer. The aim of this study is to provide some basic and comprehensive information on gene expression of EPCs to characterize the cells in molecular terms. Here, we focus on EPCs derived from CD34-positive mononuclear cells of human umbilical cord blood. The EPCs were purified and expanded in culture and analyzed by a high-density oligonucleotide microarray and real-time RT-PCR analysis. We identified 169 up-regulated and 107 down-regulated genes in the EPCs compared with three differentiated endothelial cells of human umbilical vein endothelial cells (HUVEC), human lung microvascular endothelial cells (LMEC) and human aortic endothelial cells (AoEC). It is expected that the obtained list include key genes which are critical for EPC function and survival and thus potential targets of EPC recognition in vivo and therapeutic modulation of vasculogenesis in cancer as well as other diseases, in which de novo vasculogenesis plays a crucial role. For instance, the list includes Syk and galectin-3, which encode protein tyrosine kinase and β-galactoside-binding protein, respectively, and are expressed higher in EPCs than the three control endothelial cells. In situ hybridization showed that the genes were expressed in isolated cells in the fetal liver at E11.5 and E14.5 of mouse development.     

Rotenoids and flavonoids with anti-invasion of HT1080, anti-proliferation of U937, and differentiation-inducing activity in HL-60 from Erycibe expansa

Principal rotenoids (deguelin, tephrosin, rotenone, and 12a-hydroxyrotenone) (3-30microM) isolated from the stems of Erycibe expansa significantly inhibited invasion of human fibrosarcoma HT1080 cells through Matrigel-coated filters and release of proMMPs-2 and 9. In addition, deguelin and tephrosin showed differentiation-inducing activity in human promyelocytic leukemia HL-60 cells. Furthermore, effects of various constituents isolated from the ethyl acetate-soluble fraction on proliferation of human leukemia U937 cells were examined. As a result, most of isoflavones and several flavans as well as rotenoids showed moderate or substantial anti-proliferative activities.