Permeabilized Escherichia coli Whole Cells Containing Co‐Expressed Two Thermophilic Enzymes Facilitate the Synthesis of scyllo‐Inositol from myo‐Inositol

Scyllo‐inositol (SI), a stereoisomer of inositol, is regarded as a promising therapeutic agent for Alzheimer's disease. Here, an in vitro cofactor‐balance biotransformation for the production of SI from myo‐inositol (MI) by thermophilic myo‐inositol 2‐dehydrogenase (IDH) and scyllo‐inositol 2‐dehydrogenase (SIDH) is presented. These two enzymes (i.e., IDH and SIDH from Geobacillus kaustophilus) are co‐expressed in Escherichia coli BL21(DE3), and E. coli cells containing the two enzymes are permeabilized by heat treatment as whole‐cell catalysts to convert MI to SI. After condition optimizations about permeabilized temperature, reaction temperature, and initial MI concentration, about 82 g L^?1 of SI is produced from 250 g L^?1 of MI within 24 h without any cofactor supplementation. This final titer of SI produced is the highest to the authors’ limited knowledge. This study provides a promising method for the large‐scale industrial production of SI.     

重组枯草芽孢杆菌全细胞催化合成2-O-α-D-甘油葡糖苷

2-O-α-D-甘油葡糖苷是一种在食品、化妆品、保健品及医药领域有着重大应用前景的高附加值产品,但国内仍未实现2-O-α-D-甘油葡糖苷的工业化生产,且鲜有关于2-O-α-D-甘油葡糖苷合成的相关报道。文中旨在开发一种利用食品安全级重组枯草芽孢杆菌全细胞催化合成2-O-α-D-甘油葡糖苷的方法,通过构建一株异源表达肠膜明串珠菌蔗糖磷酸化酶(Sucrose phosphorylase,SPase)的重组枯草芽孢杆菌Bacillus subtilis 168/pMA5-gtfA,并将其用作全细胞催化剂合成2-O-α-D-甘油葡糖苷,通过优化培养温度、时间及全细胞转化条件,提高其转化合成2-O-α-D-甘油葡糖苷的产量。结果表明,重组枯草芽孢杆菌B. subtilis 168/pMA5-gtfA在30℃下培养20 h,菌体裂解物酶活力最大达1.43 U/mL,并且在1 mol/L蔗糖、2.5 mol/L甘油、pH 7.0、菌体OD600为40、30℃下全细胞转化反应48h,共生成2-O-α-D-甘油葡糖苷189.3g/L,平均转化速率为15.6mmol/(L·h),蔗糖转化率约为75.1%,...     

Casein kinase 2 promotes the TGF-β-induced activation of α-tubulin acetyltransferase 1 in fibroblasts cultured on a soft matrix

Cell signals for growth factors depend on the mechanical properties of the extracellular matrix (ECM) surrounding the cells. Microtubule acetylation is involved in the transforming growth factor (TGF)-β-induced myofibroblast differentiation in the soft ECM. However, the mechanism of activation of α-tubulin acetyltransferase 1 (α-TAT1), a major α-tubulin acetyltransferase, in the soft ECM is not well defined. Here, we found that casein kinase 2 (CK2) is required for the TGF-β-induced activation of α-TAT1 that promotes microtubule acetylation in the soft matrix. Genetic mutation and pharmacological inhibition of CK2 catalytic activity specifically reduced microtubule acetylation in the cells cultured on a soft matrix rather than those cultured on a stiff matrix. Immunoprecipitation analysis showed that CK2α, a catalytic subunit of CK2, directly bound to the C-terminal domain of α-TAT1, and this interaction was more prominent in the cells cultured on the soft matrix. Moreover, the substitution of alanine with serine, the 236th amino acid located at the C-terminus, which contains the CK2-binding site of α-TAT1, sig-nificantly abrogated the TGF-β-induced microtubule acetylation in the soft matrix, indicating that the successful binding of CK2 and the C-terminus of α-TAT1 led to the phosphorylation of serine at the 236th position of amino acids in α-TAT1 and regulation of its catalytic activity. Taken together, our findings provide novel insights into the molecular mechanisms underlying the TGF-β-induced activation of α-TAT1 in a soft matrix.     

Interactions between EGFR and EphA2 promote tumorigenesis through the action of Ephexin1

The cell signaling factors EGFR, EphA2, and Ephexin1 are associated with lung and colorectal cancer and play an important role in tumorigenesis. Although the respective functional roles of EGFR and EphA2 are well known, interactions between these proteins and a functional role for the complex is not understood. Here, we showed that Ephexin1, EphA2, and EGFR are each expressed at higher levels in lung and colorectal cancer patient tissues, and binding of EGFR to EphA2 was associated with both increased tumor grade and metastatic cases in both cancer types. Treatment with Epidermal Growth Factor (EGF) induced binding of the RR domain of EGFR to the kinase domain of EphA2, and this binding was promoted by Ephexin1. Additionally, the AKT-mediated phosphorylation of EphA2 (at Ser897) promoted interactions with EGFR, pointing to the importance of this pathway. Two mutations in EGFR, L858R and T790M, that are frequently observed in lung cancer patients, promoted binding to EphA2, and this binding was dependent on Ephexin1. Our results indicate that the formation of a complex between EGFR, EphA2, and Ephexin1 plays an important role in lung and colorectal cancers, and that inhibition of this complex may be an effective target for cancer therapy.Subject terms: Oncogenes, Cancer models     

Seasonal and interannual variations of ecosystem photosynthetic characteristics in a semi-arid grassland of Northern China

北方半干旱草原生态系统光合参数的季节和年际变异 生态系统表观量子效率(α)、最大光合速率(P_max)和暗呼吸速率(R_d)不仅反映了生态系统水平 光合生理特征,同时也是碳循环模型中光合过程模拟的关键参数。气候和植被因子都会影 响光合参数的季节和年际变异,但二者在光合参数调控过程中的相对贡献和作用途径尚不清晰。本研究基于连续12年(2006–2017)的涡度相关观测数据,分析了内蒙古半干旱典型草原光合参数的季节和年际变化规律;利用回归分析和结构方程模型(SEM)方法明晰了环境和生理调控的作用途径及相对贡献。结果发现,光合参数(α、P_max和R_d)均表现出单峰的季节变化趋势,并呈现明显的年际波动。温度(T_a)和土壤含水量(SWC)的变化共同影响光合参数的季节变化,而SWC主导了其年际变异。α和R_d的变化主要由T_a决定,而Pmax的变化主要受SWC的影响。SEM模型分析表明,除了直接作用外,环境因子主要通过影响冠层水平气孔导度(gc)对光合参数和碳同化生理过程进行调控。此外,叶面积指数对光合参数特别是Pmax的季节和年际变异起主要调控作用。以上结果明确了环境和植被共同决定了生态系统水平光合参数的季节和年际变异,并强调了在水分受限的草原生态系统中,植被生理调控在光合碳同化能力和碳汇功能评估中的重要作用。     

The EGFR/ErbB3 Pathway Acts as a Compensatory Survival Mechanism upon c-Met Inhibition in Human c-Met+ Hepatocellular Carcinoma

Backgroundc-Met, a high-affinity receptor for Hepatocyte Growth Factor (HGF), plays a critical role in tumor growth, invasion, and metastasis. Hepatocellular carcinoma (HCC) patients with activated HGF/c-Met signaling have a significantly worse prognosis. Targeted therapies using c-Met tyrosine kinase inhibitors are currently in clinical trials for HCC, although receptor tyrosine kinase inhibition in other cancers has demonstrated early success. Unfortunately, therapeutic effect is frequently not durable due to acquired resistance.MethodsWe utilized the human MHCC97-H c-Met positive (c-Met⁺) HCC cell line to explore the compensatory survival mechanisms that are acquired after c-Met inhibition. MHCC97-H cells with stable c-Met knockdown (MHCC97-H c-Met KD cells) were generated using a c-Met shRNA vector with puromycin selection and stably transfected scrambled shRNA as a control. Gene expression profiling was conducted, and protein expression was analyzed to characterize MHCC97-H cells after blockade of the c-Met oncogene. A high-throughput siRNA screen was performed to find putative compensatory survival proteins, which could drive HCC growth in the absence of c-Met. Findings from this screen were validated through subsequent analyses.ResultsWe have previously demonstrated that treatment of MHCC97-H cells with a c-Met inhibitor, PHA665752, results in stasis of tumor growth in vivo. MHCC97-H c-Met KD cells demonstrate slower growth kinetics, similar to c-Met inhibitor treated tumors. Using gene expression profiling and siRNA screening against 873 kinases and phosphatases, we identified ErbB3 and TGF-α as compensatory survival factors that are upregulated after c-Met inhibition. Suppressing these factors in c-Met KD MHCC97-H cells suppresses tumor growth in vitro. In addition, we found that the PI3K/Akt signaling pathway serves as a negative feedback signal responsible for the ErbB3 upregulation after c-Met inhibition. Furthermore, in vitro studies demonstrate that combination therapy with PHA665752 and Gefitinib (an EGFR inhibitor) significantly reduced cell viability and increased apoptosis compared with either PHA665752 or Gefitinib treatment alone.Conclusionc-Met inhibition monotherapy is not sufficient to eliminate c-Met⁺ HCC tumor growth. Inhibition of both c-Met and EGFR oncogenic pathways provides superior suppression of HCC tumor growth. Thus, combination of c-Met and EGFR inhibition may represent a superior therapeutic regimen for c-Met⁺ HCC.     

Different antigen presentation tendencies of granulocyte-macrophage colony-stimulating factor-induced bone marrow-derived macrophages and peritoneal macrophages

Granulocyte-macrophage colony-stimulating factor (GM-CSF)-induced bone marrow-derived cells (BMCs) and primary peritoneal exudate cells (PECs) are usually used for antigen presentation in in vitro experiments. In order to expound their tendency for uptake and antigen presentation, we compared differences in the degree of phagocytosis, the expression of co-stimulatory molecules, and the activation of T lymphocytes between these two cell types. These assays used the F4/80 marker expression, as it is the general marker for macrophages. The BMC population was found to contain both F4/80(bright) and F4/80(dim) subtypes, while PECs were mainly composed of the F4/80(bright) subtype. Expression levels of cell surface co-stimulatory molecules, CD80, CD86, CD54, and CD40, were significantly higher for F4/80(+)BMCs than F4/80(+)PECs. Their expressions were further upregulated for F4/80(+)BMCs than for F4/80(+)PECs after stimulation with flagellin. F4/80(+)BMCs had a weaker ability to phagocytize microbeads than F4/80(+)PECs (P?相似文献   

Genome-wide SNP discovery in walnut with an AGSNP pipeline updated for SNP discovery in allogamous organisms

ABSTRACT: BACKGROUND: A genome-wide set of single nucleotide polymorphisms (SNPs) is a valuable resource in genetic research and breeding and is usually developed by re-sequencing a genome. If a genome sequence is not available, an alternative strategy must be used. We previously reported the development of a pipeline (AGSNP) for genome-wide SNP discovery in coding sequences and other single-copy DNA without a complete genome sequence in self-pollinating (autogamous) plants. Here we updated this pipeline for SNP discovery in outcrossing (allogamous) species and demonstrated its efficacy in SNP discovery in walnut (Juglans regia L.). RESULTS: The first step in the original implementation of the AGSNP pipeline was the construction of a reference sequence and the identification of single-copy sequences in it. To identify single-copy sequences, multiple genome equivalents of short SOLiD reads of another individual were mapped to shallow genome coverage of long Sanger or Roche 454 reads making up the reference sequence. The relative depth of SOLiD reads was used to filter out repeated sequences from single-copy sequences in the reference sequence. The second step was a search for SNPs between SOLiD reads and the reference sequence. Polymorphism within the mapped SOLiD reads would have precluded SNP discovery; hence both individuals had to be homozygous. The AGSNP pipeline was updated here for using SOLiD or other type of short reads of a heterozygous individual for these two principal steps. A total of 32.6X walnut genome equivalents of SOLiD reads of vegetatively propagated walnut scion cultivar 'Chandler' were mapped to 48,661 'Chandler' bacterial artificial chromosome (BAC) end sequences (BESs) produced by Sanger sequencing during the construction of a walnut physical map. A total of 22,799 putative SNPs were initially identified. A total of 6,000 Infinium II type SNPs evenly distributed along the walnut physical map were selected for the construction of an Infinium BeadChip, which was used to genotype a walnut mapping population having 'Chandler' as one of the parents. Genotyping results were used to adjust the filtering parameters of the updated AGSNP pipeline. With the adjusted filtering criteria, 69.6% of SNPs discovered with the updated pipeline were real and could be mapped on the walnut genetic map. A total of 13,439 SNPs were discovered by BES re-sequencing. BESs harboring SNPs were in 677 FPC contigs covering 98% of the physical map of the walnut genome. CONCLUSION: The updated AGSNP pipeline is a versatile SNP discovery tool for a high-throughput, genome-wide SNP discovery in both autogamous and allogamous species. With this pipeline, a large set of SNPs were identified in a single walnut cultivar.     

Laminin-411 is a vascular ligand for MCAM and facilitates TH17 cell entry into the CNS

TH17 cells enter tissues to facilitate pathogenic autoimmune responses, including multiple sclerosis (MS). However, the adhesion molecules involved in the unique migratory capacity of TH17 cells, into both inflamed and uninflamed tissues remain unclear. Herein, we characterize MCAM (CD146) as an adhesion molecule that defines human TH17 cells in the circulation; following in vitro restimulation of human memory T cells, nearly all of the capacity to secrete IL-17 is contained within the population of cells expressing MCAM. Furthermore, we identify the MCAM ligand as laminin 411, an isoform of laminin expressed within the vascular endothelial basement membranes under inflammatory as well as homeotstatic conditions. Purified MCAM-Fc binds to laminin 411 with an affinity of 27 nM, and recognizes vascular basement membranes in mouse and human tissue. MCAM-Fc binding was undetectable in tissue from mice with targeted deletion of laminin 411, indicating that laminin 411 is a major tissue ligand for MCAM. An anti-MCAM monoclonal antibody, selected for inhibition of laminin binding, as well as soluble MCAM-Fc, inhibited T cell adhesion to laminin 411 in vitro. When administered in vivo, the antibody reduced TH17 cell infiltration into the CNS and ameliorated disease in an animal model of MS. Our data suggest that MCAM and laminin 411 interact to facilitate TH17 cell entry into tissues and promote inflammation.