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.     

4′,6-Dimethoxyisoflavone-7-<Emphasis Type="Italic">O</Emphasis>-β-<Emphasis Type="SmallCaps">d</Emphasis>-glucopyranoside (wistin) is a peroxisome proliferator-activated receptor α (PPARα) agonist in mouse hepatocytes

Human calcium/calmodulin-dependent protein kinase IV (CAMKIV) is a member of Ser/Thr kinase family, and is associated with different types of cancer and neurodegenerative diseases. Vanillin is a natural compound, a primary component of the extract of the vanilla bean which possesses varieties of pharmacological features including anti-oxidant, anti-inflammatory, anti-bacterial and anti-tumor. Here, we have investigated the binding mechanism and affinity of vanillin to the CAMKIV which is being considered as a potential drug target for cancer and neurodegenerative diseases. We found that vanillin binds strongly to the active site cavity of CAMKIV and stabilized by a large number of non-covalent interactions. We explored the utility of vanillin as anti-cancer agent and found that it inhibits the proliferation of human hepatocyte carcinoma (HepG2) and neuroblastoma (SH-SY5Y) cells in a dose-dependent manner. Furthermore, vanillin treatment resulted into the significant reduction in the mitochondrial membrane depolarization and ROS production that eventually leads to apoptosis in HepG2 and SH-SY5Y cancer cells. These findings may offer a novel therapeutic approach by targeting the CAMKIV using natural product and its derivative with a minimal side effect.     

Isolation and Identification of Gibberellins A4 and A9 from a Fungus Phaeosphaeria sp.

Quinolactacide isolated from Penicillium citrinum F 1539 was synthesized and evaluated for its insecticidal activities. The key steps of the total synthesis were an acyl migration reaction of the enol ester intermediate and dehydrogenation of tetrahydroquinolactacide with manganese dioxide. The synthesized quinolactacide showed 100% and 42% mortality against the green peach aphid (Myzus persicae) and diamondback moth (Plutella xylostella) at 500 ppm, respectively.