A novel thermophilic endo-β-1,4-mannanase from Aspergillus nidulans XZ3: functional roles of carbohydrate-binding module and Thr/Ser-rich linker region

The gene man5XZ3 from Aspergillus nidulans XZ3 encodes a multimodular β-mannanase of glycoside hydrolase family 5 that consists of a family 1 carbohydrate-binding module (CBM1), a Thr/Ser-rich linker region, and a catalytic domain. Recombinant Man5XZ3 and its two truncated derivatives, Man5ΔCBM (removing the CBM1) and Man5ΔCL (removing both the CBM1 and linker region), were produced in Pichia pastoris and showed significant variance in the secondary structure. The three enzymes had similar biochemical properties, such as optimal pH and temperature (pH 5.0 and 80 °C) and excellent pH stability at pH 4.0–10.0. Removal of the CBM1 alone could improve the thermostability of Man5XZ3, but further removal of the linker region resulted in worse thermostability. Man5XZ3 retained greater enzyme activity in the presence of an organic solvent (acetone), two detergents (SDS and Triton X-100), and a chaotropic agent (urea) compared with Man5ΔCBM and Man5ΔCL. This study provides an excellent β-mannanase candidate favorable for various industries and primarily demonstrates the relationship between enzyme structure and function.     

Aerosol Performance and Long-Term Stability of Surfactant-Associated Liposomal Ciprofloxacin Formulations with Modified Encapsulation and Release Properties

Previously, we showed that the encapsulation and release properties of a liposomal ciprofloxacin formulation could be modified post manufacture, by addition of surfactant in concert with osmotic swelling of the liposomes. This strategy may provide more flexibility and convenience than the alternative of manufacturing multiple batches of liposomes differing in composition to cover a wide range of release profiles. The goal of this study was to develop a surfactant-associated liposomal ciprofloxacin (CFI) formulation possessing good long-term stability which could be delivered as an inhaled aerosol. Preparations of 12.5 mg/ml CFI containing 0.4% polysorbate 20 were formulated between pH 4.7 and 5.5. These formulations, before and after mesh nebulization, and after refrigerated storage for up to 2 years, were characterized in terms of liposome structure by cryogenic transmission electron microscopy (cryo-TEM) imaging, vesicle size by dynamic light scattering, pH, drug encapsulation by centrifugation-filtration, and in vitro release (IVR) performance. Within the narrower pH range of 4.9 to 5.2, these formulations retained their physicochemical stability after 2-year refrigerated storage, were robust to mesh nebulization, and formed respirable aerosols with a volume mean diameter (VMD) of 3.7 μm and a geometric standard deviation (GSD) of 1.7. This study demonstrates that it may be possible to provide a range of release profiles by simple addition of surfactant to a liposomal formulation post manufacture, and that these formulations may retain their physicochemical properties after long-term refrigerated storage and following aerosolization by mesh nebulizer.KEY WORDS: ciprofloxacin, drug delivery, liposome, nebulized aerosol, surfactant     

Ubiquitination of Inositol-requiring Enzyme 1 (IRE1) by the E3 Ligase CHIP Mediates the IRE1/TRAF2/JNK Pathway

Deciphering the inositol-requiring enzyme 1 (IRE1) signaling pathway is fundamentally important for understanding the unfolded protein response (UPR). The ubiquitination of proteins residing on the endoplasmic reticulum (ER) membrane has been reported to be involved in the UPR, although the mechanism has yet to be fully elucidated. Using immunoprecipitation and mass spectrometry, IRE1 was identified as a substrate of the E3 ligase CHIP (carboxyl terminus of HSC70-interacting protein) in HEK293 cells under geldanamycin-induced ER stress. Two residues of IRE1, Lys⁵⁴⁵ and Lys⁸²⁸, were targeted for Lys⁶³-linked ubiquitination. Moreover, in CHIP knockdown cells, IRE1 phosphorylation and the IRE1-TRAF2 interaction were nearly abolished under ER stress, which may be due to lacking ubiquitination of IRE1 on Lys⁵⁴⁵ and Lys⁸²⁸, respectively. The cellular responses were evaluated, and the data indicated that CHIP-regulated IRE1/TRAF2/JNK signaling antagonized the senescence process. Therefore, our findings suggest that CHIP-mediated ubiquitination of IRE1 contributes to the dynamic regulation of the UPR.     

A Thermostable Glucoamylase from Bispora sp. MEY-1 with Stability over a Broad pH Range and Significant Starch Hydrolysis Capacity

Background

Glucoamylase is an exo-type enzyme that converts starch completely into glucose from the non-reducing ends. To meet the industrial requirements for starch processing, a glucoamylase with excellent thermostability, raw-starch degradation ability and high glucose yield is much needed. In the present study we selected the excellent Carbohydrate-Activity Enzyme (CAZyme) producer, Bispora sp. MEY-1, as the microbial source for glucoamylase gene exploitation.

Methodology/Principal Findings

A glucoamylase gene (gla15) was cloned from Bispora sp. MEY-1 and successfully expressed in Pichia pastoris with a high yield of 34.1 U/ml. Deduced GLA15 exhibits the highest identity of 64.2% to the glucoamylase from Talaromyces (Rasamsonia) emersonii. Purified recombinant GLA15 was thermophilic and showed the maximum activity at 70°C. The enzyme was stable over a broad pH range (2.2–11.0) and at high temperature up to 70°C. It hydrolyzed the breakages of both α-1,4- and α-1,6-glycosidic linkages in amylopectin, soluble starch, amylose, and maltooligosaccharides, and had capacity to degrade raw starch. TLC and H¹-NMR analysis showed that GLA15 is a typical glucoamylase of GH family 15 that releases glucose units from the non-reducing ends of α-glucans. The combination of Bacillus licheniformis amylase and GLA15 hydrolyzed 96.14% of gelatinized maize starch after 6 h incubation, which was about 9% higher than that of the combination with a commercial glucoamylase from Aspergillus niger.

Conclusion/Significance

GLA15 has a broad pH stability range, high-temperature thermostability, high starch hydrolysis capacity and high expression yield. In comparison with the commercial glucoamylase from A. niger, GLA15 represents a better candidate for application in the food industry including production of glucose, glucose syrups, and high-fructose corn syrups.     

RNAi-Directed Downregulation of Vacuolar H+ -ATPase Subunit A Results in Enhanced Stomatal Aperture and Density in Rice

Stomatal movement plays a key role in plant development and response to drought and salt stress by regulating gas exchange and water loss. A number of genes have been demonstrated to be involved in the regulation of this process. Using inverse genetics approach, we characterized the function of a rice (Oryza sativa L.) vacuolar H⁺-ATPase subunit A (OsVHA-A) gene in stomatal conductance regulation and physiological response to salt and osmotic stress. OsVHA-A was constitutively expressed in different rice tissues, and the fusion protein of GFP-OsVHA-A was exclusively targeted to tonoplast when transiently expressed in the onion epidermal cells. Heterologous expression of OsVHA-A was able to rescue the yeast mutant vma1Δ (lacking subunit A activity) phenotype, suggesting that it partially restores the activity of V-ATPase. Meanwhile, RNAi-directed knockdown of OsVHA-A led to a reduction of vacuolar H⁺-ATPase activity and an enhancement of plasma membrane H⁺-ATPase activity, thereby increasing the concentrations of extracellular H⁺ and intracellular K⁺ and Na⁺ under stress conditions. Knockdown of OsVHA-A also resulted in the upregulation of PAM3 (plasma membrane H⁺-ATPase 3) and downregulation of CAM1 (calmodulin 1), CAM3 (calmodulin 3) and YDA1 (YODA, a MAPKK gene). Altered level of the ion concentration and the gene expression by knockdown of OsVHA-A probably resulted in expanded aperture of stomatal pores and increased stomatal density. In addition, OsVHA-A RNAi plants displayed significant growth inhibition under salt and osmotic stress conditions. Taken together, our results suggest that OsVHA-A takes part in regulating stomatal density and opening via interfering with pH value and ionic equilibrium in guard cells and thereby affects the growth of rice plants.     

过氧亚硝基阴离子通过JAK/STAT信号途径促进系膜细胞纤连蛋白的合成

氧化应激是糖尿病肾病的重要发病机制之一。过氧亚硝基阴离子(peroxynitrite,ONOO–)是参与氧化应激损伤的重要成员,与糖尿病及其并发症密切相关。该文观察高糖环境下ONOO–对系膜细胞合成纤连蛋白(fibronectin,FN)的影响,并探讨其作用机制。实验中,人肾小球系膜细胞分为4组:正常对照组、高糖组、高糖+尿酸组及高糖+AG490组。培养12,24,48 h后收集细胞及其上清液、并提取细胞总蛋白。采用酶联免疫吸附实验(ELISA)检测细胞上清液中FN的含量,采用免疫细胞化学和Western blot检测NT总蛋白(ONOO–生成的生物标志物)、p-JAK2及p-STAT3蛋白的表达。结果显示,与同期正常组相比,高糖组NT总蛋白、p-JAK2及p-STAT3的表达及FN含量明显增高(P<0.05),并且随着时间的延长表达逐渐增多,以48 h组最为显著;高糖+尿酸组,NT、p-JAK2、p-STAT3及FN较高糖组明显减少(P<0.05);高糖+AG490组,p-JAK2、p-STAT3及FN较高糖组明显减少(P<0.05),但NT表达与高糖组差异无统计学意义(P>0.05)。由此可见,高糖环境下系膜细胞中存在ONOO–的过量表达,ONOO–通过JAK/STAT信号途径促进系膜细胞FN的合成。     

海拔梯度变化对武夷山黄山松林土壤磷组分和有效性的影响

土壤磷（P）是植物生长必需的养分元素,也是亚热带森林生产力的主要限制元素。目前,关于不同海拔土壤P组分和P有效性的变化规律尚无统一定论,其原因主要是忽略了植被类型变化导致的P组分和P有效性对海拔的响应更为复杂。因此,以武夷山不同海拔黄山松林为研究对象,通过测定土壤环境因素、理化性质、微生物生物量（SMB）、酸性磷酸单酯酶（ACP）和磷酸双酯酶（PD）活性以及土壤P组分,探究土壤P组分和P有效性的变化及其影响因素。结果表明,随海拔降低,速效P含量显著增加,而易分解P、中等易分解P、难利用P和总磷含量显著减少。冗余分析结果表明,微生物生物量磷和微生物生物量氮是影响土壤P组分和P有效性变化的关键因素。研究表明,随海拔降低,黄山松林土壤微生物通过提高ACP、PD活性和降低SMB含量的能量分配策略,促进更多较难分解P组分的矿化,从而提高速效P含量,以满足微生物对P的需求。因此,在低海拔地区,微生物通过能量分配策略获取更多有效P,可能有利于提高武夷山黄山松林土壤速效P的供应,但从长期来看,可能使P矿化速率提高和P损耗增加,导致P库的储备不足,不利于土壤P素养分的可持续供应。     

重组阳离子抗肿瘤肽AIK的原核表达、纯化及活性测定

利用Gateway克隆技术构建重组抗瘤肽AIK的原核表达体系,建立表达及纯化重组AIK的最优条件,为深入研究和利用AIK奠定基础。首先,设计含AttB重组位点的引物,通过重叠PCR技术扩增出Att B-TEV-FLAG-AIK序列,利用BP重组反应将目的序列TEV-FLAG-AIK克隆到供体载体pDONR223中,构建入门载体,再通过LR重组反应,将目的序列转移到目的载体pDEST15中,构建GST-AIK融合蛋白原核表达质粒。随后,在BL21(DE3)工程菌中优化诱导融合蛋白表达的条件。以谷胱甘肽磁珠纯化GST-AIK融合蛋白,再以rTEV酶切除GST,获得FLAG-AIK重组蛋白。最后以MTS法检测FLAG-AIK对白血病细胞HL-60的细胞毒性。菌液PCR验证和测序分析表明成功构建了重组抗瘤肽AIK的入门质粒和原核表达质粒。在BL21(DE3)工程菌中实现了GST-AIK融合蛋白的高效可溶性表达。并测得在37℃下以0.1 mmol/L IPTG诱导工程菌(OD600=1.0)4 h,重组蛋白表达量占菌体总蛋白的30%以上。经GST亲和层析、rTEV酶切除GST标签及二次GST亲和层析获得纯度高于95%的FLAG-AIK蛋白。MTS法测得所制备的FLAG-AIK蛋白抑瘤活性与化学合成的AIK相当。总之,本课题应用Gateway克隆系统成功构建了抗瘤肽AIK的原核表达质粒,实现了GST-AIK融合蛋白的高效可溶性表达,经亲和层析获得了有生物活性的重组AIK多肽,为后续深入研究和大规模制备奠定了基础。     

噬菌体与宿主菌间的角逐

噬菌体和它们的宿主菌组成了地球上教目最庞大的微生物种群,噬菌体靠寄生宿主菌来扩增繁衍。但在漫长的进化中,噬菌体与宿主菌间不单是捕食关系,它们间还形成了复杂的相互对抗机制,其中抵抗防御机制的抗防御机制也会促使抗．抗防御机制的产生。从生态学角度来看,噬菌体和宿主菌间的共进化保持着动态平衡。本文综述近年来这一领域的研究,为更清楚地了解噬菌体与宿主菌间的关系和应用提供了参考。     

活疫苗及其生产基质中Sendai病毒RT-PCR检测方法的研究

目的建立检测Sendai病毒的RT-PCR方法并应用于活疫苗及其生产基质中Sendai病毒的检测.方法将Sendai病毒E17株接种9日龄鸡胚尿囊腔,72h后收集尿囊液,用于提取病毒RNA,并逆转录成cDNA,用两对针对Sendai病毒NP基因设计的外引物和内引物分别进行扩增.扩增产物克隆于T-载体,并测序.尿囊液按10倍倍比稀释,进行敏感性实验.将该方法用于检测乙脑减毒活疫苗和用于生产疫苗用的普通级乳地鼠肾中的Sendai病毒.结果外引物和内引物的PCR分别扩增出684bp和248bp的片段,外引物PCR产物的测序结果与Genbank报告的序列完全一致.敏感性实验结果表明,第一次PCR可检测到10-4病毒滴度,巢式PCR可检测到10-7病毒滴度.乙脑减毒活疫苗和乳地鼠肾的检测结果为阴性.结论建立检测Sendai病毒的RT-PCR方法具有很高的特异性和敏感性.