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The tyrosyl radicals generated in reactions of ethyl hydrogen peroxide with both native and indomethacin-pretreated prostaglandin H synthase 1 (PGHS-1) were examined by low-temperature electron paramagnetic resonance (EPR) and electron nuclear double resonance (ENDOR) spectroscopies. In the reaction of peroxide with the native enzyme at 0 degrees C, the tyrosyl radical EPR signal underwent a continuous reduction in line width and lost intensity as the incubation time increased, changing from an initial, 35-G wide doublet to a wide singlet of slightly smaller line width and finally to a 25-G narrow singlet. The 25-G narrow singlet produced by self-inactivation was distinctly broader than the 22-G narrow singlet obtained by indomethacin treatment. Analysis of the narrow singlet EPR spectra of self-inactivated and indomethacin-pretreated enzymes suggests that they reflect conformationally distinct tyrosyl radicals. ENDOR spectroscopy allowed more detailed characterization by providing hyperfine couplings for ring and methylene protons. These results establish that the wide doublet and the 22-G narrow singlet EPR signals arise from tyrosyl radicals with different side-chain conformations. The wide-singlet ENDOR spectrum, however, is best accounted for as a mixture of native wide-doublet and self-inactivated 25-G narrow-singlet species, consistent with an earlier EPR study [DeGray et al. (1992) J. Biol. Chem. 267, 23583-23588]. We conclude that a tyrosyl residue other than the catalytically essential Y385 species is most likely responsible for the indomethacin-inhibited, narrow-singlet spectrum. Thus, this inhibitor may function by redirecting radical formation to a catalytically inactive side chain. Either radical migration or conformational relaxation at Y385 produces the 25-G narrow singlet during self-inactivation. Our ENDOR data also indicate that the catalytically active, wide-doublet species is not hydrogen bonded, which may enhance its reactivity toward the fatty-acid substrate bound nearby.

Acetylcholine (ACh), which can be synthesized and secreted by cancer cells, has been reported to play an important role in tumor progression. ACh acts its role through activation of its receptors, muscarinic receptor (mAChR), and nicotinic receptor (nAChR). As a member of mAChR, M3 muscarinic receptor (M3R) is often highly expressed in many cancers. Activation of M3R by ACh participates in the proliferation, differentiation, transformation, and carcinogenesis of cancer. However, the effect of M3R activation on non-small cell lung cancer (NSCLC) remains unclear. Here, our study found that ACh dose-dependently promoted the proliferation, invasion, and migration of NSCLC cells. After silencing of M3R, the biological functions of ACh in NSCLC cells were greatly attenuated. Furthermore, ACh stimulation increased the production of IL-8 and time-dependently induced the activation of EGFR, PI3K, and AKT through M3R. In addition, ACh stimulated the activation of PI3K and AKT via EGFR activity, and blocking of PI3K/AKT pathway by special inhibitor LY294002 suppressed the ACh-mediated proliferation, invasion, and migration of NSCLC cells. Taken together, these findings indicate that activation of M3R by ACh enhances the proliferation, invasion, and migration of NSCLC cells. ACh-induced activation of EGFR/PI3K/AKT pathway and subsequent IL-8 upregulation may be one of the important mechanisms of M3R function. Thus, M3R could be a potential therapeutic target for the treatment of NSCLC.

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The effect and mechanism of preharvest and postharvest ultraviolet (UV) irradiation on anthocyanin biosynthesis during blueberry development were investigated. The results showed that preharvest UV-B,C and postharvest UV-A,B,C irradiation significantly promoted anthocyanin biosynthesis and the transcripts of late biosynthetic genes (LBG) VcDFR, VcANS, VcUFGT, and VcMYB transcription factor as well as DFR and UFGT activities in anthocyanin pathway in a UV wavelength- and developmental stage-dependent manner. VcMYB expression was positively correlated with that of VcANS and VcUFGT and coincided with anthocyanin biosynthesis responding to the UV radiation. Sugar decreased during postharvest but increased during preharvest UV radiation in mature fruit. Our results indicate that UV-responsive production of anthocyanins is mainly caused by the activation of anthocyanin downstream pathway genes, which could be upregulated by VcMYB. Furthermore, different potential response mechanisms may exist between preharvest and postharvest UV radiation in blueberries, involving a systemic response in living plants and a nonsystemic response in postharvest fruit.