蓝细菌ORF469的分子克隆和缺失突变工程株的构建

PCR扩增了蓝细菌Synechocystis sp．PCC 6803的ORF469(编码469个氨基酸的开放阅读框),进一步以pUC118为载体将其克隆到E．Coli中,构建了pOQ2质粒。通过DNA体外重组,以红霉素抗性基因取代部分克隆化ORF469片段,又构建丁缺失ORF469片段(保留部分上游和下游序列)的pOQ22质粒。用pOQ22质粒转化Synechocystis sp．PCC 6803野生株细胞,获ORF489缺失突变工程株,它在红霉素抗性培养基上生长正常。对缺失突变工程株DNA的PCR和Southern blot分析证明,Synechocystis sp．PCC 6803的ORF469已被删除。色素测定结果揭示Synechocystis sp．PCC 6803中ORF469表达产物控制细胞内不依赖光的叶绿素生物合成。     

Normalizing the bone marrow microenvironment with p38 inhibitor reduces multiple myeloma cell proliferation and adhesion and suppresses osteoclast formation

The multiple myeloma (MM) bone marrow (BM) microenvironment plays a critical role in supporting tumor growth and survival as well as in promoting formation of osteolytic lesions. Recent results suggest that the p38 mitogen-activated protein kinase (MAPK) is an important factor in maintaining this activated environment. In this report, we demonstrate that the p38alpha MAPK inhibitor, SCIO-469, suppresses secretion of the tumor-supportive factors IL-6 and VEGF from BM stromal cells (BMSCs) as well as cocultures of BMSCs with MM cells, resulting in reduction in MM cell proliferation. Additionally, we show that SCIO-469 prevents TNFalpha-induced adhesion of MM cells to BMSCs through an ICAM-1- and VCAM-1-independent mechanism. Microarray analysis revealed a novel set of TNFalpha-induced chemokines in BMSCs that is strongly inhibited by SCIO-469. Furthermore, reintroduction of chemokines CXCL10 and CCL8 to BMSCs overcomes the inhibitory effect of SCIO-469 on TNFalpha-induced MM adhesion. Lastly, we show that SCIO-469 inhibits secretion and expression of the osteoclast-activating factors IL-11, RANKL, and MIP-1alpha as well as prevents human osteoclast formation in vitro. Collectively, these results suggest that SCIO-469 treatment can suppress factors in the bone marrow microenvironment to inhibit MM cell proliferation and adhesion and also to alleviate osteolytic activation in MM.     

Fibril modelling by sequence and structure conservation analysis combined with protein docking techniques: β2-microglobulin amyloidosis

Obtaining atomic resolution structural models of amyloid fibrils is currently impossible, yet crucial for our understanding of the amyloid mechanism. Different pathways in the transformation of a native globular domain to an amyloid fibril invariably involve domain destabilization. Hence, locating the unstable segments of a domain is important for understanding its amyloidogenic transformation and possibly control it. Since relative conservation is suggested to relate to local stability [H. Benyamini, K. Gunasekaran, H. Wolfson, R. Nussinov, Conservation and amyloid formation: a study of the gelsolin-like family, Proteins 51 (2003) 266–282. [24]], we performed an extensive, sequence and structure conservation analysis of the β₂-microglobulin (β₂-m) domain. Our dataset include 51 high resolution structures belonging to the “C1 set domain” family and 132 clustered PSI-BLAST search results. Segments of the β₂-m domain corresponding to strands A (residues 12–18), D (45–55) and G (91–95) were found to be less conserved and stable, while the central strands B (residues 22–28), C (36–41), E (62–70) and F (78–83) were found conserved and stable. Our findings are supported by accumulating observations from various experimental methods, including urea denaturation, limited proteolysis, H/D exchange and structure determination by both NMR and X-ray crystallography. We used our conservation findings together with experimental literature information to suggest a structural model for the polymerized unit of β₂-m. Pairwise protein docking and subsequent monomer stacking in the same manner suggest a fibril model consistent with the cross-β structure.     

Nitric oxide inhibits ATPase activity and induces resistance to topoisomerase II-poisons in human MCF-7 breast tumor cells

BackgroundTopoisomerase poisons are important drugs for the management of human malignancies. Nitric oxide (^?NO), a physiological signaling molecule, induces nitrosylation (or nitrosation) of many cellular proteins containing cysteine thiol groups, altering their cellular functions. Topoisomerases contain several thiol groups which are important for their activity and are also targets for nitrosation by nitric oxide.MethodsHere, we have evaluated the roles of ^?NO/^?NO-derived species in the stability and activity of topo II (α and β) both in vitro and in human MCF-7 breast tumor cells. Furthermore, we have examined the effects of ^?NO on the ATPase activity of topo II.ResultsTreatment of purified topo IIα and β with propylamine propylamine nonoate (PPNO), an NO donor, resulted in inhibition of the catalytic activity of topo II. Furthermore, PPNO significantly inhibited topo II-dependent ATP hydrolysis. ^?NO-induced inhibition of these topo II (α and β) functions resulted in a decrease in cleavable complex formation in MCF-7 cells in the presence of m-AMSA and XK469 and induced significant resistance to both drugs in MCF-7 cells.ConclusionPPNO treatment resulted in the nitrosation of the topo II protein in MCF-7 cancer cells and inhibited both catalytic-, and ATPase activities of topo II. Furthermore, PPNO significantly affected the DNA damage and cytotoxicity of m-AMSA and XK469 in MCF-7 tumor cells.General significanceAs tumors express nitric oxide synthase and generate ^?NO, inhibition of topo II functions by ^?NO/^?NO-derived species could render tumors resistant to certain topo II-poisons in the clinic.