Purification of phosphoproteins by immobilized metal affinity chromatography and its application to phosphoproteome analysis

Prefractionation procedures facilitate the identification of lower-abundance proteins in proteome analysis. Here we have optimized the conditions for immobilized metal affinity chromatography (IMAC) to enrich for phosphoproteins. The metal ions, Ga(III), Fe(III), Zn(II), and Al(III), were compared for their abilities to trap phosphoproteins; Ga(III) was the best. Detailed analyses of the pH and ionic strength for IMAC enabled us to determine the optimal conditions (pH 5.5 and 0.5 m NaCl). When whole cell lysates were fractionated in this way, about one-tenth of the total protein was recovered in the eluate, and the recovery of phosphorylated extracellular signal-regulated kinase (ERK) was more than 90%. Phosphorylated forms of ribosomal S6 kinase (RSK) and Akt were also enriched efficiently under the same conditions. Our Ga(III) IMAC and a commercially available purification kit for phosphoproteins performed similarly, with a slight difference in the spectrum of phosphoproteins. When phosphoproteins enriched from NIH3T3 cells in which ERK was either activated or suppressed were analyzed by two-dimensional fluorescence difference gel electrophoresis, phosphorylated ERK was detected as discrete spots unique to ERK-activated cells, which overlapped with surrounding spots in the absence of prefractionation. We applied the same technique to search for Akt substrates and identified Abelson interactor 1 as a novel potential target. These results demonstrate the efficacy of phosphoprotein enrichment by IMAC and suggest that this procedure will be of general use in phosphoproteome research.     

Changes in herbaceous plants in an urban habitat garden in Kyoto city, Japan, 9 years after construction

To understand the characteristics and problems of artificial urban ecological environments, we investigated the changes in herbaceous plants in an urban habitat garden for 9 years after construction and compared the results with 15 remnant semi-natural green spaces in Kyoto city, Japan. The area of the habitat garden is 0.6 ha and it was constructed approximately 3 km from the nearest mountains in 1996. From 1996 to 2004, 301 unplanted species, including 218 native species and 83 alien species, were recorded. Most newly recorded species were recorded in 1998 and the timing of colonization was different from ferns. The species turnover rate per year decreased from 30.8% in 1998 to 18.5% in 2004. This indicates that established species in the habitat garden have been gradually determined. In the species recorded in the habitat garden in 2004, the percentage of high-temperature species, which were relatively departed from the nested pattern of species composition for the 15 green spaces, was significantly high. It was considered that herbaceous plant succession in the habitat garden was at an early stage, even though it was 9 years ago since construction. Buried seeds and various environments are likely to contribute to higher species richness in the habitat garden as compared to remnant green spaces of approximately the same area. On the other hand, the high percentage of alien species and the low percentages of forest and forest edge species in the habitat garden indicate the problems of a habitat garden constructed in the center of a city.     

The independent detection of drought stress and leaf density using hyperspectral resolution data

Measurement of vegetation drought stress or leaf density is essential in ecosystem and agronomic studies. The normalized differential vegetation index (NDVI), a widely used vegetation index in remote sensing, seems to have some limitations as it is known to be affected by both drought stress and leaf density. A field experiment was conducted, using two-year-old potted Quercus serrata (a deciduous tree) and Q. glauca (an evergreen tree), to determine the optimal indices of vegetation drought stress or leaf density that have the least a simultaneous effect, and to test if the existing vegetation indices are useful for independently detecting drought stress or leaf density. The results showed that NDVI and similar indices, which utilize the difference or ratio between the reflectance of red and near infrared bands, such as the ratio vegetation index (RVI), the difference vegetation index (DVI), the atmospherically resistant vegetation index (ARVI), the renormalized difference vegetation index (RDVI), the enhanced vegetation index (EVI), the perpendicular vegetation index (PVI), soil-adjusted vegetation index (SAVI) and the improved variants of SAVI, were effective for the independent detection of leaf density but relatively ineffective for drought stress because they were significantly affected by leaf area index (LAI). Similarly, vegetation indices developed as detectors of vegetation stress, such as the water index (WI), the stress index (SI) and the derivative chlorophyll index (DCI), showed weak correlation (r) and partial correlation (r _p) with leaf water content (LWC). The optimal hyperspectral indices were proposed as (F _502.8 − F _852.0)/(F _502.8 + F _852.0) for LWC (r = 0.847, r _p = 0.849) and R ₇₅₀/R ₅₅₀ (R750R550; Lichtenthaler et al. in J Plant Physiol 148:483–493, 1996) for LAI (r = 0.926, r _p = 0.940) where R _λ and F _λ represent reflectance and first derivatives at wavelength λ nm, respectively. A simulation of lower spectral sampling intervals (ca. 3-nm intervals of original to 10-nm intervals) indicated that it will be necessary to check the appropriateness of the derivative indices approximate to the proposed indices before application because derivative spectra are less smooth as a function of wavelength than reflectance spectra.     

On-fiber display of a functional peptide at sites distant from the cell surface using a long bacterionanofiber of a trimeric autotransporter adhesin

In the cell surface display system, the distance of a surface-displayed molecule from the cell surface should influence its functionality due to the interference by other surface structures. For the purpose of developing this distance-variable surface display system, we utilized a long fibrous adhesin, Acinetobacter trimeric autotransporter adhesin (AtaA) of the strain Tol 5. We constructed His-tagged full-length and shorter AtaA fibers designed by N-terminal deletion and expressed them in the ΔataA mutant. Immunoelectron microscopy clearly showed that they formed fibers on the cell surface and the His-tag was displayed on the fiber tip located at fixed distances from the cell surface. N-terminal deletion of AtaA shortened the distance between the His-tag and the cell surface, as designed. Time-course analyses of the cell-to-Ni-Sepharose beads binding revealed that cells producing the longer fibers bound more rapidly to the beads. The His-tagged AtaA derivatives were also displayed on Escherichia coli cells, and a similar tendency was shown; the His-tag on the longer fiber was more functional than that on the shorter one. Thus, we developed an on-fiber display system of a functional peptide using a long trimeric autotransporter adhesin (TAA) fiber, which can vary the distance between the displayed molecule and the cell surface.     

Three new species in the genus Tulasnella isolated from orchid mycorrhiza of Spiranthes sinensis var. amoena (Orchidaceae)

We isolated Tulasnella spp. from Spiranthes sinensis var. amoena, a Japanese native winter green terrestrial orchid collected in Tsukuba City, Ibaraki Pref., Japan. These isolates were classified into four morphotypes according to morphological characters, i.e., shape of monilioid cells and branch type of monilioid cell chains, while they were separated into five clades by molecular phylogenetic analysis based on the rDNA 5.8S and D1/D2 regions. The four morphotypes and five clades were correlated, and four morpho-phylogenetic groups were identified. Thus, Tulasnella deliquescens including two phylogenetic subgroups and three new species, Tulasnella dendritica sp. nov., Tulasnella ellipsoidea sp. nov., and Tulasnella cumulopuntioides sp. nov., were recognized in this study. In this study, the monilioid cell chain morphology is newly defined, and is suggested as a useful taxonomic characteristic in the asexual stage of Tulasnella spp.     

Individual spawning performance and mating pair combinations in captive grouper aggregations

Environmental Biology of Fishes - Spawning aggregations have been widely observed in fish. However, individual reproductive performance in aggregations has rarely been measured because of the lack...     

Use of an Escherichia coli recombinant producing thermostable polyphosphate kinase as an ATP regenerator to produce fructose 1,6-diphosphate

Heat-treated Escherichia coli producing Thermus polyphosphate kinase regenerated ATP by using exogenous polyphosphate. This recombinant could be used as a platform to produce valuable compounds in combination with thermostable phosphorylating or energy-requiring enzymes. In this work, we demonstrated the production of fructose 1,6-diphosphate from fructose and polyphosphate.     

Inhibition of quorum sensing in Pseudomonas aeruginosa by N-acyl cyclopentylamides

N-octanoyl cyclopentylamide (C8-CPA) was found to moderately inhibit quorum sensing in Pseudomonas aeruginosa PAO1. To obtain more powerful inhibitors, a series of structural analogs of C8-CPA were synthesized and examined for their ability to inhibit quorum sensing in P. aeruginosa PAO1. The lasB-lacZ and rhlA-lacZ reporter assays revealed that the chain length and the ring structure were critical for C8-CPA analogs to inhibit quorum sensing. N-decanoyl cyclopentylamide (C10-CPA) was found to be the strongest inhibitor, and its concentrations required for half-maximal inhibition for lasB-lacZ and rhlA-lacZ expression were 80 and 90 microM, respectively. C10-CPA also inhibited production of virulence factors, including elastase, pyocyanin, and rhamnolipid, and biofilm formation without affecting growth of P. aeruginosa PAO1. C10-CPA inhibited induction of both lasI-lacZ by N-(3-oxododecanoyl)-L-homoserine lactone (PAI1) and rhlA-lacZ by N-butanoyl-L-homoserine lactone (PAI2) in the lasI rhlI mutant of P. aeruginosa PAO1, indicating that C10-CPA interferes with the las and rhl quorum-sensing systems via inhibiting interaction between their response regulators (LasR and RhlR) and autoinducers.     

START-GAP3/DLC3 is a GAP for RhoA and Cdc42 and is localized in focal adhesions regulating cell morphology

In the human genome there are three genes encoding RhoGAPs that contain the START (steroidogenic acute regulatory protein (StAR)-related lipid transfer)—domain. START-GAP3/DLC3 is a tumor suppressor gene similar to two other human START-GAPs known as DLC1 or DLC2. Although expression of START-GAP3/DLC3 inhibits the proliferation of cancer cells, its molecular function is not well understood. In this study we carried out biochemical characterization of START-GAP3/DLC3, and explored the effects of its expression on cell morphology and intracellular localization. We found that START-GAP3/DLC3 serves as a stimulator of PLCδ1 and as a GAP for both RhoA and Cdc42 in vitro. Moreover, we found that the GAP activity is responsible for morphological changes. The intracellular localization of endogenous START-GAP3/DLC3 was explored by immunocytochemistry and was revealed in focal adhesions. These results indicate that START-GAP3/DLC3 has characteristics similar to other START-GAPs and the START-GAP family seems to share common characteristics.     

Production of bioactive lysophosphatidic acid by lysophospholipase D in hen egg white

Lysophosphatidic acid (LPA), a lysophospholipid mediator, is produced extracellularly by lysophospholipase D (lysoPLD) secreted in several animal body fluids including blood plasma. Previously, we reported that hen egg white contains polyunsaturated fatty acid-rich LPA. In this study, we examined whether lysoPLD is involved in the production of LPA in hen egg white. LysoPLD activity was measured by determining LPA and choline by mass spectrometric and enzyme-linked fluorometric analyses, respectively. LysoPLD increased with increased dilution of egg white, indicating that one or more components of egg white strongly inhibit its lysoPLD activity. This dilution-dependent increase in the lysoPLD activity was masked by co-incubation of the egg white with lysozyme, a major protein in hen egg white. Furthermore, addition of Zn(2+), Mn(2+), Ni(2+), or Co(2+) to diluted egg white altered preference patterns of lysoPLD toward choline-containing substrates. In particular, the egg white lysoPLD activity was greatly increased when Co(2+) was added. The cation-requirement of lysoPLD activity in hen egg white resembled that of plasma autotaxin (ATX)/lysoPLD. Western blot analysis revealed that egg white contained a protein that was immunostained with anti-ATX antibody. These results suggested that LPA in hen egg white is produced from lysophospholipids, especially LPC, by the action of ATX/lysoPLD, possibly originating from hen oviduct fluid.