Exosome-encapsulated microRNAs (miRNAs) have been identified as potential biomarkers in autoimmune diseases. However, little is known about the role of exosome-delivered miRNAs in rheumatoid arthritis (RA). In this study, we investigated the profile of specific exosomal miRNAs by microarray analysis of serum exosomes from three patients with RA and three healthy controls. Quantitative real-time PCR (qRT-PCR) was performed to validate the aberrantly expressed exosomal miRNAs. A total of 20 exosome-encapsulated miRNAs were identified to be differently expressed in the serum of patients with RA compared with controls. Interestingly, we found that exosome-encapsulated miR-6089 was significantly decreased after validation by qRT-PCR in serum exosomes from 76 patients with RA and 20 controls. Besides, miR-6089 could inhibit lipopolysaccharide (LPS)-induced cell proliferation and activation of macrophage-like THP-1 cells. TLR4 was a direct target for miR-6089. MiR-6089 regulated the generation of IL-6, IL-29, and TNF-α by targetedly controlling TLR4 signaling. In conclusion, exosome-encapsulated miR-6089 regulates LPS/TLR4-mediated inflammatory response, which may serve as a novel, promising biomarker in RA.
4-Nitrophenol (PNP), is generally regarded as an environmental endocrine disruptor (EED). Phytosterin (PS), a new feed additive, possesses highly efficient antioxidant activities. The transcription factor, nuclear factor-erythroid 2-related factor 2 (Nrf2), is an important regulator of cellular oxidative stress. Using rats, this study examined PNP-induced testicular oxidative damage and PS-mediated protection against that damage. The generation of MDA and H2O2 upon PNP and PS treatment was milder than that upon treatment with PNP alone. This mitigation was accompanied by partially reversed changes in SOD, CAT, GSH and GSH-Px. Moreover, PNP significantly reduced the caudal epididymal sperm counts and serum testosterone levels. Typical morphological changes were also observed in the testes of PNP-treated animals. PNP reduced the transcriptional level of Nrf2, as evaluated by RT-PCR, but it promoted the dissociation from the Nrf2 complex, stabilization and translocation into the nucleus, as evaluated by immunohistochemistry and Western blotting. In addition, PNP enhanced the Nrf2-dependent gene expression of heme oxygenase-1 (HO-1) and glutamate–cysteine ligase catalytic subunit (GCLC). These results suggest that the Nrf2 pathway plays an important role in PNP-induced oxidative damage and that PS possesses modulatory effects on PNP-induced oxidative damage in rat testes.
We have identified and characterized A bacteriophage clones containing genomic DNA encoding rat malic enzyme [(S)-malate:NADP+ oxidoreductase (oxaloacetatedecarboxylating); EC 1.1.1.40]. The malic enzyme gene is unexpectedly large, spanning at least 95 kilobases. It is divided into 14 exons that range in size from 76 to 1513 base pairs. The sizes and boundaries of the exons were determined by Southern blotting and DNA sequencing. The sequences at the 5' and 3' ends of each intron conformed to the consensus sequence for mammalian introns. S1 nuclease and primer-extension assays showed that transcription of the malic enzyme gene initiates at multiple sites, the strongest one at position -31 relative to the ATG. "TATA and CCAAT box" homologies are not present in the proximal promoter region. Analysis of the 3' end of the gene showed that the utilization of alternate polyadenylylation signals in exon 14 results in two mRNAs with 3' untranslated regions of 345 and 1345 nucleotides, respectively.Cytosolic malic enzyme [ME; (S)-malate:NADP+ oxidoreductase (oxaloacetate-decarboxylating; EC 1.1.1.40] catalyzes the NADP-dependent oxidative decarboxylation of malate to pyruvate and CO2 generating NADPH. In liver the enzyme is under nutritional and hormonal regulation. Studies from this laboratory indicate that the dietary, thyroid hormone, and dehydroepiandrosterone regulation of rat hepatic ME mRNA occur by different mechanisms. The accumulation of ME mRNA after feeding a high-carbohydrate diet to euthyroid rats results solely from increased cytoplasmic stability of the ME mRNAs (1). The increase of ME gene expression in response to 3,5,3'-triiodo-L-thyronine (T3) is due to both transcriptional activation of the gene and stabilization of the ME pre-mRNA (2). Whereas the =8-fold increase in cytoplasmic ME mRNA in liver of rats treated with dehydroepiandrosterone can be fully accounted for by changes in the rate of transcription (unpublished results).To understand the molecular mechanisms by which ME is regulated, to identify sequences in the ME gene that are involved in these regulations, and to disclose the structural basis for the generation of the two ME mRNAs, we have characterized genomic DNA encoding ME. In the present study, we report the isolation and characterization of cloned sequences representing the rat ME gene. § By restriction endonuclease mapping and direct sequencing of exon-intron boundaries, we have found that this gene is unexpectedly large and contains at least 14 exons. Analysis of the transcription start sites by S1 nuclease and primer-extension analyses revealed multiple sites of transcription initiation. This is in accord with the absence of two frequently identified promoter elements, the so-called "TATA and CCAAT boxes," which are usually located in the vicinity of cap sites and are necessary for precise and efficient initiation of transcription (4, 5). Comparison of the genomic and the cDNA sequences downstream from the last exon in conjunction with S1 nuclease analysis of this region re...
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