Engineering of Plasmin-Resistant Forms of Streptokinase and Their Production in Bacillus subtilis: Streptokinase with Longer Functional Half-Life

The short in vivo half-life of streptokinase limits its efficacy as an efficient blood clot-dissolving agent. During the clot-dissolving process, streptokinase is processed to smaller intermediates by plasmin. Two of the major processing sites are Lys59 and Lys386. We engineered two versions of streptokinase with either one of the lysine residues changed to glutamine and a third version with both mutations. These mutant streptokinase proteins (muteins) were produced by secretion with the protease-deficient Bacillus subtilis WB600 as the host. The purified muteins retained comparable kinetics parameters in plasminogen activation and showed different degrees of resistance to plasmin depending on the nature of the mutation. Muteins with double mutations had half-lives that were extended 21-fold when assayed in a 1:1 molar ratio with plasminogen in vitro and showed better plasminogen activation activity with time in the radial caseinolysis assay. This study indicates that plasmin-mediated processing leads to the inactivation of streptokinase and is not required to convert streptokinase to its active form. Plasmin-resistant forms of streptokinase can be engineered without affecting their activity, and blockage of the N-terminal cleavage site is essential to generate engineered streptokinase with a longer in vitro functional half-life.     

Bisphenol A exposure at an environmentally relevant dose induces meiotic abnormalities in adult male rats

Whether environmental exposure to bisphenol A (BPA) may induce reproductive disorders is still controversial but certain studies have reported that BPA may cause meiotic abnormalities in C. elegans and female mice. However, little is known about the effect of BPA on meiosis in adult males. To determine whether BPA exposure at an environmentally relevant dose could induce meiotic abnormalities in adult male rats, we exposed 9-week-old male Wistar rats to BPA by gavage at 20 μg/kg body weight (bw)/day for 60 consecutive days. We found that BPA significantly increased the proportion of stage VII seminiferous epithelium and decreased the proportion of stage VIII. Consequently, spermiation was inhibited and spermatogenesis was disrupted. Further investigation revealed that BPA exposure delayed meiosis initiation in the early meiotic stage and induced the accumulation of chromosomal abnormalities and meiotic DNA double-strand breaks (DSBs) in the late meiotic stage. The latter event subsequently activated the phosphatidylinositol 3-kinase-related protein kinase (ATM). Our results suggest that long-term exposure to BPA may lead to continuous meiotic abnormalities and ultimately put mammalian reproductive health at risk.     

Expression and characterization of an <Emphasis Type="Italic">α</Emphasis>-glucosidase from <Emphasis Type="Italic">Thermoanaerobacter ethanolicus</Emphasis> JW200 with potential for industrial application

In this study, a new α-glucosidase gene from Thermoanaerobacter ethanolicus JW200 was cloned and expressed in Escherichia coli by a novel heat-shock vector pHsh. The recombinant α-glucosidase exhibited its maximum hydrolytic activity at 70°C and pH 5.0∼5.5. With p-nitrophenyl-α-D-glucoside as a substrate and under the optimal condition (70°C, pH 5.5), K _m and V _max of the enzyme was 1.72 mM and 39 U/mg, respectively. The purified α-glucosidase could hydrolyze oligosaccharides with both α-1,4 and α-1,6 linkages. The enzyme also had strong transglycosylation activity when maltose was used as sugar donor. The transglucosylation products towards maltose are isomaltose, maltotriose, panose, isomaltotriose and tetrasaccharides. The enzyme could convert 400 g/L maltose to oligosaccharides with a conversion rate of 52%, and 83% of the oligosaccharides formed were prebiotic isomaltooligosaccharides (containing isomaltose, panose and isomaltotriose).     

DDPH对低氧内皮细胞条件培养液诱导肺动脉平滑肌细胞增殖及α-SM-actin表达的影响

目的探讨1-(2,6-二甲基苯氧基)-2-(3,4-二甲氧基苯乙氨基)丙烷盐酸盐(DDPH)抑制低氧内皮细胞条件培养液(HECCM)诱导肺动脉平滑肌细胞增殖及对α-SM-actin表达的影响.方法利用低氧内皮细胞条件培养液建立猪肺动脉平滑肌细胞(PASMC)的增殖模型;以四甲基偶氮唑盐(MTT)比色法、α平滑肌肌动蛋白(α-SM-actin)为指标,采用免疫细胞化学染色法观察低氧内皮细胞条件培养液对肺动脉平滑肌细胞增殖的影响以及DDPH对低氧内皮细胞条件培养液促肺动脉平滑肌细胞增殖后的逆转效应.结果低氧内皮细胞条件培养液显著促进肺动脉平滑肌细胞增殖,低氧内皮细胞条件培养液促肺动脉平滑肌细胞增殖后,肺动脉平滑肌细胞的表型发生转化,由收缩表型转化为合成表型,肺动脉平滑肌细胞胞浆内的α-SM-actin含量下降;DDPH能显著抑制低氧内皮细胞条件培养液对肺动脉平滑肌细胞的增殖作用,并使肺动脉平滑肌细胞的表型发生逆转,即由合成表型逆转为具有执行正常收缩功能的收缩表型,肺动脉平滑肌细胞胞浆内的α-SM-actin含量回升.结论提示DDPH能显著抑制低氧内皮细胞条件培养液促肺动脉平滑肌细胞的增殖作用,其作用机制可能是通过肺动脉平滑肌细胞的表型发生逆转来实现的.     

Influence of pulsed electromagnetic field with different pulse duty cycles on neurite outgrowth in PC12 rat pheochromocytoma cells

The influence of low frequency (50 Hz repetition rate) pulsed electromagnetic field (EMF) on PC12 cell neurite outgrowth in vitro was investigated in this study. We studied the percentage of neurite bearing cells, average length of neurites, and directivity of neurite outgrowth in PC12 cells cultured for 96 h in the presence of nerve growth factor (NGF). PC12 cells were exposed in one incubator to pulsed EMF at 1.36 mT (peak value) generated by a pair of Helmholtz coils, and the control samples were placed in another identical incubator. We found that the pulse duty cycle had significant effect on neurite outgrowth. Low (10%) pulse on-time significantly inhibited the percentage of neurite bearing cells, but at the same time increased the average length of neurites, while 100% on-time (DC) had exactly the opposite effects. Furthermore, we found that neurites were prone to extend along the direction of pulsed EMF with 10% pulse on-time. Our studies show that neurite outgrowth in PC12 cells is sensitive to the pulse duty and this sensitivity was associated with NGF concentration.     

Change in floral orientation in Anisodus luridus (Solanaceae) protects pollen grains and facilitates development of fertilized ovules

? Premise of the study: Some floral traits could be selected by pollinators and nonpollinator agents, and studying the floral traits shaped by physical agents could reveal adaptive mechanisms to the environment. We explored the adaptive significance of the change in floral orientation, from pendulous flowers to erect fruits that have a persistent calyx, in Anisodus luridus, a perennial native to the Qinghai-Tibet Plateau (QTP). ? Methods: We examined the effect of flower orientation on pollinator visitation rates, pollen deposition efficiency, pollen quantity, and pollen viability to estimate whether pendulous flowers have improved male fitness. We then measured seed production and seed germination rate to assess whether erect fruits have enhanced female fitness. ? Key results: Pendulous flowers did not have any preferred pollinators or increased pollen deposition. In artificially erected flowers, the number of pollen grains greatly decreased after rainwash. Pollen germination experiments indicated that pollen damage by water and exposure to solar radiation is serious. In the persistent calyx that holds water within it, the temperature inside the calyx changed slower than in the calyx that had the water removed. After supplemental pollination, the seed number, seed set, and seed mass of fruits that had water removed from the calyx were reduced greatly in both years. ? Conclusions: We conclude that the change in floral orientation could enhance male and female fitness of A. luridus and is effectively adaptive to the alpine environments, indicating a strong selection by the combined pressure from various abiotic nonpollinator agents in shaping the floral traits of this alpine plant.     

miRConnect: identifying effector genes of miRNAs and miRNA families in cancer cells

micro(mi)RNAs are small non-coding RNAs that negatively regulate expression of most mRNAs. They are powerful regulators of various differentiation stages, and the expression of genes that either negatively or positively correlate with expressed miRNAs is expected to hold information on the biological state of the cell and, hence, of the function of the expressed miRNAs. We have compared the large amount of available gene array data on the steady state system of the NCI60 cell lines to two different data sets containing information on the expression of 583 individual miRNAs. In addition, we have generated custom data sets containing expression information of 54 miRNA families sharing the same seed match. We have developed a novel strategy for correlating miRNAs with individual genes based on a summed Pearson Correlation Coefficient (sPCC) that mimics an in silico titration experiment. By focusing on the genes that correlate with the expression of miRNAs without necessarily being direct targets of miRNAs, we have clustered miRNAs into different functional groups. This has resulted in the identification of three novel miRNAs that are linked to the epithelial-to-mesenchymal transition (EMT) in addition to the known EMT regulators of the miR-200 miRNA family. In addition, an analysis of gene signatures associated with EMT, c-MYC activity, and ribosomal protein gene expression allowed us to assign different activities to each of the functional clusters of miRNAs. All correlation data are available via a web interface that allows investigators to identify genes whose expression correlates with the expression of single miRNAs or entire miRNA families. miRConnect.org will aid in identifying pathways regulated by miRNAs without requiring specific knowledge of miRNA targets.     

Potent inhibition of human immunodeficiency virus type 1 in primary T cells and alveolar macrophages by a combination anti-Rev strategy delivered in an adeno-associated virus vector.

The rate of viral replication appears to play a pivotal role in human immunodeficiency virus type 1 (HIV-1) pathogenesis and disease progression as it outstrips the capacity of the immune system to respond. Important cellular sites for HIV-1 production include T lymphocytes and tissue macrophages. Antiviral strategies, including newer treatment modalities such as gene therapy of HIV-1-susceptible cell populations, must be capable of engendering durable inhibitory effects to HIV-1 replication in both of these primary cell types in order to be effective. Among the potential genetic targets for intervention in the HIV-1 life cycle, the Rev regulatory system, consisting of Rev and its binding site, the Rev-responsive element (RRE), stands out as particularly attractive. Rev is essential for maintaining the stability of the viral genomic RNA as well as viral mRNAs encoding key structural and regulatory proteins. Moreover, it exhibits favorable threshold kinetics, in that Rev concentrations must rise above a critical level to exert their effect. To disable Rev function, primary T cells or macrophages were transduced with anti-Rev single-chain immunoglobulin (SFv) or RRE decoy genes either singly or in combination by employing adeno-associated virus vectors and then challenged with HIV-1. By directing both a protein and a nucleic acid against the normal interaction between Rev and the RRE, this genetic antiviral strategy effectively inhibited infection by either clinical or laboratory virus isolates. These results provide a framework for novel interventions to reduce virus production in the infected host.