The FBXL10/KDM2B Scaffolding Protein Associates with Novel Polycomb Repressive Complex-1 to Regulate Adipogenesis

Polycomb repressive complex 1 (PRC1) plays an essential role in the epigenetic repression of gene expression during development and cellular differentiation via multiple effector mechanisms, including ubiquitination of H2A and chromatin compaction. However, whether it regulates the stepwise progression of adipogenesis is unknown. Here, we show that FBXL10/KDM2B is an anti-adipogenic factor that is up-regulated during the early phase of 3T3-L1 preadipocyte differentiation and in adipose tissue in a diet-induced model of obesity. Interestingly, inhibition of adipogenesis does not require the JmjC demethylase domain of FBXL10, but it does require the F-box and leucine-rich repeat domains, which we show recruit a noncanonical polycomb repressive complex 1 (PRC1) containing RING1B, SKP1, PCGF1, and BCOR. Knockdown of either RING1B or SKP1 prevented FBXL10-mediated repression of 3T3-L1 preadipocyte differentiation indicating that PRC1 formation mediates the inhibitory effect of FBXL10 on adipogenesis. Using ChIP-seq, we show that FBXL10 recruits RING1B to key specific genomic loci surrounding the key cell cycle and the adipogenic genes Cdk1, Uhrf1, Pparg1, and Pparg2 to repress adipogenesis. These results suggest that FBXL10 represses adipogenesis by targeting a noncanonical PRC1 complex to repress key genes (e.g. Pparg) that control conversion of pluripotent cells into the adipogenic lineage.     

Discovery of spiropiperidine-based potent and selective Orexin-2 receptor antagonists

To generate novel human Orexin-2 Receptor (OX2R) antagonists, a spiropiperidine based scaffold was designed and a SAR study was carried out. Compound 4f possessed the highest OX2R antagonistic activity with an IC(50) value of 3nM with 450-fold selectivity against Orexin-1 Receptor (OX1R).     

Discovery of potent, selective, orally active benzoxazepine-based Orexin-2 receptor antagonists

During our efforts to identify a series of potent, selective, orally active human Orexin-2 Receptor (OX2R) antagonists, we elucidated structure-activity relationship (SAR) on the 7-position of a benzoxazepine scaffold by utilizing Hammett σ(p) and Hansch-Fujita π value as aromatic substituent constants. The attempts led to the discovery of compound 1m, possessing good in vitro potency with over 100-fold selectivity against OX1R, good metabolic stability in human and rat liver microsome, good oral bioavailability in rats, and in vivo antagonistic activity in rats by oral administration.     

Design and synthesis of 3-pyridylacetamide derivatives as dipeptidyl peptidase IV (DPP-4) inhibitors targeting a bidentate interaction with Arg125

We have previously discovered nicotinic acid derivative 1 as a structurally novel dipeptidyl peptidase IV (DPP-4) inhibitor. In this study, we obtained the X-ray co-crystal structure between nicotinic acid derivative 1 and DPP-4. From these X-ray co-crystallography results, to achieve more potent inhibitory activity, we targeted Arg125 as a potential amino acid residue because it was located near the pyridine core, and some known DPP-4 inhibitors were reported to interact with this residue. We hypothesized that the guanidino group of Arg125 could interact with two hydrogen-bond acceptors in a bidentate manner. Therefore, we designed a series of 3-pyridylacetamide derivatives possessing an additional hydrogen-bond acceptor that could have the desired bidentate interaction with Arg125. We discovered the dihydrochloride of 1-{[5-(aminomethyl)-2-methyl-4-(4-methylphenyl)-6-(2-methylpropyl)pyridin-3-yl]acetyl}-l-prolinamide (13j) to be a potent and selective DPP-4 inhibitor that could interact with the guanidino group of Arg125 in a unique bidentate manner.     

The habitat disruption induces immune-suppression and oxidative stress in honey bees

The honey bee is a major insect used for pollination of many commercial crops worldwide. Although the use of honey bees for pollination can disrupt the habitat, the effects on their physiology have never been determined. Recently, honey bee colonies have often collapsed when introduced in greenhouses for pollination in Japan. Thus, suppressing colony collapses and maintaining the number of worker bees in the colonies is essential for successful long-term pollination in greenhouses and recycling of honey bee colonies. To understand the physiological states of honey bees used for long-term pollination in greenhouses, we characterized their gene expression profiles by microarray. We found that the greenhouse environment changes the gene expression profiles and induces immune-suppression and oxidative stress in honey bees. In fact, the increase of the number of Nosema microsporidia and protein carbonyl content was observed in honey bees during pollination in greenhouses. Thus, honey bee colonies are likely to collapse during pollination in greenhouses when heavily infested with pathogens. Degradation of honey bee habitat by changing the outside environment of the colony, during pollination services for example, imposes negative impacts on honey bees. Thus, worldwide use of honey bees for crop pollination in general could be one of reasons for the decline of managed honey bee colonies.     

Rapid isolation of antigen-specific antibody-secreting cells using a chip-based immunospot array

Here we report a new method for isolating antigen-specific antibody-secreting cells (ASCs) using a microwell array chip, which offers a rapid, efficient and high-throughput (up to 234,000 individual cells) system for the detection and retrieval of cells that secrete antibodies of interest on a single-cell basis. We arrayed a large population of lymphoid cells containing ASCs from human peripheral blood on microwell array chips and detected spots with secreted antibodies. This protocol can be completed in less than 7 h, including 3 h of cell culture. The method presented here not only has high sensitivity and specificity comparable with enzyme-linked immunospot (ELISPOT) but it also overcomes the limitations of ELISPOT in recovering ASCs that can be used to produce antigen-specific human monoclonal antibodies. This method can also be used to detect cells secreting molecules other than antibodies, such as cytokines, and it provides a tool for cell analysis and clinical diagnosis.     

Mode of action on deacetylation of acetylated methyl glycoside by cellulose acetate esterase from Neisseria sicca SB

The regioselective deacetylation of purified cellulose acetate esterase from Neisseria sicca SB was investigated on methyl 2,3,4,6-tetra-O-acetyl-beta-D-glucopyranoside and 2,3,4,6-tetra-O-acetyl-beta-D-galactopyranoside. The substrates were used as model compounds of cellulose acetate in order to estimate the mechanism for deacetylation of cellulose acetate by the enzyme. The enzyme rapidly deacetylated at position C-3 of methyl 2,3,4,6-tetra-O-acetyl-beta-D-glucopyranoside to accumulate 2,4,6-triacetate as the main initial reaction product in about 70% yield. Deacetylation was followed at position C-2, and generated 4,6-diacetate in 50% yield. The enzyme deacetylated the product at positions C-4 and C-6 at slower rates, and generated 4- and 6-monoacetates at a later reaction stage. Finally, it gave a completely deacetylated product. For 2,3,4,6-tetra-O-acetyl-beta-D-galactopyranoside, CA esterase deacetylated at positions C-3 and C-6 to give 2,4,6- and 2,3,4-triacetate. Deacetylation proceeded sequentially at positions C-3 and C-6 to accumulate 2,4-diacetate in 55% yield. The enzyme exhibited regioselectivity for the deacetylation of the acetylglycoside.     

Interaction analysis between tmRNA and SmpB from Thermus thermophilus

Small protein B, SmpB, is a tmRNA-specific binding protein essential for trans-translation. We examined the interaction between SmpB and tmRNA from Thermus thermophilus, using biochemical and NMR methods. Chemical footprinting analyses using full-length tmRNA demonstrated that the sites protected upon SmpB binding are located exclusively in the tRNA-like domain (TLD) of tmRNA. To clarify the SmpB binding sites, we constructed several segments derived from TLD. Optical biosensor interaction analyses and melting profile analyses with mutational studies showed that SmpB efficiently binds to only a 30-nt segment that forms a stem and loop, with the 5' and 3' extensions composed of the D-loop and variable-loop analogues. The conserved sequences, 16UCGA and 319GAC, in the extensions are responsible for the SmpB binding. These results agree with the those visualized by the cocrystal structure of TLD and SmpB from Aquifex aeolicus. In addition, NMR chemical shift mapping analyses, using the 30-nt segment and (15)N-labeled SmpB, revealed the characteristic RNA binding mode. The hydrogen bond pattern around beta2 changes, with the Gly in beta2, which acts as a hinge, showing the largest chemical shift change. It appears that SmpB undergoes structural changes indicating an induced fit upon binding to the specific region of TLD.     

Time-related mapping of quantitative trait loci controlling grain-filling in rice (Oryza sativa L.)

Grain-filling is a crucial process that determines final grain yield in rice (Oryza sativa L.). To understand the genetic basis of dynamics of grain-filling, quantitative trait locus (QTL) analysis was conducted using time-related phenotypic data on grain-filling collected from a population of 155 recombinant inbred lines (F12), derived from a cross between Milyang 23 and Akihikari. Two QTLs detected on chromosomes 8 and 12 were strongly associated with increased filling percentage per panicle. These QTLs were not linked with those controlling spikelet numbers per panicle. This result confers the possibility of improving grain-filling together with an enlargement of sink size. The QTL for filling percentage per panicle on chromosome 8 exactly overlapped that for non-structural carbohydrate (NSC) content in the culm and leaf sheaths during grain-filling, and the Milyang 23 allele associated with increased grain-filling percentage per panicle was associated with decreased NSC content. Therefore, this QTL may be directly involved in NSC translocation from the culm and leaf sheaths to panicle. In addition, the Milyang 23 alleles of QTLs associated with greater spikelet number per panicle on chromosomes 1 and 6 were also related with a reduction in NSC content in the culm and leaf sheaths during grain-filling. These results indicate that NSC dynamics during grain-filling is partly dependent on sink size. NSC accumulation in the culm and leaf sheaths at the heading stage was mainly controlled by different genetic regulations from NSC dynamics during grain-filling. Nitrogen dynamics during grain-filling may also be involved in carbohydrate dynamics.