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.     

Novel autotrophic nitrogen removal system using gel entrapment technology

A pilot plant involving a nitritation-anammox process was operated for treating digester supernatant. In the preceding nitritation process, ammonium-oxidizing bacteria were immobilized in gel carriers, and the growth of nitrite-oxidizing bacteria was suppressed by heat-shock treatment. For the following anammox process, in order to maintain the anammox biomass in the reactor, a novel process using anammox bacteria entrapped in gel carriers was also developed. The nitritation performance was stable, and the average nitrogen loading and nitritation rates were 3.0 and 1.7 kg N m⁻³ d⁻¹, respectively. In the nitritation process, nitrate production was completely suppressed. For the anammox process, the startup time was about two months. Stable nitrogen removal was achieved, and an average nitrogen conversion rate of 5.0 kg N m⁻³ d⁻¹ was obtained. Since the anammox bacteria were entrapped in gel carriers, stable nitrogen removal performance was attained even at an influent suspended solids concentration of 1500 mg L⁻¹.     

Larvicidal activity of (-)-dihydroguaiaretic acid derivatives against Culex pipiens

The larvicidal activity against Culex pipiens of all stereoisomers of dihydroguaiaretic acid (DGA) and secoisolariciresinol was measured, and these DGAs were found to be potent. Sixteen (-)-DGA derivatives were then newly synthesized to analyze their structure-activity relationship. Two derivatives monohydroxylated at the 3- or 4-position of the 7-phenyl group of DGA induced acute paralytic activity in the mosquitoes. Derivatives with several hydroxyl groups had lower activity than the natural compound, suggesting that hydrophobicity was probably an important factor for their insecticidal activity.     

Artificial extracellular matrix proteins containing phenylalanine analogues biosynthesized in bacteria using T7 expression system and the PEGylation

In vivo incorporation of phenylalanine (Phe) analogues into an artificial extracellular matrix protein (aECM-CS5-ELF) was accomplished using a bacterial expression host that harbors the mutant phenylalanyl-tRNA synthetase (PheRS) with an enlarged binding pocket. Although the Ala294Gly/Thr251Gly mutant PheRS (PheRS**) under the control of T5 promoter allows incorporation of some Phe analogues into a protein, the T5 system is not suitable for material science studies because the amount of materials produced is not sufficient due to the moderate strength of the T5 promoter. This limitation can be overcome by using a pair of T7 promoter and T7 RNA polymerase instead. In the T7 expression system, it is difficult, however, to achieve a high incorporation level of Phe analogues, due to competition of Phe analogues for incorporation with the residual Phe that is required for synthesis of active T7 RNA polymerase. In this study, we prepared the PheRS** under T7 promoter and optimized culture condition to improve both the incorporation level of recombinant aECM protein and the incorporation level of Phe analogues. Incorporation and expression levels tend to increase in the case of p-azidophenylalanine, p-iodophenylalanine, and p-acetylphenylalanine. We evaluated the lower critical transition temperature, which is dependent on the incorporation ratio and the turbidity decreased when the incorporation level increased. Circular dichromism measurement indicated that this tendency is based on conformational change from random coil to β-turn structure. We demonstrated that polyethylene glycol (PEG) can be conjugated at reaction site of Phe analogues incorporated. We also demonstrated that the increased hydrophilicity of elastin-like sequences in the aECM-CS5-ELF made by PEG conjugation could suppress nonspecific adhesion of human umbilical vein endothelial cells (HUVEC).     

Rapid identification of protein-protein interfaces for the construction of a complex model based on multiple unassigned signals by using time-sharing NMR measurements

Protein-protein interactions are necessary for various cellular processes, and therefore, information related to protein-protein interactions and structural information of complexes is invaluable. To identify protein-protein interfaces using NMR, resonance assignments are generally necessary to analyze the data; however, they are time consuming to collect, especially for large proteins. In this paper, we present a rapid, effective, and unbiased approach for the identification of a protein-protein interface without resonance assignments. This approach requires only a single set of 2D titration experiments of a single protein sample, labeled with a unique combination of an (15)N-labeled amino acid and several amino acids (13)C-labeled on specific atoms. To rapidly obtain high resolution data, we applied a new pulse sequence for time-shared NMR measurements that allowed simultaneous detection of a ω(1)-TROSY-type backbone (1)H-(15)N and aromatic (1)H-(13)C shift correlations together with single quantum methyl (1)H-(13)C shift correlations. We developed a structure-based computational approach, that uses our experimental data to search the protein surfaces in an unbiased manner to identify the residues involved in the protein-protein interface. Finally, we demonstrated that the obtained information of the molecular interface could be directly leveraged to support protein-protein docking studies. Such rapid construction of a complex model provides valuable information and enables more efficient biochemical characterization of a protein-protein complex, for instance, as the first step in structure-guided drug development.     

Acetylation regulates monopolar attachment at multiple levels during meiosis I in fission yeast

In fission yeast, meiotic mono-orientation of sister kinetochores is established by cohesion at the core centromere, which is established by a meiotic cohesin complex and the kinetochore protein Moa1. The cohesin subunit Psm3 is acetylated by Eso1 and deacetylated by Clr6. We show that in meiosis, Eso1 is required for establishing core centromere cohesion during S phase, whereas Moa1 is required for maintaining this cohesion after S phase. The clr6-1 mutation suppresses the mono-orientation defect of moa1Δ cells, although the Clr6 target for this suppression is not Psm3. Thus, several acetylations are crucial for establishing and maintaining core centromere cohesion.     

Effects of menstrual cycle and physical training on heat loss responses during dynamic exercise at moderate intensity in a temperate environment

We evaluated the effects of the menstrual cycle and physical training on heat loss (sweating and cutaneous vasodilation) responses during moderate exercise in a temperate environment. Ten untrained (group U) and seven endurance-trained (group T) women (maximal O2 uptake of 36.7+/-1.1 vs. 49.4+/-1.7 ml.kg-1.min-1, respectively; P<0.05) performed a cycling exercise at 50% maximal O2 uptake for 30 min during both the midfollicular and midluteal menstrual phase in a temperate environment (ambient temperature of 25 degrees C, relative humidity of 45%). In group U, plasma levels of estrone, estradiol, and progesterone at rest and esophageal temperature (Tes) during exercise were significantly higher during the midluteal than during the midfollicular phase (P<0.05). Sweating rate and cutaneous blood flow (measured via laser-Doppler flowmetry) on the chest, back, forearm, and thigh were lower during the midluteal than during the midfollicular phase during exercise. Tes threshold for heat loss responses was significantly higher and sensitivity of the heat loss responses was significantly lower in the midluteal than in the midfollicular phase, regardless of body site. These effects of the menstrual cycle in group U were not observed in group T. The sweating rate and cutaneous blood flow were significantly higher in group T than in group U, regardless of menstrual phase or body site. Tes threshold for heat loss responses was significantly lower and sensitivity of heat loss responses was significantly greater in group T than in group U in the midluteal phase; however, sensitivity of the sweating response was significantly greater in the midfollicular phase. These results suggest that heat loss responses in group U were inhibited in the midluteal phase compared with in the midfollicular phase. Menstrual cycle had no remarkable effects in group T. Physical training improved heat loss responses, which was more marked in the midluteal than in the midfollicular phase.