Aluminum (Al) tolerance in Arabidopsis is a genetically complex trait, yet it is mediated by a single physiological mechanism based on Al-activated root malate efflux. We investigated a possible molecular determinant for Al tolerance involving a homolog of the wheat Al-activated malate transporter, ALMT1. This gene, named AtALMT1 (At1g08430), was the best candidate from the 14-member AtALMT family to be involved with Al tolerance based on expression patterns and genomic location. Physiological analysis of a transferred DNA knockout mutant for AtALMT1 as well as electrophysiological examination of the protein expressed in Xenopus oocytes showed that AtALMT1 is critical for Arabidopsis Al tolerance and encodes the Al-activated root malate efflux transporter associated with tolerance. However, gene expression and sequence analysis of AtALMT1 alleles from tolerant Columbia (Col), sensitive Landsberg erecta (Ler), and other ecotypes that varied in Al tolerance suggested that variation observed at AtALMT1 is not correlated with the differences observed in Al tolerance among these ecotypes. Genetic complementation experiments indicated that the Ler allele of AtALMT1 is equally effective as the Col allele in conferring Al tolerance and Al-activated malate release. Finally, fine-scale mapping of a quantitative trait locus (QTL) for Al tolerance on chromosome 1 indicated that AtALMT1 is located proximal to this QTL. These results indicate that AtALMT1 is an essential factor for Al tolerance in Arabidopsis but does not represent the major Al tolerance QTL also found on chromosome 1. abiotic stress ͉ electrophysiology ͉ genetics ͉ organic acid exudation

A novel intracellular pH-sensitive polymeric micelle drug carrier that controls the systemic, local, and subcellular distributions of pharmacologically active drugs has been developed in this study. The micelles were prepared from self-assembling amphiphilic block copolymers, poly(ethylene glycol)-poly(aspartate hydrazone adriamycin), in which the anticancer drug, adriamycin, was conjugated to the hydrophobic segments through acid-sensitive hydrazone linkers. By this polymer design, the micelles can stably preserve drugs under physiological conditions (pH 7.4) and selectively release them by sensing the intracellular pH decrease in endosomes and lysosomes (pH 5-6). In vitro and in vivo studies show that the micelles have the characteristic properties, such as an intracellular pH-triggered drug release capability, tumor-infiltrating permeability, and effective antitumor activity with extremely low toxicity. The acquired experimental data clearly elucidate that the optimization of both the functional and structural features of polymeric micelles provides a promising formulation not only for the development of intracellular environment-sensitive supramolecular devices for cancer therapeutic applications but also for the future treatment of intractable cancers with limited vasculature.

Acid soil syndrome causes severe yield losses in various crop plants because of the rhizotoxicities of ions, such as aluminum (Al 3؉ ). Although protons (H ؉ ) could be also major rhizotoxicants in some soil types, molecular mechanisms of their tolerance have not been identified yet. One mutant that was hypersensitive to H ؉ rhizotoxicity was isolated from ethyl methanesulfonate mutagenized seeds, and a single recessive mutation was found on chromosome 1. Positional cloning followed by genomic sequence analysis revealed that a missense mutation in the zinc finger domain in a predicted Cys2His2-type zinc finger protein, namely sensitive to proton rhizotoxicity (STOP)1, is the cause of hypersensitivity to H ؉ rhizotoxicity. The STOP1 protein belongs to a functionally unidentified subfamily of zinc finger proteins, which consists of two members in Arabidopsis based on a Blast search. The stop1 mutation resulted in no effects on cadmium, copper, lanthanum, manganese and sodium chloride sensitivitities, whereas it caused hypersensitivity to Al 3؉ rhizotoxicity. This stop1 mutant lacked the induction of the AtALMT1 gene encoding a malate transporter, which is concomitant with Al-induced malate exudation. There was no induction of AtALMT1 by Al 3؉ treatment in the stop1 mutant. These results indicate that STOP1 plays a critical role in Arabidopsis tolerance to major stress factors in acid soils. aluminum toxicity ͉ Arabidopsis thaliana ͉ Cys2His2-type zinc finger protein ͉ proton-rhizotoxicity ͉ sensitive to proton rhizotoxicity

Polyplexes assembled from poly(aspartamide) derivatives bearing 1,2-diaminoethane side chains, [PAsp(DET)] display amplified in vitro and in vivo transfection activity with minimal cytotoxicity. To elucidate the molecular mechanisms involved in this unique function of PAsp(DET) polyplexes, the physicochemical and biological properties of PAsp(DET) were thoroughly evaluated with a control bearing 1,3-diaminopropane side chains, PAsp(DPT). Between PAsp(DET) and PAsp(DPT) polyplexes, we observed negligible physicochemical differences in particle size and zeta-potential. However, the one methylene variation between 1,2-diaminoethane and 1,3-diaminopropane drastically altered the transfection profiles. In sharp contrast to the constantly high transfection efficacy of PAsp(DET) polyplexes, even in regions of excess polycation to plasmid DNA (pDNA) (high N/P ratio), PAsp(DPT) polyplexes showed a significant drop in the transfection efficacy at high N/P ratios due to the progressively increased cytotoxicity with N/P ratio. The high cytotoxicity of PAsp(DPT) was closely correlated to its strong destabilization effect on cellular membrane estimated by hemolysis, leakage assay of cytoplasmic enzyme (LDH assay), and confocal laser scanning microscopic observation. Interestingly, PAsp(DET) revealed minimal membrane destabilization at physiological pH, yet there was significant enhancement in the membrane destabilization at the acidic pH mimicking the late endosomal compartment (pH approximately 5). Apparently, the pH-selective membrane destabilization profile of PAsp(DET) corresponded to a protonation change in the flanking diamine unit, i.e., the monoprotonated gauche form at physiological pH and diprotonated anti form at acidic pH. These significant results suggest that the protonated charge state of 1,2-diaminoethane may play a substantial role in the endosomal disruption. Moreover, this novel approach for endosomal disruption neither perturbs the membranes of cytoplasmic vesicles nor organelles at physiological pH. Thus, PAsp(DET) polyplexes, residing in late endosomal or lysosomal states, smoothly exit into the cytoplasm for successful transfection without compromising cell viability.

The Arabidopsis (Arabidopsis thaliana) mutant stop1 (for sensitive to proton rhizotoxicity1) carries a missense mutation at an essential domain of the histidine-2-cysteine-2 zinc finger protein STOP1. Transcriptome analyses revealed that various genes were down-regulated in the mutant, indicating that STOP1 is involved in signal transduction pathways regulating aluminum (Al)- and H+-responsive gene expression. The Al hypersensitivity of the mutant could be caused by down-regulation of AtALMT1 (for Arabidopsis ALUMINUM-ACTIVATED MALATE TRANSPORTER1) and ALS3 (ALUMINUM-SENSITIVE3). This hypothesis was supported by comparison of Al tolerance among T-DNA insertion lines and a transgenic stop mutant carrying cauliflower mosaic virus 35S∷AtALMT1. All T-DNA insertion lines of STOP1, AtALMT1, and ALS3 were sensitive to Al, but introduction of cauliflower mosaic virus 35S∷AtALMT1 did not completely restore the Al tolerance of the stop1 mutant. Down-regulation of various genes involved in ion homeostasis and pH-regulating metabolism in the mutant was also identified by microarray analyses. CBL-INTERACTING PROTEIN KINASE23, regulating a major K+ transporter, and a sulfate transporter, SULT3;5, were down-regulated in the mutant. In addition, integral profiling of the metabolites and transcripts revealed that pH-regulating metabolic pathways, such as the γ-aminobutyric acid shunt and biochemical pH stat pathways, are down-regulated in the mutant. These changes could explain the H+ hypersensitivity of the mutant and would make the mutant more susceptible in acid soil stress than other Al-hypersensitive T-DNA insertion lines. Finally, we showed that STOP1 is localized to the nucleus, suggesting that the protein regulates the expression of multiple genes that protect Arabidopsis from Al and H+ toxicities, possibly as a transcription factor.