A simple, fast, and orientation-controllable technology for preparing antibody-modified liposomes.

Modification with antibodies is a useful strategy for the delivery of nanoparticles to target cells. However, the complexity of the required chemical modifications makes them time-consuming and low efficiency, and the orientation of the antibody is challenging to control. To develop a simple, fast, effective, and orientation-controllable technology, we employed staphylococcal protein A, which can bind to the Fc region of antibodies, as a tool for conjugating antibodies to nanoparticles. Specifically, we modified the C-domain dimer of protein A to contain a lysine cluster to create a molecule, DPACK, that would electrostatically bind to anionic liposomes. Using this protein, antibody-modified liposomes can be prepared in 35 min with two steps: (1) interaction of DPACK with liposomes and (2) interaction of an antibody with DPACK-modified liposomes. Binding efficiencies of DPACK with liposomes and IgG with DPACK-modified liposomes were 75% and 72-84%, respectively. Uptake of liposomes modified with anti-epidermal growth factor receptor (EGFR) antibodies via DPACK by EGFR-expressing cancer cells was significantly higher than that of unmodified liposomes, and the liposomes accumulated in tumors and colocalized with EGFR. This simple, fast, effective and orientation-controllable technology for preparing antibody-modified liposomes will be useful for active targeting drug delivery.

Effective capture of proteins inside living cells by antibodies indirectly linked to a novel cell-penetrating polymer-modified protein A derivative.

Antibodies against cytoplasmic proteins are useful tools that can control cellular function and clarify signaling mechanisms. However, it is difficult to capture proteins inside living cells, and thus appropriate methods for antibody delivery to the cytoplasm of living cells are required. Cell-penetrating materials, such as the TAT-peptide, have received attention for their ability to deliver various cargos into living cells. However, the direct modification of cargos with cell-penetrating materials is time-consuming and lacks versatility. Therefore, we conceived that protein A, which can bind to the fragment crystallizable region of an antibody, could indirectly link antibodies with cell-penetrating materials, creating an efficient and simple antibody delivery system. Here, we constructed a novel antibody delivery system using a cell-penetrating polymer-modified protein A derivative (CPP-pAd). Living cells treated with CPP-pAd/antibody complexes showed significantly higher antibody levels than those achieved with the commercially available reagent HVJ-E. Pre-treatment with sucrose prevented cellular uptake of the CPP-pAd/antibody complex, suggesting that the CPP-pAd/antibody internalization mechanism occurs through clathrin-dependent endocytosis. Interestingly, intracellularly delivered antibodies did not colocalize with endosome/lysosome markers, further suggesting that antibodies were delivered to the cytoplasm by escape from endosome/lysosome. Moreover, we observed that anti-nuclear pore complex antibodies, delivered to cells using CPP-pAd, localized to the nuclear membrane and inhibited nuclear factor κB dependent luciferase activity. Together, these results suggest that the antibodies delivered by CPP-pAd captured functional proteins, making CPP-pAd a promising strategy for effective capture of proteins inside living cells.

Localization of myo-inositol-1-phosphate synthase to the endosperm in developing seeds of Arabidopsis.

Expression and localization of myo-inositol-1-phosphate synthase (MIPS) in developing seeds of Arabidopsis thaliana was investigated. MIPS is an essential enzyme for production of inositol and inositol phosphates via its circularization of glucose-6-phosphate as the initial step. myo-inositol-6-phosphate (InsP(6) or phytic acid) is the predominant form of phosphorus found in seeds and accumulates as a consequence of MIPS action. Three MIPS genes have been identified in Arabidopsis, all of which were expressed not only in siliques but in both leaves and roots. Immunoelectron microscopy using a MIPS antibody showed that MIPS localizes to the cytosol primarily in the endosperm during seed development and not in the embryo. This is consistent with results obtained using fluorescent microscopy and western blot analysis that showed a similar pattern of localization. However, InsP(6), which is the final product of inositol phosphate metabolism, was present mainly in the embryo. This suggests that a complex interaction between the endosperm and embryo occurs during the synthesis and subsequent accumulation of InsP(6) in developing seeds of Arabidopsis.

Inorganic phosphate uptake in intact vacuoles isolated from suspension-cultured cells of Catharanthus roseus (L.) G. Don under varying Pi status.

Inorganic phosphate (Pi) uptake across the vacuolar membrane of intact vacuoles isolated from Catharanthus roseus suspension-cultured cells was measured. Under low Pi status, Pi uptake into the vacuole was strongly activated compared to high Pi status. Since Pi uptake across the vacuolar membrane is correlated with H+ pumping, we examined the dependency of H+ pumping on plant Pi status. Both H+ pumping and the activities of the vacuolar H+-pumps, the V-type H+-ATPase and the H+-PPase were enhanced under low Pi status. Despite this increase in H+ pumping, Western blot analysis showed no distinct increase in the amount of proton pump proteins. Possible mechanisms for the activation of Pi uptake into the vacuole under low Pi status are discussed.

Development of a comprehensive analytical method for phosphate metabolites in plants by ion chromatography coupled with tandem mass spectrometry.

This paper describes the development of a practical method for the analysis of phosphorus compounds with a focus on sugar phosphates from the model higher plant Arabidopsis thaliana by ion chromatography coupled to electrospray ionization tandem mass spectrometry (IC-ESI-MS-MS). After the analytical separation, the potassium hydroxide eluent was converted to water with an anion suppressor allowing the effluent from the IC to be connected to the mass spectrometer directly. In the optimized method, 17 phosphorous compounds (adenosine diphosphate (ADP), fructose 1,6-bisphosphate, fructose 2,6-bisphosphate, fructose 6-phosphate, galactose 1-phosphate, glucose 1-phosphate, glucose 1,6-bisphosphate, glucose 6-phosphate, mannose 6-phosphate, phosphoenol pyrvate, 3-phosphoglyceric acid, ribulose 1,5-bisphosphate, ribulose 5-phosphate, ribose 5-phosphate, sucrose 6-phosophate and uridine 5'-diphosphate-glucose (UDPG)) were determined. The linearity of response for these phosphorous compounds over the concentration range of 0 and 10 microM was better than 0.9993 in all cases. The minimum detection limit was between 0.01 and 2.50 microM for a 25 microL injection, and recovery rates for standard addition to the sample were within the range from 93% to 110%.

Phytic acid synthesis and vacuolar accumulation in suspension-cultured cells of Catharanthus roseus induced by high concentration of inorganic phosphate and cations.

We have established a new system for studying phytic acid, myo-inositol hexakisphosphate (InsP(6)) synthesis in suspension-cultured cells of Catharanthus. InsP(6) and other intermediates of myo-inositol (Ins) phosphate metabolism were measured using an ion chromatography method. The detection limit for InsP(6) was less than 50 nM, which was sufficient to analyze Ins phosphates in living cells. Synthesis of Ins phosphates was induced by incubation in high inorganic phosphate medium. InsP(6) was mainly accumulated in vacuoles and was enhanced when cells were grown in high concentration of inorganic phosphates with the cations K(+), Ca(2+), or Zn(2+). However, there was a strong tendency for InsP(6) to accumulate in the vacuole in the presence of Ca(2+) and in nonvacuolar compartments when supplied with Zn(2+), possibly due to precipitation of InsP(6) with Zn(2+) in the cytosol. A vesicle transport inhibitor, brefeldin A, stimulated InsP(6) accumulation. The amounts of both Ins(3)P(1) myo-inositol monophosphate synthase, a key enzyme for InsP(6) synthesis, and Ins(1,4,5)P(3) kinase were unrelated to the level of accumulation of InsP(6). The mechanisms for InsP(6) synthesis and localization into vacuoles in plant cells are discussed.

Analysis of sugar phosphates in plants by ion chromatography on a titanium dioxide column with pulsed amperometric detection.

This paper describes the development of a practical method for the analysis of sugar phosphates from the model higher plant Arabidopsis thaliana by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). The extraction method of sugar phosphates from higher plants was first optimized for HPAEC-PAD analysis. In order to improve the resolution in HPAEC-PAD, a column packed with titanium dioxide resin was used. The titanium dioxide column was used as a trap-column for sugar phosphates and nucleotides, for the removal of sample matrices. Sample pretreatment was achieved in-line and automatically using a six-port valve placed after the injection valve.

Isolation of intact vacuoles and proteomic analysis of tonoplast from suspension-cultured cells of Arabidopsis thaliana.

A large number of proteins in the tonoplast, including pumps, carriers, ion channels and receptors support the various functions of the plant vacuole. To date, few proteins involved in these activities have been identified at the molecular level. In this study, proteomic analysis was used to identify new tonoplast proteins. A primary requirement of any organelle analysis by proteomics is that the purity of the isolated organelle needs to be high. Using suspension-cultured Arabidopsis cells (Arabidopsis Col-0 cell suspension), a method was developed for the isolation of intact highly purified vacuoles. No plasma membrane proteins were detected in Western blots of the isolated vacuole fraction, and only a few proteins from the Golgi and endoplasmic reticulum. The proteomic analysis of the purified tonoplast involved fractionation of the proteins by SDS-PAGE and analysis by LC-MS/MS. Using this approach, it was possible to identify 163 proteins. These included well-characterized tonoplast proteins such as V-type H+ -ATPases and V-type H+ -PPases, and others with functions reasonably expected to be related to the tonoplast. There were also a number of proteins for which a function has not yet been deduced.