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We have previously reported the identification of a novel splicing variant of the mouse peroxisome proliferator‐activated receptor‐γ (Pparγ), referred to as Pparγ1sv. This variant, encoding the PPARγ1 protein, is abundantly and ubiquitously expressed, playing a crucial role in adipogenesis. Pparγ1sv possesses a unique promoter and 5 ′ untranslated region (5 ′UTR), distinct from those of the canonical mouse Pparγ1 and Pparγ2 mRNAs. We observed a significant increase in DNA methylation at two CpG sites within the proximal promoter region (‐733 to ‐76) of Pparγ1sv during adipocyte differentiation. Concurrently, chromatin immunoprecipitation‐quantitative PCR (ChIP‐qPCR) using antibodies against H3K4me3 and H3K27ac indicated marked elevations in both methylation and acetylation of histone H3, while the repressive histone mark H3K9me2 significantly decreased, at the transcription start sites of both Pparγ1sv and Pparγ2 following differentiation. Knocking down Pparγ1sv using specific siRNA also led to a decrease in Pparγ2 mRNA and PPARγ2 protein levels; conversely, knocking down Pparγ2 resulted in reduced Pparγ1sv mRNA and PPARγ1 protein levels, suggesting synergistic transcriptional regulation of Pparγ1sv and Pparγ2 during adipogenesis. Furthermore, our experiments utilizing the CRISPR‐Cas9 system identified crucial PPARγ‐binding sites within the Pparγ gene locus, underscoring their significance in adipogenesis. Based on these findings, we propose a model of positive feedback regulation for Pparγ1sv and Pparγ2 expression during the adipocyte differentiation process in 3T3‐L1 cells. [ABSTRACT FROM AUTHOR]

Background: Elongation of the spinal cord is dependent on neural development from neuromesodermal progenitors in the tail bud. We previously showed the involvement of the Oct4‐type gene, pou5f3, in this process in zebrafish mainly by dominant‐interference gene induction, but, to compensate for the limitation of this transgene approach, mutant analysis was indispensable. pou5f3 involvement in the signaling pathways was another unsolved question. Results: We examined the phenotypes of pou5f3 mutants and the effects of Pou5f3 activation by the tamoxifen‐ERT2 system in the posterior neural tube, together confirming the involvement of pou5f3. The reporter assays using P19 cells implicated tail bud‐related transcription factors in pou5f3 expression. Regulation of tail bud development by retinoic acid (RA) signaling was confirmed by treatment of embryos with RA and the synthesis inhibitor, and in vitro reporter assays further showed that RA signaling regulated pou5f3 expression. Importantly, the expression of the RA degradation enzyme gene, cyp26a1, was down‐regulated in embryos with disrupted pou5f3 activity. Conclusions: The involvement of pou5f3 in spinal cord extension was supported by using mutants and the gain‐of‐function approach. Our findings further suggest that pou5f3 regulates the RA level, contributing to neurogenesis in the posterior neural tube. Key Findings: We confirmed possible involvement of Oct4‐type POU transcription factor (PouV) in elongation of the spinal cord in zebrafish embryos.Phenotypes of pou5f3 mutation and pou5f3 activation were assessed in the neural tube.pou5f3 regulates retinoic acid metabolism, contributing to neurogenesis in the posterior neural tube. [ABSTRACT FROM AUTHOR]

In this paper electrical properties of a novel linear low-density polyethylene synthesized with a catalyst, developed by our unique technique, were measured. It was found that this polyethylene had a higher volume resistivity, a higher breakdown strength and less space charge accumulation than conventional high-pressure low-density polyethylene and linear low-density polyethylene. We also measured electrical properties of blends of the new polyethylene and the conventional low-density polyethylene and found that the blends which contained up to 90wt% of the low-density polyethylene had nearly the same value as the new polyethylene. The morphologies of the polyethylene were observed by using transmission electron microscope (TEM) and found that the blends kept the characteristics shown in the new polyethylene up to 90wt% of the low-density polyethylene content. The relationship between morphology and electrical properties is discussed and comparison is made between the new polyethylene with those of the conventional low-density polyethylenes. Index Terms--single-site catalyst, polyethylene, insulation, volume resistivity, breakdown, morphology, TEM.

Proteolytic activity declines with age, resulting in the accumulation of aggregated proteins in aged organisms. To investigate how disturbance in proteostasis causes cellular senescence, we developed a stress‐induced premature senescence (SIPS) model, in which normal human fibroblast MRC‐5 cells were treated with the proteasome inhibitor MG132 or the vacuolar‐type ATPase inhibitor bafilomycin A1 (BAFA1) for 5 days. Time‐course studies revealed a significant increase in intracellular reactive oxygen species (ROS) and mitochondrial superoxide during and after drug treatment. Mitochondrial membrane potential initially decreased, suggesting temporal mitochondrial dysfunction during drug treatment, but was restored along with mitochondrial accumulation after drug treatment. AMP‐activated protein kinase alpha was notably activated during treatment; thereafter, intracellular ATP levels significantly increased. SIPS induction by MG132 or BAFA1 was partially attenuated by co‐treatment with vitamin E or rapamycin, in which the levels of ROS, mitochondrial accumulation, and protein aggregates were suppressed, implying the critical involvement of oxidative stress and mitochondrial function in SIPS progression. Rapamycin co‐treatment also augmented the expression of HSP70 and activation of AKT, which could recover proteostasis and promote cell survival, respectively. Our study proposes a possible pathway from the disturbed proteostasis to cellular senescence via excess ROS production as well as functional and quantitative changes in mitochondria. [ABSTRACT FROM AUTHOR]

The five β-like globin genes (ε, Gγ, Aγ, δ and β) at the human β-globin gene locus are known to be expressed at specific developmental stages, although details of the underlying mechanism remain to be uncovered. Here we used an in vitro transcription assay to clarify the mechanisms that control this gene expression. We first tested nuclear RNA from HeLa cells using RT-qPCR and discovered a long noncoding RNAs (lncRNAs) within a 5.2-kb region beginning 4.4 kb downstream of the β-globin gene coding region. We investigated nuclear RNA from K562 cells using a primer-extension assay and determined the transcription start sites (TSSs) of these lncRNAs. To clarify their functional role, we performed knockdown (KD) of these lncRNAs in K562 cells. Hydroxyurea (HU), which induces differentiation of K562 cells, increased haemoglobin peptide production, and the effect was enhanced by KD of these lncRNAs, which also enhanced upregulation of the γ-globin expression induced by HU. To confirm these results, we performed an in vitro transcription assay. Noncoding single-stranded RNAs inhibited β-globin expression, which was upregulated by GATA1. Furthermore, lncRNAs interacted with GATA1 without sequence specificity and inhibited its binding to its target DNA response element in vitro. Our results suggest that lncRNAs downstream of the β-globin gene locus are key factors regulating globin gene expression. [ABSTRACT FROM AUTHOR]

The relation between morphological change, induced by copolymerized benzene rings or by kneading, and the dielectric strength of ethylene-styrene random copolymer was studied. Random distribution of styrene in the copolymer was confirmed by [C.sup.13] Fourier-transform nuclear magnetic resonance. With increasing the styrene content of the unkneaded copolymer, the crystallinity, the size of spherulite and the lamellar thickness decrease, while the lattice constant increases. However, in kneaded samples, the lamellar thickness and the lattice constant remain unchanged up to the styrene content of 3.10 wt%. Styrene content of the sample showing the highest dielectric strength increases as the temperature decreases. It is considered that the effect of benzene rings, which hinder the acceleration of electrons, becomes pronounced in the low temperature region where the breakdown process is electronic. Kneaded samples, especially the kneaded sample with the styrene content of 3.10 wt%, have higher dielectric strength than unkneaded samples of the same styrene content. It is considered that benzene rings are moved to the interfacial region between lamellar and amorphous parts by kneading and hinder the acceleration of electrons along the lamellae more effectively.

Abstract Background Luciferase assay systems enable the real-time monitoring of gene expression in living cells. We have developed a dual-color luciferase assay system in which the expression of multiple genes can be tracked simultaneously using green- and red-emitting beetle luciferases. We have applied the system to monitoring independent gene expressions in two types of cocultured fibroblasts in real time. Results Two Rat-1 cell lines were established that stably express either green- or red-emitting luciferases under the control of the mBmal1 promoter, a canonical clock gene. We cocultured these cell lines, and gene expression profiles in both were monitored simultaneously. The circadian rhythms of these cell lines are independent, oscillating following their intrinsic circadian phases, even when cocultured. Furthermore, the independent rhythms were synchronized by medium change as an external stimulus. Conclusion Using this system, we successfully monitored independent gene expression patterns in two lines of cocultured fibroblasts.