ALBERT

Description: The spatial clustering of active galactic nuclei (AGNs) is considered to be one of the important diagnostics for the understanding of the underlying processes behind their activities complementary to measurements of the luminosity function (LF). We analyse the AGN clustering from a recent semi-analytic model performed on a large cosmological N-body simulation covering a cubic gigaparsec comoving volume. We have introduced a new time-scale of gas accretion on to the supermassive black holes to account for the loss of the angular momentum on small scales, which is required to match the faint end of the observed X-ray LF. The large simulation box allows us accurate determination of the autocorrelation function of the AGNs. The model prediction indicates that this time-scale plays a significant role in allowing massive haloes to host relatively faint population of AGNs, leading to a higher bias factor for those AGNs. The model predictions are in agreement with observations of X-ray selected AGNs in the luminosity range $10^{41.5}~mathrm{erg} mathrm{s}^{-1} le L_{2{-}10mathrm{keV}} le 10^{44.5}~mathrm{erg} mathrm{s}^{-1}$, with the typical host halo mass of $10^{12.5-13.5} h^{-1}, { m M}_{odot }$ at $z lesssim 1$. This result shows that the observational clustering measurements impose an independent constraint on the accretion time-scale complementary to the LF measurements. Moreover, we find that not only the effective halo mass corresponding to the overall bias factor, but the extended shape of the predicted AGN correlation function shows remarkable agreement with those from observations. Further observational efforts towards the low-luminosity end at $z$ ∼ 1 would give us stronger constraints on the triggering mechanisms of AGN activities through their clustering.

Description: Biological roles of most protocadherins (Pcdhs) are a largely unsolved problem. Therefore, we cloned cDNA for Xenopus laevis protocadherin-9 and characterized its properties to elucidate the role. The deduced amino acid sequence was highly homologous to those of mammalian protocadherin-9 s. X. laevis protocadherin-9 expressed from the cDNA in L cells showed basic properties similar to those of mammalian Pcdhs. Expression of X. laevis protocadherin-9 was first detected in stage-31 embryos and increased as the development proceeded. In the later stage embryos and the adults, the retina strongly expressed protocadherin-9, which was mainly localized at the plexiform layers. Injection of morpholino anti-sense oligonucleotide against protocadherin-9 into the fertilized eggs inhibited eye development; and eye growth and formation of the retinal laminar structure were hindered. Moreover, affected retina showed abnormal extension of neurites into the ganglion cell layer. Co-injection of protocadherin-9 mRNA with the morpholino anti-sense oligonucleotide rescued the embryos from the defects. These results suggest that X. laevis protocadherin-9 was involved in the development of retina structure possibly through survival of neurons, formation of the lamina structure and neurite localization.

Description: With the aim of broadening the versatility of lentiviral vectors as a tool in nucleic acid research, we expanded the genetic code in the propagation of lentiviral vectors for site-specific incorporation of chemical moieties with unique properties. Through systematic exploration of the structure–function relationship of lentiviral VSVg envelope by site-specific mutagenesis and incorporation of residues displaying azide- and diazirine-moieties, the modifiable sites on the vector surface were identified, with most at the PH domain that neither affects the expression of envelope protein nor propagation or infectivity of the progeny virus. Furthermore, via the incorporation of such chemical moieties, a variety of fluorescence probes, ligands, PEG and other functional molecules are conjugated, orthogonally and stoichiometrically, to the lentiviral vector. Using this methodology, a facile platform is established that is useful for tracking virus movement, targeting gene delivery and detecting virus–host interactions. This study may provide a new direction for rational design of lentiviral vectors, with significant impact on both basic research and therapeutic applications.

Description: Ganglioside GM3 (Siaα2-3Galβ1-4Glcβ1-1Cer) has been known to participate in insulin signaling by regulating the association of the insulin receptor in caveolae microdomains (lipid rafts), which is essential for the execution of the complete insulin metabolic signaling in adipocytes. Macrophage-secreted factors including proinflammatory cytokines, tumor necrosis factor-α and interleukin-1β, in adipose tissues have been known to limit the local adipogenesis and induce insulin resistance; however, the interplay between adipocytes and macrophages upon regulation of GM3 expression is not clear. GM3 was virtually absent in primary adipocytes differentiated from macrophage-depleted mesenteric stromal vesicular cells, which accompanies enhancement of insulin signaling and adipogenesis. We found that the expression of GM3 is governed by soluble factors including steady-state levels of proinflammatory cytokines secreted from resident macrophages. The direct involvement of GM3 in insulin signaling is demonstrated by the fact that embryonic fibroblasts obtained from GM3 synthase ( GM3S )-deficient mice have increased insulin signaling, when compared with wild-type embryonic fibroblasts, which in turn leads to enhanced adipogeneis. In addition, GM3 expression in primary adipocytes is increased under proinflammatory conditions as well as in adipose tissue of diet-induced obese mice. Moreover, GM3S -deficient mice fed high-fat diets become obese but are resistant to the development of insulin resistance and chronic low-grade inflammatory states. Thus, GM3 functions as a physiological regulatory factor of the balance between homeostatic and pathological states in adipocytes by modulating insulin signaling in lipid rafts.

Description: Strategies to regulate gene function frequently use small interfering RNAs (siRNAs) that can be made from their shRNA precursors via Dicer. However, when the duplex components of these siRNA effectors are expressed from their respective coding genes, the RNA interference (RNAi) activity is much reduced. Here, we explored the mechanisms of action of shRNA and siRNA and found the expressed siRNA, in contrast to short hairpin RNA (shRNA), exhibits strong strand antagonism, with the sense RNA negatively and unexpectedly regulating RNAi. Therefore, we altered the relative levels of strands of siRNA duplexes during their expression, increasing the level of the antisense component, reducing the level of the sense component, or both and, in this way we were able to enhance the potency of the siRNA. Such vector-delivered siRNA attacked its target effectively. These findings provide new insight into RNAi and, in particular, they demonstrate that strand antagonism is responsible for making siRNA far less potent than shRNA.

Description: Tri- N -acetylchitotriosyl moranoline, (GlcNAc) 3 -M, was previously shown to strongly inhibit lysozyme (Ogata M, Umemoto N, Ohnuma T, Numata T, Suzuki A, Usui T, Fukamizo T. 2013. A novel transition-state analogue for lysozyme, 4-O-β-tri-Nacetylchitotriosyl moranoline, provided evidence supporting the covalent glycosyl-enzyme intermediate. J Biol Chem . 288:6072–6082). The findings prompted us to examine the interaction of di- N -acetylchitobiosyl moranoline, (GlcNAc) 2 -M, with a family GH19 chitinase from moss, Bryum coronatum ( Bc Chi19A). Thermal unfolding experiments using Bc Chi19A and the catalytic acid-deficient mutant ( Bc Chi19A-E61A) revealed that the transition temperature ( T m ) was elevated by 4.3 and 5.8°C, respectively, upon the addition of (GlcNAc) 2 -M, while the chitin dimer, (GlcNAc) 2 , elevated T m only by 1.0 and 1.4°C, respectively. By means of isothermal titration calorimetry, binding free energy changes for the interactions of (GlcNAc) 3 and (GlcNAc) 2 -M with Bc Chi19A-E61A were determined to be –5.2 and –6.6 kcal/mol, respectively, while (GlcNAc) 2 was found to interact with Bc Chi19A-E61A with markedly lower affinity. nuclear magnetic resonance titration experiments using 15 N-labeled Bc Chi19A and Bc Chi19A-E61A revealed that both (GlcNAc) 2 and (GlcNAc) 2 -M interact with the region surrounding the catalytic center of the enzyme and that the interaction of (GlcNAc) 2 -M is markedly stronger than that of (GlcNAc) 2 for both enzymes. However, (GlcNAc) 2 -M was found to moderately inhibit the hydrolytic reaction of chitin oligosaccharides catalyzed by Bc Chi19A (IC 50 = 130–620 μM). A molecular dynamics simulation of BcChi19A in complex with (GlcNAc) 2 -M revealed that the complex is quite stable and the binding mode does not significantly change during the simulation. The moranoline moiety of (GlcNAc) 2 -M did not fit into the catalytic cleft (subsite –1) but was rather in contact with subsite +1. This situation may result in the moderate inhibition toward the Bc Chi19A-catalyzed hydrolysis.

Description: Growth factor receptor-bound protein 14 (Grb14) interacts with insulin receptor (IR) through the between PH and SH2 (BPS) domain. Grb14–IR complex formation is initiated by insulin stimulation, and the binding event results in the inhibition of insulin signalling. Thus, Grb14 is regarded as an endogenous suppressor of insulin signal transduction; however, there are no studies describing the mechanism whereby Grb14–IR complex formation is suppressed in the absence of insulin stimulation. In the present study, multiple phosphorylation motifs for glycogen synthase kinase 3 (GSK-3) were identified within the Grb14 BPS domain (Ser 358 , Ser 362 and Ser 366 of human Grb14). Pharmacological inhibition as well as knockdown of GSK-3 facilitated complex formation between Grb14 and IR, implicating GSK-3 activity in regulating Grb14–IR binding. In situ proximity ligation assay and in vitro kinase assays of phosphopeptides suggested that serine residues in the BPS domain would be substrates for GSK-3. The kinase assays also indicated phosphoserine 370 (in human Grb14) was required for the phosphorylation of Ser 358 , Ser 362 and Ser 366 by GSK-3. Grb14–IR binding was also facilitated by replacement of the serines with Ala. We also observed that Ser 366 of endogenous Grb14 in Hep G2 cell was phosphorylated and the phosphorylation was influenced by treatments with insulin, as well as the GSK-3 inhibitor.

Description: High mobility group box 1 (HMGB1), a non-histone chromosomal protein, is a proinflammatory cytokine. There are two known pathways for the release of HMGB1 into the extracellular milieu—passive and active. The passive pathway is attributable to cell death from damage or necrosis, and the active pathway is secretion from immunocompetent cells activated by proinflammatory stimuli. Recent studies have shown that post-translational modifications of HMGB1, including phosphorylation, are involved in the relocation of HMGB1 to the cytoplasm and subsequent secretion. With regard to the HMGB1 phosphorylation, Youn and Shin [Nucleocytoplasmic shuttling of HMGB1 is regulated by phosphorylation that redirects it toward secretion. J Immunol 2006;177:7889–97] reported that treatment of the murine macrophage RAW264.7 with okadaic acid resulted in nucleocytoplasmic translocation and secretion of HMGB1. Herein, we demonstrate the physical interaction between HMGB1 and protein phosphatase 2A (PP2A) in the RAW264.7. The results of in vitro phosphatase assay further indicate that PP2A dephosphorylates specific phosphoserine residues within one of the two nuclear localization signals (NLSs) of HMGB1. The cytoplasmic relocation of HMGB1 through PP2A inhibition was markedly suppressed by replacement of the Ser residues within the NLS with Ala. These consequences imply that PP2A correlates in the nucleocytoplasmic shuttling of HMGB1.

Description: DNA alkylation damage can be repaired by nucleotide excision repair (NER), base excision repair (BER) or by direct removal of alkyl groups from modified bases by O 6 -alkylguanine DNA alkyltransferase (AGT; E.C. 2.1.1.63). DNA mismatch repair (MMR) is also likely involved in this repair. We have investigated alkylation-induced mutagenesis in a series of NER- or AGT-deficient Escherichia coli strains, alone or in combination with defects in the MutS, MutL or MutH components of MMR. All strains used contained the F' prolac from strain CC102 (F'CC102) episome capable of detecting specifically lac GC to AT reverse mutations resulting from O 6 -alkylguanine. The results showed the repair of O 6 -methylguanine to be performed by AGT 〉〉 MMR 〉 NER in order of importance, whereas the repair of O 6 -ethylguanine followed the order NER 〉 AGT 〉 MMR. Studies with double mutants showed that in the absence of AGT or NER repair pathways, the lack of MutS protein generally increased mutant frequencies for both methylating and ethylating agents, suggesting a repair or mutation avoidance role for this protein. However, lack of MutL or MutH protein did not increase alkylation-induced mutagenesis under these conditions and, in fact, reduced mutagenesis by the N -alkyl- N -nitrosoureas MNU and ENU. The combined results suggest that little or no alkylation damage is actually corrected by the mutHLS MMR system; instead, an as yet unspecified interaction of MutS protein with alkylated DNA may promote the involvement of a repair system other than MMR to avoid a mutagenic outcome. Furthermore, both mutagenic and antimutagenic effects of MMR were detected, revealing a dual function of the MMR system in alkylation-exposed cells.