Silicon intake to vertebral columns of mice after dietary supply

Vertebral columns were dissected and analyzed after birth with oral administration of silicon for 4 wk and for 8 wk. The silicon level was lower (20 μg/g) at the beginning. It remains unchanged after 4 wk and then increases twice as much as that for those mice bred for 8 wk than those bred for 4 wk. This increase depends remarkably on the mass ratio of Si/Ca (M/M). The ratio increases to three times higher than that of the control at the beginning of the experiments (5 wk after birth). Although the S and P contents appeared to be lower, these increased when Si was administered in combination with phosphopeptide. Other elements, such as Ca, Mg, Fe, and Zn, appeared to be unchanged as the weeks proceeded. These findings seem to support a proposal that silicon is necessary for the growth of backbones in mice.     

Differential inhibitory effects of mu-opioids on substance P- and capsaicin-induced nociceptive behavior in mice

The antinociceptive mechanisms of the selective mu-opioid receptor agonists [D-Ala2,NMePhe4,Gly(ol)5]enkephalin (DAMGO), H-Tyr-D-Arg-Phe-beta-Ala-OH (TAPA) or H-Tyr-D-Arg-Phe-beta-Ala-NH2 (TAPA-NH2) against substance P (SP)- or capsaicin-elicited nociceptive behaviors was investigated in mice. DAMGO, TAPA or TAPA-NH2 given intrathecally inhibited the nociceptive behaviors elicited by intrathecally administered SP or capsaicin, and these antinociceptive effects were completely eliminated by intrathecal co-administration with D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 (CTOP), a selective mu-opioid receptor antagonist. Pretreatment subcutaneously with naloxonazine, a selective mu1-opioid receptor antagonist, partially attenuated the antinociceptive effect of TAPA-NH2, but not DAMGO and TAPA, against SP. However, the antinociception induced by TAPA, but not DAMGO and TAPA-NH2, against capsaicin was significantly inhibited by naloxonazine. On the other hand, co-administration intrathecally with Tyr-D-Pro-Trp-Gly-NH2 (D-Pro2-Tyr-W-MIF-1), a selective mu2-opioid receptor antagonist, significantly attenuated the antinociceptive effects of DAMGO, but not TAPA and TAPA-NH2, against capsaicin, while the antinociceptions induced by three opioid peptides against SP were significantly inhibited by D-Pro2-Tyr-W-MIF-1. These results suggest that differential inhibitory mechanisms on pre- and postsynaptic sites in the spinal cord contribute to the antinociceptive effects of the three mu-opioid peptides.     

EGFR signaling pathway negatively regulates PSA expression and secretion via the PI3K-Akt pathway in LNCaP prostate cancer cells

Epidermal growth factor (EGF) and its receptor (EGFR) are involved in hormone-refractory growth and poor prognosis of a subgroup of human prostate cancer. In this communication, we investigated the regulation of PSA by the EGFR signaling pathway using LNCaP C-81 prostate cancer cells. Administration of EGF stimulated the growth of LNCaP C-81 cells, however, PSA expression and secretion were suppressed. An EGFR inhibitor, AG1478, abrogated the PSA suppression effect by EGF, in concurrence with the suppression of tyro-phosphorylation levels of EGFR. Interestingly, the AR level was also decreased in EGF-treated LNCaP C-81 cells. Moreover, LY294002, but not PD98059, inhibited the PSA and AR suppression effect by EGF in concurrence with the suppression of phosphorylation levels of Akt. In conclusion, our results strongly suggest the existence of a novel androgen-independent PSA regulatory mechanism, i.e., the EGFR signaling pathway negatively regulates PSA expression which may be induced by the alteration of AR expression via the PI3K-Akt pathway in LNCaP C-81 cells.     

O-linked-N-acetylglucosamine modification of mammalian Notch receptors by an atypical O-GlcNAc transferase Eogt1

O-linked-β-N-acetylglucosamine (O-GlcNAc) modification is a unique cytoplasmic and nuclear protein modification that is common in nearly all eukaryotes, including filamentous fungi, plants, and animals. We had recently reported that epidermal growth factor (EGF) repeats of Notch and Dumpy are O-GlcNAcylated by an atypical O-GlcNAc transferase, EOGT, in Drosophila. However, no study has yet shown whether O-GlcNAcylation of extracellular proteins is limited to insects such as Drosophila or whether it occurs in other organisms, including mammals. Here, we report the characterization of A130022J15Rik, a mouse gene homolog of Drosophila Eogt (Eogt 1). Enzymatic analysis revealed that Eogt1 has a substrate specificity similar to that of Drosophila EOGT, wherein the Thr residue located between the fifth and sixth conserved cysteines of the folded EGF-like domains is modified. This observation is supported by the fact that the expression of Eogt1 in Drosophila rescued the cell-adhesion defect caused by Eogt downregulation. In HEK293T cells, Eogt1 expression promoted modification of Notch1 EGF repeats by O-GlcNAc, which was further modified, at least in part, by galactose to generate a novel O-linked-N-acetyllactosamine structure. These results suggest that Eogt1 encodes EGF domain O-GlcNAc transferase and that O-GlcNAcylation reaction in the secretory pathway is a fundamental biochemical process conserved through evolution.     

Diverse lectin-binding specificity of four ZP3 glycoprotein isoforms with a discrete isoelectric point in chicken egg coat

The vertebrate egg coat corresponding to mammalian zona pellucida is a filamentous matrix composed of highly and heterogeneously glycosylated proteins designated ZP glycoproteins including ZP1 to 4, ZPD and ZPAX, and play important roles in species-specific egg-sperm interactions. Recent advance in structural biology of chicken ZP3 provided new insights into molecular mechanisms of the egg-coat function involving its carbohydrate moieties. In this study, chicken ZP3 was separated into four major and distinct isoforms with different pI in 2D-PAGE. To investigate the meanings of the ZP3 heterogeneity in egg-sperm interactions, we preliminary analyzed glycan diversity on the molecules by using lectin-staining assays. The four major ZP3 isoforms 4-7 (from acidic to basic) were recognized equally with PNA (Galβ1-3GalNAc), but the isoforms 5-7 were recognized dominantly with WGA ((β-GlcNAc)n, clustered Sia), PHA-E (bi- and triantennary N-glycan containing Galβ1-4GlcNAcβ1-2Manα1-6) and RCA I (terminal Galβ1-4GlcNAc), respectively. Despite such sugar chain diversity among the ZP3 isoforms, a partner in the egg coat, ZP1, showed specific binding to each isoform equally. Localization of ZP1 and ZP3 in the egg-coat matrix were also analyzed.     

Experimental challenges of wild Manila clams with Perkinsus species isolated from naturally infected wild Manila clams

Manila clams, Ruditapes philippinarum, are widely harvested in the coastal waters in Japan. However, there have been significant decreases in the populations of Manila clams since the 1980s. It is thought that infection with the protozoan Perkinsus species has contributed to these decreases. A previous study demonstrated that high infection levels of a pure strain of Perkinsus olseni (ATCC PRA-181) were lethal to hatchery-raised small Manila clams, however, the pathogenicity of wild strain Perkinsus species to wild Manila clam is unclear. To address this, we challenged large (30-40mm in shell length) and small (3-15mm in shell length) wild Manila clams with Perkinsus species isolated from naturally infected wild Manila clams. We report high mortalities among the small clams, but not among the large ones. This is the first report to confirm the pathogenicity of wild isolate of Perkinsus species to wild Manila clams.     

The toll-like receptor 3-mediated antiviral response is important for protection against poliovirus infection in poliovirus receptor transgenic mice

RIG-I-like receptors and Toll-like receptors (TLRs) play important roles in the recognition of viral infections. However, how these molecules contribute to the defense against poliovirus (PV) infection remains unclear. We characterized the roles of these sensors in PV infection in transgenic mice expressing the PV receptor. We observed that alpha/beta interferon (IFN-α/β) production in response to PV infection occurred in an MDA5-dependent but RIG-I-independent manner in primary cultured kidney cells in vitro. These results suggest that, similar to the RNA of other picornaviruses, PV RNA is recognized by MDA5. However, serum IFN-α levels, the viral load in nonneural tissues, and mortality rates did not differ significantly between MDA5-deficient mice and wild-type mice. In contrast, we observed that serum IFN production was abrogated and that the viral load in nonneural tissues and mortality rates were both markedly higher in TIR domain-containing adaptor-inducing IFN-β (TRIF)-deficient and TLR3-deficient mice than in wild-type mice. The mortality rate of MyD88-deficient mice was slightly higher than that of wild-type mice. These results suggest that multiple pathways are involved in the antiviral response in mice and that the TLR3-TRIF-mediated signaling pathway plays an essential role in the antiviral response against PV infection.     

STT3B-dependent posttranslational N-glycosylation as a surveillance system for secretory protein

The chemical identity and integrity of the genome is challenged by the incorporation of ribonucleoside triphosphates (rNTPs) in place of deoxyribonucleoside triphosphates (dNTPs) during replication. Misincorporation is limited by the selectivity of DNA replicases. We show that accumulation of ribonucleoside monophosphates (rNMPs) in the genome causes replication stress and has toxic consequences, particularly in the absence of RNase H1 and RNase H2, which remove rNMPs. We demonstrate that postreplication repair (PRR) pathways-MMS2-dependent template switch and Pol ζ-dependent bypass-are crucial for tolerating the presence of rNMPs in the chromosomes; indeed, we show that Pol ζ efficiently replicates over 1-4 rNMPs. Moreover, cells lacking RNase H accumulate mono- and polyubiquitylated PCNA and have a constitutively activated PRR. Our findings describe a crucial function for RNase H1, RNase H2, template switch, and translesion DNA synthesis in overcoming rNTPs misincorporated during DNA replication, and may be relevant for the pathogenesis of Aicardi-Goutières syndrome.