Effect of transferrin as a ligand of pH-sensitive fusogenic liposome-lipoplex hybrid complexes

We previously developed potent nonviral vectors based on complexation of lipoplexes and pH-sensitive fusogenic liposomes, which achieve efficient transfection through membrane fusion with intracellular acidic compartments such as endosomes. Because transferrin receptor is known to be overexpressed in cancer cells, in this study, we investigated the effect of transferrin as a ligand for transfection of various cancer-derived cell lines mediated by the liposome-lipoplex hybrid complexes. Results showed that these hybrid complexes with transferrin exhibited higher transfection efficiency toward these cells than complexes without transferrin, but the extent of the transferrin-induced enhancement was dependent on the cell line. Conjugation of transferrin increased their transfection activity for HeLa and KB cells, although it only slightly enhanced transfection for HT1080, HepG2, and K562. Transferrin receptors in HT1080, HepG2, and K562 cells were internalized slowly, whereas those in HeLa and KB cells were internalized quickly and actively. These results indicate that transfection mediated by the ligand-attached hybrid complex does not correlate with the amount of transferrin receptor in the cell surface but correlate with the activity of internalization of transferrin receptor into the cells.     

Catalytic features and eradication ability of antibody light-chain UA15-L against Helicobacter pylori

We have successfully developed a catalytic antibody capable of degrading the active site of the urease of Helicobacter pylori and eradicating the bacterial infection in a mouse stomach. This monoclonal antibody UA15 was generated using a designed recombinant protein UreB, which contained the crucial region of the H. pylori urease beta-subunit active site, for immunization. The light chain of this antibody (UA15-L) by itself showed a proteolytic activity to substantially degrade both UreB and the intact urease. Oral administration of UA15-L also significantly reduced the number of H. pylori in a mouse stomach. This is the first example of a monoclonal catalytic antibody capable of functioning in vivo, and such an antibody may have a therapeutic utility in the future.     

Diacylglycerol specifically blocks spontaneous integration of membrane proteins and allows detection of a factor-assisted integration

We recently found that the spontaneous integration of M13 procoat is blocked by diacylglycerol (DAG) (Nishiyama, K., Ikegami, A., Moser, M., Schiltz, E., Tokuda, H., and Muller, M. (2006) J. Biol. Chem. 281, 35667-35676). Here, we demonstrate that the spontaneous integration of Pf3 coat, another membrane protein that has been thought to be integrated spontaneously into liposomes, can be blocked by DAG at physiological concentrations. Moreover, the spontaneous integration of the membrane potential-independent version of Pf3 coat (3L-Pf3 coat), which is independent of YidC, was also blocked by DAG. To clarify the mechanism by which DAG blocks spontaneous integration, we examined lipid compounds similar to DAG and DAG derivatives. The blockage of spontaneous integration was specific to DAG, as fatty acids, monoacylglycerol, and phosphatidic acids were not effective for the blockage. When the acyl chains in DAG were shortened even to octanoyl residues, it still blocked spontaneous integration, whereas diheptanoylglycerol did not block it at all. Triacylglycerol was more effective than DAG. However, the lipid A-derivative-dependent integration of M13 procoat could not be reconstituted when triacylglycerol was included in the liposomes. On the other hand, when DAG was included in the liposomes, we found that the integration of 3L-Pf3 coat was strictly dependent on the lipid A-derived integration factor. We propose that the bulky structure of DAG rather than changes in membrane curvature is essential for the blockage of spontaneous integration. We also demonstrated that the blockage of spontaneous integration by DAG is also operative in native membrane vesicles.     

An FGF1:FGF2 chimeric growth factor exhibits universal FGF receptor specificity, enhanced stability and augmented activity useful for epithelial proliferation and radioprotection

Structural instability of wild-type fibroblast growth factor (FGF)-1 and its dependence on exogenous heparin for optimal activity diminishes its potential utility as a therapeutic agent. Here we evaluated FGFC, an FGF1:FGF2 chimeric protein, for its receptor affinity, absolute heparin-dependence, stability and potential clinical applicability. Using BaF3 transfectants overexpressing each FGF receptor (FGFR) subtype, we found that, like FGF1, FGFC activates all of the FGFR subtypes (i.e., FGFR1c, FGFR1b, FGFR2c, FGFR2b, FGFR3c, FGFR3b and FGFR4) in the presence of heparin. Moreover, FGFC activates FGFRs even in the absence of heparin. FGFC stimulated keratinocytes proliferation much more strongly than FGF2, as would be expected from its ability to activate FGFR2b. FGFC showed greater structural stability, biological activity and resistance to trypsinization, and less loss in solution than FGF1 or FGF2. When FGFC was intraperitoneally administered to BALB/c mice prior to whole body gamma-irradiation, survival of small intestine crypts was significantly enhanced, as compared to control mice. These results suggest that FGFC could be useful in a variety of clinical applications, including promotion of wound healing and protection against radiation-induced damage.     

Morphological changes in woody stem of Prunus jamasakura under simulated microgravity.

When the four-week-old woody stem of Prunus jamasakura was grown under simulated microgravity condition on a three-dimensional clinostat, it bent at growth, and width of its secondary xylem decreased due to the reduction of fiber cell numbers and a smaller microfibril angle in the secondary cell wall, as reported in our previous paper. Gravity induces the development of the secondary xylem that supports the stem upward against the action of gravity. In this study, morphological changes of the tissues and cells were microscopically observed. Disorder was found in the concentric structure of tissues that organize the stem. The radial arrangement of the cells was also disturbed in the secondary xylem, and in the secondary phloem secondary cell walls of the bast fiber cells were undeveloped. These findings suggest that differentiation and development of the secondary xylem and the bast fiber cells are strongly controlled by terrestrial gravity. These tissue and cells functions to support the stem under the action of gravity. Furthermore, clinorotation induced disorder in the straight joint of vessel elements and the lattice-like structure of radial parenchyma cells, which is responsible for water transportation and storage, respectively. Gravity is an essential factor for keeping the division and differentiation normal in woody stem.     

Development of bioluminescent pyrophosphate assay using pyruvate phosphate dikinase and its application to single-nucleotide polymorphism analysis

DNA analysis is an important technology with respect to diagnosis of infectious disease and tailored medication. In this study, we developed a novel bioluminescent assay for pyrophosphate, and it was applied to single-nucleotide polymorphism (SNP) analysis using one-base extension reaction. The principle of this method is as follows. A specific primer within each aliquot possessing a short 3′ end of the base of interest was hybridized to the single-stranded DNA template. Subsequently, (exo-)Klenow DNA polymerase and one of either α-thio-dATP, dTTP, dGTP, or dCTP were added and incubated for 1 min. Pyrophosphate released by DNA polymerase is converted to ATP by pyruvate phosphate dikinase (PPDK), and the concentration of ATP is determined using the firefly luciferase reaction. This method, which does not require expensive equipment, can be used to rapidly monitor one point mutation in the gene.     

Transient gene expression mediated by integrase-defective retroviral vectors

Nonintegrating retroviral vectors were produced from a Moloney murine leukemia virus (MoMLV)-based retroviral vector system by introducing a point mutation into the integrase (IN) gene of the packaging plasmid. The efficacy of IN-defective retroviral vectors was measured through the transient expression of ZsGreen or luciferase in human cell lines. The IN-defective retroviral vectors could transduce target cells efficiently, but their gene expression was transient and lower than that seen with the integrating vectors. IN-defective retroviral vector gene expression decreased to background levels in fewer than 10 days. Southern blot analysis of transduced K562 cells confirmed the loss of a detectable vector sequence by 15 days. The residual integration activity of the IN-defective vector was 1000- to 10,000-fold lower than that of the integrating vector. These results demonstrate that the IN-defective retroviral vectors can provide a useful tool for efficient transient gene expression targeting of primary hematopoietic stem cells and lymphoid cells.     

Isolation of stable (alphabeta)4-Tetraprotomer from Na+/K+-ATPase solubilized in the presence of short-chain fatty acids

Previously, it was demonstrated that acetate anions increase the higher oligomer (H), consuming (alphabeta) 2-diprotomer (D) and alphabeta-protomer (P) of solubilized dog kidney Na (+)/K (+)-ATPase [ Kobayashi, T. et al. (2007) J. Biochem. 142, 157-173 ]. Presently, short-chain fatty acids, such as propionate (Prop) and butyrate, have been substituted effectively for acetate. The molecular weight of 6.01 x 10 (5) for H and quantitative Na (+)/K (+)-dependent interconversion among H, D, and P showed that H was an (alphabeta) 4-tetraprotomer (T). T was optimally isolated from the enzyme solubilized in aqueous 40 mM K (+)Prop at pH 5.6 by gel chromatography performed at 0 degrees C with elution buffer containing synthetic dioleoyl phosphatidylserine (PS). K 0.5 values of K (+)-congeners constituting K (+)Prop for the maximal amount of T were NH 4 (+) > Rb (+) congruent with K (+) > Tl (+), while Na (+) had no effect. The oligomers of T, D, and P were simultaneously assayed for ATPase upon elution from the gel column, resulting in a specific activity ratio of 1:2:2. The activity of the chromatographically isolated T increased with an increasing dioleoyl PS, giving a saturated activity of 2.38 units/mg at pH 5.6 and 25 degrees C, and the active enzyme chromatography of T showed 34% dissociation into D by exposing it at 25 degrees C. On the basis of these data, the specific ATPase activities of T, D, and P were concluded to be 32, 65, and 65 units/mg, respectively, under the conventionally optimal conditions of pH 7.3 and 37 degrees C, suggesting an equivalence to a fully active enzyme for D and P but half activity for T. The physiological significance of the stable form of T remains to be investigated.     

Functional cooperation among Ras, STAT5, and phosphatidylinositol 3-kinase is required for full oncogenic activities of BCR/ABL in K562 cells.

BCR/ABL tyrosine kinase generated from the chromosomal translocation t(9;22) causes chronic myelogenous leukemia and acute lymphoblastic leukemia. To examine the roles of BCR/ABL-activated individual signaling molecules and their cooperation in leukemogenesis, we inducibly expressed a dominant negative (DN) form of Ras, phosphatidylinositol 3-kinase, and STAT5 alone or in combination in p210 BCR/ABL-positive K562 cells. The inducibly expressed DN Ras (N17), STAT5 (694F), and DN phosphatidylinositol 3-kinase (Delta p85) inhibited the growth by 90, 55, and 40%, respectively. During the growth inhibition, the expression of cyclin D2 and cyclin D3 was suppressed by N17, 694F, or Delta p85; that of cyclin E by N17; and that of cyclin A by Delta p85. In addition, N17 induced apoptosis in a small proportion of K562, whereas 694F and Delta p85 were hardly effective. In contrast, coexpression of two DN mutants in any combinations induced severe apoptosis. During these cultures, the expression of Bcl-2 was suppressed by N17, 694F, or Delta p85, and that of Bcl-XL by N17. Furthermore, although K562 was resistant to interferon-alpha- and dexamethasone-induced apoptosis, disruption of one pathway by N17, 694F, or Delta p85 sensitized K562 to these reagents. These results suggested that cooperation among these molecules is required for full leukemogenic activities of BCR/ABL.     

Recovery and purification of aprotinin from industrial insulin-processing effluent by immobilized chymotrypsin and negative IMAC chromatographies

Aprotinin, a bovine protease inhibitor currently also produced in recombinant bacteria, yeast, and corn, has valuable applications as a human therapeutic and in tissue culture. The objective of this work was to develop the basis of a large-scale aprotinin purification process centered on immobilized metal ion affinity chromatography (IMAC). This technique uses ligands—metal ions—of a lower cost and higher stability than those traditionally used in affinity chromatography. Since aprotinin does not interact with IMAC ligands, collection is from the nonretained fractions (negative chromatography). Stirred-tank batch IMAC adsorption experiments indicated that one-step aprotinin purification could not be successful. Immobilized chymotrypsin chromatography was then used as a prepurification step, yielding a suitable feed for IMAC (with purification factors as high as 476). IMAC column fed with these prepurified materials produced purified aprotinin in the nonretained fractions with purification factors as high as 952.