Copper ions inhibit S-adenosylhomocysteine hydrolase by causing dissociation of NAD+ cofactor

S-Adenosylhomocysteine hydrolase (SAHH) regulates biomethylation and homocysteine metabolism and thus is an attractive target in drug design studies. SAHH has been shown to be a copper binding protein in vivo; however, the structure and catalytic mechanism of SAHH exclude a role for Cu2+. In the present work, we studied the mechanism of inhibition of SAHH activity by Cu2+. The experimental results showed that Cu2+ inhibited SAHH activity in a noncompetitive manner. Binding of Cu2+ to SAHH resulted in the release of NAD+ cofactors, explaining the loss of the enzymatic activity of SAHH. Further investigation by an ESR probe and computational simulation suggested that Cu2+ could bind at the central channel and interrupt the subunit interactions of SAHH, resulting in a large decrease in affinity to the NAD+ cofactor. This effect of Cu2+ resembled that of enzyme mutations at the C-terminal domain or Asp244 [Komoto, J., Huang, Y., Gomi, T., Ogawa, H., Takata, Y., Fujioka, M., and Takusagawa, F. (2000) Effects of site-directed mutagenesis on structure and function of recombinant rat liver S-adenosylhomocysteine hydrolase. Crystal structure of D244E mutant enzyme, J. Biol. Chem. 275, 32147-32156]. The mechanism of action of Cu2+ on SAHH suggested a possible regulative role for Cu2+ on the intracellular activity of SAHH. This could be helpful in understanding the biological effects of copper compounds and suggest a potential coupling mechanism between biomethylation and the redox states of cells.     

The variant at TGFBRAP1 is significantly associated with type 2 diabetes mellitus and affects diabetes‐related miRNA expression

While the transforming growth factor‐β1 (TGF‐β1) regulates the growth and proliferation of pancreatic β‐cells, its receptors trigger the activation of Smad network and subsequently induce the insulin resistance. A case‐control was conducted to evaluate the associations of the polymorphisms of TGF‐β1 receptor‐associated protein 1 (TGFBRAP1) and TGF‐β1 receptor 2 (TGFBR2) with type 2 diabetes mellitus (T2DM), and its genetic effects on diabetes‐related miRNA expression. miRNA microarray chip was used to screen T2DM‐related miRNA and 15 differential expressed miRNAs were further validated in 75 T2DM and 75 normal glucose tolerance (NGT). The variation of rs2241797 (T/C) at TGFBRAP1 showed significant association with T2DM in case‐control study, and the OR (95% CI) of dominant model for cumulative effects was 1.204 (1.060‐1.370), Bonferroni corrected P < 0.05. Significant differences in the fast glucose and HOMA‐β indices were observed amongst the genotypes of rs2241797. The expression of has‐miR‐30b‐5p and has‐miR‐93‐5p was linearly increased across TT, TC, and CC genotypes of rs2241797 in NGT, P_trend values were 0.024 and 0.016, respectively. Our findings suggest that genetic polymorphisms of TGFBRAP1 may contribute to the genetic susceptibility of T2DM by mediating diabetes‐related miRNA expression.     

Ethanol-mediated regulation of cytochrome P450 2A6 expression in monocytes: role of oxidative stress-mediated PKC/MEK/Nrf2 pathway

Cytochrome P450 2A6 (CYP2A6) is known to metabolize nicotine, the major constituent of tobacco, leading to the production of toxic metabolites and induction of oxidative stress that result in liver damage and lung cancer. Recently, we have shown that CYP2A6 is induced by ethanol and metabolizes nicotine into cotinine and other metabolites leading to generation of reactive oxygen species (ROS) in U937 monocytes. However, the mechanism by which CYP2A6 is induced by ethanol is unknown. In this study, we have examined the role of the PKC/Nrf2 pathway (protein kinase C-mediated phosphorylation and translocation of nuclear erythroid 2-related factor 2 to the nucleus) in ethanol-mediated CYP2A6 induction. Our results showed that 100 mM ethanol significantly induced CYP2A6 mRNA and protein (~150%) and increased ROS formation, and induction of gene expression and ROS were both completely blocked by treatment with either a CYP2E1 inhibitor (diallyl sulfide) or an antioxidant (vitamin C). The results suggest the role of oxidative stress in the regulation of CYP2A6 expression. Subsequently, we investigated the role of Nrf2 pathway in oxidative stress-mediated regulation of CYP2A6 expression in U937 monocytes. Our results showed that butylated hydroxyanisole, a stabilizer of nuclear Nrf2, increased CYP2A6 levels >200%. Staurosporine, an inhibitor of PKC, completely abolished ethanol-induced CYP2A6 expression. Furthermore, our results showed that a specific inhibitor of mitogen-activated protein kinase kinase (MEK) (U0126) completely abolished ethanol-mediated CYP2A6 induction and Nrf2 translocation. Overall, these results suggest that CYP2E1-mediated oxidative stress produced as a result of ethanol metabolism translocates Nrf2 into the nucleus through PKC/MEK pathway, resulting in the induction of CYP2A6 in monocytes. An increased level of CYP2A6 in monocytes is expected to further increase oxidative stress in smokers through CYP2A6-mediated nicotine metabolism. Thus, this study has clinical relevance because of the high incidence of alcohol use among smokers, especially in HIV-infected individuals.     

苹果花期至幼果期花朵和果实对低温的敏感性研究北大核心CSCD

为了确立不同苹果品种在花蕾期到幼果期的抗冻性类型,建立精准苹果晚霜抗冻性评价方法,以宁夏2个主栽苹果品种‘嘎啦’和‘富士’花蕾期、盛花期、坐果期和幼果期的花朵和果实为试验材料,利用野外霜冻试验箱分别模拟不同时期自然降温过程,在20%、50%和80%受冻率下分别建立Logistic方程,确定轻、中、重度受冻的临界温度;并检测各品种不同物候期的过冷却点、结冰点、电导率、可溶性糖和可溶性蛋白等抗寒性指标,结合多元线性回归法综合判断苹果花期至幼果期抗寒能力差异的主要影响因素。结果显示,(1)‘嘎啦’各个物候期的抗寒能力均强于‘富士’;(2)同一苹果品种不同物候期抗寒能力不同,表现为花蕾期>盛花期>坐果期>幼果期;(3)2个苹果品种各物候期轻度、中度、重度受冻临界温度随着物候期推移呈现升高的趋势;(4)受冻率与半致死温度呈极显著正相关(0.909**),与可溶性蛋白含量呈极显著负相关(-0.874**)。研究表明,‘嘎啦’和‘富士’4个物候期花器官相对电导率和可溶性蛋白含量对子房受冻率有较强的响应关系,过冷却点和结冰点温度等对子房受冻率也有一定的响应,可将相对电导率、可溶性蛋白含量、过冷却点和结冰点作为评价苹果抗寒性状的重要指标。     

Pranlukast,a novel binding ligand of human Raf1 kinase inhibitory protein

Objectives

To study the binding of pranlukast to hRKIP and its regulatory role in the Raf1/MEK/ERK signal pathway.

Results

NMR and fluorescence experiments demonstrated hRKIP could bind pranlukast with a binding constant of 1016 mM^?1. Residues (Y81, S109 and Y181) on the conserved ligand-binding pocket of hRKIP played a crucial role in binding pranlukast, and their mutations reduced the binding affinity more than 85 %. Furthermore, 25 μM pranlukast could up-regulate the ERK phosphorylation by about 17 %.

Conclusion

Pranlukast may be used as a potential drug precursor for treating hRKIP involved diseases.

     

3‐photon fluorescence imaging of sulforhodamine B‐labeled elastic fibers in the mouse skin in vivo

Elastic fibers are key constituents of the skin. The commonly adopted optical technique for visualizing elastic fibers in the animal skin in vivo is 2‐photon microscopy (2 PM) of autofluorescence, which typically suffers from low signal level. Here we demonstrate a new optical methodology to image elastic fibers in animal models in vivo: 3‐photon microscopy (3 PM) excited at the 1700‐nm window combining with preferential labeling of elastic fibers using sulforhodamine B (SRB). First, we demonstrate that intravenous injection of SRB can circumvent the skin barrier (encountered in topical application) and preferentially label elastic fibers, as verified by simultaneous 2 PM of both autofluorescence and SRB fluorescence from skin structures. Then through 3‐photon excitation property characterization, we show that 3‐photon fluorescence can be excited from SRB at the 1700‐nm window, and 1600‐nm excitation is most efficient according to our 3‐photon action cross section measurement. Based on these results and using our developed 1600‐nm femtosecond laser source, we finally demonstrate 3 PM of SRB‐labeled elastic fibers through the whole dermis in the mouse skin in vivo, with only 3.7‐mW optical power deposited on the skin surface. We expect our methodology will provide novel optical solution to elastic fiber research.     

Visualizing the “sandwich” structure of osteocytes in their native environment deep in bone in vivo

Osteocytes are the most abundant cells in bone and always the focus of bone research. They are embedded in the highly scattering mineralized bone matrix. Consequently, visualizing osteocytes deep in bone with subcellular resolution poses a major challenge for in vivo bone research. Here we overcome this challenge by demonstrating 3‐photon imaging of osteocytes through the intact mouse skull in vivo. Through broadband transmittance characterization, we establish that the excitation at the 1700‐nm window enables the highest optical transmittance through the skull. Using label‐free third‐harmonic generation (THG) imaging excited at this window, we visualize osteocytes through the whole 140‐μm mouse skull and 155 μm into the brain in vivo. By developing selective labeling technique for the interstitial space, we visualize the “sandwich” structure of osteocytes in their native environment. Our work provides novel imaging methodology for bone research in vivo.