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.     

Conformational analysis in solution of gastrin releasing peptide

Gastrin releasing peptide (GRP) is the first peptide isolated from porcine gastric and intestinal tissues and is homologous to the carboxyl terminus of bombesin (Bn) isolated from the skin of the frog Bombina bombina. It is a member of the Bn-like peptides, which are important in numerous biological and pathological processes. The Bn-like peptides show high sequence homology in their C-terminal regions, but they have different selectivity for their receptors. In particular, GRP selectively binds to the GRP receptor (GRPR). However, the molecular basis for this selectivity remains largely unknown. Here, we report the three-dimensional structure of GRP. Hopefully, it could be helpful in a better understanding of the binding selectivity between GRP and GRPR.     

Production of three O-methhylated esculetins with Escherichia coli expressing O-methyltransferase from poplar

O-Methyltransferase, POMT-9 was expressed in Escherichia coli. HPLC analysis of reaction products revealed three peaks corresponding to isoscopoletin, scopoletin, and scoparone, and their structures were determined using NMR. Biotransformation of esculetin with E. coli expressing POMT-9 generated scopoletin, isoscopoletin, and scoparone at 30.3, 21, and 31 microM respectively. POMT-9 is the first O-methyltransferase that produces three different O-methylated products.     

Protein tyrosine phosphatase 1B inhibitors from Morus root bark

An organic layer prepared from the Chinese crude drug 'Sang-Bai-Pi' (Morus root bark) was studied in order to identify the inhibitory compounds for protein tyrosine phosphatase 1B (PTP1B). Bioassay-guided fractionation resulted in the isolation of sanggenon C (1), sanggenon G (2), mulberrofuran C (3) and kuwanon L (4) as PTP1B inhibitors, along with moracin O (5) and moracin P (6). Compounds 1-4 inhibited PTP1B with IC(50) values ranging from 1.6+/-0.3 microM to 16.9+/-1.1 microM.     

Isolation and functional characterization of the Arabidopsis salt-tolerance 32 (AtSAT32) gene associated with salt tolerance and ABA signaling

Recently, we have isolated salt-tolerance genes (SATs) on the basis of the overexpression screening of yeast with a maize cDNA library from kernels. One of the selected genes [ salt-tolerance 32 ( SAT32 )] appears to be a key determinant for salt stress tolerance in yeast cells. Maize SAT32 cDNA encodes for a 49-kDa protein, which is 41% identity with the Arabidopsis salt-tolerance 32 ( AtSAT32 ) unknown gene. Arabidopsis Transfer-DNA (T-DNA) knockout AtSAT32 ( atsat32 ) altered root elongation, including reduced silique length and reduced seed number. In an effort to further assess salinity tolerance in Arabidopsis , we have functionally characterized the AtSAT32 gene and determined that salinity and the plant hormone ABA induced the expression of AtSAT32 . The atsat32 mutant was more sensitive to salinity than the wild-type plant. On the contrary, Arabidopsis overexpressing AtSAT32 (35S:: AtSAT32 ) showed enhanced salt tolerance and increased activity of vacuolar H⁺-pyrophosphatase (V-PPase, EC 3.6.1.1) under high-salt conditions. Consistent with these observations, 35S:: AtSAT32 plants exhibited increased expression of salt-responsive and ABA-responsive genes, including the Rd29A , Erd15 , Rd29B , Rd22 and RAB18 genes. Therefore, our results indicate that AtSAT32 is involved in both salinity tolerance and ABA signaling as a positive regulator in Arabidopsis .     

Molecular characterization of flavonoid malonyltransferase from Oryza sativa

In this study, a flavonoid malonyltransferase (OsMaT-2) was cloned from Oryza sativa, and the recombinant protein OsMaT-2 was purified via affinity chromatography. OsMaT-2 utilized a variety of flavonoid glucosides, including flavanone glucosides, flavone glucosides, flavonol glucosides, and isoflavone glucosides as substrates, but did not utilize anthocyanin. As an acyl donor, OsMaT-2 utilized only malonyl-CoA. Based on reactions with various quercetin 3-O-sugars, we identified the probable position of malonylation as the 6″-hydroxyl group of the sugar. This is the first report, to the best of our knowledge, of the cloning of a flavonoid malonyltransferase from O. sativa.     

A critical role for AKT activation in protecting cells from ionizing radiation-induced apoptosis and the regulation of acinus gene expression

Although AKT activation leads to the activation of various pathways related to cell survival, the roles of AKT in modulating cellular responses induced by ionizing radiation in normal human cells remain unclear. Here we show that low-dose radiation of 0.05 Gy did not affect cell death, but high-dose radiation (> 0.2 Gy) induced apoptosis through the activation of caspases and acinus cleavage. Ionizing radiation induced acinus phosphorylation via AKT activation. Thus, we examined the effect of AKT activation on radiation-induced cell death using CCD-18Lu cells transduced with a retroviral vector expressing constitutively active AKT (CA-AKT). The overexpression of CA-AKT rendered the cells resistant to ionizing radiation and prevented the proteolytic cleavage of acinus via phosphorylation. In addition, overexpression of CA-AKT resulted in the upregulation of acinus expression by activation of the NF-κB pathway. On the other hand, suppression of endogenous AKT expression by siRNA resulted in the reduction of acinus expression and enhanced the radiation-induced apoptosis in both CCD-18Lu and IM-9 cells. Our results suggest that AKT activation inhibits cell death during radiation-induced apoptosis through the regulation of phosphorylation and expression of acinus. The AKT/NF-κB/acinus pathway functions as one of the important regulatory mechanisms required for modulating ionizing radiation sensitivity.     

Nutrient removal from polluted stream water by artificial aquatic food web system

For the removal of nutrients from eutrophic stream water polluted by non-point sources, an artificial aquatic food web (AAFW) system comprising processes of phytoplankton growth and Daphnia magna grazing was developed. The AAFW system was a continuous-flow system constructed with one storage basin of 3 m³ capacity, one phytoplankton tank of 3 m³ capacity, and one zooplankton growth chamber of 1.5 m³ capacity. The system was optimized by setting hydraulic retention time of phytoplankton tank as 3 days and D. magna density as 740–1000 individual l⁻¹. When the system was operated on eutrophic stream water that was delivering 471 g of total nitrogen (TN) and 29 g of total phosphorus (TP) loadings for 45 days, 250 g (53%) of TN and 16 g (54%) of TP were removed from the water during its passage through the phytoplankton tank. In addition, 64 g (14%) of TN and 4 g (13%) of TP were removed from the water by harvesting zooplankton biomass in the zooplankton growth chamber, resulting in significant overall removal rates of TN (69%), nitrate (78%), TP (73%), and dissolved inorganic phosphorus (94%). While the removal efficiency of the AAFW system is comparable to those of other ecotechnologies such as constructed wetlands, its operation is less limited by the availability of space or seasonal shift of temperature. Therefore, it was concluded that AAFW system is a highly efficient, flexible system for reducing nutrient levels in tributary streams and hence nutrient loading to large aquatic systems receiving the stream water. Handling editor: J. Padisak     

An assessment of soil bacterial community structure and physicochemistry in two microtopographic locations of a palustrine forested wetland

We studied redoximorphic features, field indicators and bacterial communities of soils in hummocks and hollows of a palustrine forested wetland in Virginia. We hypothesized that presence of hydric soils, soil physicochemistry and soil bacterial community structure would differ between hummocks and hollows. We fingerprinted soils collected from different microtopographic locations using Length Heterogeneity Polymerase Chain Reaction (LH-PCR) to study their bacterial community structures. Two hummocks had silty/sandy loam soils with mean chroma values of > 4, showing no indication of ‘hydric soils’ (i.e., wetland soils). Two hollows, however, had clay loam soils with mean chroma values of 2 with gleying and redox concentrations observed, indicative of seasonally inundated wetlands. The soils of hollows also had higher organic matter content and soil moisture compared to the soils of hummocks (P < 0.05). Multidimensional scaling (MDS) and Analysis of similarity (ANOSIM) of the fingerprints revealed differences in soil microbial community structures between hummocks and hollows (Global R = 0.30, P < 0.01). The diversity measures of the fingerprints (Shannon’s H′) were also different by microtopography with higher diversity in hollows relative to hummocks (P < 0.05). LH-PCR proves to be a useful tool in examining bacterial community composition of wetland soils in this study. However, cloning and sequencing of specific community LH-PCR profiles of interest is necessary to fully characterize the community down to genus/species level. With species identities we should be able to not only better explain differences observed in the community profiles, but study their relations to hydrologic and/or physicochemical conditions of wetlands.     

Inhibitory effect of luteolin on osteoclast differentiation and function

Osteoclasts are multinucleated cells that play a crucial role in bone resorption, and are formed by the fusion of mononuclear osteoclasts derived from osteoclast precursors of the macrophage lineage. Compounds that specifically target functional osteoclasts would be ideal candidates for anti-resorptive agents for clinical applications. In the present study, we investigated the effects of luteolin, a flavonoid, on the regulation of receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclastogenesis, functions and signaling pathway. Addition of luteolin to a coculture system of mouse bone marrow cells and ST2 cells in the presence of 10⁻⁸ M 1α,25(OH)₂D₃ caused significant inhibition of osteoclastogenesis. Luteolin had no effects on the 1α,25(OH)₂D₃-induced expressions of RANKL, osteoprotegerin and macrophage colony-stimulating factor mRNAs. Next, we examined the direct effects of luteolin on osteoclast precursors using bone marrow macrophages and RAW264.7 cells. Luteolin completely inhibited RANKL-induced osteoclast formation. Moreover, luteolin inhibited the bone resorption by mature osteoclasts accompanied by the disruption of their actin rings, and these effects were reversely induced by the disruption of the actin rings in mature osteoclasts. Finally, we found that luteolin inhibited RANKL-induced osteoclastogenesis through the suppression of ATF2, downstream of p38 MAPK and nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 1 (NFATc1) expression, respectively. Taken together, the present results indicate that naturally occurring luteolin has inhibitory activities toward both osteoclast differentiation and functions through inhibition of RANKL-induced signaling pathway as well as actin ring disruption, respectively.