Akt phosphorylation is essential for nuclear translocation and retention in NGF-stimulated PC12 cells

Nerve growth factor (NGF) elicits Akt translocation into the nucleus, where it phosphorylates nuclear targets. Here, we describe that Akt phosphorylation can promote the nuclear translocation of Akt and is necessary for its nuclear retention. Overexpression of Akt-K179A, T308A, S473A-mutant failed to show either nuclear translocation or nuclear Akt phosphorylation, whereas expression of wild-type counterpart elicited profound Akt phosphorylation and induced nuclear translocation under NGF stimulation. Employing the PI3K inhibitor and a variety of mutants PI3K, we showed that nuclear translocation of Akt was mediated by activation of PI3K, and Akt phosphorylation status in the nucleus required PI3K activity. Thus the activity of PI3K might contribute to the nuclear translocation of Akt, and that Akt phosphorylation is essential for its nuclear retention under NGF stimulation conditions.     

An affinity-based scoring scheme for predicting DNA-binding activities of modularly assembled zinc-finger proteins

Zinc-finger proteins (ZFPs) have long been recognized for their potential to manipulate genetic information because they can be engineered to bind novel DNA targets. Individual zinc-finger domains (ZFDs) bind specific DNA triplet sequences; their apparent modularity has led some groups to propose methods that allow virtually any desired DNA motif to be targeted in vitro. In practice, however, ZFPs engineered using this ‘modular assembly’ approach do not always function well in vivo. Here we report a modular assembly scoring strategy that both identifies combinations of modules least likely to function efficiently in vivo and provides accurate estimates of their relative binding affinities in vitro. Predicted binding affinities for 53 ‘three-finger’ ZFPs, computed based on energy contributions of the constituent modules, were highly correlated (r = 0.80) with activity levels measured in bacterial two-hybrid assays. Moreover, K_d values for seven modularly assembled ZFPs and their intended targets, measured using fluorescence anisotropy, were also highly correlated with predictions (r = 0.91). We propose that success rates for ZFP modular assembly can be significantly improved by exploiting the score-based strategy described here.     

Tyrosinase inhibitory effects of 1,3-diphenylpropanes from Broussonetia kazinoki

Six 1,3-diphenylpropanes exhibiting inhibitory activities against both the monophenolase and diphenolase actions of tyrosinase were isolated from the methanol (95%) extract of Broussonetia kazinoki. These compounds, 1–6, were identified as kazinol C (1), D (2), F (3), broussonin C (4), kazinol S (5) and kazinol T (6). The latter two species (5 and 6) emerged to be new 1,3-diphenylpropanes which we fully spectroscopically characterized. The IC₅₀ values of compounds (1, 3–5) for monophenolase inhibition were determined to range between 0.43 and 17.9 μM. Compounds 1 and 3–5 also inhibited diphenolase significantly with IC₅₀ values of 22.8, 1.7, 0.57, and 26.9 μM, respectively. All four active tyrosinase inhibitors (1, 3–5) were competitive inhibitors. Interestigly they all mainfested simple reversible slow-binding inhibition against diphenolase. The most potent inhibitor, compound 4 diplayed the following kinetic parameters k₃ = 0.0993 μM^?1 min^?1, k₄ = 0.0048 min^-1, and K_i^app = 0.0485 μM.     

Long-term gus expression from Gladiolus callus lines containing either a bar-uidA fusion gene or bar and uidA delivered on separate plasmids

Gus expression was determined for 19 lines of embryogenic Gladiolus callus that contained the 35S-bar-uidA-nos fusion gene and for 21 callus lines that had been cobombarded with the 35S-bar-nos and 35S-uidA-nos plasmid DNAs. These lines were selected for analysis because they grew vigorously on Murashige and Skoog’s medium supplemented with 6 mg l⁻¹ phosphinothricin. All 19 lines that contained the 35S-bar-uidA-nos fusion gene expressed gus compared to only 15 (71%) of the lines that had been cobombarded as determined by enzyme assay. The level of gus expression was significantly higher the first year for 12 callus lines containing the bar-uidA fusion gene as compared to 2 years later in culture. Southern hybridization confirmed integration of the uidA gene in all callus lines that had been bombarded with the 35S-bar-uidA-nos fusion gene. Two of the callus lines that had been cobombarded lacked the uidA gene, and another cobombarded line that did not express gus contained a truncated uidA gene. Two callus lines resulting from cobombardment showed gus expression in only a few cells indicating that gus expression was not completely silenced in these lines. Gus expression could not be reversed using 5-azacytidine in these two low-expressing lines, and Southern hybridization supported that methylation of the genomic DNA had not occurred. Average levels of gus expression were significantly higher, 8.9× , in cells with the 35S-bar-uidA-nos fusion gene compared to the cobombarded callus lines indicating the advantage of using a bar-uidA fusion gene for obtaining higher levels of gus expression in Gladiolus.     

Characteristics of cold-adaptive endochitinase from Antarctic bacterium Sanguibacter antarcticus KOPRI 21702

The psychrotrophic Sanguibacter antarcticus KOPRI 21702^T, isolated from Antarctic seawater, produced a cold-adapted chitinolytic enzyme that is a new 55 kDa family 18 chitinase (Chi21702). Chi21702 exhibited high activities toward pNP-(GlcNAc)₂ and pNP-(GlcNAc)₃ with no activity for pNP-GlcNAc, indicating that it prefers chitin chains longer than dimers, just as endochitinases do. A mixture of GlcNAc and GlcNAc₂ was produced as a main product by Chi21702 activity from chitin oligosaccharides and swollen chitin, while less GlcNAc₃ was produced. These results show that Chi21702 has an endochitinase activity, randomly hydrolyzing chitin at internal sites. Chi21702 displayed chitinase activity at 0–40 °C (optimal temperature of 37 °C), maintained its activity at pH 4–11 (optimal pH of 7.6). Interestingly, Chi21702 exhibited relative activities of 40% and 60% at 0 and 10 °C, respectively, in comparison to 100% at 37 °C, which is higher than those of the previously characterized, cold-adapted, chitinases from bacterial strains.     

Introducing pulsed low-intensity ultrasound to culturing human umbilical cord-derived mesenchymal stem cells

The human umbilical cord (hUC) is a source of adult tissue-derived mesenchymal stem cells (MSCs). A pulsed low-intensity ultrasound (PLIUS) method is described for increasing the yield of MSCs from whole hUC without enzymatic digestion or growth factor supplementation. Analysis of the immunophenotype of cells and a differentiation study were performed to show the compatibility of MSCs. The mean number of cells recovered from primocultures of hUC was 6 × 10⁵ cells/cm. PLIUS resulted in a 3.3-fold increase in MSC yield at passage 0. PLIUS exposure increases the yield of hUC-MSCs by promoting release and enhancing proliferation. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Metabolic profiles of genetically modified potatoes using a combination of metabolite fingerprinting and multivariate analysis

Comprehensive metabolite fingerprinting of transgenic potatoes that constitutively express human beta amyloid, curdlan synthase (CRDS), and glycogen synthase (glgA); and of wild-type potatoes was carried out using FT-IR and ¹H NMR spectroscopy in combination with multivariate analyses. Comparison of metabolic patterns between transgenic and wild-type potatoes revealed that there were neither quantitative nor qualitative differences in metabolites between transgenic potatoes expressing human beta amyloid, CRDS or glgA, and non-transformed control potatoes. However, there were metabolic differences between two control potato lines — one that was fresh and the other stored. After 1 week of storage, comprehensive metabolite patterns were significantly modified. Although the differences between CRDS and glgA transgenic and control potato lines were small, PCA analysis of FT-IR and ¹H NMR spectral data identified two distinct control lines. These results suggest that the comprehensive metabolite changes in control potato lines, which occurred after 1 week of storage, were greater than the differences between CRDS and glgA transgenic and wild-type potato lines. Thus, the combination of FT-IR and ¹H NMR spectral data and multivariate analysis was valuable for the detection of comprehensive differences in metabolic profiles between transgenic and non-transformed control plants, even though peak-signal overlap prevented assignment of pure compounds. The combination of FT-IR and ¹H NMR spectral data and multivariate analysis is a simple and rapid method for evaluation of the metabolic equivalence of GM crops.