β-Arrestins facilitate ubiquitin-dependent degradation of apoptosis signal-regulating kinase 1 (ASK1) and attenuate H2O2-induced apoptosis

β-Arrestins are ubiquitously expressed proteins that play important roles in receptor desensitization, endocytosis, proteosomal degradation, apoptosis and signaling. It has been reported that β-Arrestin2 acts as a scaffold by directly interacting with the JNK3 isoform and recruiting MKK4 and the apoptosis-signaling kinase-1 (ASK1). Here, we report a novel function of β-Arrestins in regulating H₂O₂-induced apoptosis. Our study demonstrates that β-Arrestins physically associate with C-terminal domain of ASK1, and moreover, both over-expression and RNA interference (RNAi) experiments indicate that β-Arrestins down-regulate ASK1 protein. In detail, β-Arrestin-induced reduction of ASK1 protein is due to ubiquitination and proteasome-dependent degradation of ASK1 in response to association of β-Arrestins and ASK1. Upon H₂O₂ stimulation, the protein binding between β-Arrestins and ASK1 increases and ASK1 degradation is expedited. In consequence, β-Arrestins prevent ASK1-JNK signaling and as a result attenuate H₂O₂-induced apoptosis. Structurally, C-terminal domain of ASK1 is essential for β-Arrestins and ASK1 association. We also found that CHIP is required for β-Arrestins-induced ASK1 degradation, which suggested that β-Arrestins function as a scaffold of ASK1 and CHIP, leading to CHIP-mediated ASK1 degradation. All these findings indicate that β-Arrestins play a negative regulatory role in H₂O₂-induced apoptosis signaling through associating with ASK1 and CHIP and facilitating ASK1 degradation, which provides a new insight for analyzing the effects of β-Arrestins on protecting cells from oxidative stress-induced apoptosis.     

Development and application of a T7 RNA polymerase-dependent expression system for antibiotic production improvement in <Emphasis Type="Italic">Streptomyces</Emphasis>

Objective

To develop a reliable and easy to use expression system for antibiotic production improvement of Streptomyces.

Results

A two-compound T7 RNA polymerase-dependent gene expression system was developed to fulfill this demand. In this system, the T7 RNA polymerase coding sequence was optimized based on the codon usage of Streptomyces coelicolor. To evaluate the functionality of this system, we constructed an activator gene overexpression strain for enhancement of actinorhodin production. By overexpression of the positive regulator actII-ORF4 with this system, the maximum actinorhodin yield of engineered strain was 15-fold higher and the fermentation time was decreased by 48 h.

Conclusion

The modified two-compound T7 expression system improves both antibiotic production and accelerates the fermentation process in Streptomyces. This provides a general and useful strategy for strain improvement of important antibiotic producing Streptomyces strains.

     

The lncRNA HNF1A‐AS1 is a negative prognostic factor and promotes tumorigenesis in osteosarcoma

Recent studies have revealed that long noncoding RNA HNF1A‐antisense 1 (HNF1A‐AS1) plays an important role in the development of several human malignancy entities. However, the expression and function of HNF1A‐AS1 in the carcinogenesis and development of osteosarcoma remains unknown. In this study, we detected the HNF1A‐AS1 levels in human osteosarcoma tissues and cell lines by quantitative real‐time polymerase chain reaction (qRT‐PCR), and investigated its role in osteosarcoma by using in vitro assays. Our study showed that HNF1A‐AS1 expression was significantly up‐regulated in human osteosarcoma tissues and cell lines compared with their normal counterparts, and its expression level was positively correlated with the distance metastasis (P = 0.009) and tumour stage (P = 0.019). Moreover, Kaplan–Meier curves with the log‐rank test showed that higher expression of HNF1A‐AS1 conferred a significantly poorer survival and multivariate Cox proportional hazards analysis revealed that HNF1A‐AS1 was an independent risk factor of overall survival. In addition, the expression of HNF1A‐AS1 in serum is correlated with patients’ status and receiver operating characteristic (ROC) curve analysis demonstrated that HNF1A‐AS1 could distinguish patients with osteosarcoma from healthy individuals (the area under curve 0.849, P < 0.001). Furthermore, in vitro knockdown of HNF1A‐AS1 by siRNA significantly inhibited cell proliferation and G₁/S transition, and suppressed migration and invasion by reducing the epithelial‐mesenchymal transition (EMT) program in osteosarcoma cells. Taken together, our data suggested that HNF1A‐AS1 is a novel molecule involved in osteosarcoma progression, which may provide as a potential diagnostic, prognostic biomarker and therapeutic target.     

黑河天涝池流域典型林分生态水文化学特征

采集了黑河天涝池流域典型林分林外雨、穿透雨、树干径流和枯透水,并检测水体pH值和12种离子(K~+、Ca~(2+)、Na~+、Mg~(2+)、NH_4~+、Cu~(2+)、Zn~(2+)、Pb~(2+)、Cd~(2+)、Cl~-、SO_4~(2-)、NO_3~3)的质量浓度。结果表明:天涝池流域大气降水pH均值为7.74,呈碱性,降水中离子绝对质量浓度较低,最高的是NO_3~-,质量浓度为1.1111 mg/L,最低的为Na~+,质量浓度为0.0108 mg/L;两种林分冠层有降低降雨pH值的作用,青海云杉林冠层对NH_4~+有升高作用,祁连圆柏林冠层对NH_4~+有降低作用,两种林冠层对NO_3~-和Cu~(2+)质量浓度有降低作用,对其它离子质量浓度均表现为升高作用;两种林分树干径流有提高穿透雨pH值的作用,与穿透雨相比,两种林分树干径流中阴离子均有升高,圆柏树干径流中所有阳离子质量浓度均有下降,云杉树干径流中Ca~(2+)、K~+、Mg~(2+)和Na~+减少,NH_4~+和Cu~(2+)增加;典型林分枯透水有提升穿透雨pH值的作用,与穿透雨相比,两种林分枯透水中阴离子质量浓度均有升高,云杉枯透水各阳离子均有降低,圆柏枯透水中Ca~(2+)、K~+和Mg~(2+)质量浓度升高,NH_4~+、Na~+和Cu~(2+)质量浓度下降;在采集的所有样本中,Pb~(2+)和Cd~(2+)均未检出,而Zn~(2+)仅在云杉树干径流中检出。     

Kinetic resolution of ketoprofen ester catalyzed by lipase from a mutant of CBS 5791

A biotransformation process was developed for the production of (S)-ketoprofen by enantioseletive hydrolysis of racemic ketoprofen ester using the mutant Trichosporon laibacchii strain CBS 5791. A satisfactory result was obtained, in which the E was 82.5, with an ee of 0.94 and a conversion of 0.47 under the optimum hydrolysis conditions [E is enantiomeric ratio, E=ln[1–X(1+ee)]/ln[1–X(1–ee)]; ee is enantiomeric excess, ee=(C_S–C_R)/(C_S+C_R): temperature of hydrolysis was 23°C]. The medium used in biotransformation was a mixture of growth broth and biotransformation broth at a ratio of 1:9, the concentration of Tween 80 was 15 g/l, the time of hydrolysis, 72 h. These results are promising for further scale-up. Tween 80 significantly improved lipase enantioselectivity and activity at the optimum concentration.     

Determination of 9-nitrocamptothecin by precolumn derivatization and its metabolite 9-aminocamptothecin in a biological fluid using reversed-phase high-performance liquid chromatography with fluorescence detection

A novel insoluble topoisomerase I inhibitor, 9-nitrocamptothecin (9-NC), is in advanced stages of clinical development and has been used to treat a diverse array of tumor types, including breast, ovarian, pancreatic and haematological malignancies. We have established a sensitive high-performance liquid chromatography method using fluorescence detection for the quantitation of 9-NC. Non-fluorescent 9-NC is converted to fluorescent 9-aminocamptothecin (9-AC) via a one-step pre-column derivative reaction. The quantitative limit of 9-NC was 1 ng/ml and the method was reproducible with the respective intra- and inter-day variability falling below 5.0 and 9.0%. The determination of both 9-NC and its metabolite 9-AC in dog plasma was also achieved using the same chromatographic and detection conditions. In dog plasma, the quantitative limits of 9-AC and 9-NC were 0.25 and 1 ng/ml, respectively. The presence of 9-AC in the samples yielded no interference with the determination of 9-NC. However, individual matrices can affect the conversion efficiency of 9-NC, thus indicating that standard samples should be run for each matrix.     

Acetylation of lysine 56 of histone H3 catalyzed by RTT109 and regulated by ASF1 is required for replisome integrity

In budding yeast, acetylation of histone H3 lysine 56 (H3-K56) is catalyzed by the Rtt109-Vps75 histone acetyltransferase (HAT) complex, with Rtt109 being the catalytic subunit, and histone chaperone Asf1 is required for this modification. Cells lacking Rtt109 are susceptible to perturbations in DNA replication. However, how Asf1 regulates acetylation of H3-K56 and how loss of H3-K56 acetylation affects DNA replication are unclear. We show that at low concentrations the Rtt109-Vps75 HAT complex acetylates H3-K56 in vitro when H3/H4 is complexed with Asf1, but not H3/H4 tetramers, recapitulating the in vivo requirement of Asf1 for H3-K56 acetylation using recombinant proteins. Moreover, the Rtt109-Vps75 complex interacts with Asf1-H3/H4 but not Asf1. In vivo, the Rtt109-Asf1 interaction is also dependent on the ability of Asf1 to bind H3/H4. Furthermore, the Rtt109 homolog in Schizosaccharomyces pombe (SpRtt109) also displayed an Asf1-dependent H3-K56 HAT activity in vitro. These results indicate that Asf1 regulates H3-K56 acetylation by presenting histones H3 and H4 to Rtt109-Vps575 for acetylation, and this mechanism is likely to be conserved. Finally, we have shown that cells lacking Rtt109 or expressing H3-K56 mutants exhibited significant reduction in the association of three proteins with stalled DNA replication forks and hyper-recombination of replication forks stalled at replication fork barriers of the ribosomal DNA locus compared with wild-type cells. Taken together, these studies provide novel insight into the role of Asf1 in the regulation of H3-K56 acetylation and the function of this modification in DNA replication.     

蓝色长盾金小蜂对橡副珠蜡蚧的控制作用

蓝色长盾金小蜂Scutellista caerulea Fonscolombe是橡胶树重要害虫橡副珠蜡蚧Parasaissetia nigra Nietner的一种外寄生性天敌。为了明确蓝色长盾金小蜂对橡副珠蜡蚧的控害潜能,为该寄生蜂的进一步利用提供数据支撑。在室内通过体视镜下解剖观察寄生蜂在寄主腹下是否产卵测定了不同温度、寄主发育阶段下蓝色长盾金小蜂的寄生功能反应、寻找效应和自身密度干扰效应。温度设置有21、24、27、30、33、36℃共6个处理,寄主发育阶段设初期成虫（1-2 d成虫）、褐色期成虫（体色褐色,产卵前3-4 d成虫）、黑色期成虫（体色黑色,产卵1-2 d成虫）三个处理。结果显示：蓝色长盾金小蜂对橡副珠蜡蚧的寄生功能反应符合Holling-Ⅱ型和Holling-Ⅲ型模型,在21-36℃范围内,33℃时该蜂寄生效能最大,为44.4201,21℃时最小,为9.2458;在橡副珠蜡蚧为初期成虫-黑色期成虫范围内,寄生效能由大到小为黑色期成虫>褐色期成虫>初期成虫,分别为18.9044、13.7410、7.2002。采用Hassell-Varley干扰模型对不同温度下蓝色长盾金小蜂受自身密度干扰的寄生作用率进行拟合,表明蓝色长盾金小蜂在寄生时存在种群内个体间自我干扰情况。温度会影响蓝色长盾金小蜂的搜寻和自我干扰,在21-33℃范围内,33℃搜寻常数和干扰常数达到最大值,分别为0.6116、0.7535。当温度为33℃,寄主发育阶段为黑色期成虫时,蓝色长盾金小蜂对橡副珠蜡蚧有较强的控制能力,自身干扰作用最强。