Structural and phylogenetic comparison of napsin genes: The duplication, loss of function and human-specific pseudogenization of napsin B

Aspartic proteinases form a widely distributed protein superfamily, including cathepsin D, cathepsin E, pepsins, renin, BACE and napsin. Human napsin genes are located on human chromosome 19q13, which comprises napsin A and napsin B. Napsin B has been annotated as a pseudogene because it lacks an in-frame stop codon; its nascent chains are cotranslationally degraded. Until recently, there have been no studies concerning the molecular evolution of the napsin protein family in the human genome. In the present study, we investigated the evolution and gene organization of the napsin protein family. Napsin B orthologs are primarily distributed in primates, while napsin A orthologs are the only napsin genes in other species. The corresponding regions of napsin B in the available sequences from primate species contain an in-frame stop codon at a position equivalent to that of human napsin A. In addition, a rare single-nucleotide polymorphism (SNP) that creates a proper stop codon in human napsin B was identified using HapMap populations. Recombinant protein expression and three-dimensional comparative modeling revealed that napsin B exhibits residual activity toward synthetic aspartic protease substrates compared with napsin A, presumably through a napsin B-specific Arg287 residue. Thus, napsin B was duplicated from napsin A during the early stages of primate evolution, and the subsequent loss of napsin B function during primate evolution reflected ongoing human-specific napsin B pseudogenization

Genetic backgrounds and redox conditions influence morphological characteristics and cell differentiation of osteoclasts in mice

Osteoclasts (OCLs) are multinucleated giant cells and are formed by the fusion of mononuclear progenitors of monocyte/macrophage lineage. It is known that macrophages derived from different genetic backgrounds exhibit quite distinct characteristics of immune responses. However, it is unknown whether OCLs from different genetic backgrounds show distinct characteristics. In this study, we showed that bone-marrow macrophages (BMMs) derived from C57BL/6, BALB/c and ddY mice exhibited considerably distinct morphological characteristics and cell differentiation into OCLs. The differentiation of BMMs into OCLs was comparatively quicker in the C57BL/6 and ddY mice, while that of BALB/c mice was rather slow. Morphologically, ddY OCLs showed a giant cell with a round shape, C57BL/6 OCLs were of a moderate size with many protrusions and BALB/c OCLs had the smallest size with fewer nuclei. The intracellular signaling of differentiation and expression levels of marker proteins of OCLs were different in the respective strains. Treatment of BMMs from the three different strains with the reducing agent N-acetylcysteine (NAC) or with the oxidation agent hydrogen peroxide (H 2O 2) induced changes in the shape and sizes of the cells and caused distinct patterns of cell differentiation and survival. Thus, genetic backgrounds and redox conditions regulate the morphological characteristics and cell differentiation of OCLs

Dual Effects of Liquiritigenin on the Proliferation of Bone Cells: Promotion of Osteoblast Differentiation and Inhibition of Osteoclast Differentiation

Bone is constantly controlled by a balance between osteoblastic bone formation and osteoclastic bone resorption. Liquiritigenin is a plant-derived flavonoid and has various pharmacological effects, such as antioxidative, antitumor, and antiinflammatory effects. Here, we show that liquiritigenin has dual effects on the proliferation of bone cells, regarding the promotion of osteoblast differentiation and the inhibition of osteoclast differentiation. Liquiritigenin-treated murine osteoblastic MC3T3-E1 cells showed an increased alkaline phosphatase activity and enhanced phosphorylation of Smad1/5 compared with untreated cells. Moreover, liquiritigenin inhibited osteoclast differentiation, its bone-resorption activity through slightly decreased the phosphorylation of extracellular signal-regulated kinase, c-Jun N-terminal kinase, and inhibitor of nuclear factor kappa Bα; however, the phosphorylation of Akt and p38 slightly increased in bone marrow-derived osteoclasts. The expression levels of the osteoclast marker proteins nuclear factor of activated T-cell cytoplasmic-1, Src, and cathepsin K diminished. These results suggest that liquiritigenin may be useful as a therapeutic and/or preventive agent for osteoporosis or inflammatory bone diseases

Interaction between gustatory depolarizing receptor potential and efferent-induced slow depolarizing synaptic potential in frog taste cell.

Electrical stimulation of parasympathetic nerve (PSN) efferent fibers in the glossopharyngeal nerve induced a slow depolarizing synaptic potential (DSP) in frog taste cells under hypoxia. The objective of this study is to examine the interaction between a gustatory depolarizing receptor potential (GDRP) and a slow DSP. The amplitude of slow DSP added to a tastant-induced GDRP of 10 mV was suppressed to 60% of control slow DSPs for NaCl and acetic acid stimulations, but to 20-30% for quinine-HCl (Q-HCl) and sucrose stimulations. On the other hand, when a GDRP was induced during a prolonged slow DSP, the amplitude of GDRPs induced by 1 M NaCl and 1 M sucrose was suppressed to 50% of controls, but that by 1 mM acetic acid and 10 mM Q-HCl unchanged. It is concluded that the interaction between GDRPs and efferent-induced slow DSPs in frog taste cells under hypoxia derives from the crosstalk between a gustatory receptor current across the receptive membrane and a slow depolarizing synaptic current across the proximal subsynaptic membrane of taste cells

Cobalt protoporphyrin represses osteoclastogenesis through blocking multiple signaling pathways

Cobalt protoporphyrin (CoPP) is a metallo-protoporphyrin that works as a powerful inducer of heme oxigenase-1 (HO-1) in various tissues and cells. Our recent studies have demonstrated that induction of HO-1 by several reagents inhibited differentiation and activation of osteoclasts (OCLs), which are multinucleated bone resorbing cells. However, the effects of CoPP on osteoclastogenesis remain to be elucidated. In this study, we report that CoPP inhibits receptor activator of nuclear factor κB ligand (RANKL)-induced OCL formation in a dose dependent manner. Importantly, CoPP had little cytotoxicity, but rather enhanced cell proliferation of OCLs. CoPP suppressed the protein levels of nuclear factor of activated T cells cytoplasmic-1 (NFATc1) as well as those of OCLs markers such as Src and cathepsin K, which are transcriptionally regulated by NFATc1 in mature OCLs. Western blot analyses also showed that CoPP abolished RANKL-stimulated phosphorylation of several major signaling pathways such as IκB, Akt, ERK, JNK and p38 MAPKs in OCL precursor cells. Thus, our results show that CoPP represses osteoclastogenesis through blocking multiple signaling pathways

Effect of gap junction blocker beta-glycyrrhetinic acid on taste disk cells in frog.

A gap junction blocker, 18beta-glycyrrhetinic acid (beta-GA), increased the membrane resistance of Ia, Ib and II/III cells of frog taste disk by 50, 160, and 300 M Omega, respectively, by blocking the gap junction channels and hemichannels. The amplitudes of gustatory depolarizing potentials in the disk cells for 4 basic taste stimuli were reduced to 40-60% after intravenous injection of beta-GA at 1.0 mg/kg. beta-GA of 1.0 mg/kg did not affect the resting potentials and the reversal potentials for tastant-induced depolarizing potentials in any taste disk cells. The percentage of cells responding to each of 4 basic taste stimuli and varying numbers of 4 taste qualities did not differ between control and beta-GA-treated taste disk cells. This implies that gustatory depolarizing response profiles for 4 basic taste stimuli were very similar in control and beta-GA-treated taste disk cells. It is concluded that beta-GA at 1.0 mg/kg reduced the amplitude of gustatory depolarizing potentials in taste disk cells by strongly blocking depolarizing currents flowing through the gap junction channels and hemichannels, but probably weakly affected the gustatory transduction mechanisms for 4 taste stimuli