An efficient method for the preparation of preferentially heterodimerized recombinant S100A8/A9 coexpressed in Escherichia coli

It is now known that multicomponent protein assemblies strictly regulate many protein functions. The S100 protein family is known to play various physiological roles, which are associated with alternative complex formations. To prepare sufficient amounts of heterodimeric S100A8 and S100A9 proteins, we developed a method for bicistronic coexpression from a single-vector system using Escherichia coli cells as a host. The complex formation between S100A8 and S100A9 appears to be dependent on the thermodynamic stability of the protein during expression. The stable S100A8/A9 heterodimer complex spontaneously formed during coexpression, and biologically active samples were purified by cation-exchange chromatography. Semi-stable homodimers of S100A8 and S100A9 were also formed when expressed individually. These results suggest that the assembly of S100 protein complexes might be regulated by expression levels of partner proteins in vivo. Because protein assembly occurs rapidly after protein synthesis, coexpression of relevant proteins is crucial for the design of multicomponent recombinant protein expression systems.     

Tailor-made approach for selective isolation and elution of low-density lipoproteins by immunoaffinity sorbent on silica

Immunoaffinity procedure was developed for isolation of low density lipoprotein (LDL) from biological samples by using silica-derived immunoaffinity sorbent. Sorbent was prepared by immobilization of monoclonal anti-apoB-100 antibody onto macroporous silica particles, using carefully optimized binding chemistry. Binding capacity of the sorbent towards LDL was determined by batch extraction experiments with solutions of isolated LDL in phosphate-buffered saline, and found to be 8 mg LDL/g. The bound LDL fraction was readily released from the sorbent by elution with ammonia at pH 11.2. The total time needed for isolation procedure was less than 1 h, with LDL recoveries being essentially quantitative for samples containing less than 0.3 mg LDL/mL. With higher concentrations, recoveries were less favorable, most probably due to irreversible adsorption caused by LDL aggreggation. However, reusability studies with isolated LDL at concentration 0.2 mg/mL indicate that the developed immunoaffinity material may be used for multiple binding-release cycles, with minor losses in binding capacity. Finally, the sorbent was successfully applied to isolation of LDL from diluted plasma. Apart from its practical implications for LDL isolation, this study provides crucial insights into issues associated with LDL-sorbent interactions, and may be useful in future efforts directed to development of lipoprotein isolation approaches.     

S100A11 plays a role in homologous recombination and genome maintenance by influencing the persistence of RAD51 in DNA repair foci

The repair of DNA double-strand breaks (DSBs) by homologous recombination (HR) is an essential process in maintenance of chromosomal stability. A key player of HR is the strand exchange factor RAD51 whose assembly at sites of DNA damage is tightly regulated. We detected an endogenous complex of RAD51 with the calcium-binding protein S100A11, which is localized at sites of DNA repair in HaCaT cells as well as in normal human epidermal keratinocytes (NHEK) synchronized in S phase. In biochemical assays, we revealed that S100A11 enhanced the RAD51 strand exchange activity. When cells expressing a S100A11 mutant lacking the ability to bind Ca²⁺, a prolonged persistence of RAD51 in repair sites and nuclear γH2AX foci was observed suggesting an incomplete DNA repair. The same phenotype became apparent when S100A11 was depleted by RNA interference. Furthermore, down-regulation of S100A11 resulted in both reduced sister chromatid exchange confirming the restriction of the recombination capacity of the cells, and in an increase of chromosomal aberrations reflecting the functional requirement of S100A11 for the maintenance of genomic stability. Our data indicate that S100A11 is involved in homologous recombination by regulating the appearance of RAD51 in DSB repair sites. This function requires the calcium-binding activity of S100A11.     

非ST段抬高型急性冠脉综合征患者血浆S100A1水平与GRACE评分及短期预后相关性的研究

目的:探讨非ST段抬高型急性冠脉综合征(NST-ACS)患者血浆S100A1水平与全球急性冠状动脉事件注册(GRACE)评分之间的关系,以及S100A1水平对NST-ACS患者30天预后的判定价值。方法:共有162例NST-ACS患者符合入选标准,收集基本临床资料,进行GRACE评分,同时收集次日清晨空腹采集肘静脉血,检测血浆S100A1浓度,与患者的GRACE评分进行比较。根据S100A1的水平进行分组随访,KM生存分析不同组患者30天预后进行评价。结果:不同GRACE分组患者间S100A1水平具有显著性差异(P0.05);相关性分析显示,NST-ACS患者S100A1与GRACE评分呈显著正相关(r=0.49,P0.01);KM生存分析显示,S100A1水平3.41 ng/mL的患者30天内心血管事件发生率显著升高(P0.05)。结论:S100A1可作为预测NST-ACE患者病情的发生发展的生化指标;在NST-ACS患者中运用S100A1有助于对患者早期危险分层及评估预后有一定的临床价值。     

Culture of transgenic Drosophila melanogaster Schneider 2 cells in serum-free media based on TC100 basal medium

Requirements of eliminating animal proteins from cell culture have intensified in recent years, with the pressure of regulatory agencies related to biopharmaceuticals production. In this work, the substitution of fetal bovine serum by yeastolate and a soy hydrolysate (Hy Soy) for the culture of Drosophila melanogaster Schneider 2 cells transfected for the production of rabies virus G glycoprotein was evaluated. TC100 supplemented with glucose, glutamine, lipid emulsion and Pluronic F68 was employed as basal medium. Results show that yeastolate was more efficient on cell growth stimulation than Hy Soy. Cells adapted in medium formulation supplemented with 3 g/L yeastolate, 1% lipid emulsion, 10 g/L glucose, 3.5 g/L glutamine and 0.1% Pluronic F68 attained a maximum concentration of 10.7 x 10(6) cells/mL, with the expression of 9.4 ng/mL G glycoprotein.     

Circular RNA circMAN2B2 facilitates glioma progression by regulating the miR-1205/S100A8 axis

The aim of this study was to research the mechanism of circMAN2B2 in the development of glioma. In our study, we found that circMAN2B2 has a higher expression in glioma tissues and cells, which was negatively related to the overall survival of glioma patients. The cell counting kit-8 assay, 5-ethynyl-2′-deoxyuridine labeling assay, transwell assay, and the nude mice assay indicated that knockdown of circMAN2B2 inhibited the cell proliferation, invasion, migration and decreased tumor size. In terms of mechanism, knockdown of circMAN2B2 increased the expression of miR-1205. Moreover, circMAN2B2 regulated S100A8 expression by inhibiting miR-1205. We also showed that knockdown of S100A8 inhibited cell proliferation, invasion, and migration. Increasing S100A8 expression rescued the effect of si-circMAN2B2. In conclusion, circMAN2B2 could improve cell proliferation, invasion, and migration of the glioma by inhibiting miR-1205 and promoting the expression of S100A8.     

Nr5a1-Cre-mediated Tspo conditional knockout mice with low growth rate and prediabetes symptoms – A mouse model of stress diabetes

Translocator protein (TSPO) is a high-affinity cholesterol- and drug-binding mitochondrial protein. Nuclear receptor subfamily 5 group A member 1 or steroidogenic factor 1 (Nr5a1)-Cre mice were previously used to generate steroidogenic cell-specific Tspo gene conditional knockout (cKO) mice. TSPO-depleted homozygotes showed no response to adrenocorticotropic hormone (ACTH) in stimulating adrenal cortex corticosterone production but showed increased epinephrine synthesis in the medulla. No other phenotype was observed under normal growth conditions. During these studies, we noted that pairing two cKO mice resulted in the generation of small pups. These pups showed low growth rate at weaning, which has been linked to the development of type 2 diabetes (T2D) in adulthood. Experimental verification of T2D symptoms via blood testing of the adult mice, including glycated hemoglobin and insulin C-peptide measurements, showed that these Tspo cKO mice exhibited sustained hyperglycemia, a sign of prediabetes, likely due to the augmentation of hepatic glucose production mediated by the increased epinephrine. We also observed increased expression of the S100a8 gene, which is upregulated after chronic glucose stimulation. Taken together, the observed prediabetes phenotype and lack of response to ACTH indicate that Tspo cKO mice (Nr5a1-Cre^+/?, Tspo^fl/fl) could provide a useful model to study the link between diabetes and stress.