Protective CD8+ T-cell responses to cytomegalovirus driven by rAAV/GFP/IE1 loading of dendritic cells

Background: Recent studies demonstrate that recombinant adeno-associated virus (rAAV)-based antigen loading of dendritic cells (DCs) generates in vitro, significant and rapid cytotoxic T-lymphocyte (CTL) responses against viral antigens. Methods: We used the rAAV system to induce specific CTLs against CVM antigens for the development of cytomegalovirus HCMV) gene therapy. As an extension of the versatility of the rAAV system, we incorporated immediate-early 1 (IE1), expressed in HCMV. Our rAAV vector induced a strong stimulation of CTLs directed against the HCMV antigen IE1. We then investigated the efficiency of the CTLs in killing IE1 targeted cells. Results: A significant MHC Class I-restricted, anti-IE1-specificCTL killing was demonstrated against IE1 positive peripheral blood mononuclear cells (PBMC) after one, in vitro, stimulation. Conclusion: In summary, single PBMC stimulation with rAAV/IE1 pulsed DCs induces strong antigen specific-CTL generation. CTLs were capable to lyse low doses of peptides pulsed into target cells. These data suggest that AAV-based antigen loading of DCs is highly effective for generating human CTL responses against HCMV antigens

AtCHIP, a U-Box-Containing E3 Ubiquitin Ligase, Plays a Critical Role in Temperature Stress Tolerance in Arabidopsis

The Arabidopsis gene AtCHIP encodes a protein with three tetratricopeptide repeats and a U-box domain, which is structurally similar to the animal CHIP proteins, a new class of E3 ubiquitin ligases. Like animal CHIP proteins, AtCHIP has E3 ubiquitin ligase activity in vitro. AtCHIP is a single-copy gene, and its transcript is up-regulated by several stress conditions such as low and high temperatures. However, increased AtCHIP expression alone was not correlated with increased stress tolerance; in fact, overexpression of AtCHIP in Arabidopsis rendered plants more sensitive to both low- and high-temperature treatments. Higher electrolyte leakage was observed in leaves of AtCHIP overexpression plants after chilling temperature treatment, suggesting that membrane function is likely impaired in these plants under such a condition. These results indicate that AtCHIP plays an important role in plant cellular metabolism under temperature stress conditions

Photosynthesis and seed production under water-deficit conditions in transgenic tobacco plants that over-express an Arabidopsis ascorbate peroxidase gene

An underlying mechanism for reductions in crop yield under stress conditions is excessive production of reactive oxygen species (ROS) that can damage lipids, nucleic acids, and proteins, leading to disruption of physiological processes. The aim of this study was to determine whether overexpression of the gene for a peroxisomal antioxidant enzyme, ascorbate peroxidase 3 (APX3), could provide protection of photosynthesis during drought when the potential rises for excessive photorespiratory H^sub 2^O^sub 2^ production. Tobacco (Nicotiana tabacum L.) plants were transformed to constitutively overexpress the Arabidopsis thaliana gene for APX3. Following repeated water-deficit cycles, fruit number and seed mass of transgenic plants were significantly higher than those of control plants. In another experiment, water deficit was developed gradually by reducing, in stages, the extent to which water lost was replenished. Genotypic differences in gas-exchange parameters were observed at the 25% replenishment stage and at 5 h after severely stressed plants were rewatered. At these times, transgenic plants exhibited greater rates of CO2 assimilation (A), stomatal conductance (g^sub s^), and internal CO2 (C^sub i^) to atmospheric CO2 (C^sub a^) concentration than control plants, suggesting that differences in A were controlled by differences in g^sub s^. Although these data did not support the idea that overexpression of the gene for APX3 enhances protection of the photosynthetic apparatus during water deficit, overexpression of APX3 may affect other cellular metabolisms that result in higher A under moderate water-deficit conditions and therefore higher seed mass after repeated water-deficit treatments

ANKYRIN REPEAT-CONTAINING PROTEIN 2A Is an Essential Molecular Chaperone for Peroxisomal Membrane-Bound ASCORBATE PEROXIDASE3 in Arabidopsis[W][OA]

ANKYRIN REPEAT-CONTAINING PROTEIN 2A (AKR2A) is known to be involved in targeting proteins to the chloroplast outer envelope membrane. This work provides evidence that AKR2A binds specifically to several single-membrane spanning proteins that are targeted to various cellular compartments, suggesting that AKR2A may serve as a molecular chaperone for a set of membrane proteins