Regulation of Activation and Processing of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) by a Complex Electrostatic Interaction between the Regulatory Domain and Cytoplasmic Loop 3

NEG2, a short C-terminal segment (817–838) of the unique regulatory (R) domain of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel, has been reported to regulate CFTR gating in response to cAMP-dependent R domain phosphorylation. The underlying mechanism, however, is unclear. Here, Lys-946 of cytoplasmic loop 3 (CL3) is proposed as counter-ion of Asp-835, Asp-836, or Glu-838 of NEG2 to prevent the channel activation by PKA. Arg-764 or Arg-766 of the Ser-768 phosphorylation site of the R domain is proposed to promote the channel activation possibly by weakening the putative CL3-NEG2 electrostatic attraction. First, not only D835A, D836A, and E838A but also K946A reduced the PKA-dependent CFTR activation. Second, both K946D and D835R/D836R/E838R mutants were activated by ATP and curcumin to a different extent. Third, R764A and R766A mutants enhanced the PKA-dependent activation. However, it is very exciting that D835R/D836R/E838R and K946D/H950D and H950R exhibited normal channel processing and activity whereas D835R/D836R/E838R/K946D/H950D was fractionally misprocessed and silent in response to forskolin. Further, D836R and E838R played a critical role in the asymmetric electrostatic regulation of CFTR processing, and Ser-768 phosphorylation may not be involved. Thus, a complex interfacial interaction among CL3, NEG2, and the Ser-768 phosphorylation site may be responsible for the asymmetric electrostatic regulation of CFTR activation and processing.     

Selective electrochemical detection of cysteine in complex serum by graphene nanoribbon

Selective detection of cysteine in serum samples was achieved on a graphene nanoribbon (GNR) and Nafion nanocomposite modified electrode with high precision. The superior conductivity and abundant amount of active chemical oxygen groups on the edge of GNR led to extremely highly electrocatalytic activity of GNR towards the electrochemical oxidation of cysteine at +0.025 V. The electrocatalytic behavior was further used for sensitive detection of cysteine by differential pulse voltammetry. Under optimized conditions, the calibration curve was linear in the range from 25 nM to 500 μM. The electrochemical sensor showed strong antifouling ability, good stability and selectivity. It could effectively exclude the interferences from other kinds of biothiols and the biological relevant species, thus had great perspective for in vivo analysis of biological samples.     

Overexpression of aldo-keto reductase 1C3 (AKR1C3) in LNCaP cells diverts androgen metabolism towards testosterone resulting in resistance to the 5α-reductase inhibitor finasteride

Type 5 17β-hydroxysteroid dehydrogenase (AKR1C3) is the major enzyme in the prostate that reduces 4-androstene-3,17-dione (Δ(4)-Adione) to the androgen receptor (AR) ligand testosterone. AKR1C3 is upregulated in prostate cancer (PCa) and castrate resistant prostate cancer (CRPC) that develops after androgen deprivation therapy. PCa and CRPC often depend on intratumoral androgen biosynthesis and upregulation of AKR1C3 could contribute to intracellular synthesis of AR ligands and stimulation of proliferation through AR signaling. To test this hypothesis, we developed an LNCaP prostate cancer cell line overexpressing AKR1C3 (LNCaP-AKR1C3) and compared its metabolic and proliferative responses to Δ(4)-Adione treatment with that of the parental, AKR1C3 negative LNCaP cells. In LNCaP and LNCaP-AKR1C3 cells, metabolism proceeded via 5α-reduction to form 5α-androstane-3,17-dione and then (epi)androsterone-3-glucuronide. LNCaP-AKR1C3 cells made significantly higher amounts of testosterone-17β-glucuronide. When 5α-reductase was inhibited by finasteride, the production of testosterone-17β-glucuronide was further elevated in LNCaP-AKR1C3 cells. When AKR1C3 activity was inhibited with indomethacin the production of testosterone-17β-glucuronide was significantly decreased. Δ(4)-Adione treatment stimulated cell proliferation in both cell lines. Finasteride inhibited LNCaP cell proliferation, consistent with 5α-androstane-3,17-dione acting as the major metabolite that stimulates growth by binding to the mutated AR. However, LNCaP-AKR1C3 cells were resistant to the growth inhibitory properties of finasteride, consistent with the diversion of Δ(4)-Adione metabolism from 5α-reduced androgens to increased formation of testosterone. Indomethacin did not result in differences in Δ(4)-Adione induced proliferation since this treatment led to the same metabolic profile in LNCaP and LNCaP-AKR1C3 cells. We conclude that AKR1C3 overexpression diverts androgen metabolism to testosterone that results in proliferation in androgen sensitive prostate cancer. This effect is seen despite high levels of uridine glucuronosyl transferases suggesting that AKR1C3 activity can surmount the effects of this elimination pathway. Treatment options in prostate cancer that target 5α-reductase where AKR1C3 co-exists may be less effective due to the diversion of Δ(4)-Adione to testosterone.     

Discovery of estrogen receptor α modulators from natural compounds in Si-Wu-Tang series decoctions using estrogen-responsive MCF-7 breast cancer cells

The binding between the estrogen receptor α (ER-α) and a variety of compounds in traditional Chinese formulae, Si-Wu-Tang (SWT) series decoctions, was studied using a stably-transfected human breast cancer cell line (MVLN). In 38 compounds tested from SWT series decoctions, the estrogen-like activity of 22 compounds was above 60% in 20 μg mL(-1). Furthermore, theoretical affinity of these compounds was certificated using the functional virtual screen of ER-α modulators by FlexX-Pharm. The accuracy of functional virtual screening of ER-α modulators could reach to 77.27%. The results showed that some compounds, such as organic acids and flavones in SWT series decoctions could be used as selective estrogen receptor modulators (SERMs) and could be selected for further development as potential agents for estrogen related diseases.     

Discovery of novel P3-oxo inhibitor of hepatitis C virus NS3/4A serine protease

A novel series of P3 oxo-modified macrocyclic hepatitis C virus NS3/4A serine protease inhibitor was designed, synthesized and biologically evaluated. The hydroxy-substituted inhibitor 10 demonstrated high potency in genotype 1a and 1b replicon and in the panel of HCV protease mutants. Interestingly, the t-butyl carbonate analog 9c, while not the most potent one in this series, exhibited a virtually flat potency profile in the panel of HCV protease mutants, thus providing opportunity for further optimization.     

天山西部野杏光合作用日变化特征与其生理生态因子的关系

在新疆天山西部伊犁野果林资源发展研究中心的野杏林选定野生成年杏树为样株,用Li-6400光合仪测定晴天和阴天两种天气状况下野杏的光合生理生态特性,并通过相关分析、通径分析、回归分析探讨其净光合速率与生理生态因子的关系。结果显示:(1)野杏的最大净光合速率(Pnmax)和光饱和点(LSP)分别为12.63和1 389.44μmol.m-2.s-1,属喜光的阳生树种。(2)晴天和多云天气下野杏的净光合速率(Pn)日变化最大值和日均值之间差异不显著,晴天Pn日变化为单峰型,而多云为双峰型,具典型的"午休"现象,而且是由非气孔限制因素造成,与叶温(Tl)的降低有关。(3)野杏气孔导度(Gs)与Pn和蒸腾速率(Tr)呈显著正相关;随着其Tl的增加,晴天Pn、Gs和Tr为单峰形;多云Pn和Tr呈线性增加,而Gs呈线性降低;Tl增加超过最适值后Gs的降低是晴天野杏产生严重光抑制的重要原因;气孔的快速关闭降低了Tr,造成Tl增加和Pn降低,在最大程度上控制了水分丢失。(4)晴天和多云天气下影响野杏叶片Pn日变化主导生理因子均为Gs,主要限制因子为胞间CO2浓度(Ci);主导生态因子为空气相对湿度(RH),限制因子为气温(Ta)。研究表明,野杏能够适应高光强环境条件,这是野杏主要分布在较为干旱阳坡上的重要原因之一;多云天气条件对野杏进行光合作用的抑制程度较弱,这为野杏种群天然更新环境条件的人工调节提供一定的理论依据。     

水土流失治理措施对小流域土壤有机碳和全氮的影响

明确综合治理条件下小流域土壤有机碳(Soil organic carbon,SOC)和全氮(Total nitrogen,TN)的空间分布特征及其影响因素,对科学评价水土流失区土壤固碳潜力具有重要意义。以黄土高原丘陵沟壑区典型小流域(砖窑沟流域)为对象,基于流域内3种典型地貌类型(梁峁坡、沟坡、沟谷)和3种典型水土流失治理措施(水平梯田、林地和草地措施,坡耕地为对照),采集土壤样品737个,研究地貌类型和水土流失治理措施对小流域SOC和TN变化的影响。结果表明,同一地貌类型上,水平梯田、林地和草地措施的SOC和TN(0—10 cm土层)含量均显著高于坡耕地(P<0.1)。梁峁坡上,水平梯田、林地和草地措施条件下的SOC和TN含量较坡耕地依次提高了18%和24%、70%和59%、25%和21%;沟坡上,林地和草地措施的SOC和TN较坡耕地依次提高了76%和54%、25%和27%。同一治理措施在不同地貌类型间对0—10 cm土层SOC和TN的影响存在显著差异(P<0.1)。水平梯田条件下,沟谷的SOC和TN含量比峁坡提高了46%和43%;林地措施条件下,沟坡的SOC和TN含量比峁坡提高了18%和6%;草地措施条件下,沟坡的SOC和TN含量比峁坡提高了14%和18%。0—100 cm土层的SOC或TN在不同地貌类型或不同治理措施间的差异与土壤水分含量(Soil moisture,SM)的变化趋势基本一致,并且SOC或TN与SM呈指数关系y=aebx(y为SOC或TN,x为SM)。