The Helicobacter pylori Protein CagM is Located in the Transmembrane Channel That is Required for CagA Translocation

Helicobacter pylori (H. pylori) is a human gastric pathogen that colonizes the stomach in more than 50 % of the world’s human population. Infection with this bacterium can induce several gastric diseases ranging from gastritis to peptic ulcer and gastric cancer. Virulent H. pylori isolates harboring the cag pathogenicity island (cag PAI), which encodes a Type IV Secretion System (T4SS), form a pilus for the injection of its major virulence protein CagA into gastric cells. Several cag PAI genes have been identified as homologues of T4SS genes from Agrobacterium tumefaciens, while the other members in cag PAI still have no known function. We studied one of such proteins with unknown function, CagM, which was predicted to have a putative N-terminal signal sequence and at least three transmembrane helices. To determine the subcellular localization of CagM, we performed a cell fractionation procedure and produced rabbit anti-CagM polyclonal antibodies for immunoblotting assays. Furthermore, we generated an isogenic ΔcagM mutant to investigate the ability of CagA translocation compared with the wild-type NCTC 11637 strain using GES-1 and MKN-45 cell infection experiments. Our results indicated that CagM was mainly located in the bacterial membrane, partially located in the periplasm, and essential for CagA translocation both in GES-1 and MKN-45 cells, which suggested that CagM was one of the core members of Cag T4SS and localized in the transmembrane channel.     

全反式维甲酸协同DAPT抑制胶质瘤干细胞的自我更新

前期研究脑表明,脑胶质瘤干细胞（glioma stem cells,GSCs）是胶质瘤发生和发展的重要因素,探索靶向干预GSCs生长有可能成为脑胶质瘤治疗的有效途径之一。该研究旨在阐明两种药物ATRA和Y-分泌酶抑制剂DAPT协同抑制GSCs自我更新的生物学效应。通过用台盼蓝排染法、克隆球形成试验和免疫印迹分析了两种药物的单独使用或联用对GSC样细胞PGCl和PGC2生长、成球能力和自我更新以及干细胞标志物表达的影响。结果发现,单独使用ATRA对PGCl生长有一定的抑制作用,而对PGC2生长几乎没有影响;DAPT对PGCs的生长抑制作用明显强于ATRA。高浓度ATRA（80μmol/L）能诱导PGCs的分化,降低PGCs成球大小,且成球效率降至5％～8％,而正常对照组为32％～35％;同样,DAPT（40μmol／L）也能降低PGCs成球大小,且成球效率降至2％～3％;低浓度ATRA（20μmol／L）和DAPT（5gmol／L）对PGCs自我更新能力和干性没有明显影响,而联合使用后其明显降低PGCs的成球大小,且成球效率降至3％～5％,促进细胞凋亡,并且明显抑制了干细胞标志物Nestin、CDl33、Sox2、Oct4的表达,提高了分化标志物GFAP的表达。该研究证明了低浓度的ATRA和DAPT能协同抑制脑胶质瘤干细胞PGCs的自我更新。研究结果将为脑胶质瘤的临床研究提供实验依据。     

A novel ultrasensitive bioluminescent receptor-binding assay of INSL3 through chemical conjugation with nanoluciferase

Insulin-like peptide 3 (INSL3) is a reproduction-related peptide hormone belonging to the insulin/relaxin superfamily, which mediates testicular descent in the male fetus, suppresses male germ cell apoptosis and promotes oocyte maturation in adults by activating the relaxin family peptide receptor 2 (RXFP2). To establish an ultrasensitive receptor-binding assay for INSL3−RXFP2 interaction studies, in the present work we labeled a recombinant INSL3 peptide with a newly developed nanoluciferase (NanoLuc) reporter through a convenient chemical conjugation approach, including the introduction of an active disulfide bond to INSL3 by chemical modification and engineering of a 6× His-Cys-NanoLuc carrying a unique exposed cysteine at the N-terminus. The bioluminescent NanoLuc-conjugated INSL3 retained high binding affinity with the target receptor RXFP2 (K_d = 2.0 ± 0.1 nM, n = 3) and was able to sensitively monitor the receptor-binding of a variety of ligands, representing a novel ultrasensitive tracer for non-radioactive receptor-binding assays. Our present chemical conjugation approach could readily be adapted for conjugation of NanoLuc with other proteins, even other macrobiomolecules, for various highly sensitive bioluminescent assays.     

Capsaicin induces apoptosis in human osteosarcoma cells through AMPK-dependent and AMPK-independent signaling pathways

Recent studies have focused on the anti-tumor activity of capsaicin. However, the potential effects of capsaicin in osteosarcoma cells and the underlying mechanisms are not fully understood. In the current study, we observed that capsaicin-induced growth inhibition and apoptosis in cultured osteosarcoma cells (U2OS and MG63), which were associated with a significant AMP-activated protein kinase (AMPK) activation. AMPK inhibition by compound C or RNA interference suppressed capsaicin-induced cytotoxicity, while AMPK activators (AICAR and A769662) promoted osteosarcoma cell death. For the mechanism study, we found that AMPK activation was required for capsaicin-induced mTORC1 (mTOR complex 1) inhibition, B cell lymphoma 2 (Bcl-2) downregulation and Bax upregulation in MG63 cells. Capsaicin administration induced p53 activation, mitochondrial translocation and Bcl-2 killer association, such effects were dependent on AMPK activation. Interestingly, we observed a significant pro-apoptotic c-Jun NH2-terminal kinases activation by capsaicin in MG63 cells, which appeared to be AMPK independent. In conclusion, capsaicin possessed strong efficacy against human osteosarcoma cells. Molecular studies revealed that capsaicin activated AMPK-dependent and AMPK-independent signalings to mediate cell apoptosis. The results of this study should have significant translational relevance in managing this deadly malignancy.     

Genetic manipulation revealing an unusual N-terminal region in a stand-alone non-ribosomal peptide synthetase involved in the biosynthesis of ramoplanins

Ramoplanins produced by Actinoplanes are new structural class of lipopeptide and are currently in phase III clinical trials for the prevention of vancomycin-resistant enterococcal infections. The depsipeptide structures of ramoplanins are synthesized by non-ribosomal peptide synthetases (NRPS). Romo-orf17, a stand-alone NRPS, is responsible for the recruitment of Thr into the linear NRPS pathways for which the corresponding adenylation domain is absent. Here, systematical gene inactivation and complementation have been carried out in a Actinoplanes sp. using homologous recombination and site-specific integration methods. A hybrid gene coding for the N-terminal region of the stand-alone NRPS and the A-PCP domains of a heterologous NRPS restored production of ramoplanins. The results elucidate the unusual N-terminal region which is essential for the biosynthesis of ramoplanins.     

Functional identification of the gene encoding the enzyme involved in mannosylation in ramoplanin biosynthesis in Actinoplanes sp.

Ramoplanin is a lipopeptide antibiotic active against multi-drug-resistant, Gram-positive pathogens. Structurally, it contains a di-mannose moiety attached to the peptide core at Hpg¹¹. The biosynthetic gene cluster of ramoplanin has already been reported and the assembly of the depsipeptide has been elucidated but the mechanism of transferring sugar moiety to the peptide core remains unclear. Sequence analysis of the biosynthetic gene cluster indicated ramo-orf29 was a mannosyltransferase candidate. To investigate the involvement of ramo-orf29 in ramoplanin biosynthesis, gene inactivation and complementation have been conducted in Actinoplanes sp. ATCC 33076 by homologous recombination. Metabolite analysis revealed that the ramo-orf29 inactivated mutant produced no ramoplanin but the ramoplanin aglycone. Thus, ramo-orf29 codes for the mannosyltransferase in the ramoplanin biosynthesis pathway. This lays the foundation for further exploitation of the ramoplanin mannosyltransferase and aglycone in combinatorial biosynthesis.