Constitutive production of human leptin by fed-batch culture of recombinant rpoS- Escherichia coli

High-level production of human leptin by fed-batch culture of recombinant Escherichia coli using constitutive promoter system was investigated. For the constitutive expression of the obese gene encoding human leptin, the strong constitutive HCE promoter cloned from the D-amino acid aminotransferase gene of Geobacillus toebii was used. To develop an optimal host-vector system, several different recombinant E. coli strains were compared for leptin production. In flask cultures, E. coli FMJ123, which is a rpoS mutant strain, showed the highest level of leptin production (41% of total proteins). By comparing the expression levels of leptin in several different rpoS- and rpoS+ strains, it could be concluded that rpoS mutation positively affected constitutive production of leptin. For the large-scale production of human leptin, fed-batch cultures of recombinant E. coli FMJ123 were carried out using three different feeding solutions--chemically defined, yeast extract-containing, and casamino acid-containing feeding solutions. Among these, the use of casamino acid-containing feeding solution allowed production of leptin up to 2.1 g/L, which was 2.1- and 1.8-fold higher than that obtained with chemically defined and yeast extract-contained feeding solutions, respectively. These results suggest that the HCE promoter can be used for the efficient production of leptin, and most likely other recombinant proteins, in a constitutive manner.     

Combinatorial polymer electrospun matrices promote physiologically-relevant cardiomyogenic stem cell differentiation

Myocardial infarction results in extensive cardiomyocyte death which can lead to fatal arrhythmias or congestive heart failure. Delivery of stem cells to repopulate damaged cardiac tissue may be an attractive and innovative solution for repairing the damaged heart. Instructive polymer scaffolds with a wide range of properties have been used extensively to direct the differentiation of stem cells. In this study, we have optimized the chemical and mechanical properties of an electrospun polymer mesh for directed differentiation of embryonic stem cells (ESCs) towards a cardiomyogenic lineage. A combinatorial polymer library was prepared by copolymerizing three distinct subunits at varying molar ratios to tune the physicochemical properties of the resulting polymer: hydrophilic polyethylene glycol (PEG), hydrophobic poly(ε-caprolactone) (PCL), and negatively-charged, carboxylated PCL (CPCL). Murine ESCs were cultured on electrospun polymeric scaffolds and their differentiation to cardiomyocytes was assessed through measurements of viability, intracellular reactive oxygen species (ROS), α-myosin heavy chain expression (α-MHC), and intracellular Ca(2+) signaling dynamics. Interestingly, ESCs on the most compliant substrate, 4%PEG-86%PCL-10%CPCL, exhibited the highest α-MHC expression as well as the most mature Ca(2+) signaling dynamics. To investigate the role of scaffold modulus in ESC differentiation, the scaffold fiber density was reduced by altering the electrospinning parameters. The reduced modulus was found to enhance α-MHC gene expression, and promote maturation of myocyte Ca(2+) handling. These data indicate that ESC-derived cardiomyocyte differentiation and maturation can be promoted by tuning the mechanical and chemical properties of polymer scaffold via copolymerization and electrospinning techniques.     

RML1 and RML2, Arabidopsis genes required for cell proliferation at the root tip.

New cells are produced from the meristematic tissues located at the shoot and root tip throughout the life of higher plants. To investigate the genetic mechanism regulating meristematic activity, we isolated and characterized four single-gene, recessive mutants in Arabidopsis thaliana called root meristemless (rml). Complementation tests identified two RML loci; RML1 maps to chromosome IV and RML2 maps to chromosome III. These mutants produce normal embryonic roots that either did not undergo or experienced limited cell division following germination, resulting in primary roots of less than 2.0 mm in length. Mutants can produce lateral and adventitious roots, which can grow to a length comparable to the embryonic root and arrest, indicating that the growth arrest is unrelated to the embryonic dormancy process. Neither the addition of growth regulators to the media nor the removal of shoots can rescue mutant roots from growth arrest, indicating that the mutant phenotype is not caused by a shortage of known growth regulators or by a transmissible shoot inhibitor. Normal cell division ability in mutant embryo, shoot, and callus cells indicates that the RML gene functions are not part of the general cell division processes; rather, they are involved specifically in activating the cell division cycle in the root apical cells.     

Fabrication of degradable carboxymethyl cellulose (CMC) microneedle with laser writing and replica molding process for enhancement of transdermal drug delivery

Transdermal drug delivery system (TDDS) may provide a more reliable method of drug delivery than oral delivery by avoiding gut absorption and first-pass metabolism, but needs a method for efficiently crossing the epidermal barrier. To enhance the delivery through the skin, we have developed a biocompatible, dissolvable microneedle array made from carboxymethyl cellulose (CMC). Using laser ablation for creating the mold greatly improved the efficiency and reduced the cost of microneedle fabrication. Mixing CMC with amylopectin (AP) enhanced the mechanical and tunable dissolution properties of the microneedle for controlled release of model compounds. Using the CMC microneedle array, we observed significant enhancement in the skin permeability of a fluorescent model compound, and also increase in the anti-oxidant activity of ascorbic acid after crossing the skin. Our dissolvable microneedle array provides a new and biocompatible method for delivery of drugs and cosmetic compounds through the skin.     

白细胞介素2-绿脓杆菌外毒素融合基因的克隆及高效表达

利用聚合酶链式反应和寡核苷酸介导的定向诱变技术构建了白细胞介素2-绿脓杆菌外毒素IL2-PE40、IL2-PE40KDEL、IL2-PE66~(4Glu)和IL2-PE66~(4Glu)KDEL融合基因的原核表达重组质粒,并实现了高效表达,表达产物占菌体总可溶蛋白的20％～30％。此外,由于这一表达质粒在IL-2cDNA与PE基因连接处引入了唯一的SmaⅠ位点,其5＇、3＇端分别含有唯一的EcoRⅠ、PstⅠ位点,因此可方便地用其它基因替换IL-2或PE基因而获得相应融合蛋白的表达质粒。     

长期施肥对双季稻田甲烷排放和关键功能微生物的影响

研究不同施肥措施对双季稻田甲烷(CH_4)排放特征的影响及其微生物学机理,对合理利用及评价不同施肥模式对水稻生长的影响具有重要意义。以长期施肥定位试验田为平台,采用静态箱-气相色谱法对施用化肥(MF:mineral fertilizer alone)、秸秆还田配施化肥(RF:rice residues plus mineral fertilizer)、30%有机肥配施70%化肥(LOM:30%organic matter plus 70%mineral fertilizer)、60%有机肥配施40%化肥(HOM:60%organic matter plus 40%mineral fertilizer)和无肥(CK:without fertilizer)条件下双季稻田CH_4排放及其微生物学机理进行了分析。结果表明,早稻和晚稻生长期,不同施肥处理稻田CH_4排放通量均显著高于CK,表现为HOMLOMRFMFCK。各处理间CH_4总排放量差异达显著水平,其大小顺序与排放通量趋势一致,以HOM处理为最高,比CK处理增加105.56%,其次是LOM和RF处理,分别比CK处理增加72.97%和54.17%。关键功能土壤微生物测定结果表明,早稻和晚稻各个主要生育时期,各处理稻田土壤产甲烷古菌的数量变化范围为(3.18—81.07)×10~3cfu/g,土壤甲烷氧化细菌的数量变化范围为(24.82—379.72)×10~3cfu/g。稻田土壤产甲烷古菌和甲烷氧化细菌数量大小顺序为HOMLOMRFMFCK,各施肥处理均显著高于CK;HOM、LOM、RF处理显著高于MF、CK处理。双季稻田CH_4排放与稻田土壤产甲烷古菌、甲烷氧化细菌数量变化关系密切。采用有机无机肥配施促进了双季稻田生态系统CH_4的排放和关键功能微生物的数量。     

禁牧条件下不同类型草地群落结构特征

利用幂乘方法则模型探讨了鄂尔多斯3种不同类型草地在禁牧情况下的群落结构特征,包括物种组成、物种多样性、生物量和空间分布规律。结果表明:幂乘方法则在解析鄂尔多斯不同类型草地的空间异质性时具有很好的吻合性;羊草(Leymuschinensis)草地、芨芨草(Achnatherum splendens)草地和油蒿(Artemisia ordosica)灌丛草地均比随机分布呈现了较强的空间异质性,群落整体的空间异质性指数表现为油蒿灌丛草地>羊草草地>芨芨草草地;群落整体的物种多样性指数为油蒿灌丛草地>羊草草地>芨芨草草地;L-样方(50cm×50cm)内的平均物种数和物种多样性指数均表现为羊草草地极显著地高于油蒿灌丛草地,油蒿灌丛草地又极显著地高于芨芨草草地(P<0.001);L-样方内的平均生物量表现为油蒿灌丛草地极显著地高于芨芨草草地(P<0.001),而羊草草地与油蒿灌丛草地以及芨芨草草地之间没有显著性差异;这3种类型的草地其L-样方内的平均生物量和物种多样性指数都随着群落整体空间异质性指数的增大而增大。     

复合诱变被孢霉原生质体获高产γ-亚麻酸的研究

以拉曼被孢霉（Mortierella remanniana）为出发菌株M₅,通过原生质体制备,经亚硝酸和激光等进行复合诱变;进行初筛,经摇瓶发酵法复筛,并测定其相关性能指标,获得一生产性能比出发菌株显著提高的突变株M_?;其干菌体收率为46.8g/L、油脂产率达34.6g/L、γ-亚麻酸的产率达12.5g/L,分别是出发菌株的1.73倍、2.02倍和2.6倍。通过基因传代实验,说明突变株的基因可稳定遗传。     

鬼斧神工南极石笔走南极（二）

风的力量究竟有多大?这是无法完全说得清楚的。 就其暴虐而言:它使大海白浪滔天,狂潮涌动,造成了一个个有泪有悲的海难;它在塔克拉玛干大沙漠塑造了雅丹地貌,然后又令其发出鬼哭狼嚎声,让人毛骨悚然;它将散在空中的人们灭虫时使用的“六六六”、“滴滴涕”雾剂,长途运往南极,弄得一些企鹅身上也发现这些有害的残留。 然而,狂风也能塑造美丽,这就是多姿多彩的南极石。是风的打磨,风的