In vivo assessment of specific cytotoxic T lymphocyte killing.

The direct killing of target cells by cytotoxic T lymphocytes (CTLs) plays a fundamental role in protective immunity to viral, bacterial, protozoan and fungi infections, as well as to tumor cells. In vivo cytotoxic assays take into account the interaction of target and effector cells in the context of the proper microenvironment making the analysis biologically more relevant than in vitro cytotoxic assays. Thus, the development, improvement and validation of in vivo methods are necessary in view of the importance of the results they may provide. We describe and discuss in this manuscript a method to evaluate in vivo specific cytotoxic T lymphocyte killing. We used as model system mice immunized with human recombinant replication-deficient adenovirus 5 (HAd5) containing different transgenes as the trigger of a CTL-mediated immune response. To these mice, we adoptively transferred syngeneic cells labeled with different vital fluorescent dyes. Donor cells were pulsed (target) or not (control non-target) with distinct CD8 T-cell epitopes, mixed in a 1:1 ratio and injected i.v. into immunized or non-immunized recipient mice. After 18-24h, spleen cells are collected and analysed by flow cytometry. A deviation from the 1:1 ratio of control and target cell populations indicates antigen specific lysis of target cells.

Evaluation of the Escherichia coli threonine deaminase gene as a selectable marker for plant transformation.

The initial step in the synthesis of isoleucine (Ile) is the conversion of threonine to alpha-ketobutyrate. This reaction is carried out by threonine deaminase (TD), which is feedback-regulated by Ile. Mutations in TD that manifest insensitivity to Ile feedback inhibition result in intracellular accumulation of Ile. Previous reports have shown that in planta expression of the wild-type Escherichia coli TD, ilvA, or an Ile-insensitive mutant designated ilvA-466, increased cellular concentrations of Ile. A structural analog of Ile, l-O-methylthreonine (OMT), is able to compete effectively with Ile during translation and induce cell death. It has been postulated that OMT could therefore be utilized as an effective selective agent in plant engineering studies. To test this concept, we designed two binary plasmids that harbored an nptII cassette and either the wild-type ilvA or mutant ilvA-466. The ilvA coding sequences were fused to a plastid transit peptide down stream of a modified 35S CaMV promoter. Tobacco transformations were set up implementing a selection protocol based on either kanamycin or OMT. The ilvA gene was effectively utilized as a selectable marker gene to identify tobacco transformants when coupled with OMT as the selection agent. However, the transformation efficiency was substantially lower than that observed with nptII using kanamycin as the selection agent. Moreover, in a subset of the ilvA transformants and in a majority of the ilvA-466 transgenic lines, a severe off-type was observed under greenhouse conditions that correlated with increased levels of expression of the ilvA transgene.

Efficient down-regulation of the major vegetative storage protein genes in transgenic soybean does not compromise plant productivity.

Soybean (Glycine max L. Merr.) contains two related and abundant proteins, VSP alpha and VSP beta, that have been called vegetative storage proteins (VSP) based on their pattern of accumulation, degradation, tissue localization, and other characteristics. To determine whether these proteins play a critical role in sequestering N and other nutrients during early plant development, a VspA antisense gene construct was used to create transgenic plants in which VSP expression was suppressed in leaves, flowers, and seed pods. Total VSP was reduced at least 50-fold due to a 100-fold reduction in VSP alpha and a 10-fold reduction in VSP beta. Transgenic lines were grown in replicated yield trials in the field in Nebraska during the summer of 1999 and seed harvested from the lines was analyzed for yield, protein, oil, and amino acid composition. No significant difference (alpha = 0.05) was found between down-regulated lines and controls for any of the traits tested. Young leaves of antisense plants grown in the greenhouse contained around 3% less soluble leaf protein than controls at the time of flowering. However, total leaf N did not vary. Withdrawing N from plants during seed fill did not alter final seed protein content of antisense lines compared with controls. These results indicate that the VSPs play little if any direct role in overall plant productivity under typical growth conditions. The lack of VSPs in antisense plants might be partially compensated for by increases in other proteins and/or non-protein N. The results also suggest that the VSPs could be genetically engineered or replaced without deleterious effects.

Abrogation of disease development in plants expressing animal antiapoptotic genes.

An emerging topic in plant biology is whether plants display analogous elements of mammalian programmed cell death during development and defense against pathogen attack. In many plant-pathogen interactions, plant cell death occurs in both susceptible and resistant host responses. For example, specific recognition responses in plants trigger formation of the hypersensitive response and activation of host defense mechanisms, resulting in restriction of pathogen growth and disease development. Several studies indicate that cell death during hypersensitive response involves activation of a plant-encoded pathway for cell death. Many susceptible interactions also result in host cell death, although it is not clear how or if the host participates in this response. We have generated transgenic tobacco plants to express animal genes that negatively regulate apoptosis. Plants expressing human Bcl-2 and Bcl-xl, nematode CED-9, or baculovirus Op-IAP transgenes conferred heritable resistance to several necrotrophic fungal pathogens, suggesting that disease development required host-cell death pathways. In addition, the transgenic tobacco plants displayed resistance to a necrogenic virus. Transgenic tobacco harboring Bcl-xl with a loss-of-function mutation did not protect against pathogen challenge. We also show that discrete DNA fragmentation (laddering) occurred in susceptible tobacco during fungal infection, but does not occur in transgenic-resistant plants. Our data indicate that in compatible plant-pathogen interactions apoptosis-like programmed cell death occurs. Further, these animal antiapoptotic genes function in plants and should be useful to delineate resistance pathways. These genes also have the potential to generate effective disease resistance in economically important crops.

Construction of a derivative of Agrobacterium tumefaciens C58 that does not mutate to tetracycline resistance.

Agrobacterium tumefaciens C58 mutates to tetracycline resistance at high frequency, complicating the use of many broad-host-range cloning and binary vectors that code for resistance to this antibiotic as the selection marker. Such mutations are associated with a resistant gene unit, tetC58, that is present in the genome of this strain. By deleting the tetC58 locus, we constructed NTL4, a derivative of C58 that no longer mutates to tetracycline resistance. The deletion had no detectable effect on genetic or physiological traits of NTL4 or on the ability of this strain to transform plants.

Autohydrolysis of plant polysaccharides using transgenic hyperthermophilic enzymes.

Commercial bioprocessing of plant carbohydrates, such as starch or cellulose, necessitates the use of commodity enzyme additives to accelerate polysaccharide hydrolysis. To simplify this procedure, transgenic plant tissues constitutively producing commodity enzymes were examined as a strategy for accelerating carbohydrate bioprocessing. Hyperthermophilic glycosyl hydrolases were selected to circumvent enzyme toxicity, because such enzymes are inactive at plant growth temperatures and are therefore physiologically benign. Transgenic tobacco lines were established that produced either a hyperthermophilic alpha-glucosidase or a beta-glycosidase using genes derived from the archaeon Sulfolobus solfataricus. Western blot and immunoprecipitation analyses were used to demonstrate the presence of recombinant enzymes in plant tissues. Transgenic enzyme levels exhibited an unusual delayed pattern of accumulation while their activities survived plant tissue preservation. Transgenic plant protein extracts released glucose from purified polysaccharide substrates at appreciable rates during incubation in high-temperature reactions. Glucose was also produced following enzymatic treatment of plant extracts enriched for endogenous polysaccharides. Direct conversion of plant tissue into free sugar was evident using whole plant extracts of either transgenic line, and could be significantly accelerated in a synergistic manner by combining transgenic line extracts.

Sequence of PHA synthase gene from two strains of Rhodospirillum rubrum and in vivo substrate specificity of four PHA synthases across two heterologous expression systems.

A 3.0-kb genomic fragment has been isolated from Rhodospirillum rubrum (ATCC 25903) that contains an open reading frame (ORF) with strong homology to other known polyhydroxyalkanoate (PHA) synthase genes. This ORF has lower homology to the R. rubrum strain Ha PHA synthase than would be expected within the same species. We have conducted a series of heterologous expression studies evaluating the in vivo substrate specificity of PHA synthase genes from Rhodobacter sphaeroides, Ralstonia eutropha (formerly Alcaligenes eutrophus), Thiocystis violacea, and Nocardia corrallina, within the PHA-synthase-negative hosts, Ralstonia eutropha DSM541 and Pseudomonas putida GpP104. The N. corrallina PHA synthase incorporated the highest percentage of C5 monomers in the polymer when fermented in medium supplemented with 0.1% heptanoate as the sole carbon source. When the T. violacea and R. sphaeroides were expressed in the PHA-negative host DSM541, a greater percentage of C5 monomer was observed in the polymer as compared to the expression of the PHA synthase of R. eutropha, when the transconjugants were fermented in medium supplemented with 0.4% propionate. Evaluation for preference of medium-chain-length monomers demonstrated the flexibility of the N. corrallina, T. violacea, and R. eutropha synthase genes to polymerize a copolyester composed of short- and medium-chain-length monomers when the respective transconjugants were fermented in medium supplemented with 0.5% octanoate. These studies demonstrate that the PHA synthase from N. corrallina, T. violacea, and R. eutropha are able to polymerize a copolyester composed of short- and medium-chain-length monomers, while the PHA synthase from R. sphaeroides lacks this ability and only produces a short-chain-length polymer. These observations suggest that the composition of the PHA from the PHA-producing organisms does not necessarily reflect the inherent specificity of the PHA synthase.

Electroconvulsive Therapy and Cerebral Venous Angioma.

A 24-year-old man with a left parietal cerebral venous angioma underwent a course of electroconvulsive therapy (ECT) for the treatment of bipolar affective disorder. We were able to treat this patient safely and successfully with ECT. There was no evidence of a complication related to his vascular malformation, as was demonstrated by follow-up MRI examination. Our experience suggests that ECT may be safely instituted in patients with venous angiomas, and changes in their lesions can be safely examined by MRI.