Combination of site-directed mutagenesis and calcium ion addition for enhanced production of thermostable MBP-fused heparinase I in recombinant Escherichia coli

Heparinase I (HepI), which specifically cleaves heparin and heparan sulfate, is one of the most extensively studied glycosaminoglycan lyases. Low productivity of HepI has largely hindered its industrial and pharmaceutical applications. Loss of bacterial HepI enzyme activity through poor thermostability during its expression and purification process in production can be an important issue. In this study, using a thermostabilization strategy combining site-directed mutagenesis and calcium ion addition during its production markedly improved the yield of maltose-binding protein-fused HepI (MBP–HepI) from recombinant Escherichia coli. Substitution of Cys297 to serine in MBP–HepI offered a 30.6 % increase in the recovered total enzyme activity due to a mutation-induced thermostabilizing effect. Furthermore, upon addition of Ca²⁺ as a stabilizer at optimized concentrations throughout its expression, extraction, and purification process, purified mutant MBP–HepI showed a specific activity of 56.3 IU/mg, 206 % higher than that of the wild type obtained without Ca²⁺ addition, along with a 177 % increase in the recovered total enzyme activity. The enzyme obtained through this novel approach also exhibited significantly enhanced thermostability, as indicated by both experimental data and the kinetic modeling. High-yield production of thermostable MBP–HepI using the present system will facilitate its applications in laboratory-scale heparin analysis as well as industrial-scale production of low molecular weight heparin as an improved anticoagulant substitute.     

Requirement of histone acetyltransferases HAM1 and HAM2 for epigenetic modification of FLC in regulating flowering in Arabidopsis

Histone acetylation is an important posttranslational modification associated with gene activation. In Arabidopsis, two MYST histone acetyltransferases HAM1 and HAM2 work redundantly to acetylate histone H4 lysine 5 (H4K5ace) in vitro. The double mutant ham1/ham2 is lethal, which suggests the critical role of HAM1 and HAM2 in development. Here, we used an artificial microRNA (amiRNA) strategy in Arabidopsis to uncover a novel function of HAM1 and HAM2. The amiRNA-HAM1/2 transgenic plants showed early flowering and reduced fertility. In addition, they responded normally to photoperiod, gibberellic acid treatment, and vernalization. The expression of flowering-repressor FLOWERING LOCUS C (FLC) and its homologues, MADS-box Affecting Flowering genes 3/4 (MAF3/4), were decreased in amiRNA-HAM1/2 lines. HAM1 overexpression caused late flowering and elevated expression of FLC and MAF3/4. Mutation of FLC almost rescued the late flowering with HAM1 overexpression, which suggests that HAM1 regulation of flowering time depended on FLC. Global H4 acetylation was decreased in amiRNA-HAM1/2 lines, but increased in HAM1-OE lines, which further confirmed the acetyltransferase activity of HAM1 in vivo. Chromatin immunoprecipitation revealed that H4 hyperacetylation and H4K5ace at FLC and MAF3/4 were less abundant in amiRNA-HAM1/2 lines than the wild type, but were enriched in HAM1-OE lines. Thus, HAM1 and HAM2 may affect flowering time by epigenetic modification of FLC and MAF3/4 chromatins at H4K5 acetylation.     

Simulations of the alternating access mechanism of the sodium symporter Mhp1

Sodium coupled cotransporters of the five-helix inverted repeat (5HIR) superfamily use an alternating access mechanism to transport a myriad of small molecules across the cell membrane. One of the primary steps in this mechanism is the conformational transition from a state poised to bind extracellular substrates to a state that is competent to deliver substrate to the cytoplasm. Here, we construct a coarse-grained model of the 5HIR benzylhydantoin transporter Mhp1 that incorporates experimental structures of the outward- and inward-open states to investigate the mechanism of this conformational change. Using the weighted ensemble path-sampling method, we rigorously sample the outward- to inward-facing transition path ensemble. The transition path ensemble reveals a heterogeneous set of pathways connecting the two states and identifies two modes of transport: one consistent with a strict alternating access mechanism and another where decoupling of the inner and outer gates causes the transient formation of a continuous permeation pathway through the transporter. We also show that the conformational switch between the outward- and inward-open states results from rigid body motions of the hash motif relative to the substrate bundle, supporting the rocking bundle hypothesis. Finally, our methodology provides the groundwork for more chemically detailed investigations of the alternating mechanism.     

A new method for multiple gene inactivations in Bacillus subtilis 168, producing a strain free of selectable markers

This study describes a novel method for repeated gene inactivation in Bacillus subtilis 168. A B. subtilis strain (BS-PS) that is conditionally auxotrophic for lysine was obtained by replacing the PlysA promoter with the Pspac promoter. The homologous recombination integration vector PLC-T was constructed to contain lacI, which encodes a Pspac promoter repressor, and the chloromycetin resistance gene. Target genes were manipulated by generating an insertion sequence with two homologous arms and the target gene in PLC-T to create a specific integrating vector. Integration into the BS-PS chromosome occurred by a single crossover at either of the two homologous arms. The resulting transitional strain (BS-PS-PI) was chloromycetin resistant and lysine auxotrophic and had an unstable genome structure because of the duplication. Excision of lacI and chloromycetin resistance gene was achieved by a second single crossover at the duplication. Recovery of a lysine prototroph functioned as counter-selection and was identified by PCR. In this work, we inactivated nprE and aprE, two protease genes secreted by B. subtilis 168 free of selectable markers.     

Development of the Drosophila entero-endocrine lineage and its specification by the Notch signaling pathway

In this paper we have investigated the developmental–genetic steps that shape the entero-endocrine system of Drosophila melanogaster from the embryo to the adult. The process starts in the endoderm of the early embryo where precursors of endocrine cells and enterocytes of the larval midgut, as well as progenitors of the adult midgut, are specified by a Notch signaling-dependent mechanism. In a second step that occurs during the late larval period, enterocytes and endocrine cells of a transient pupal midgut are selected from within the clusters of adult midgut progenitors. As in the embryo, activation of the Notch pathway triggers enterocyte differentiation and inhibits cells from further proliferation or choosing the endocrine fate. The third step of entero-endocrine cell development takes place at a mid-pupal stage. Before this time point, the epithelial layer destined to become the adult midgut is devoid of endocrine cells. However, precursors of the intestinal midgut stem cells (pISCs) are already present. After an initial phase of symmetric divisions which causes an increase in their own population size, pISCs start to spin off cells that become postmitotic and express the endocrine fate marker, Prospero. Activation of Notch in pISCs forces these cells into an enterocyte fate. Loss of Notch function causes an increase in the proliferatory activity of pISCs, as well as a higher ratio of Prospero-positive cells.     

化石植物中文名的现状、问题与建议

化石植物的中文名, 即化石植物学名的中文译名, 在古植物学、植物演化生物学专业领域和科学普及等方面起着重要的作用。然而, 长期以来对化石植物中文名重视程度不够且拟订时缺乏统一的标准, 导致各种文本中化石植物的中文名比较混乱, 不利于古植物学知识的传播及科学普及。本文通过统计中文古植物学综合性文献和教材中的化石植物中文名, 梳理出化石植物中文名拟订方面出现的一些包括同物异中文名、中文名重名、音译拗口和存在生僻字的使用等常见问题。针对这些问题, 本文提出应尽快制定出一套规范统一的化石植物中文名拟定方案, 编写和出版化石植物拉汉词典及相应网络查询系统等, 从而统一和规范化石植物的中文名, 同时也可为化石动物中文名的拟订方案提供相关参考。     

野生蛹虫草菌株培养特性及子实体多糖含量比较分析

蛹虫草(Cordyceps militaris)是一种药食两用真菌,为获得高产优质的菌种资源,以采集分离的5株蛹虫草野生菌株(XY002、XY008、XY011、XY029、XY032)为研究对象,通过对5株菌株的分子鉴定、交配型基因分子检测、培养特性及子实体多糖含量测定,确定5株菌株皆为蛹虫草菌株,除XY008只含有MAT1-1-1交配型基因外,其他菌株都含有两种交配型基因MAT1-1-1和MAT1-2-1,5株菌株在菌丝生长速度、分生孢子数量、子实体形态、产量及子实体多糖含量均存在较大差异。综合培养特性及多糖含量分析的结果表明,菌株XY011子实体产量较高达29.60 g/瓶,多糖含量最高达100.79 mg/g,子实体长度最长达11.41 cm,而且子实体粗壮,发育周期短,确定为优势菌株,具有较好的开发价值。     

FCRL3 promotes IL‐10 expression in B cells through the SHP‐1 and p38 MAPK signaling pathways

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system that is caused by the interaction of genetic and environmental factors. Current studies have shown that Fc‐receptor like‐3 (FCRL3) is closely related to MS, but the specific role of FCRL3 in MS has not yet been clarified. This study further found that FCRL3 and interleukin 10 (IL‐10) expression was downregulated in MS patients, but the expression of these proteins was higher in the remission phase than that in the acute phase. The C allele of rs7528684 was associated with MS, and the CC genotype could lead to the upregulation of FCRL3 expression and the increase in IL‐10 secretion. Further in vitro experiments with B cells found that lipopolysaccharide (LPS) promoted FCRL3 expression in a dose‐ and time‐dependent manner, thereby promoting IL‐10 secretion. LPS regulated Src homology region 2 domain‐containing phosphatase‐1 (SHP‐1) expression and p38 mitogen‐activated protein kinase (MAPK) pathway activation through FCRL3, and FCRL3 upregulated the SHP‐1 expression and p38 phosphorylation levels. When SHP‐1 small interfering RNA or a p38 pathway inhibitor was added, the effect of FCRL3 on IL‐10 secretion was significantly inhibited. In addition, FCRL3 inhibited the secretion of inflammatory factors (tumor necrosis factor‐α, IL‐1β, IL‐6, and IL‐8); after inhibiting the expression of IL‐10, the abovementioned effects of FCRL3 were blocked. These results suggest that FCRL3 can activate the SHP‐1 and p38 MAPK pathways and then promote the secretion of IL‐10 in B cells, thus inhibiting the secretion of inflammatory factors. Therefore, FCRL3 may play an immunoprotective role in MS, and it will be an effective target for the diagnosis and treatment of MS.