Background: Clock genes are expressed in different peripheral organs. Results: Rhythmic expression was lost with Ihh in the growth plate from mice defective of BMAL1 with a small body size. Conclusion: Endochondral ossification is under the control by clock genes in chondrocytes. Significance: Peripheral clocks are a target for treating cartilaginous diseases relevant to abnormal postnatal chondrogenesis.
Adrenaline is believed to play a dual role as a neurotransmitter in the central nervous system and an adrenomedullary hormone in the peripheral tissues. In contrast to accumulating evidence for the involvement in endochondral ossification, osteoblastogenesis, and osteoclastogenesis, little attention has been paid to the role of adrenergic signals in the mechanisms underlying proliferation and differentiation of mesenchymal stem cells with self-renewal capacity and multi-potentiality to differentiate into osteoblast, chondrocyte, adipocyte, and myocyte lineages. Expression of mRNA was seen for different adrenergic receptor (AdR) subtypes, including beta(2)AdR, in the mesenchymal stem cell line C3H10T1/2 cells and mouse bone marrow mesenchymal stem cells before differentiation. Exposure to adrenaline not only increased cAMP formation, phosphorylation of cAMP responsive element (CRE) binding protein (CREB) on serine133 and CRE reporter activity in a manner sensitive to propranolol, but also rendered C3H10T1/2 cells resistant to the cytotoxicity of hydrogen peroxide, but not of either 2,4-dinitirophenol or tunicamycin. Adrenaline induced a rapid but transient increase in mRNA expression of the antioxidative gene nuclear factor E2 p45-related factor-2 (Nrf2) along with an increase in the cystine/glutamate antiporter subunit xCT mRNA expression. Hydrogen peroxide was less cytotoxic in cells overexpressing Nrf2, moreover, while adrenaline significantly increased xCT promoter activity with an increase in endogenous glutathione levels. These results suggest that adrenaline may selectively protect mesenchymal C3H10T1/2 cells from oxidative stress through a mechanism related to the promoted biosynthesis of glutathione in association with transient Nrf2 expression after activation of beta(2)AdR.
Messenger RNAs (mRNAs) with phosphorothioate modification (PS‐mRNA) to the phosphate site of A, G, C, and U with all 16 possible combinations were prepared, and the translation reaction was evaluated using an E. coli cell‐free translation system. Protein synthesis from PS‐mRNA increased in 12 of 15 patterns when compared with that of unmodified mRNA. The protein yield increased 22‐fold when the phosphorothioate modification at A/C sites was introduced into the region from the 5′‐end to the initiation codon. Single‐turnover analysis of PS‐mRNA translation showed that phosphorothioate modification increases the number of translating ribosomes, thus suggesting that the rate of translation initiation (rate of ribosome complex formation) is positively affected by the modification. The method provides a new strategy for improving translation by using non‐natural mRNA.
Abstract. Tacrolimus (FK506) has been used as a therapeutic drug beneficial for the treatment of rheumatoid arthritis in humans. In this study, we investigated the effects of FK506 on cellular differentiation in cultured chondrogenic cells. Culture with FK506 led to a significant and concentration-dependent increase in Alcian blue staining for matrix proteoglycan at 0.1 to 1,000 ng/ml, but not in alkaline phosphatase (ALP) activity, in ATDC5 cells, a mouse prechondrogenic cell line, cultured for 7 to 28 days, while the non-steroidal anti-inflammatory drug indomethacin significantly decreased Alcian blue staining in a concentration-dependent manner, without altering ALP activity. FK506 significantly increased the expression of mRNA for both type II and type X collagen, but not for osteopontin, in ATDC5 cells. Similar promotion was seen in chondrogenic differentiation in both mouse metatarsals and chondrocytes cultured with FK506. However, FK506 failed to significantly affect transcriptional activity of the reporter construct for either sry-type HMG box 9 (Sox9) or runt-related transcription factor-2 (Runx2), which are both transcription factors responsible for chondrocytic maturation as a master regulator. These results suggest that FK506 may predominantly promote cellular differentiation into proliferating chondrocytes through a mechanism not relevant to the transactivation by either Sox9 or Runx2 in chondrogenic cells.
This chapter describes a simple and straightforward way to obtain single-stranded circular RNA sequences in vitro. Linear RNA that is phosphorylated at the 5' end is first prepared by a chemical or enzymatic method, then circularized using ligase. The function of the prepared circular RNA molecule, such as an ability to induce translation, can then be investigated.
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