Expression, Purification, and Mass Spectrometric Analysis of (15)N, (13)C-Labeled RGD-Hirudin, Expressed in Pichia pastoris, for NMR Studies

A novel recombinant hirudin, RGD-hirudin, inhibits the activity of thrombin and the aggregation of platelets. Here, we successfully expressed (15)N, (13)C-labeled RGD-hirudin in Pichia pastoris in a fermenter. The protein was subsequently purified to yield sufficient quantities for structural and functional studies. The purified protein was characterized by HPLC and MALDI-TOF mass spectroscopy. Analysis revealed that the protein was pure and uniformly labeled with (15)N and (13)C. A bioassay showed that the anti-thrombin activity and the anti-platelet aggregation ability of the labeled protein were the same as those of unlabeled RGD-hirudin. Multidimensional heteronuclear NMR spectroscopy has been used to determine almost complete backbone (15)N, (13)C and (1)H resonance assignments of the r-RGD-Hirudin. The (15)N-(1)H HSQC spectrum of uniformly (15)N, (13)C-labeled RGD-hirudin allowed successful assignment of the signals. Examples of the quality of the data are provided for the (15)N-(l)H correlation spectrum, and by selected planes of the CBCA(CO)NH, CBCANH, and HNCO experiments. These results provide a basis for further studies on the structure-function relationship of RGD-hirudin with thrombin and platelets.     

Cryopreserved mesenchymal stromal cells display impaired immunosuppressive properties as a result of heat-shock response and impaired interferon-γ licensing

Human mesenchymal stromal cells (MSC) can suppress T-cell activation in vitro in an indoleamine 2,3-dioxygenase (IDO)-dependent manner. However, their clinical effects on immune ailments have been inconsistent, with a recent phase III study showing no benefit in acute graft-versus-host disease (GvHD). We here tested the hypothesis that the banked, cryopreserved MSC often used in clinical trials display biologic properties distinct from that of MSC in the log phase of growth typically examined in pre-clinical studies. In freshly thawed cryopreserved MSC derived from normal human volunteers, we observed that MSC up-regulate heat-shock proteins, are refractory to interferon (IFN)-γ-induced up-regulation of IDO, and are compromised in suppressing CD3/CD28-driven T cell proliferation. Immune suppressor activity, IFN-γ responsiveness and induction of IDO were fully restored following 24 h of MSC tissue culture post-thaw. These results highlight a possible cause for the inefficacy of MSC-based immunotherapy reported in clinical trials using cryopreserved MSC thawed immediately prior to infusion.     

Navigating the epigenetic landscape of pluripotent stem cells

Pluripotent stem cells, which include embryonic stem cells and induced pluripotent stem cells, use a complex network of genetic and epigenetic pathways to maintain a delicate balance between self-renewal and multilineage differentiation. Recently developed high-throughput genomic tools greatly facilitate the study of epigenetic regulation in pluripotent stem cells. Increasing evidence suggests the existence of extensive crosstalk among epigenetic pathways that modify DNA, histones and nucleosomes. Novel methods of mapping higher-order chromatin structure and chromatin-nuclear matrix interactions also provide the first insight into the three-dimensional organization of the genome and a framework in which existing genomic data of epigenetic regulation can be integrated to discover new rules of gene regulation.     

TLR4在气道上皮细胞诱导的哮喘气道平滑肌细胞迁移中的作用

目的:探讨Toll样受体4(TLR4)的激活在气道上皮细胞诱导的哮喘气道平滑肌细胞(ASMCs)迁移中的作用。方法:细胞消化法培养原代哮喘ASMCs,TNF-α刺激上皮细胞系RTE细胞收集细胞培养上清液,检测上清液中IL-8和RANTES的含量,改良Boyden趋化小室检测哮喘ASMCs的跨膜迁移,以TLR4抗体作为工具药,观察其在上皮细胞诱导的哮喘ASMCs跨膜迁移中的作用。结果:各TNF-α组培养上清液中IL-8和RANTES水平均显著增高,20 ng/ml组较其他组显著增高(P<0.01)。各组哮喘ASMCs跨膜迁移较正常组均增加(P<0.01);哮喘组和TNF-α+TLR4抗体组哮喘ASMCs跨膜迁移数较TNF-α组显著减少(P<0.01)。TLR4抗体组哮喘ASMCs跨膜迁移数较哮喘组增加(P<0.05)。结论:气道上皮细胞可能通过分泌细胞因子激活哮喘ASMCs表面的TLR4,诱导增强ASMCs的跨膜迁移,在哮喘的气道重构中发挥一定的作用。     

Identification and expansion of cancer stem cells in tumor tissues and peripheral blood derived from gastric adenocarcinoma patients

Gastric cancer is the fourth most common cancer worldwide, with a high rate of death and low 5-year survival rate. To date, there is a lack of efficient therapeutic protocols for gastric cancer. Recent studies suggest that cancer stem cells (CSCs) are responsible for tumor initiation, invasion, metastasis, and resistance to anticancer therapies. Thus, therapies that target gastric CSCs are attractive. However, CSCs in human gastric adenocarcinoma (GAC) have not been described. Here, we identify CSCs in tumor tissues and peripheral blood from GAC patients. CSCs of human GAC (GCSCs) that are isolated from tumor tissues and peripheral blood of patients carried CD44 and CD54 surface markers, generated tumors that highly resemble the original human tumors when injected into immunodeficient mice, differentiated into gastric epithelial cells in vitro, and self-renewed in vivo and in vitro. Our findings suggest that effective therapeutic protocols must target GCSCs. The capture of GCSCs from the circulation of GAC patients also shows great potential for identification of a critical cell population potentially responsible for tumor metastasis, and provides an effective protocol for early diagnosis and longitudinal monitoring of gastric cancer.     

Correlation between biological activity and binding energy in systems of integrin with cyclic RGD-containing binders: a QM/MM molecular dynamics study

We here report a combined quantum mechanical/molecular mechanical (QM/MM) molecular dynamics (MD) study on the binding interactions between the α(V)β(3) integrin and eight cyclic arginine-glycine-aspartate (RGD) containing peptides. The initial conformation of each peptide within the binding site of the integrin was determined by docking the ligand to the reactive site of the integrin crystal structure with the aid of docking software FRED. The subsequent QM/MM MD simulations of the complex structures show that these eight cyclic RGD-peptides have a generally similar interaction mode with the binding site of the integrin to the cyclo(RGDf-N[M]V) analog found in the crystal structure. Still, there are subtle differences in the interactions of peptide ligands with the integrin, which contribute to the different inhibition activities. The averaged QM/MM protein-ligand interaction energy (IE) is remarkably correlated to the biological activity of the ligand. The IE, as well as a three-variable model which is somewhat interpretable, thus can be used to predict the bioactivity of a new ligand quantitatively, at least within a family of analogs. The present study establishes a helpful protocol for advancing lead compounds to potent inhibitors.     

Structural Basis for Type VI Secretion Effector Recognition by a Cognate Immunity Protein

The type VI secretion system (T6SS) has emerged as an important mediator of interbacterial interactions. A T6SS from Pseudomonas aeruginosa targets at least three effector proteins, type VI secretion exported 1–3 (Tse1–3), to recipient Gram-negative cells. The Tse2 protein is a cytoplasmic effector that acts as a potent inhibitor of target cell proliferation, thus providing a pronounced fitness advantage for P. aeruginosa donor cells. P. aeruginosa utilizes a dedicated immunity protein, type VI secretion immunity 2 (Tsi2), to protect against endogenous and intercellularly-transferred Tse2. Here we show that Tse2 delivered by the T6SS efficiently induces quiescence, not death, within recipient cells. We demonstrate that despite direct interaction of Tsi2 and Tse2 in the cytoplasm, Tsi2 is dispensable for targeting the toxin to the secretory apparatus. To gain insights into the molecular basis of Tse2 immunity, we solved the 1.00 Å X-ray crystal structure of Tsi2. The structure shows that Tsi2 assembles as a dimer that does not resemble previously characterized immunity or antitoxin proteins. A genetic screen for Tsi2 mutants deficient in Tse2 interaction revealed an acidic patch distal to the Tsi2 homodimer interface that mediates toxin interaction and immunity. Consistent with this finding, we observed that destabilization of the Tsi2 dimer does not impact Tse2 interaction. The molecular insights into Tsi2 structure and function garnered from this study shed light on the mechanisms of T6 effector secretion, and indicate that the Tse2–Tsi2 effector–immunity pair has features distinguishing it from previously characterized toxin–immunity and toxin–antitoxin systems.     

一株脂肪酶产生菌的筛选及产酶条件优化研究

通过利用溴甲酚紫显色培养基初筛和酶活测定法复筛得到产脂肪酶的一株细菌HP2,经形态学观察和生理生化测定初步鉴定该菌株为不动杆菌属。并对该菌株的摇床培养产酶条件进行了初步研究,采用正交试验对HP2菌株发酵产脂肪酶的条件进行了优化,得到最佳发酵条件为初始pH为7.7,培养温度为35℃,接种量(V/V)为1.5%,发酵周期为48 h,酶活力达到129.7 U/mL。