Isolation and characterization of <i>cat8-1</i> and 35S::CAT8 overexpressor lines.

<p>(A) Schematic drawing of the position of the T-DNA insertion (black triangle) and the used primers in the cat8 gene. Verification of the T-DNA insertion on genomic DNA (left panel) and identification of homozygous plants by RT-PCR confirmed by amplification of a 1739 bp fragment (right panel). Actin served as control. In all cases, 30 cycles were used. (B) Growth on 1 µM MSX (upper panel) and 0.5 µM MSX (lower panel). From left to right: <i>35S::CAT8</i>, wild type, <i>cat8-1</i>.</p

Sub-cellular localization of CAT8 expressed from the endogenous promoter in the plasma membrane and the tonoplast.

<p>(A–D) Subcellular localization of pCAT8::CAT8-GFP (A) in the root tip, (B) in the meristematic zone, (C) from the center to epidermal cells, (D) and guard cell. Scaling bars: 10 µm. (E) Ultrastructural analysis of pCAT8::CAT8-GFP plants with transmission electron microscopy and immunogold labeling using the GFP antibody. Tonoplast and plasma membrane localization of CAT8 is visible as black dots. The plasma membrane localization was highlighted by small arrows. Vacuole, v; plasmodesmata, pd; cell wall, cw. Scaling bar: 100 nm. (F) Immunogold labeling of CAT8-GFP in membranes of autophagocytotic structures (as). Scaling bar: 100 nm. (G, H) Quantitative analysis of the fluorescence intensity in plasma membranes and the tonoplast along the orange line. Fluorescence intensity histograms indicate similar fluorescence strength in the membranes.</p

Freeze-Dissolving Method: A Fast Green Technology for Producing Nanoparticles and Ultrafine Powder

A new technology, a freeze-dissolving method, has been developed to isolate nanoparticles or ultrafine powder and is a more efficient and sustainable method than the traditional freeze-drying method. In this work, frozen spherical ice particles were produced with an aqueous solution of sodium bicarbonate or ammonium dihydrogen phosphate at various concentrations to generate nanoparticles of NaHCO3 or (NH4)­(H2PO4). The freeze-drying method sublimates ice, and nanoparticles of NaHCO3 or (NH4)­(H2PO4) in the ice templates remain. The freeze-dissolving method dissolves ice particles in a low freezing point solvent at temperatures below 0 °C, and then, nanoparticles of NaHCO3 or (NH4)­(H2PO4) can be isolated after filtration. The freeze-dissolving method is 100 times faster with about 100 times less energy consumption than the freeze-drying method as demonstrated in this work with a much smaller facility footprint and produces the same quantity of nanoparticles with a more uniform size distribution

Influence of solvent and solid-state structure on nucleation of parabens

In the present work, the induction time for nucleation of ethyl paraben (EP) and propyl paraben (PP) in ethanol, ethyl acetate, and acetone has been measured at different levels of supersaturation. The induction time shows a wide variation among repeat experiments, indicative of the stochastic nature of nucleation. The solid–liquid interfacial energy and the size of the critical nucleus have been determined according to the classical nucleation theory. Combined with previous results for butyl paraben (BP), the nucleation behavior is analyzed with respect to differences in the solid phase of the three pure compounds, and with respect to differences in the solution. The results indicate that the difficulty of nucleation in ethanol and acetone increases in the order BP < PP < EP but is approximately the same in ethyl acetate. For each of the three parabens, the difficulty of nucleation increases in the order acetone < ethyl acetate < ethanol. The Gibbs energy of melting increases in the order BP < PP < EP, but the crystal structures are quite similar resulting in the basic crystal shape being very much the same. The solid–liquid interfacial energy is reasonably well correlated to the solvation energy, and even better correlated to the deformation energy, of the solute molecule within the first solvation shell as obtained by density functional theory calculations

Construction of a FRET reporter for glutamine and <i>in vitro</i> properties of the purified protein.

<p>(A) Schematic design of the reporter. (B,C) FRET reporter with mutation D157N. (B) K_d^app = 6.9 mM Gln, dynamic range <i>r</i>_max−<i>r</i>_min = 2.21 (n = 3). (D) Specificity to all proteinogenic amino acids. Ratio change upon addition of 3 mM (black bars) or 10 mM (white bars) of the amino acids given in tree letter code (n = 3). (D,E) FRET reporter with mutations D157N and T70A. (D) K_d^app = 18.8 mM Gln, dynamic range <i>r</i>_max−<i>r</i>_min = 2.31 (n = 3). (E) Specificity and ratio change upon addition of 3 mM (black bar) or 10 mM (white bar) of the amino acids given in tree letter code (n = 3).</p

Functional characteristics of CAT8 in yeast and oocytes.

<p>(A) Uptake rates in 22Δ8AA yeast with 1 mM Gln. White bars: pH 5.5, black bars pH 7.5 (n = 4). The uptake by CAT8 was marginally significant at the p<0.05 level at pH 7.5 (*), using the Mann-Whitney test. (B) Uptake rates in oocytes at 1 mM Gln at pH 5.5 and pH 7.5. In the indicated experiments, Arg and Leu, or Glu and Phe, were added at 2 mM as competitors (n = 3).The uptake by CAT8 was significant at the p<0.05 level (Mann-Whitney test). (C) Concentration dependence of Gln uptake by CAT8. Uptake from water-injected oocytes was subtracted from CAT8-injected oocytes at pH 7.5. A linear background uptake was observed in both oocyte batches. (D) Current-voltage relations of CAT8-expressing oocytes in the absence (black circle) and presence (open triangle) of 1 mM Gln at pH 5.5 (n = 4, solid lines). The dashed line corresponds to the current-voltage relation of water-injected control oocytes from the same batch (closed triangles). Similar data were obtained at pH 7.5.</p

Wet Milling, Seeding, and Ultrasound in the Optimization of the Oiling-Out Crystallization Process

Complicated solution environments in oiling-out crystallization can lead to particle agglomeration with wide size distribution and low purity of the products, due to complex interactions among two liquid phases and one solid phase during the oiling-out crystallization. This research mainly focuses on the optimization of size distribution by controlling particle agglomeration during the oiling-out crystallization process in a model system of propyl paraben–ethanol–water. Nucleation control technologies, wet milling, seeding, and ultrasound were used to limit the agglomeration. Further investigations of wet milling were performed before the nucleation or in the crystal growth stages with different geometries, such as coarse, medium, and fine rotor–stator tooth pairs. An integrated process analytical technology tools (PAT) array, including focused beam reflectance measurement (FBRM), particle visual monitoring (PVM), and attenuated total reflectance ultraviolet/visible (ATR-UV/vis), was used to observe the droplet formation of the dispersed phase, size distributions, and particle shapes during the nucleation and crystal growth. The results demonstrate that wet milling, seeding, and ultrasound technologies can help to optimize the particle size distribution in complex solution environments with different levels of efficiencies

Localization of CAT8 to the plasma membrane and the tonoplast in the root tip.

<p>35S::CAT8-GFP (A,D) and co-localization with FM4-64 (B,E, 15 min.) in the plasma membrane, but not in early endocytic vesicles, overlay (C,F). (G) Partial co-localization with FM4-64 (red, H) in 35S::CAT8-GFP plants after 2 h of BFA treatment. (I) Sub-cellular localization of 35S::GFP-CAT8 in the apical root zone. Scaling bars: 10 µm.</p

Similar Gln transport at pH 7.5 and partial reduction by a protonophore.

<p>(A) Fluorescence changes that correspond to an influx of Gln at pH 7.5. Superfusion by Gln (in mM) is indicated by the black bars. (B) Fluorescence changes in the presence of the protonophore CCCP (100 µM). Gln is given at times marked with black bars. Numbers indicate Glutamine concentrations in mM. (C) Concentration dependence of the fluorescence. Half maximal ratio change was K_d^app = 8.2 mM (n = 2) at pH 7.5 (closed squares) and K_d^app = 17.9 at pH 5.5 in the presence of CCCP (100 µM, n = 3, closed circles). (D) Maximal ratio changes at pH 7.5 and pH 5.5 with CCCP relative to those at pH 5.5.</p

<i>In vivo</i> function of the Gln (D157N) reporter in <i>rdr6</i> plants.

<p>(A) Confocal snapshots of a responding root tip (gray) with blue (CFP) and yellow (YFP) fluorescence. Higher magnification pictures are shown in the lower panels. (B) Normalized fluorescence ratio changes upon superfusion with Gln-containing nutrient solution of <i>Arabidopsis rdr6</i> root tips. Numbers indicate Glutamine concentrations in mM. (C) Apparent saturation kinetics of the Ratio changes revealed a K_d of 8.5 mM.</p