Control of passive permeability of chinese hamster ovary cells by external and intracellular ATP

External ATP causes passive permeability change in several transformed cells, but not in untransformed cells. We studied the effect of external ATP on the passive permeability of CHO-K1 cells, a transformed clone of Chinese hamster ovary cells. Treatment of the cells with external ATP alone did not produce a permeability change, and this was observed only when a mitochondrial inhibitor, such as rotenone or oligomycin, was present together with ATP. These inhibitors reduced the concentration of intracellular ATP and a permeability change by external ATP was observed when intracellular ATP was decreased more than 70%. This requirement for permeability change of CHO-K1 cells was quite unique, since passive permeability change of other transformed cells so far tested was induced by ATP alone. Treatment of CHO-K1 cells with cyclic AMP analogues increased their sensitivity to external ATP about 2-fold. The roles of external and intracellular ATP in controlling passive permeability are discussed.     

Anti-viral activity of 3-deazaadenosine and 5'-deoxy-5'-isobutylthio-3-deazaadenosine (3-deaza-SIBA)

3-Deazaadenosine and 5′-deoxy-5′-isobutylthio-3-deazaadenosine (3-deaza-SIBA) inhibits replication of both herpes simplex type 1 virus and the RNA type C virus, HL-23. Oncogenic transformation caused by SV40 and HL-23 are also blocked by either compound. Both compounds exhibit relatively low cytotoxicity at the anti-viral concentrations.     

The role of YKL-40 in the pathogenesis of autoimmune diseases: a comprehensive review

YKL-40, a chitinase-3-like protein 1 (CHI3L1) or human cartilage glycoprotein 39 (HC gp-39), is expressed and secreted by various cell-types including macrophages, chondrocytes, fibroblast-like synovial cells and vascular smooth muscle cells. Its biological function is not well elucidated, but it is speculated to have some connection with inflammatory reactions and autoimmune diseases. Although having important biological roles in autoimmunity, there were only attempts to elucidate relationships of YKL-40 with a single or couple of diseases in the literature. Therefore, in order to analyze the relationship between YKL-40 and the overall diseases, we reviewed 51 articles that discussed the association of YKL-40 with rheumatoid arthritis, psoriasis, systemic lupus erythematosus, Behçet disease and inflammatory bowel disease. Several studies showed that YKL-40 could be assumed as a marker for disease diagnosis, prognosis, disease activity and severity. It is also shown to be involved in response to disease treatment. However, other studies showed controversial results particularly in the case of Behçet disease activity. Therefore, further studies are needed to elucidate the exact role of YKL-40 in autoimmunity and to investigate its potential in therapeutics.     

Biotechnological challenges of bioartificial kidney engineering

With the world-wide increase of patients with renal failure, the development of functional renal replacement therapies have gained significant interest and novel technologies are rapidly evolving. Currently used renal replacement therapies insufficiently remove accumulating waste products, resulting in the uremic syndrome. A more preferred treatment option is kidney transplantation, but the shortage of donor organs and the increasing number of patients waiting for a transplant warrant the development of novel technologies. The bioartificial kidney (BAK) is such promising biotechnological approach to replace essential renal functions together with the active secretion of waste products. The development of the BAK requires a multidisciplinary approach and evolves at the intersection of regenerative medicine and renal replacement therapy. Here we provide a concise review embracing a compact historical overview of bioartificial kidney development and highlighting the current state-of-the-art, including implementation of living-membranes and the relevance of extracellular matrices. We focus further on the choice of relevant renal epithelial cell lines versus the use of stem cells and co-cultures that need to be implemented in a suitable device. Moreover, the future of the BAK in regenerative nephrology is discussed.     

恒河猴外周血及肾组织SV40病毒基因分析

SV40即猿猴病毒40(simian virus 40),是DNA肿瘤病毒的原型代表,其基因结构为共价闭合环状双股DNA分子,标准参考株SV40-776含5243个核甘酸,不同分离株bp数略有差异.     

GST pull-down验证流感病毒PB1-F2蛋白与热休克蛋白40的相互作用

为体外验证流感病毒PB1-F2与热休克蛋白Hsp40相互作用,通过两个方向的GST pull-down试验验证PB1-F2与Hsp40的相互作用。构建GST-多肽融合蛋白原核表达载体pGEX-6P-1-PB1-F2和pGEX-6P-1-Hsp40,并在大肠杆菌(E.co-li)BL21中诱导表达;构建真核表达载体pLEGFP-Hsp40及pCAGGS-PB1-F2,并分别转染293T细胞使其表达Hsp40及PB1-F2融合蛋白,然后进行GST pull-down试验验证二者的相互作用。成功地构建了两种蛋白的各种表达载体,经表达、纯化获得了可溶性的GST-多肽融合蛋白,GST pull-down试验正反两方向都证实了PB1-F2与Hsp40的相互作用,初步证实了流感病毒PB1-F2在体外能与Hsp40发生相互作用。     

Interdomain salt‐bridges in the Ebola virus protein VP40 and their role in domain association and plasma membrane localization

The Ebola virus protein VP40 is a transformer protein that possesses an extraordinary ability to accomplish multiple functions by transforming into various oligomeric conformations. The disengagement of the C‐terminal domain (CTD) from the N‐terminal domain (NTD) is a crucial step in the conformational transformations of VP40 from the dimeric form to the hexameric form or octameric ring structure. Here, we use various molecular dynamics (MD) simulations to investigate the dynamics of the VP40 protein and the roles of interdomain interactions that are important for the domain–domain association and dissociation, and report on experimental results of the behavior of mutant variants of VP40. The MD studies find that various salt‐bridge interactions modulate the VP40 domain dynamics by providing conformational specificity through interdomain interactions. The MD simulations reveal a novel salt‐bridge between D45‐K326 when the CTD participates in a latch‐like interaction with the NTD. The D45‐K326 salt‐bridge interaction is proposed to help domain–domain association, whereas the E76‐K291 interaction is important for stabilizing the closed‐form structure. The effects of the removal of important VP40 salt‐bridges on plasma membrane (PM) localization, VP40 oligomerization, and virus like particle (VLP) budding assays were investigated experimentally by live cell imaging using an EGFP‐tagged VP40 system. It is found that the mutations K291E and D45K show enhanced PM localization but D45K significantly reduced VLP formation.     

The role of polar interactions in the molecular recognition of CD40L with its receptor CD40.

CD40 Ligand (CD40L) is transiently expressed on the surface of T-cells and binds to CD40, which is expressed on the surface of B-cells. This binding event leads to the differentiation, proliferation, and isotype switching of the B-cells. The physiological importance of CD40L has been demonstrated by the fact that expression of defective CD40L protein causes an immunodeficiency state characterized by high IgM and low IgG serum levels, indicating faulty T-cell dependent B-cell activation. To understand the structural basis for CD40L/CD40 association, we have used a combination of molecular modeling, mutagenesis, and X-ray crystallography. The structure of the extracellular region of CD40L was determined by protein crystallography, while the CD40 receptor was built using homology modeling based upon a novel alignment of the TNF receptor superfamily, and using the X-ray structure of the TNF receptor as a template. The model shows that the interface of the complex is composed of charged residues, with CD40L presenting basic side chains (K143, R203, R207), and CD40 presenting acidic side chains (D84, E114, E117). These residues were studied experimentally through site-directed mutagenesis, and also theoretically using electrostatic calculations with the program Delphi. The mutagenesis data explored the role of the charged residues in both CD40L and CD40 by switching to Ala (K143A, R203A, R207A of CD40L, and E74A, D84A, E114A, E117A of CD40), charge reversal (K143E, R203E, R207E of CD40L, and D84R, E114R, E117R of CD40), mutation to a polar residue (K143N, R207N, R207Q of CD40L, and D84N, E117N of CD40), and for the basic side chains in CD40L, isosteric substitution to a hydrophobic side chain (R203M, R207M). All the charge-reversal mutants and the majority of the Met and Ala substitutions led to loss of binding, suggesting that charged interactions stabilize the complex. This was supported by the Delphi calculations which confirmed that the CD40/CD40L residue pairs E74-R203, D84-R207, and E117-R207 had a net stabilizing effect on the complex. However, the substitution of hydrophilic side chains at several of the positions was tolerated, which suggests that although charged interactions stabilize the complex, charge per se is not crucial at all positions. Finally, we compared the electrostatic surface of TNF/TNFR with CD40L/CD40 and have identified a set of polar interactions surrounded by a wall of hydrophobic residues that appear to be similar but inverted between the two complexes.