藜磷酸肌醇5-磷酸酶基因(CaP5P)的表达分析和胁迫响应检测

植物通过多种信号途径感知外界环境变化,从而引发一系列的信号级联反应,其中磷脂信号途径起着重要的作用。本研究从藜盐胁迫差减文库中获得了一个EST05序列,利用RT-PCR检测了其在非生物胁迫下表达趋势,显示EST05序列在非生物胁迫下的表达有升高。随后利用RACE技术获得其全长编码序列,测序结果显示其与蓖麻(Ricinus communis) 的磷酸肌醇5-磷酸酶基因(P5P)的同源性达56%,遂将其命名为CaP5P。最后过量表达CaP5P在拟南芥中初步验证了功能,转基因植株表现对盐胁迫耐受性降低的趋势,呈现负调控的特征。本研究对磷酸肌醇5-磷酸酶基因功能的初步研究为磷脂信号途径中负调控组分的分析提供了参考依据。     

The influence of hybridization between African and European honeybees, Apis mellifera, on asymmetries in wing size and shape

Abstract We examined the possible role of hybridization in the invasion process of the African honeybee by testing two hypotheses regarding fluctuating asymmetry (FA), a measure of developmental stability, in wing characteristics: (1) FA should be higher in hybrid versus parental genotypes of African and European races; (2) FA should be lower in African bees compared to hybrid and European workers. Parental and reciprocal hybrid worker genotypes were cross fostered in common-hive rearing environments. We did not find greater FA for wing size and shape in the hybrids compared to both parental types. However, we did find significantly lower FA of shape in the African workers compared to the European and hybrid workers, suggesting that European bees and their hybrids may have compromised fitness relative to African bees. We also found that the two hybrid genotypes significantly differed in overall wing size and shape. If these differences affect wing aerodynamics, then the paternity of hybrids may influence worker performance and could potentially contribute to the loss of European matrilines. Hybridization had few consistent effects on directional asymmetry for wing size and shape. Genotypic factors played a far greater role in determining the effect of hybridization on wing morphology than did differences in rearing environment. Thus, African bees may have lower FA for wing shape (and by inference greater developmental stability) relative to European and hybrid workers, which may contribute to the ability of African bees to displace European honeybee races in invaded regions.     

Expression differences of miRNAs and genes on NF-κB pathway between the healthy and the mastitis Chinese Holstein cows

In order to discover the variation of microRNAs and genes associated with NF-κB signaling pathway between the healthy and the mastitis Chinese Holstein cows, Illumina Deep Sequencing and qRT-PCR are applied to detect 25 kinds of miRNAs (miR-16, miR-125b, miR-15, miR-29a, miR-23b, miR-146, miR-301a, miR-181b, let-7, miR-30b, miR-21, miR-223, miR-27b, miR-10a, miR-143, etc.) expression levels in blood samples and 14 genes (RelA, RelB, Rel, p105, p100, IκBα, IκBβ, IκBδ, IκBε, IκBζ, Bcl-3, IKKα, IKKβ, IKKγ/NEMO) relative expression levels in nine tissues. The total number of miRNAs is declining, and RelA, Rel, p105, p100, IκBα, IκBβ, IκBδ, IκBζ, Bcl-3, and IKKα expressions are rising in mastitis individuals. So, we suppose that NF-κB pathway is active in mastitis individuals as a result of the decrease inhibition of miRNAs. While in healthy ones, the NF-κB pathway is inactive, because of the miRNAs enhanced inhibition action. However, the specific regulatory mechanism of miRNAs on NF-κB pathway in mastitis Holstein cows needs further investigation. Moreover, due to obvious expression differences, some miRNAs, especially miR-16 and miR-223, may be used as new markers for the dairy mastitis prognosing.     

A missense mutant myostatin causes hyperplasia without hypertrophy in the mouse muscle

Myostatin, which is a member of the TGF-beta superfamily, is a negative regulator of skeletal muscle formation. Double-muscled Piedmontese cattle have a C313Y mutation in myostatin and show increased skeletal muscle mass which resulted from an increase of myofiber number (hyperplasia) without that of myofiber size (hypertrophy). To examine whether this mutation in myostatin gene affects muscle development in a dominant negative manner, we generated transgenic mice overexpressing the mutated gene. The transgenic mice exhibited dramatic increases in the skeletal muscle mass resulting from hyperplasia without hypertrophy. In contrast, it has been reported that a myostatin mutated at its cleavage site produces hypertrophy without hyperplasia in the muscle. Thus, these results suggest that (1) the myostatin containing the missense mutation exhibits a dominant negative activity and that (2) there are two types in the dominant negative form of myostatin, causing either hypertrophy or hyperplasia.     

The p38 MAPK inhibitor, PD169316, inhibits transforming growth factor beta-induced Smad signaling in human ovarian cancer cells

Transforming growth factor beta (TGFbeta) can signal through a variety of Smad-independent pathways, including the p38 MAPK pathway. Recent work has shown that inhibitors of p38 MAPK, such as SB203580 and SB202190, can inhibit signaling induced by TGFbeta. Here we show that another p38 MAPK inhibitor, PD169316, abrogates signaling initiated by both TGFbeta and Activin A, but not bone morphogenetic protein (BMP) 4. Inhibition of TGFbeta signaling is dose dependent and results in reduced Smad2 and Smad3 phosphorylation, nuclear translocation, and up-regulation of the TGFbeta target gene Smad7. Reduced TGFbeta signaling is not due to abrogation of p38 MAPK activity, since blocking p38 MAPK activity with a dominant negative form of p38 MAPK has no effect on TGFbeta/Smad signaling. Our results show that use of PD169316 at 5 MICROM or higher can block TGFbeta signaling activity and thus caution must be used when attributing cellular activities exclusively to p38 MAPK signaling when these inhibitors are used experimentally.     

Evidence for interplay between genes and maternal stress in utero: monoamine oxidase A polymorphism moderates effects of life events during pregnancy on infant negative emotionality at 5 weeks

The low activity variant of the monoamine oxidase A (MAOA) functional promoter polymorphism, MAOA‐LPR, in interaction with adverse environments (G × E) is associated with child and adult antisocial behaviour disorders. MAOA is expressed during foetal development so in utero G × E may influence early neurodevelopment. We tested the hypothesis that MAOA G × E during pregnancy predicts infant negative emotionality soon after birth. In an epidemiological longitudinal study starting in pregnancy, using a two stage stratified design, we ascertained MAOA‐LPR status (low vs. high activity variants) from the saliva of 209 infants (104 boys and 105 girls), and examined predictions to observed infant negative emotionality at 5 weeks post‐partum from life events during pregnancy. In analyses weighted to provide estimates for the general population, and including possible confounders for life events, there was an MAOA status by life events interaction (P = 0.017). There was also an interaction between MAOA status and neighbourhood deprivation (P = 0.028). Both interactions arose from a greater effect of increasing life events on negative emotionality in the MAOA‐LPR low activity, compared with MAOA‐LPR high activity infants. The study provides the first evidence of moderation by MAOA‐LPR of the effect of the social environment in pregnancy on negative emotionality in infancy, an early risk for the development of child and adult antisocial behaviour disorders .     

假单胞菌Pseudomonas fluorescens Pf0-1中转录调控因子SpfB负调控DNA磷硫酰化修饰

[目的]DNA磷硫酰化修饰是DNA骨架上非桥接的氧原子以序列选择性和R-构型被硫取代的一种新型DNA修饰。目前,磷硫酰化修饰在多种细菌、古生菌以及人类致病菌中多有发现,但其分子调控机制尚不清楚。为了全面解析磷硫酰化修饰的调控机制,本文选择荧光假单胞菌Pf0-1为研究对象,开展了其DNA磷硫酰化修饰的调控机制研究。[方法]首先,构建了spfB基因缺失和回补菌株,使用碘能特异性断裂磷硫酰化修饰DNA的方法,研究了该基因缺失对修饰表型的影响。利用cDNA在相邻同方向的基因间隔区进行PCR,确定了磷硫酰化修饰基因簇spfBCDE内的共转录单元。通过荧光定量RT-PCR,分析了spfB基因缺失突变株中磷硫酰化修饰基因的转录量。利用异源表达并纯化得到的重组蛋白SpfB进行了体外功能研究。通过EMSA实验,验证了SpfB蛋白具有与spfB启动子序列结合活性。通过DNase I footprinting实验,精确定位了SpfB蛋白与DNA结合序列。[结果]spfB基因的缺失加剧了磷硫酰化修饰DNA断裂所致电泳条带弥散的表型,spfB基因的回补能够恢复该表型,证明spfB基因负调控磷硫酰化修饰。鉴定了spf基因簇中只含有1个共转录单元,且该共转录单元在△spfB突变株中转录水平明显上升。通过EMSA和DNase I footprint实验,检测了SpfB蛋白与磷硫酰化修饰基因spfBCDE的启动子区域5''-TGTTTGT-3''相结合。[结论]SpfB作为转录调控因子负调控磷硫酰化修饰基因spfBCDE的表达,为解析磷硫酰化修饰的调控机制和全面理解基因组上的部分修饰特征奠定了基础。     

Inhibition of endogenous thioredoxin-1 in the heart of transgenic mice does not confer cardioprotection in ischemic postconditioning

Thioredoxin-1 maintains the cellular redox status and decreases the infarct size in ischemia/reperfusion injury. However, whether the increase of thioredoxin-1 expression or its lack of activity modifies the protection conferred by ischemic postconditioning has not been yet elucidated. The aim was to evaluate if the thioredoxin-1 overexpression enhances the posctconditioning protective effect, and whether the lack of the activity abolishes the reduction of the infarct size. Wild type mice hearts, transgenic mice hearts overexpressing thioredoxin-1, and a dominant negative mutant (C32S/C35S) of thioredoxin-1 were used. The hearts were subjected to 30 min of ischemia and 120 min of reperfusion (Langendorff) (I/R group) or to postconditioning protocol (PostC group). The infarct size in the Wt-PostC group decreased in comparison to the Wt-I/R group (54.6 ± 2.4 vs. 39.2 ± 2.1%, p < 0.05), but this protection was abolished in DN-Trx1-PostC group (49.7 ± 1.1%). The ischemia/reperfusion and postconditioning in mice overexpressing thioredoxin-1 reduced infarct size at the same magnitude (35.9 ± 2.1 and 38.4 ± 1.3%, p < 0.05 vs. Wt-I/R). In Wt-PostC, Trx1-I/R and Trx1- PostC, Akt and GSK3β phosphorylation increased compared to Wt-I/R, without changes in DN-Trx1 groups. In conclusion, given that the cardioprotection conferred by thioredoxin-1 overexpression and postconditioning, is accomplished through the activation of the Akt/GSK3β survival pathway, no synergic effect was evidenced. Thioredoxin-1 plays a key role in the postconditioning, given that when this protein is inactive the cardioprotective mechanism was abolished. Thus, diverse comorbidities or situations modifying the thioredoxin activity, could explain the absence of this strong mechanism of protection in different clinical situations.     

Factors Affecting Events During Oxidation of Low Density Lipoprotein: Correlation of Multiple Parameters of Oxidation

The present study shows that copper oxidation of LDL is a tightly-ordered process which can be finely controlled by appropriate selection of duration of oxidation and of concentrations of LDL and copper. Oxidation of LDL (0.1-2.0 mg LDL protein/ml) was carried out by copper catalysis (in the ratio of 2.5 μM Cu²⁺ to 0.1 mg LDL protein/ml) in phosphate-buffered saline, and was monitored by agarose gel electro-phoresis, gas chromatography (GC), anion exchange fast protein liquid chromatography (FPLC), fluorescence spectroscopy and dynamic light scattering. Analysis of the data showed strong cross correlations between many of the parameters of oxidation. Oxidation was more rapid for lower concentrations than for higher concentrations of LDL, despite the same ratio of copper to LDL being employed. Chemical kinetics analysis of the GC data suggested that 7β-hydroxycholesterol formation occurred as a first order (or pseudo first order) consecutive reaction to the oxidation of linoleate. The first order rate constants for decomposition of lioleate and production of 7β-hydroxycholesterol correlated closely with the theoretically-calculated times between collision of LDL particles. LDL particle diameter, measured by dynamic light scattering, increased by ca. 50% over 24 h oxidation, suggesting unfolding of apo B-100.

Prolonged oxidation of LDL at low concentration suggested that the radical chain reaction was able to propagate, albeit slowly, on cholesterol after all the polyunsaturated fatty acid was consumed. For higher concentrations of LDL, prolonged oxidation resulted in partial aggregation. These findings are applicable to preparing oxidised LDL with different degrees of oxidation, under controlled conditions, for studying its biological properties.